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type 2 diabetes mellitus ( t2 dm ) is associated with an increased risk of cardiovascular disease ( cvd ) mortality . approximately half of all deaths in t2 dm patients are attributable to coronary heart disease ( chd ) , and 15% are caused by stroke . in addition , the prevalence of diabetic kidney disease among adults with diabetes mellitus is reported to be 34.5% . annual assessments of urine albumin excretion and estimated glomerular filtration rate ( egfr ) are recommended to detect and monitor kidney damage in patients with t2 dm . the classification of chronic kidney disease ( ckd ) is based on both egfr and urine albumin excretion . in a randomized , controlled , multicenter trial ( advance study ) , high albuminuria and low egfr were proved to be independent risk factors for cardiovascular events among t2 dm patients . moreover , in another prospective cohort study in the united states , ckd was confirmed to be associated with an increase in stroke risk . however , the association of ckd with the risks of chd and stroke in chinese patients with t2 dm has seldom been thoroughly investigated . to evaluate these two risks , we chose the uk prospective diabetes study ( ukpds ) risk engine , which is a t2dm - specific risk calculator from the ukpds . a cross - sectional study has shown that the ukpds risk engine has good feasibility , convergent validity , and sensitivity in predicting chd risk in chinese diabetic patients . the present study sought to determine whether ckd was correlated with the chd and stroke risk scores computed using the ukpds risk engine in t2 dm patients from zhejiang , china . a total of 1401 inpatients with t2 dm ( 802 men and 599 women , aged 58.4 12.9 years [ 2194 years ] ) at the second affiliated hospital of zhejiang university school of medicine between april 2008 and november 2013 were enrolled in this cross - sectional study . individuals with obvious symptoms were diagnosed with diabetes mellitus if one of the following was present : ( 1 ) fasting blood glucose ( fbg ) 7.0 mmol / l at 2 h after an oral glucose tolerance test ; or ( 3 ) random plasma glucose 11.1 mmol / l . the exclusion criteria included the following : type 1 diabetes mellitus ; gestational diabetes mellitus ; specific types of diabetes attributable to other causes ; urinary tract infections ; cushing disease ; cancer ; hematological diseases including multiple myeloma , leukemia and lymphoma ; systemic lupus erythematosus ; and the use of drugs those affect urine albumin - to - creatinine radio ( uacr ) and egfr . this study was approved by the ethics committee of the second affiliated hospital of zhejiang university school of medicine ( zhejiang , china ) . smoking status was divided into three categories : current smoker if the individual had regularly smoked in the past 1 year ; nonsmoker if the individual had never regularly smoked ; and ex - smoker if the individual had stopped smoking for at least 1 year . body mass index was calculated as the ratio of weight ( kg ) and height squared ( m ) . waist circumference was measured at the umbilicus at the end of a normal expiration while standing . diastolic and systolic blood pressures were measured with a standard sphygmomanometer after at least 5 min of supine rest after patients came to the in - patient department . hypertension was defined as a systolic blood pressure 140 mmhg ( 1 mmhg = 0.133 kpa ) , diastolic blood pressure 90 mmhg , and/or the use of antihypertensive drugs . fbg , creatinine , total cholesterol , triglyceride , low - density lipoprotein cholesterol , and high - density lipoprotein cholesterol ( hdl - c ) were measured using the beckman coulter chemistry analyzer au5400 series ( beckman coulter biomedical k.k , tokyo , japan ) . glycosylated hemoglobin ( hba1c ) was measured by high - performance liquid chromatography using tosoh hlc-723g8 ( tosoh corporation , yamaguchi 746 - 0042 , japan ) . screening for uacr was performed using a random , spot urine sample , and the results were expressed in milligrams per gram of creatinine . urine albumin was measured by nephelometry immunoassay with the siemens bn ii system ( siemens healthcare diagnostics products gmbh , 35041 marburg , germany ) . according to the american diabetic association classification of microalbuminuria , patients were divided into three groups : normal albumin group , spot uacr < 30 mg / gcr ; microalbuminuria , spot uacr 30299 mg / gcr ; and macroalbuminuria , spot uacr 300 mg / gcr . gfr was calculated using the modified ckd - epidemiology collaboration ( ckd - epi ) equation for asians as follows . scr 9 mg / l ; egfrckd - epi= 141 ( scr/0.9 ) 0.993 1.057 scr > 9 mg / l ; egfrckd - epi= 141 ( scr/0.9 ) 0.993 1.057 scr 7 mg / l ; egfrckd - epi= 144 ( scr/0.7 ) 0.993 1.049 scr > 7 mg / l ; egfrckd - epi= 144 ( scr/0.7 ) 0.993 1.049 where scr = serum creatinine . the patients were classified into five ckd stages according to the 2002 kidney disease outcomes quality initiative ( kdoqi ) guidelines : stage 1 , kidney damage ( indicated by albuminuria 30 mg/24 h or 20 mg / l , clinical proteinuria 300 mg/24 h or 200 mg / l , or a positive dipstick test for proteinuria ) and egfr 90 mlmin1.73 m ; stage 2 , kidney damage and egfr 6089 mlmin1.73 m ; stage 3 , egfr 3059 mlmin1.73 m ; stage 4 , egfr 1529 mlmin1.73 m ; and stage 5 , egfr < 15 mlmin1.73 m. the ukpds risk engine version 2.0 for windows was used to evaluate chd and stroke risks . this scale has been validated specifically for patients with diabetes and provides an estimate of the risks for a new chd event and stroke ( fatal and nonfatal ) . the data used to calculate the ukpds risk scores ( available at http://www.dtu.ox.ac.uk/riskengine/download.php ) included hba1c , systolic blood pressure , serum total cholesterol and hdl - c , atrial fibrillation , sex , age , ethnicity , smoking status , and diabetes duration . the risks of chd , fatal chd , stroke , and fatal stroke were estimated for 10 years . chd was defined as the occurrence of a fatal or nonfatal myocardial infarction or sudden death . in this study , cases with risk higher than 20% were classified into the high - risk group whereas those with a risk score lower than 20% were classified into the low - to - moderate risk group . continuous variables were expressed as mean standard deviation , median ( 2575 percentiles ) , or mean ( 95% confidence interval [ ci ] ) , and categorical variables were reported as percentages . normally distributed continuous variables were compared using one - way analysis of variance and the least - significant difference test was used for comparisons between pairs of groups . the method of rank transform analysis was used for the data of abnormal distributions and comparisons between pairs of groups were analyzed with scheffe post hoc comparison . difference between groups were analyzed by the chi - square test for unordered categorical variables and fisher 's exact test was used if cells had expected count < 5 . binary logistic regression analysis was performed to obtain odds ratios ( ors ) and their 95% cis to predict the presence of high chd and stroke risks . all statistical tests were two - tailed , and p < 0.05 was considered statistically siginficant . a total of 1401 inpatients with t2 dm ( 802 men and 599 women , aged 58.4 12.9 years [ 2194 years ] ) at the second affiliated hospital of zhejiang university school of medicine between april 2008 and november 2013 were enrolled in this cross - sectional study . individuals with obvious symptoms were diagnosed with diabetes mellitus if one of the following was present : ( 1 ) fasting blood glucose ( fbg ) 7.0 mmol / l at 2 h after an oral glucose tolerance test ; or ( 3 ) random plasma glucose 11.1 mmol / l . the exclusion criteria included the following : type 1 diabetes mellitus ; gestational diabetes mellitus ; specific types of diabetes attributable to other causes ; urinary tract infections ; cushing disease ; cancer ; hematological diseases including multiple myeloma , leukemia and lymphoma ; systemic lupus erythematosus ; and the use of drugs those affect urine albumin - to - creatinine radio ( uacr ) and egfr . this study was approved by the ethics committee of the second affiliated hospital of zhejiang university school of medicine ( zhejiang , china ) . smoking status was divided into three categories : current smoker if the individual had regularly smoked in the past 1 year ; nonsmoker if the individual had never regularly smoked ; and ex - smoker if the individual had stopped smoking for at least 1 year . body mass index was calculated as the ratio of weight ( kg ) and height squared ( m ) . waist circumference was measured at the umbilicus at the end of a normal expiration while standing . diastolic and systolic blood pressures were measured with a standard sphygmomanometer after at least 5 min of supine rest after patients came to the in - patient department . hypertension was defined as a systolic blood pressure 140 mmhg ( 1 mmhg = 0.133 kpa ) , diastolic blood pressure 90 mmhg , and/or the use of antihypertensive drugs . fbg , creatinine , total cholesterol , triglyceride , low - density lipoprotein cholesterol , and high - density lipoprotein cholesterol ( hdl - c ) were measured using the beckman coulter chemistry analyzer au5400 series ( beckman coulter biomedical k.k , tokyo , japan ) . glycosylated hemoglobin ( hba1c ) was measured by high - performance liquid chromatography using tosoh hlc-723g8 ( tosoh corporation , yamaguchi 746 - 0042 , japan ) . screening for uacr was performed using a random , spot urine sample , and the results were expressed in milligrams per gram of creatinine . urine albumin was measured by nephelometry immunoassay with the siemens bn ii system ( siemens healthcare diagnostics products gmbh , 35041 marburg , germany ) . according to the american diabetic association classification of microalbuminuria , patients were divided into three groups : normal albumin group , spot uacr < 30 mg / gcr ; microalbuminuria , spot uacr 30299 mg / gcr ; and macroalbuminuria , spot uacr 300 mg / gcr . gfr was calculated using the modified ckd - epidemiology collaboration ( ckd - epi ) equation for asians as follows . scr 9 mg / l ; egfrckd - epi= 141 ( scr/0.9 ) 0.993 1.057 scr > 9 mg / l ; egfrckd - epi= 141 ( scr/0.9 ) 0.993 1.057 scr 7 mg / l ; egfrckd - epi= 144 ( scr/0.7 ) 0.993 1.049 scr > 7 mg / l ; egfrckd - epi= 144 ( scr/0.7 ) 0.993 1.049 where scr = serum creatinine . the patients were classified into five ckd stages according to the 2002 kidney disease outcomes quality initiative ( kdoqi ) guidelines : stage 1 , kidney damage ( indicated by albuminuria 30 mg/24 h or 20 mg / l , clinical proteinuria 300 mg/24 h or 200 mg / l , or a positive dipstick test for proteinuria ) and egfr 90 mlmin1.73 m ; stage 2 , kidney damage and egfr 6089 mlmin1.73 m ; stage 3 , egfr 3059 mlmin1.73 m ; stage 4 , egfr 1529 mlmin1.73 m ; and stage 5 , egfr < 15 mlmin1.73 m. the ukpds risk engine version 2.0 for windows was used to evaluate chd and stroke risks . this scale has been validated specifically for patients with diabetes and provides an estimate of the risks for a new chd event and stroke ( fatal and nonfatal ) . the data used to calculate the ukpds risk scores ( available at http://www.dtu.ox.ac.uk/riskengine/download.php ) included hba1c , systolic blood pressure , serum total cholesterol and hdl - c , atrial fibrillation , sex , age , ethnicity , smoking status , and diabetes duration . the risks of chd , fatal chd , stroke , and fatal stroke were estimated for 10 years . chd was defined as the occurrence of a fatal or nonfatal myocardial infarction or sudden death . in this study , cases with risk higher than 20% were classified into the high - risk group whereas those with a risk score lower than 20% were classified into the low - to - moderate risk group . continuous variables were expressed as mean standard deviation , median ( 2575 percentiles ) , or mean ( 95% confidence interval [ ci ] ) , and categorical variables were reported as percentages . normally distributed continuous variables were compared using one - way analysis of variance and the least - significant difference test was used for comparisons between pairs of groups . the method of rank transform analysis was used for the data of abnormal distributions and comparisons between pairs of groups were analyzed with scheffe post hoc comparison . difference between groups were analyzed by the chi - square test for unordered categorical variables and fisher 's exact test was used if cells had expected count < 5 . binary logistic regression analysis was performed to obtain odds ratios ( ors ) and their 95% cis to predict the presence of high chd and stroke risks . all statistical tests were two - tailed , and p < 0.05 was considered statistically siginficant . in this cohort of 1401 patients ( 802 men and 599 women ) with t2 dm , 60.9% patients had no ckd , 30.5% were in ckd stages 12 , which suggested mild renal dysfunction , and 8.6% were in stages 35 , which suggested moderate - to - severe renal dysfunction . table 1 compares the clinical and metabolic parameters of patients according to the ckd stages as defined by the kdoqi classification . patients at higher ckd stages were generally older , had diabetes for a longer duration , had higher systolic blood pressure , and had higher uacr . anthropometric and biochemical characteristics of patients with t2 dm according to ckd stage * p<0.001 ; p<0.05 , compared to cases without ckd ; p<0.05 ; p<0.001 compared to cases in ckd stages 12 . data are expressed as mean sd , median ( 2575 percentiles ) or percentages . ckd : chronic kidney disease ; dm : diabetes mellitus ; bmi : body mass index ; wc : waist circumference ; sbp : systolic blood pressure ; dbp : diastolic blood pressure ; fpg : fasting plasma glucose ; hba1c : glycosylated hemoglobin ; tc : total cholesterol ; tg : triglyceride ; hdl c : high - density lipoprotein cholesterol ; ldl c : low - density lipoprotein cholesterol ; uacr : urine albumin to creatinine ratio ; cr : creatinine ; af : atrial fibrillation ; sd : standard deviation ; t2 dm : type 2 diabetes mellitus . compared to those without ckd , patients in ckd stages 12 and ckd stages 35 had higher risks of chd , fatal chd , stroke , and fatal stroke , as calculated by the ukpds risk engine [ p < 0.001 for all ; table 2 ] . as the ckd stage deteriorated , the percentage of individuals with high chd and stroke risks also increased . the ors of chd in patients in ckd stages 12 and 35 , compared with patients without ckd , were 1.7 ( 95% ci , 1.42.2 ) and 3.5 ( 95% ci , 2.35.3 ) , respectively [ p < 0.001 for both ; table 3 ] . similarly , the ors for high stroke risk in patients in ckd stages 12 and 35 were 1.9 ( 95% ci , 1.42.7 ) and 8.2 ( 95% ci , 5.412.5 ) , respectively [ p < 0.001 for both ; table 3 ] . the percentage of individuals with high chd and stroke risks was also increased in the patients with higher uacr [ table 3 ] . risks of chd and stroke among patients with t2 dm according to ckd stage ( % ) * p<0.001 compared to cases no ckd ; p<0.001 compared to cases in ckd stages 12 . chd : coronary heart disease ; ckd : chronic kidney disease ; t2 dm : type 2 diabetes mellitus . presence and ors of high risks of chd and stroke according to ckd groups * p<0.001 compared to cases no ckd . the 10-year chd and stroke risks were estimated using the uk prospective diabetes study risk engine , and individuals were categorized as having low ( < 10% ) , intermediate ( 1020% ) or high ( > 20% ) 10-year risk . chd : coronary heart disease ; ckd : chronic kidney disease ; or : odds ratio ; ci : confidence interval ; uacr : urine albumin to creatinine ratio ; : not applicable . as the uacr elevated , the percentage of individuals with a high risk of chd and stroke also increased [ table 4 ] . compared to patients with normal urinary albumin , the ors of chd in the microalbuminuria and macroalbuminuria groups were 1.8 ( 95% ci , 1.42.4 ) and 2.0 ( 95% ci , 1.42.7 ) , respectively ( p < 0.001 for both ) . in the case of stroke risk , the ors for the above uacr groups were 2.4 ( 95% ci , 1.83.4 ) and 2.4 ( 95% ci , 1.63.5 ) , respectively ( p < 0.001 for both ) . presence and ors of high risks of chd and stroke according to uacr value * p<0.001 compared to cases with normal uacr . the 10-year chd and stroke risks were estimated using the uk prospective diabetes study risk engine , and individuals were categorized as having low ( < 10% ) , intermediate ( 1020% ) or high ( > 20% ) 10-year risk . chd : coronary heart disease ; uacr : urine albumin to creatinine ratio ; or : odds ratio ; age is known to be one of the traditional risk factors of chd . as shown in figures 1 and 2 , age - related changes in risks of coronary heart disease ( chd , a ) and stroke ( b ) according to chronic kidney disease ( ckd ) stage . age - related changes in risks of coronary heart disease ( chd , a ) and stroke ( b ) according to urine albumin - to - creatinine ratio ( uacr ) . in this study , we examined the association of ckd with risks of chd and stroke in chinese patients with t2 dm . our results showed that patients in more advanced ckd stages were generally older , with longer diabetes duration , higher systolic blood pressure , and higher uacr . furthermore , with development in the ckd stage , the percentage of patients with atrial fibrillation and hypertension also increased . we also noticed that as the ckd progressed , the percentage of current smokers decreased and the percentage of ex - smokers increased , that may be attributed to efforts to educate the patients to quit smoking . in terms of cvd risks , worsening ckd stage was associated with increasing risks of chd and stroke . of the 1401 t2 dm patients in this study , 8.6% had evidence of advanced ckd , defined as an egfr < 60 mlmin1.73 m ( stage 3 or higher ) . a national cross - sectional survey in china in 2012 showed that the prevalence of ckd ( defined as an egfr < 60 mlmin1.73 m or the presence of albuminuria ) was 10.8% , and that diabetes was the major cause of ckd . the prevalence of advanced ckd in the present study is lower than that reported in a hong kong study , which found a prevalence of 15.8% of advanced ckd in t2 dm patients and that reported in western countries such as norway ( 10.2% ) and the usa ( 17.7.0% ) , but similar to the prevalence rate reported in the mainland of china . two studies in chinese individuals with t2 dm showed that the prevalence of advanced ckd was 6% and 10.5% in hospitalized patients . in our study , 30.5% of patients were in ckd stages 12 , and a total of 39.1% of our patients had renal impairment . uacr measurements showed that 14.4% of all patients had macroalbuminuria , and 24.1% had microalbuminuria . these rates are similar to those in a study in urban chinese patients with t2 dm , which reported that the frequency of ckd ( egfr < 60 mlmin1.73 m ) and albuminuria ( uacr 30 mg / gcr ) was 31.0% and 28.9% . a study in an asian hypertensive population with t2 dm showed that the prevalence of macro- and micro - albuminuria was 18.8% and 39.8% , respectively . differences in sample selection , study populations , and egfr evaluation methods might account for the variation in the prevalence of ckd and albuminuria among different studies . with aggravation of ckd , the risks of chd and stroke , as calculated using the ukpds risk engine , increased correspondingly . the mean chd risks were 20.1% , 24.8% , and 34.3% among patients with no ckd , ckd stage 12 , and ckd stage 35 , while the corresponding stroke risks were 8.6% , 12.7% , and 25.4% , respectively . recently , a prospective cohort study in multi - ethnic asian adults showed that decreased egfr and increased albuminuria were independently associated with incident cvd . the ors of cvd and all - cause mortality were 1.54 and 2.21 , respectively , in patients with egfrs of < 45 versus 60 mlmin1.73 m , and 2.81 and 2.34 , respectively , in patients with uacrs of 300 versus < 30 mg / gcr . in an earlier study in the general population , a 2-fold increase in urine albumin concentration was found to be associated with a 1.29-fold increase in the risk for cardiovascular mortality . it was reported that cardiovascular mortality was found to be two to three times higher in patients with stage 3 or higher ckd than in individuals without renal dysfunction . in addition , a prospective study in the netherlands showed that there appears to be no threshold effect in the association of albuminuria with cardiovascular risk , unlike the nonlinear relationship between egfr and cardiovascular risk . in a population with diabetes , a prospective study in australia showed that the multivariate - adjusted hazard ratio for cardiovascular events was 2.48 for every 10-fold increase in the baseline uacr , and 2.20 for every halving of baseline egfr . moreover , patients with both uacr > 300 mg / gcr and egfr < 60 mlmin1.73 m had an obviously higher risk of cardiovascular events compared with patients with normal gfr and uacr , but no evidence of an interaction between the effects of increased uacr and decreased egfr was found . while the impact of ckd on cvd risk is less understood , data from epidemiological studies indicate that reduced egfr and increased uacr are independent risk factors for stroke . a cohort study in taiwan ( china ) reported that beyond the traditional cardiovascular risk factors , ckd was a risk factor for stroke . furthermore , we defined high risk as a chd or stroke risk of above 20% . the ors of high chd risk were 1.73.5 times higher in patients with ckd than in patients with normal kidney function . similarly , the ors of high stroke risk were 1.98.8 times higher in patients with renal impairment than in those without renal impairment . moreover , the stroke risk was 2.42.4 times higher in patients with micro- or macro - albuminuria than in patients with normal urine albumin . it has been suggested that there exist racial differences in albuminuria , kidney function , and stroke risk . high uacr is independently associated with high stroke risk in black people , and reduced egfr is more closely associated with the incident stroke risk in white people . furthermore , the chronic renal insufficiency cohort study recently reported that compared to egfr , albuminuria is a better predictor of stroke risk in patients with ckd . although the exact mechanism is not clear , hypertension , left ventricular hypertrophy , dyslipidemia , low - grade inflammation , increased activity of the renin angiotensin system , sympathetic nervous system activity , and deficiency of active vitamin d have been implicated as the mechanisms that mediate the relationship between ckd and cvd risks . furthermore , it has been confirmed that increased uacr and impaired egfr are independently and additively associated with increased chd risk . in addition , a multicenter , observational , prospective cohort study in china indicates that renal insufficiency is an important independent predictor of cardiovascular outcome in patients with acute stemi . there are some limitations to our study . in this study , we measured renal impairment through uacr and egfr . both these indexes have been used to evaluate the occurrence and development of renal dysfunction in patients with diabetes . however , a retrospective analysis of biopsy data has indicated that approximately 8% of chinese t2 dm patients may have co - existing diabetic nephropathy and nondiabetic renal disease . such comorbidities may affect prognosis and treatment , and the evaluation of chd and stroke risks . in general , diabetic nephropathy is diagnosed on the basis of the duration of diabetes , uacr , and egfr rather than renal biopsy . as we excluded urinary tract infections , systemic lupus erythematosus , and hepatitis b virus - associated nephropathy , we considered our results would still be reliable . second , this is a cross - sectional study , and a further prospective study is required to investigate the development and progression of nephropathy and its association with cvd events . in conclusion , the present study showed that worsening ckd stage in patients with t2 dm was associated with increases in risks of chd and stroke . chd and stroke risks were greater in t2 dm patients with ckd than those without ckd . similar results were found for uacr as patients with increased uacr were proven with a higher proportion of high risks of chd and stroke . the assessments of ckd stages could be useful in identifying t2 dm patients with high risks of chd and stroke . this study was supported in part by the grants from the national natural science foundation of china ( no . 81370968 , no . 81670744 ) , the chinese society of endocrinology ( no . 13040620447 ) , and the science technology department of zhejiang province of china ( no . 2012r10038 ) . there are no conflicts of interest . this study was supported in part by the grants from the national natural science foundation of china ( no . 81370968 , no . 81670744 ) , the chinese society of endocrinology ( no . 13040620447 ) , and the science technology department of zhejiang province of china ( no .

background : chronic kidney disease ( ckd ) is an independent risk factor for cardiovascular disease ( cvd ) . however , the association between ckd and cvd risk in patients with type 2 diabetes mellitus ( t2 dm ) in china has not yet been well investigated . this study aimed to determine the association of ckd with the risks of coronary heart disease ( chd ) and stroke in a chinese population with t2dm.methods:a total of 1401 inpatients with t2 dm at the second affiliated hospital of zhejiang university school of medicine between april 2008 and november 2013 were included in this study . the ckd - epidemiology collaboration equation for asians was used to classify ckd . the uk prospective diabetes study risk engine was used to estimate the risks of chd and stroke.results:chd risk was significantly increased with ckd stage ( 20.1% , 24.8% , and 34.3% in t2 dm patients with no ckd , ckd stage 12 , and stage 35 , respectively ; p < 0.001 for all ) . the stroke risk was also increased with ckd stage ( 8.6% , 12.7% , and 25.4% in t2 dm patients with no ckd , ckd stage 12 , and stage 35 , respectively ; p < 0.001 for all ) . compared with no - ckd group , the odds ratios ( ors ) for high chd risk were 1.7 ( p < 0.001 ) in the ckd stage 12 group and 3.5 ( p < 0.001 ) in the ckd stage 35 group . the corresponding ors for high stroke risk were 1.9 ( p < 0.001 ) and 8.2 ( p < 0.001 ) , respectively.conclusion:in patients with t2 dm , advanced ckd stage was associated with the increased risks of chd and stroke .

I M Subjects Biochemical parameters Urine albumin-to-creatinine radio groups Chronic kidney disease subgroups UK Prospective Diabetes Study risk engine Statistical analysis R D Financial support and sponsorship Conflicts of interest

type 2 diabetes mellitus ( t2 dm ) is associated with an increased risk of cardiovascular disease ( cvd ) mortality . approximately half of all deaths in t2 dm patients are attributable to coronary heart disease ( chd ) , and 15% are caused by stroke . in addition , the prevalence of diabetic kidney disease among adults with diabetes mellitus is reported to be 34.5% . annual assessments of urine albumin excretion and estimated glomerular filtration rate ( egfr ) are recommended to detect and monitor kidney damage in patients with t2 dm . the classification of chronic kidney disease ( ckd ) is based on both egfr and urine albumin excretion . in a randomized , controlled , multicenter trial ( advance study ) , high albuminuria and low egfr were proved to be independent risk factors for cardiovascular events among t2 dm patients . moreover , in another prospective cohort study in the united states , ckd was confirmed to be associated with an increase in stroke risk . however , the association of ckd with the risks of chd and stroke in chinese patients with t2 dm has seldom been thoroughly investigated . to evaluate these two risks , we chose the uk prospective diabetes study ( ukpds ) risk engine , which is a t2dm - specific risk calculator from the ukpds . a cross - sectional study has shown that the ukpds risk engine has good feasibility , convergent validity , and sensitivity in predicting chd risk in chinese diabetic patients . the present study sought to determine whether ckd was correlated with the chd and stroke risk scores computed using the ukpds risk engine in t2 dm patients from zhejiang , china . a total of 1401 inpatients with t2 dm ( 802 men and 599 women , aged 58.4 12.9 years [ 2194 years ] ) at the second affiliated hospital of zhejiang university school of medicine between april 2008 and november 2013 were enrolled in this cross - sectional study . the exclusion criteria included the following : type 1 diabetes mellitus ; gestational diabetes mellitus ; specific types of diabetes attributable to other causes ; urinary tract infections ; cushing disease ; cancer ; hematological diseases including multiple myeloma , leukemia and lymphoma ; systemic lupus erythematosus ; and the use of drugs those affect urine albumin - to - creatinine radio ( uacr ) and egfr . this study was approved by the ethics committee of the second affiliated hospital of zhejiang university school of medicine ( zhejiang , china ) . gfr was calculated using the modified ckd - epidemiology collaboration ( ckd - epi ) equation for asians as follows . the patients were classified into five ckd stages according to the 2002 kidney disease outcomes quality initiative ( kdoqi ) guidelines : stage 1 , kidney damage ( indicated by albuminuria 30 mg/24 h or 20 mg / l , clinical proteinuria 300 mg/24 h or 200 mg / l , or a positive dipstick test for proteinuria ) and egfr 90 mlmin1.73 m ; stage 2 , kidney damage and egfr 6089 mlmin1.73 m ; stage 3 , egfr 3059 mlmin1.73 m ; stage 4 , egfr 1529 mlmin1.73 m ; and stage 5 , egfr < 15 mlmin1.73 m. the ukpds risk engine version 2.0 for windows was used to evaluate chd and stroke risks . this scale has been validated specifically for patients with diabetes and provides an estimate of the risks for a new chd event and stroke ( fatal and nonfatal ) . the risks of chd , fatal chd , stroke , and fatal stroke were estimated for 10 years . continuous variables were expressed as mean standard deviation , median ( 2575 percentiles ) , or mean ( 95% confidence interval [ ci ] ) , and categorical variables were reported as percentages . binary logistic regression analysis was performed to obtain odds ratios ( ors ) and their 95% cis to predict the presence of high chd and stroke risks . all statistical tests were two - tailed , and p < 0.05 was considered statistically siginficant . a total of 1401 inpatients with t2 dm ( 802 men and 599 women , aged 58.4 12.9 years [ 2194 years ] ) at the second affiliated hospital of zhejiang university school of medicine between april 2008 and november 2013 were enrolled in this cross - sectional study . the exclusion criteria included the following : type 1 diabetes mellitus ; gestational diabetes mellitus ; specific types of diabetes attributable to other causes ; urinary tract infections ; cushing disease ; cancer ; hematological diseases including multiple myeloma , leukemia and lymphoma ; systemic lupus erythematosus ; and the use of drugs those affect urine albumin - to - creatinine radio ( uacr ) and egfr . this study was approved by the ethics committee of the second affiliated hospital of zhejiang university school of medicine ( zhejiang , china ) . gfr was calculated using the modified ckd - epidemiology collaboration ( ckd - epi ) equation for asians as follows . the patients were classified into five ckd stages according to the 2002 kidney disease outcomes quality initiative ( kdoqi ) guidelines : stage 1 , kidney damage ( indicated by albuminuria 30 mg/24 h or 20 mg / l , clinical proteinuria 300 mg/24 h or 200 mg / l , or a positive dipstick test for proteinuria ) and egfr 90 mlmin1.73 m ; stage 2 , kidney damage and egfr 6089 mlmin1.73 m ; stage 3 , egfr 3059 mlmin1.73 m ; stage 4 , egfr 1529 mlmin1.73 m ; and stage 5 , egfr < 15 mlmin1.73 m. the ukpds risk engine version 2.0 for windows was used to evaluate chd and stroke risks . this scale has been validated specifically for patients with diabetes and provides an estimate of the risks for a new chd event and stroke ( fatal and nonfatal ) . the risks of chd , fatal chd , stroke , and fatal stroke were estimated for 10 years . in this study , cases with risk higher than 20% were classified into the high - risk group whereas those with a risk score lower than 20% were classified into the low - to - moderate risk group . binary logistic regression analysis was performed to obtain odds ratios ( ors ) and their 95% cis to predict the presence of high chd and stroke risks . all statistical tests were two - tailed , and p < 0.05 was considered statistically siginficant . in this cohort of 1401 patients ( 802 men and 599 women ) with t2 dm , 60.9% patients had no ckd , 30.5% were in ckd stages 12 , which suggested mild renal dysfunction , and 8.6% were in stages 35 , which suggested moderate - to - severe renal dysfunction . anthropometric and biochemical characteristics of patients with t2 dm according to ckd stage * p<0.001 ; p<0.05 , compared to cases without ckd ; p<0.05 ; p<0.001 compared to cases in ckd stages 12 . ckd : chronic kidney disease ; dm : diabetes mellitus ; bmi : body mass index ; wc : waist circumference ; sbp : systolic blood pressure ; dbp : diastolic blood pressure ; fpg : fasting plasma glucose ; hba1c : glycosylated hemoglobin ; tc : total cholesterol ; tg : triglyceride ; hdl c : high - density lipoprotein cholesterol ; ldl c : low - density lipoprotein cholesterol ; uacr : urine albumin to creatinine ratio ; cr : creatinine ; af : atrial fibrillation ; sd : standard deviation ; t2 dm : type 2 diabetes mellitus . compared to those without ckd , patients in ckd stages 12 and ckd stages 35 had higher risks of chd , fatal chd , stroke , and fatal stroke , as calculated by the ukpds risk engine [ p < 0.001 for all ; table 2 ] . as the ckd stage deteriorated , the percentage of individuals with high chd and stroke risks also increased . the ors of chd in patients in ckd stages 12 and 35 , compared with patients without ckd , were 1.7 ( 95% ci , 1.42.2 ) and 3.5 ( 95% ci , 2.35.3 ) , respectively [ p < 0.001 for both ; table 3 ] . similarly , the ors for high stroke risk in patients in ckd stages 12 and 35 were 1.9 ( 95% ci , 1.42.7 ) and 8.2 ( 95% ci , 5.412.5 ) , respectively [ p < 0.001 for both ; table 3 ] . the percentage of individuals with high chd and stroke risks was also increased in the patients with higher uacr [ table 3 ] . risks of chd and stroke among patients with t2 dm according to ckd stage ( % ) * p<0.001 compared to cases no ckd ; p<0.001 compared to cases in ckd stages 12 . chd : coronary heart disease ; ckd : chronic kidney disease ; t2 dm : type 2 diabetes mellitus . presence and ors of high risks of chd and stroke according to ckd groups * p<0.001 compared to cases no ckd . the 10-year chd and stroke risks were estimated using the uk prospective diabetes study risk engine , and individuals were categorized as having low ( < 10% ) , intermediate ( 1020% ) or high ( > 20% ) 10-year risk . chd : coronary heart disease ; ckd : chronic kidney disease ; or : odds ratio ; ci : confidence interval ; uacr : urine albumin to creatinine ratio ; : not applicable . as the uacr elevated , the percentage of individuals with a high risk of chd and stroke also increased [ table 4 ] . compared to patients with normal urinary albumin , the ors of chd in the microalbuminuria and macroalbuminuria groups were 1.8 ( 95% ci , 1.42.4 ) and 2.0 ( 95% ci , 1.42.7 ) , respectively ( p < 0.001 for both ) . in the case of stroke risk , the ors for the above uacr groups were 2.4 ( 95% ci , 1.83.4 ) and 2.4 ( 95% ci , 1.63.5 ) , respectively ( p < 0.001 for both ) . presence and ors of high risks of chd and stroke according to uacr value * p<0.001 compared to cases with normal uacr . the 10-year chd and stroke risks were estimated using the uk prospective diabetes study risk engine , and individuals were categorized as having low ( < 10% ) , intermediate ( 1020% ) or high ( > 20% ) 10-year risk . chd : coronary heart disease ; uacr : urine albumin to creatinine ratio ; or : odds ratio ; age is known to be one of the traditional risk factors of chd . as shown in figures 1 and 2 , age - related changes in risks of coronary heart disease ( chd , a ) and stroke ( b ) according to chronic kidney disease ( ckd ) stage . age - related changes in risks of coronary heart disease ( chd , a ) and stroke ( b ) according to urine albumin - to - creatinine ratio ( uacr ) . in this study , we examined the association of ckd with risks of chd and stroke in chinese patients with t2 dm . furthermore , with development in the ckd stage , the percentage of patients with atrial fibrillation and hypertension also increased . we also noticed that as the ckd progressed , the percentage of current smokers decreased and the percentage of ex - smokers increased , that may be attributed to efforts to educate the patients to quit smoking . in terms of cvd risks , worsening ckd stage was associated with increasing risks of chd and stroke . of the 1401 t2 dm patients in this study , 8.6% had evidence of advanced ckd , defined as an egfr < 60 mlmin1.73 m ( stage 3 or higher ) . a national cross - sectional survey in china in 2012 showed that the prevalence of ckd ( defined as an egfr < 60 mlmin1.73 m or the presence of albuminuria ) was 10.8% , and that diabetes was the major cause of ckd . the prevalence of advanced ckd in the present study is lower than that reported in a hong kong study , which found a prevalence of 15.8% of advanced ckd in t2 dm patients and that reported in western countries such as norway ( 10.2% ) and the usa ( 17.7.0% ) , but similar to the prevalence rate reported in the mainland of china . two studies in chinese individuals with t2 dm showed that the prevalence of advanced ckd was 6% and 10.5% in hospitalized patients . in our study , 30.5% of patients were in ckd stages 12 , and a total of 39.1% of our patients had renal impairment . these rates are similar to those in a study in urban chinese patients with t2 dm , which reported that the frequency of ckd ( egfr < 60 mlmin1.73 m ) and albuminuria ( uacr 30 mg / gcr ) was 31.0% and 28.9% . a study in an asian hypertensive population with t2 dm showed that the prevalence of macro- and micro - albuminuria was 18.8% and 39.8% , respectively . differences in sample selection , study populations , and egfr evaluation methods might account for the variation in the prevalence of ckd and albuminuria among different studies . with aggravation of ckd , the risks of chd and stroke , as calculated using the ukpds risk engine , increased correspondingly . the mean chd risks were 20.1% , 24.8% , and 34.3% among patients with no ckd , ckd stage 12 , and ckd stage 35 , while the corresponding stroke risks were 8.6% , 12.7% , and 25.4% , respectively . the ors of cvd and all - cause mortality were 1.54 and 2.21 , respectively , in patients with egfrs of < 45 versus 60 mlmin1.73 m , and 2.81 and 2.34 , respectively , in patients with uacrs of 300 versus < 30 mg / gcr . in an earlier study in the general population , a 2-fold increase in urine albumin concentration was found to be associated with a 1.29-fold increase in the risk for cardiovascular mortality . it was reported that cardiovascular mortality was found to be two to three times higher in patients with stage 3 or higher ckd than in individuals without renal dysfunction . in addition , a prospective study in the netherlands showed that there appears to be no threshold effect in the association of albuminuria with cardiovascular risk , unlike the nonlinear relationship between egfr and cardiovascular risk . in a population with diabetes , a prospective study in australia showed that the multivariate - adjusted hazard ratio for cardiovascular events was 2.48 for every 10-fold increase in the baseline uacr , and 2.20 for every halving of baseline egfr . moreover , patients with both uacr > 300 mg / gcr and egfr < 60 mlmin1.73 m had an obviously higher risk of cardiovascular events compared with patients with normal gfr and uacr , but no evidence of an interaction between the effects of increased uacr and decreased egfr was found . while the impact of ckd on cvd risk is less understood , data from epidemiological studies indicate that reduced egfr and increased uacr are independent risk factors for stroke . a cohort study in taiwan ( china ) reported that beyond the traditional cardiovascular risk factors , ckd was a risk factor for stroke . the ors of high chd risk were 1.73.5 times higher in patients with ckd than in patients with normal kidney function . similarly , the ors of high stroke risk were 1.98.8 times higher in patients with renal impairment than in those without renal impairment . moreover , the stroke risk was 2.42.4 times higher in patients with micro- or macro - albuminuria than in patients with normal urine albumin . it has been suggested that there exist racial differences in albuminuria , kidney function , and stroke risk . high uacr is independently associated with high stroke risk in black people , and reduced egfr is more closely associated with the incident stroke risk in white people . furthermore , the chronic renal insufficiency cohort study recently reported that compared to egfr , albuminuria is a better predictor of stroke risk in patients with ckd . although the exact mechanism is not clear , hypertension , left ventricular hypertrophy , dyslipidemia , low - grade inflammation , increased activity of the renin angiotensin system , sympathetic nervous system activity , and deficiency of active vitamin d have been implicated as the mechanisms that mediate the relationship between ckd and cvd risks . furthermore , it has been confirmed that increased uacr and impaired egfr are independently and additively associated with increased chd risk . in addition , a multicenter , observational , prospective cohort study in china indicates that renal insufficiency is an important independent predictor of cardiovascular outcome in patients with acute stemi . both these indexes have been used to evaluate the occurrence and development of renal dysfunction in patients with diabetes . however , a retrospective analysis of biopsy data has indicated that approximately 8% of chinese t2 dm patients may have co - existing diabetic nephropathy and nondiabetic renal disease . such comorbidities may affect prognosis and treatment , and the evaluation of chd and stroke risks . in conclusion , the present study showed that worsening ckd stage in patients with t2 dm was associated with increases in risks of chd and stroke . chd and stroke risks were greater in t2 dm patients with ckd than those without ckd . similar results were found for uacr as patients with increased uacr were proven with a higher proportion of high risks of chd and stroke . the assessments of ckd stages could be useful in identifying t2 dm patients with high risks of chd and stroke . 13040620447 ) , and the science technology department of zhejiang province of china ( no . 81670744 ) , the chinese society of endocrinology ( no . 13040620447 ) , and the science technology department of zhejiang province of china ( no .

the state of non - rem sleep ( nrem ) , or slow wave sleep , is associated with a synchronized eeg pattern in which specific electrographic events take place . these events are sleep spindles and/or k complexes and high - voltage slow wave activity ( swa ) within the delta frequency band ( between 0.5 and 4.0 hz ) that can be recorded over the entire cortical surface . in humans , nrem is subdivided into stages 2 and 34 depending on the proportions of each of these polygraphic events . stage 2 , with sleep spindles and k complexes and less than 20% of swa in the recording , corresponds , from the behavioral point of view , to a light sleep with a low - threshold to awakening . behaviorally , stage 34 , with swa occupying at least 20% of the recording , is a deeper sleep . recently , in the new sleep scoring classification of the aasm ( iber et al . , 2007 ) the former stage 34 is identified as only one stage , named n3 . in young adults stage 34 ( n3 ) is especially abundant in the first third of the night ( figure 1 ) . its proportions show notable stability in long and short sleepers , and it is the first stage to recover after sleep deprivation ; it is considered to be the main component of the core sleep necessary for normal physical and intellectual performance and behavior . recently , in a revision of the publications describing the anatomical connections and effects of lesions and electrical stimulation of specific brain structures on the sleep wakefulness cycle ( swc ) we outlined the brain structures located at practically all levels of the encephalon that participate in the organization of the nrem state ( reinoso - surez et al . , 2011 ; figure 2 ) . also , different findings have shown that impulses from peripheral nerves and spinal cord can modify brain electrical activity increasing eeg synchronization and producing swa , as occurs after stimulation of cutaneous nerves ( pompeiano and swett , 1962 ) , after repetitive visual stimuli or diffused and permanent illumination of the retina ( mancia et al . , 1959 ) , or after afferent volleys originating in the vagus nerve ( puizillout et al . conversely , decrease of swa in the eeg ( and therefore enhancement of desynchronized eeg ) takes place after suppression of trigeminal nerve afferents ( vies - morros , 1959 ) , and after lesion or blockage of the spinal cord , thus indicating that ascending synchronizing impulses can also be generated in the spinal cord ( hodes , 1964 ; de andrs et al . , 1976 ) . a parasagittal section of cat brain shows a shaded area representing the brain structures where lesion decreases nrem sleep and/or eeg synchronization or stimulation increases nrem sleep . the main connections between these structures and the structures responsible of the organization of wakefulness ( encompassed in red line ) or rem sleep ( blue lines ) with the exception of the efferent hypothalamic and midbrain connections and the brainstem connections from the basal forebrain are shown . the thalamus cerebral cortex complex or unit is darker to emphasize that these structures are necessary for the behavioral and bioelectric signs that characterize nrem sleep . ac , anterior commissure ; cc , corpus callosum ; dcn , deep cerebellar nuclei ; fo , fornix ; g7 , genu of the facial nerve ; ic , inferior colliculus ; io , inferior olive ; lv , lateral ventricle ; mrf ; midbrain reticular formation ; mt , medullary tegmentum ; och , optic chiasm ; pgs , periaqueductal gray substance ; rpc , caudal pontine reticular nucleus ; rpo , oral pontine reticular nucleus ; sc , superior colliculus ; sc and pn , spinal cord and peripheral nerves ; sn , solitary nucleus ; tb , trapezoid body . modified from reinoso - surez et al . ( 2011 ) . when the medulla and the caudal pons are separated from more cranially located brainstem structures , active and resting periods are still organized in a manner that can resemble very elemental waking and sleep states ( siegel et al . , but , in the absence of influence from higher brainstem structures , the medulla , and caudal pons are unable to generate true nrem or rem sleep signs since the full expression of these states does not occur in chronic medullary or midpontine cats ( reinoso - surez et al . , 2011 ) . however , lesion and stimulation experiments in our and other laboratories demonstrated that eeg synchronizing hypnogenic influences on the rostral brainstem and prosencephalon originate in the caudal pons and medulla at the level of the nucleus of the solitary tract ( magnes et al . reinoso - barbero and de andrs , 1995 ) and caudal pontine reticular nucleus ( batini et al . , 1958 ; cordeau and mancia , 1959 ; reinoso - surez et al . , 1962 ; rossi et al . , 1963 ; camacho - evangelista and reinoso - surez , 1964 ; zarranz and reinoso - surez , 1971 ; reinoso - surez and de andrs , 1976 ; garzn , 1996 ) these findings , together with the rostral projections of these lower brainstem structures ( reinoso - surez et al . , 1977 ) , support the assertion by moruzzi ( 1972 ) that the deactivating structures of the lower brainstem may inhibit the ascending reticular system and counteract its influence at the level of the diencephalon . our group reported that lesions in the long ascending tracts at the level of the oral pontine tegmentum produce eeg desynchronization ; bilateral desynchronization of the eeg also followed uni- or bilateral lesions in the superior cerebellar peduncle that destroyed the brachium conjunctivum tract ( camacho - evangelista and reinoso - surez , 1964 , 1965 ) . this led us to infer that these lesions suppressed ascendant hypnogenic synchronizing impulses from more caudal levels of the brainstem and the deep cerebellar nuclei . in the case of lesion to the brachium conjunctivum tract , the suppression of the synchronizing impulses was associated to a bilateral increase of desynchronized eeg but also to the generation of bursts of intermediate- to high - voltage theta band eeg activity recorded in cortical areas ( motor and parietal cortices ) that are targets of the thalamic nuclei where cerebellar projections end ( camacho - evangelista and reinoso - surez , 1965 ; reinoso - surez , 1992 , 1993 ) . in agreement with these results , unilateral lesions of the brachium conjunctivum in cats increased wakefulness and drowsiness while decreasing nrem and rem sleep ( de andrs and reinoso - surez , 1979 ) . sleep spindles and delta swa can not be observed in decerebrate animals in which the brainstem has been released from prosencephalic influences ( villablanca , 1966 ; de andrs and corpas , 1991 ) , even though , these preparations show the behavioral and bioelectric rem sleep signs . on the other hand , the isolated forebrain ( or cerveau isol preparation ) of cats transected at the intercollicular level , which lacks brainstem influences , is able to express the full bioelectric activity patterns of nrem sleep ( villablanca , 1965 ; corpas and de andrs , 1991 ) . in addition , when the third cranial nerves and nuclei are not affected by the transection in the cerveau isol preparation , the bioelectric eeg activities in the isolated forebrain are coupled to pupil size changes , that is , myosis during synchronized eeg and mydriasis during desynchronized eeg , that occur in intact cats during nrem sleep and wakefulness , respectively ( villablanca , 2004 ) . furthermore , prosencephalic mechanisms are sufficient for homeostatic regulation of nrem sleep : pharmacologic deprivation of this state in the cerveau isol preparation is followed as in intact animals by an nrem sleep rebound ( corpas and de andrs , 1991 ) . in addition , clinical , and lesion and stimulation experiments made it possible to describe a rostral nrem hypnogenic system that includes the basal forebrain , and preoptic and anterior hypothalamic regions ( von economo , 1930 ; hess , 1931 ; nauta , 1946 ; hernndez - pen , 1962 ; sterman and clemente , 1962 ; madoz and reinoso - surez , 1968 ; mcginty and sterman , 1968 ; szymusiak and mcginty , 1986 ) . the ventral lateral preoptic area and the median preoptic nucleus show a higher density of neurons with increased activity during nrem episodes than do other anterior hypothalamic regions ( szymusiak et al . , 1998 ) . also , lesion , stimulation , and clinical studies demonstrate that the thalamus is necessary to generate the electrophysiological and behavioral manifestations that characterize nrem sleep ( hess , 1931 , 1968 ; morrison and dempsey , 1942 ; villablanca , 1974 ; steriade et al . the chronic athalamic cat shows wakefulness and rem sleep but not typical nrem sleep ( villablanca , 2004 ) . also villablanca ( 1972 , 2004 ) affirmed that true nrem sleep is absent in chronic cats without telencephalon ( diencephalic cats ) because neither delta swa nor sleep spindles could be recorded in the thalamus . in summary , it appears that the thalamus and the cerebral cortex are absolutely necessary for the expression of the most significant bioelectric and behavioral events of nrem sleep . other structures like the basal forebrain , cerebellum and caudal brain stem , and impulses from the spinal cord and peripheral nerves modulate nrem sleep but are not required for the actual generation of the bioelectric and behavioral signs accompanying the expression of nrem sleep ( figure 2 ) . the cerebral cortex is the main afferent and efferent structure for the thalamus , and , in turn , the thalamus is the main subcortical afferent and efferent structure for the cerebral cortex ( reinoso - surez et al . , 2011 ; figures 3 and 4 ) . in both cases these connections are made of two types of projection neurons ( jones , 2001 ; rubio - garrido et al . , 2009 ; figures 3 and 4 ) : the thalamus sends projections from core- ( c ) and matrix- ( m ) type thalamocortical neurons and the cerebral cortex sends projections from layer vi and layer v neurons . thalamic nuclei , cell types , and thalamocortical relationships . the lower part of the figure represents a schematic coronal section of the primate diencephalon and the upper drawing depicts a schematic section of the primary and association cerebral cortices . thalamic lateral posterior ( lp ) and ventral posterior ( vp ) nuclei are represented as examples of high - order and first - order thalamic nuclei , respectively . c , core - type cells ; intr , thalamic intralaminar nuclei ; it , gabaergic interneurons ; m , matrix - type cells ; md , medial dorsal thalamic nucleus ; nr , thalamic reticular nucleus ; i vi , cortical layers . modified from reinoso - surez et al . the projection neurons , pyramidal neurons in layers ii iii , v , and vi , are represented in different colors according to their origin and targets . the two types of interneurons are represented in different colors : ( 1 ) the excitatory interneuron , a spiny stellate cell , is in pink ; and ( 2 ) the inhibitory interneurons are in light green . there are four specific examples of inhibitory interneuron : two dendrite - and tuft - targeting cells [ cajal - retzius ( c - r ) and martinotti ( m ) neurons ] , one dendrite targeting cell [ double bouquet ( db ) neuron ] , and one axon targeting cell [ chandelier ( c ) neuron ] . afferent fibers from cortical and subcortical origins are represented in different colors and specific distributions . the wide distributions of dopaminergic ( da ) , serotonergic ( 5-ht ) , noradrenergic ( na ) , histaminergic ( his ) , orexinergic ( orx ) , and gabaergic ( gaba ) fibers originating in brainstem , diencephalic , and basal prosencephalic structures are represented by their terminals , as is the topographically organized terminals of the basal forebrain cholinergic ( ach ) fibers . thalamocortical fibers targeting cortical layers i ( m - type ) and iv ( c - type ) are also shown . open triangles , excitatory terminals ; solid triangles , inhibitory terminals . from reinoso - surez et al . the axons of both types ( m and c ) of thalamocortical projection neurons provide collateral branches to the thalamic reticular nucleus when traversing it on their way to the cerebral cortex . the gabaergic thalamic reticular neurons that receive these collaterals project back , modulating both the thalamic projection neurons that provide the thalamocortical axons as well as the related interneurons ( figure 3 ) . thalamic innervation in cortical layer i is at least as heavy as that in layer iv ; every part of cortical layer i receives combined input from multiple thalamic nuclei ( rubio - garrido et al . , 2009 ) . since the 1980s , the thalamocortical and corticothalamic cellular bases of sleep spindles and delta swa generation have been extensively studied by steriade et al . these authors showed that the combination of the intrinsic electrophysiological properties of the thalamic and cortical neurons , together with their synaptic connections , is responsible for the generation of sleep spindles and delta swa oscillations in the eeg during nrem sleep ( for review see nez - molina and amzica , 2004 ; fuentealba and steriade , 2005 ) . thus , the eeg activities , which appear during stages 2 and 34 ( n3 ) of nrem sleep are essentially generated by the activity of neurons in the thalamic reticular nucleus , in the thalamocortical projecting nuclei , and in the cerebral cortex . 1985 ) proposed , early on , that the neurons of the thalamic reticular nucleus are pacemakers for sleep spindles , based on the absence of the sleep spindle oscillation in thalamocortical systems when they are disconnected from the thalamic reticular nucleus and the presence of spindle rhythmicity in the deafferentized reticular thalamic nucleus . bal and mccormick ( 1993 ) demonstrated that reticular nucleus neurons have an intrinsic rhythmic activity at spindle frequencies ( 712 hz ) in slice preparations . the thalamic reticular nucleus is a relatively thin sheet of neurons that surrounds the anterior , lateral and , to some extent , ventral surfaces of the dorsal thalamus , and it is entirely composed of gabaergic cells . because of its anatomical position the thalamic reticular nucleus is traversed by virtually all axons connecting the dorsal thalamus with the neocortex , giving the nucleus its reticulated appearance and name ; it also receives collateral branches from corticothalamic fibers originating in cortical layer vi ( figures 3 and 5 ) . thalamic reticular nucleus neurons do not project directly to the cortex although they widely project to the relay thalamic nuclei . simultaneous intracellular recordings in reticular , thalamocortical , and cortical neurons ( steriade and deschenes , 1988 ) indicated that sleep spindle generation was produced by a chain of events with three main consecutive steps : ( 1 ) repetitive spike - bursts at spindle frequency generated by gabaergic thalamic reticular nucleus neurons ; ( 2 ) transfer of this activity to relay thalamocortical nuclei producing rhythmic ( at spindle frequency ) inhibitory postsynaptic potentials ( ipsps ) in thalamocortical neurons ; and ( 3 ) transfer to the cerebral cortex of postinhibitory rebound spike - bursts of the thalamocortical cells producing excitatory postsynaptic potentials ( epsps ) in cortical cells at spindle frequency . the repetitive spike - bursts of the reticular nucleus neurons are na action potentials generated by low - threshold ca spikes at spindle frequency ( bal and mccormick , 1993 ) . since the reticular nucleus projection is gabaergic , the reticular nucleus activities are transmitted to the thalamocortical relay nuclei generating rhythmic ipsps in these cells . thalamocortical relay cells are able to generate ca spikes at membrane hyperpolarizing levels ( jahnsen and llins , 1984 ) , these ca spike makes it possible to generate na potentials in the re - polarizing phase of the ipsps that can reach the cerebral cortex and produce rhythmic epsps in cortical neurons at the sleep spindle frequencies ( steriade and deschenes , 1988 ) since the thalamic afferents to the cerebral cortex are glutamatergic ( figures 3 and 5 ) . finally , in relation with the cellular basis of the sleep spindles , it is noteworthy that spindle oscillations in the membrane potential of the reticular and thalamocortical neurons arise from sustained hyperpolarized membrane potential levels ( llins and steriade , 2006 ) , and that thalamocortical or corticothalamic collaterals to the thalamic reticular nucleus can reinforce the generation of sleep spindles . synaptic activation of the thalamic reticular nucleus neurons by stimulation of these afferents invariably determines the generation of a sequence of spindle waves crowned with spike - bursts . schematic representation of the anatomical bases for the sleep spindles of nrem sleep and the intermediate- to high - voltage theta eeg activity of drowsiness . from reinoso - surez et al . the recruiting responses are the neurophysiological activity that can be experimentally elicited in the cat cortex after low - frequency stimulation of the intralaminar and related nuclei ( morrison and dempsey , 1942 ) ; these responses resemble the eeg activity recorded in superficial layer i of the cortex during sleep spindles ( hess , 1968 ) . the frontal and posterior parietal cortices are the cortical regions that are the prime location for recruiting responses and spontaneous spindling in the cat ( morrison and dempsey , 1942 ; starzl and magoun , 1951 ; reinoso - surez , 1954 ) . the superficial thalamocortical projection system to layer i in the frontal and posterior parietal cortex of the cat does not arise from intralaminar nuclei but from the paralaminar region of ventral medial , ventral anterior , and ventral lateral nuclei ( oka et al . in addition , the m - type cells of these nuclei innervate wider zones of layer i of the cerebral hemisphere , as it also happens in rats ( rubio - garrido et al . , 2009 ) . consequently this layer i projection system to the frontal and posterior parietal cortices may be an important leading path for recruiting responses and spontaneous spindling activities ( oka et al . in relation with the possible thalamocortical route for sleep spindle generation , jimnez - castellanos and reinoso - surez ( 1985 ) , velayos et al . ( 1990 ) proposed a circuit that involves the thalamic reticular nucleus and its projection to the paralaminar ventral medial , ventral anterior , and ventral lateral nuclei ( figure 5 ) . the thalamic reticular nucleus gabaergic pacemaker neurons of the spindle oscillations would impose their rhythmicity on the paralaminar m - type neurons that project to layer i of the frontal and parietal cortices ( reinoso - surez et al . for this to occur , as happens in stage 2 of nrem sleep , it is necessary to decrease the brainstem activating system action on both the thalamic reticular nucleus neurons , which consequently fire at their intrinsic burst activity , and the paralaminar neurons , which then transfer the slow burst activity to the cortex . altogether this permits the thalamic paralaminar m - type neurons projecting to layer i of the cerebral cortex , in their turn , impose this activity on the cortex , which has also been released from the influence of the ascending activating system ( figure 5 ; reinoso - surez , 1992 , 1993 ; reinoso - surez et al . , 2011 ) . 2011 ) have concluded that spindle oscillation initiation depends on activity by cortical pyramidal neurons on thalamic reticular nucleus neurons , which would then fire synchronizing discharges . the intermediate phase would be the interaction of thalamic reticular nucleus neurons with the thalamic projection neurons as we have described above . spindle firing is terminated by the combination of ih current and corticothalamic input depolarizing actions . consequently corticothalamic activity is involved not only in the long - range synchronization of spindles but also in the initiation and termination of individual spindle sequences , and this controls spindle duration . ( 2011 ) have investigated human hippocampal functional connectivity with neocortex in wakefulness and during nrem sleep . they found that the strongest functional connectivity between the hippocampal formation and neocortex was observed in stage 2 sleep spindles . the hippocampal structure dominating functional connectivity to frontal neocortex regions during sleep stage 2 was the subiculum . this increased connectivity between the hippocampal formation and neocortical regions suggests an increased capacity for possibly global information transfer in sleep stage 2 and would support the hypothesis of sleep - dependent memory consolidation . cortical and thalamic mechanisms are also involved in the generation of eeg delta swa that appear in deep nrem sleep stage 34 . eeg with swa can be registered in the cortex of cats without thalamus ( villablanca , 1974 ) ; therefore , the cortex has classically been considered to be the structure that generates this activity . however , steriade et al . ( 1991 , 1993 ) showed that delta oscillations can also be generated in thalamocortical neurons . these cells express this property intrinsically when their membrane potential is at more strongly hyperpolarized levels than during sleep spindles . in thalamic cells , delta is an intrinsic oscillation consisting of low - threshold spikes alternating with post - hyperpolarizing potentials . action potentials at delta frequency that can be transferred to the cortex are generated in the depolarizing phase of the ca low - threshold spikes . therefore , there is a degree of incompatibility in simultaneously generating thalamic spindles and delta rhythms ; this may explain the distinct prevalences of these two types of oscillations during the different stages of nrem [ stages 2 and 34 ( n3 ) respectively ] with synchronization of the electroencephalogram ( nuez et al . in fact , the thalamus and the cerebral cortex are close structures and abundantly interconnected , and they form a non - divisible unit in brain functions . during nrem sleep oscillating cortical activities can be transferred to specific thalamic structures via cortical efferents from layer vi and v pyramidal neurons ; in turn , thalamic oscillating activities can reach cortical neurons principally through m - type , but also c - type , thalamocortical neurons , since the former innervate extensive zones of layer i of the cerebral cortex and can convey oscillatory information activity to the distal apical tufts of pyramidal neurons from layers ii , iii , and v , neurons which are source for corticocortical and subcortical connections . the different oscillatory activities and spike - bursting mode in thalamic and cortical neurons that we have just described are completely absent in wakefulness and/or rem where the associated eeg is desynchronized . during the later states , thalamic and cortical neurons are depolarized and show a tonic firing discharge pattern ( llins and steriade , 2006 ) . the level of the membrane potential of thalamic neurons is mainly controlled by cholinergic input and , in wakefulness by additional glutamatergic and aminergic inputs , from the mesopontine cholinergic , glutamatergic , and aminergic cell groups . stimulation of cholinergic mesopontine nuclei produces an opposite effect in thalamic cells to the effects of corticothalamic volleys , readily suppressing their slow oscillatory bursts due to the generation of excitatory depolarizing discharges that produce a tonic firing pattern of activity in thalamic neurons ( steriade et al . , 1991 ) . the firing rate of the cholinergic laterodorsal and pedunculopontine mesopontine nuclei neurons is increased during wakefulness and rem sleep in comparison with firing rate during nrem sleep ( steriade et al . , 1982 ) ; therefore , when the activity of the cholinergic mesopontine , hypothalamic and basal forebrain , and glutamatergic and aminergic mesopontine structures decreases during nrem , the membrane potential of thalamic and cortical cells is hyperpolarized . this allows the generation of oscillatory patterns in the thalamic and cortical neurons , leading to expression of the specific eeg events of the nrem stages ( steriade and mccarley , 1990 ) . finally , intracellular recording experiments of cortical and thalamic neurons during synchronized eeg states detected that delta membrane oscillations were accompanied by a slower < 1 hz membrane rhythm ( dossi et al . this oscillation consists of a depolarized ( up ) state in which neurons fire followed by a hyperpolarized ( down ) state with neuronal silence . there is a close temporal relationship between the > 1 hz cellular oscillation and the 1- to 4-hz delta eeg waves since the negative component of the eeg signal corresponds to the down state of the cortical neurons ( contreras and steriade , 1995 ) . the cortical origin of these slow < 1 hz rhythm waves was demonstrated by the presence of the oscillation in the cerebral cortex after thalamectomy ( steriade et al . , 1993 ) and its absence in the thalamus of decorticated animals ( timofeev and steriade , 1996 ) . the cortical slow < 1 hz activity is expressed synchronously in wide areas of the cerebral cortex ; disconnection of intracortical synaptic linkages does not abolish the < 1 hz activity , but it does determine the loss of its synchronization between the different cortical areas ( amzica and steriade , 1995 ) . these findings indicate a possible role for < 1 hz cortical activity in synchronous excitation of wide brain territories ( cortical and thalamic ) during nrem , which would allow the simultaneous expression of the same eeg activities in all territories ( nez - molina and amzica , 2004 ) . interestingly , the generation of k complexes during stage 2 of nrem sleep seems to be related with the < 1 hz rhythm of the cortical pyramidal cells as the k complexes can be generated by stimulation of a cortical area other than the one in which they are recorded . also , k complexes can be evoked by sensory stimuli arriving at the cerebral cortex ( nez - molina and amzica , 2004 ) . k complexes , during nrem stage 2 , are usually followed by a sleep spindle ; in these circumstances , it is possible that the < 1 hz cortical activity would be sufficient to induce thalamic reticular nucleus neuron oscillation at the sleep spindle frequencies , since , as we have indicated above , stimulation of corticothalamic afferents to the reticular nucleus is one the most efficient stimuli in evoking rhythmic activity in reticular thalamic cells at sleep spindle frequencies . beside thalamus and cerebral cortex , the preoptic region and the anterior hypothalamus are other prosencephalic regions involved in nrem mechanisms ( figure 2 ) . as we have mentioned before , von economo ( 1930 ) was the first to suggest that the anterior hypothalamus was a sleep - promoting structure ( reinoso - surez et al . , 2011 ) ; later on in the sixties , the importance of this region in the mechanisms for nrem generation was confirmed after behavioral sleep associated with synchronized eeg was obtained with electrical stimulation of the preoptic region and of the basal forebrain ( sterman and clemente , 1962 ) and by the insomnia that followed electrolytic or diathermocoagulation lesions in these areas ( madoz and reinoso - surez , 1968 ; mcginty and sterman , 1968 ) . also , selective neurotoxic lesions of the preoptic neurons induce insomnia ( szymusiak and mcginty , 1986 ) thus indicating that the loss of local neurons rather than of fibers of passage is critical for sleep control . single unit recordings have shown that the preoptic region as well as the basal forebrain there hold neurons whose discharge rate is higher during nrem than in w ; in addition , many preoptic- and diagonal band - sleep active neurons are also heat - sensitive ( alam et al . , 1995 ) . the same local thermal stimuli that activate preoptic heat - sensitive neurons produce a sleep propensity and nrem with delta swa ( see szymusiak et al . , 2007 for a review of the close interactions between sleep regulation and thermoregulation together with the numerous factors that modulate the activity of the sleep - sensitive neurons in the anterior hypothalamus ) . clusters of neurons exhibiting sleep - related c - fos protein immunostaining were found in the ventral lateral preoptic area and in the median preoptic nucleus ( sherin et al . , 1996 ) . neuronal density with increased activity during nrem episodes is higher in these regions than in other anterior hypothalamic sites ( szymusiak et al . , neurons of the ventral lateral preoptic area contain the inhibitory neuromodulator galanin and the inhibitory neurotransmitter gaba ( sherin et al . , 1998 ) ; gaba is also found in most cells in the median preoptic nucleus ( gong et al . , 2004 ) . anatomical tracer studies have shown that the ventral lateral preoptic area and the median preoptic nucleus project to arousal - related regions in the posterior hypothalamus and midbrain including the histaminergic tuberomammillary nucleus ( sherin et al . , 1998 ) , the hypocretinergic / orexinergic perifornical hypothalamic area ( uschakov et al . , 2006 ; yoshida et al . , 2006 ) , the serotonergic dorsal raphe , and the noradrenergic locus coeruleus ( steininger et al . , 2001 ) . all of these findings indicate that the preoptic area may promote sleep onset and maintenance by producing an inhibitory modulation of multiple arousal systems ( szymusiak et al . , 2007 ) . in fact , gaba - mediated inhibitory actions in histaminergic tuberomammillary neurons were demonstrated after electrical stimulation of the ventral lateral preoptic area ( yang and hatton , 1997 ) . also , local warming of the preoptic region ( a stimulus that as we have mentioned before activates preoptic warmth - sensitive neurons and generates nrem ) causes suppression of waking related firing in both dorsal presumedly serotonergic raphe nucleus neurons ( guzmn - marn et al . , 2000 ) and in hypothalamic perifornical neurons ( krilowicz et al . , 1994 ) . recently , it has been demonstrated that sleep - regulatory molecules like interleukin-1 , tumor necrosis factor , and growth hormone - releasing hormone , stimulate nrem sleep by acting on neurons in the preoptic region . in addition , nrem sleep - promoting actions also take place through interleukin-1 and tumor necrosis factor inhibitory actions on locus coeruleus neurons and by interleukin-1 action on serotonergic raphe cells . what is more , the facilitation of nrem sleep can occur when the lateral hypothalamic perifornical area is subject to a endogenous adenosinergic inhibition that restrains the wakefulness - generating hypocretinergic / orexinergic neurons ( krueger et al . anatomical studies have demonstrated that most of the posterior hypothalamic- and monoaminergic- arousal systems send feedback projections to the ventral lateral preoptic area ( chou et al . , 2002 ; reinoso - surez et al . , 2011 ) . in vitro experiments have indicated that the wake - promoting neurotransmitters serotonin , noradrenalin , and acetylcholine inhibit identified preoptic gaba neurons ( gallopin et al . , 2000 ) ; therefore , mutually inhibitory interactions between the sleep - promoting preoptic region and the arousal - related hypothalamic and midbrain structures may provide a substrate for a sleep wakefulness switch thus , activation of preoptic sleep - promoting cells could lead to sleep onset by inhibiting arousal structures ; in turn , activation of arousal hypothalamic and midbrain structures could suppress activity by preoptic nrem sleep - promoting cells as well as rem - promoting neurons ( reinoso - surez et al . , 2010 ) and facilitate the switch to wakefulness . finally , the involvement of the preoptic region in homeostatic sleep regulation has been reported in recent experiments examining the patterns of c - fos immunoreactivity in preoptic neurons following acute total sleep deprivation or selective rem sleep restriction ( gvilia et al . these studies indicate that the gabaergic ventral lateral preoptic neurons are more strongly activated in response to increasing sleep amounts during sleep rebound , whereas gabaergic cells of the median preoptic nucleus are more strongly activated in response to sleep pressure during sleep deprivation . thus , within the prosencephalon , the preoptic region seems to be deeply involved in homeostatic nrem and rem sleep mechanisms . prosencephalic mechanisms underlying sleep homeostasis have been indicated by the capacity of the isolated forebrain to present nrem sleep rebound after pharmacological deprivation ( corpas and de andrs , 1991 ) and the absence of rem rebound in rem sleep - deprived decerebrate animals ( de andrs and corpas , 1991 ; de andrs et al . as we have mentioned in the introduction , early work with brainstem transections or lesions below the oral pontine tegmentum indicated that sleep - generating structures were located in the lower brainstem . nrem with eeg synchronization could be obtained in behaving cats by low - frequency electrical stimulation of the medullary reticular formation at the level of the solitary tract nucleus ( magnes et al . , 1961 ) , but lesion and stimulation studies in the peripheral nerves , spinal cord , caudal pontine tegmentum , and deep cerebellar nuclei can also induce hypnogenic synchronizing impulses ( figure 2 ) . the solitary tract nucleus region in the dorsal medulla is thought to provide a link between visceral activities such as respiratory , cardiovascular and gastrointestinal functions , and the sleep wakefulness states . biochemically , the solitary tract nucleus is a rather heterogeneous region where most viscerosensory afferent fibers from the viith , ixth , and xth cranial nerves terminate ; therefore , this region has important roles in the central regulation of visceral functions , but it also influences sleep not only does electrical stimulation of the solitary tract nucleus result in behavioral sleep with slow - eeg waves , these responses have also been obtained after the local administration of a variety of substances including serotonin ( laguzzi et al . , 1984 ) , morphine and other opiates agonists of mu or delta opioid receptors ( reinoso - barbero and de andrs , 1995 ; figure 6 ) , and glutamate ( golanov and reis , 2001 ) . these latter findings indicate that it is the activation of intrinsic solitary tract nucleus neurons and not of passing fibers that is responsible for the sleep and eeg responses . unit activity studies have shown that there is a population of neurons in the solitary tract nucleus with an increased firing rate during nrem sleep ( eguchi and satoh , 1980 ) . the solitary tract nucleus does not directly project to the cerebral cortex ( saper , 1995 ) , although it does project to several brainstem , thalamic , and hypothalamic areas that innervate the cortex and can mediate eeg and sleep responses . these areas include : locus coeruleus , raphe and parabrachial nuclei , lateral hypothalamus , and nuclei of the midline thalamus . top : microphotographs illustrating the location of the saline and opioid microinjections in two cats injected in the medial nucleus ( arrow ) of the solitary tract ( sol ) . wakefulness states during the first hour of recordings in controls ( saline , 40 nl ) and after morphiceptin ( mu opioid agonist ) or dpdpe ( delta opioid agonist ) microinjections ( 2.4 nmol , 40 nl ) . respiratory rate ( triangles ) in breaths per minute and heart rate ( circles ) in beats per minute are also shown for each sleep wakefulness state during the first hour of recordings . note the decreased respiratory and heart rates in sleep states in comparison with values during wakefulness in the control saline experiments ; both opioid treatments produced a further decrease in both autonomic variables . w , wakefulness ; d , drowsiness ; nrem , non - rem sleep ; rem , rem sleep . results from our group s early work with lesions showed the existence of ascending eeg synchronizing influences arising from the rostral and intermediate parts of the nucleus reticularis pontis caudalis . lesions in these areas decreased swa in the cerebral cortex and the bioelectrical and behavioral signals of nrem and rem sleep ( figure 7 ; camacho - evangelista and reinoso - surez , 1964 ; zarranz and reinoso - surez , 1971 ; reinoso - surez and de andrs , 1976 ) . more recent experiments microinjecting small amounts of a carbachol solution in rostral and intermediate parts of the nucleus reticularis pontis caudalis produced a synchronized eeg pattern associated with some rem sleep signs such as isolated and/or clustered pgo waves ; the most rostral microinjections were also associated with muscular atonia ( garzn , 1996 ; reinoso - surez et al . power spectra analyses indicated that the synchronized eeg pattern obtained in these experiments did not consist of delta waves , instead , the high - voltage swa activity was produced by enhancement of theta and alpha band frequencies ( garzn , 1996 ) . therefore , this cortical electrical pattern does not correspond to fully developed nrem sleep ; instead , these electrical features are more similar to those expressed in the eeg during the transitional period between nrem and rem sleep ( gottesmann , 1996 ) . recently we have reported ( moreno - balandrn et al . , 2008 ) that low - doses and small carbachol microinjections delivered in the dorsal part of the oral pontine tegmentum in cats , at the level of the perilocus coeruleus alpha , produce a similar synchronized eeg without delta wave enhancement . these results indicate that not only is the pontine tegmentum the neural substrate for rem sleep and desynchronized eeg mechanisms , but that its caudal region especially the rostral and intermediate areas of the nucleus reticularis pontis caudalis ; together with the dorsal part of the oral region seems to be involved in generating the pattern of bioelectrical activity that announces rem onset from nrem sleep . activating and synchronizing eeg centers in the pontine tegmentum . ( a , c e ) show diagrams of the combined extent of lesions in the pontine tegmentum of cats producing either eeg activation ( diagonal dashed lines encircled by heavy dashed lines ) or eeg synchronization ( horizontal lines encircled by heavy solid lines ) . ( a ) parasagittal section 2 mm from the midline ; circled numbers indicate frontal plane levels on the reinoso - surez ( 1961 ) atlas corresponding to sections ( c e ) , respectively . ( b ) graph showing the percent of increase in activation ( dashed line ) and decrease in activation ( solid line ) relative to the pre - lesion control - based zero for each animal . note that lesions that produced eeg synchronization are located at the level of the oral pontine reticular nucleus and those that produced eeg activation are located caudal , dorsal , and lateral to this nucleus . bc , brachium conjunctivum ; bp , brachium pontis ; cp , colliculus inferior ; fp , fasciculus longitudinalis medialis ; lm , lemniscus medialis ; mv , tractus mesencephalicus nervi trigemini ; nll , nucleus lemnisci lateralis ; np , nuclei pontis ; nr , nucleus ruber ; nrv , nucleus reticularis ventralis ; ntr , nucleus corporis trapezoidei ; os , oliva superior ; p , tractus pyramidalis ; rpc , nucleus reticularis pontis caudalis ; rpo , nucleus reticularis pontis oralis ; scv , tractus spinocerebellaris anterior ; td , nucleus tegmenti dorsalis ; tr , corpus trapezoideum ; tf , tractus tectospinalis . from camacho - evangelista and reinoso - surez ( 1964 ) . the major role of the cerebellum in motor functions has undervalued its possible involvement in sleep regulation . cerebellar participation in sleep can be related to motor activities during sleep but also to intrinsic sleep mechanisms . the former activities involve postural adjustment and control of muscle tone and movements during sleep that , as in wakefulness , must be controlled by the cerebellum ; in fact , early experimental studies showed the existence of cerebellar components in muscle atonia and phasic activities of the eyes lateral rectus muscle during rem ( jouvet , 1962 ; guglielmino and strata , 1971 ; gadea - ciria and fuentes , 1976 ; cunchillos and de andrs , 1982 ) . together with these actions , the cerebellum has important influences on intrinsic sleep mechanisms as was first indicated by the changes observed in cortical and thalamic electrical activity that follow electrical stimulation and lesion of different cerebellar structures . moruzzi ( 1958 ) reported that diffuse eeg effects could be obtained through the efferent of the nucleus dentatus , this effect was also obtained with low - frequency electrical stimulation of the nucleus fatigius , while high frequency stimulation of this nucleus produced diffuse eeg activation ( fadiga et al . , 1968 ) . specific superficial thalamocortical responses ( surface negative - deep positive potentials ) in the frontal and parietal cortices were also produced by stimulation of the cerebellar nuclei ( sasaki et al . , 1976 ) . suppression of the impulses from the deep cerebellar nuclei through lesion of the brachium conjunctivum tract produce , together with a bilateral activation of the eeg , the appearance of synchronized bursts of intermediate- to high - voltage eeg activity in the theta band ( 713 hz , bigger of 250 mv ) that can be recorded in frontal and parietal cortical areas that are targets of the projections from the thalamic nuclei that receive cerebellar afferents ( camacho - evangelista and reinoso - surez , 1965 ; reinoso - surez , 1992 , 1993 ) . the bioelectrical features of these synchronized bursts are similar to the relaxation rhythms described in cats during periods of relaxed wakefulness or drowsiness ( ursin and sterman , 1981 ) . a similar eeg synchronization , most conspicuously recorded in the ipsilateral frontal and parietal cortices , appears especially after the lesion of the brachium conjunctivum tract at the level of the red nucleus ( reinoso - surez , 1954 ) . this intermediate- to high - voltage swa in the theta band can also be recorded in the thalamic paralaminar nuclei that receive afferents from the cerebellum ; it looks as if suppression of the cerebellar afferents releases these nuclei and allows them to fire at a possible intrinsic theta frequency , modulated by thalamic and other extrathalamic connections that , projecting to layer i , impose their bioelectrical activity on the frontal and parietal cortices ( figure 5 ; reinoso - surez , 1992 , 1993 ; reinoso - surez et al . , the two bioelectric phenomena after the lesion on the superior cerebellar peduncle ( increased eeg activation and synchronized bursts of eeg activity in the theta band ) are coherent with the swc modifications after lesion of the superior cerebellar peduncle that significantly increase wakefulness and drowsiness while decreasing rem and nrem sleep ( de andrs and reinoso - surez , 1979 ) . the sleep suppression was a consequence of the decrease in both the duration and the number of the nrem episodes ( figure 8) . all these studies point to an important participation by the cerebellum in swc mechanisms through a functional antagonism in the regulation of the proportions of sleep and wakefulness between cerebellar cortex and deep cerebellar nuclei ; that is , like the changes occurring after the brachium conjunctivum tract lesions , increased wakefulness , and decreased nrem and rem sleep were reported after bilateral lesions of the fastigial nuclei ( giannazzo et al . , 1968 ) . however , these effects were just the opposite after lesions in the middle cerebellar peduncle ( raffaele et al . , 1971 ) or in the cortex of vermis and cerebellar hemispheres that produced increased nrem and rem sleep ( de andrs and reinoso - surez , 1979 ; garca - ura et al . , 1980 ) . however and surprisingly , in spite of the severe neurological deficits produced by cerebellectomy , the quantitative alterations in the proportions of swc states in cerebellectomized animals are smaller than those produced by localized subcortical or cortical cerebellar lesions . in our experiments ( cunchillos and de andrs , 1982 ) with cerebellectomized cats , the main change after cerebellectomy was an increase in drowsiness that persisted during the entire survival time ( 5 weeks ) and that was associated with small decreases in both w and nrem sleep as well as an increase in rem sleep . these results confirm cerebellar involvement in mechanisms of synchronized eeg and support the hypothesis that the cerebellum , through dual opposite cortical and subcortical effects , would have an important regulatory action in the fine adjustments in the proportions of swc states for maintaining the sleep modifications in the total duration of wakefulness , drowsiness , nrem ( slow sleep ) , and rem ( paradoxical ) sleep , as well as their episode mean duration and number per recording after different cerebellar lesions . the cats with lesions in the brachium conjunctivum showed a significant decrease of nrem ( slow wave ) and rem ( paradoxical ) sleep . in contrast , the cats with lesions in the cerebellar cortex and white matter of the anterior vermis showed a significant decrease of drowsiness and a significant increase in rem sleep . modified from de andrs and reinoso - surez ( 1979 ) . a recent study ( dang - vu et al . , 2008 ) with functional magnetic resonance imaging has reported that the slow oscillation < 1 hz rhythm of nrem sleep is associated with significantly increased local activity in specific cerebral regions : the parahippocampal gyrus and cerebellum ( in both cerebellar hemispheres and vermis ) . on the basis of parallel changes that occur in forebrain and cerebellum during nrem sleep , andre and arrighi ( 2003 ) have proposed that forebrain and cerebellum may cooperate strongly in information processing during sleep . as yet no further studies have examined cerebellar participation at the cellular level in the generation of the slow < 1 hz nrem sleep rhythm , but the dang - vu et al . ( 2008 ) findings may be indicative of parallel operating capabilities in the cerebellum and hippocampal formation during nrem sleep . the growing data on hippocampal activation during nrem sleep after memory task training ( peigneux et al . , 2004 ; ji and wilson , 2007 ; rasch et al . , 2007 ) and recent compelling evidence that slow < 1 hz oscillations have a causal role in consolidating memories during nrem sleep ( marshall et al . , 2006 ) the current dominant theory in sleep research considers that the connections between structures putatively responsible for each of the three phases of the swc ( wakefulness , rem , and nrem sleep ) would provide the anatomical basis for the central organization of reciprocal interactions among these structures and result in the alternation of the different phases of the swc under the circadian control of the suprachiasmatic nucleus ( reinoso - surez et al . , 2001 ) . in addition , we would note that , in humans and other mammals , sleep is not only regulated by the circadian clock , but also by sleep homeostasis . humans are awake in the morning because sleep pressure is low after a night of rest ( mignot and huguenard , 2009 ) . throughout the day increased wakefulness - promoting signals , partly driven by the circadian clock , counter the growing sleep debt or sleep pressure , keeping the subject awake . sleep would be necessary to normalize the synapses to a basal condition that is sustainable and ensures cellular homeostasis ( cirelli and tononi , 2008 ) . nrem sleep homeostasis depends not only on the duration of prior wakefulness but also on intensity , and sleep intensity increases when wakefulness is associated with learning ( cirelli , 2009 ) . the metabolic factors that initiate sleep are molecules produced by neuronal activity during wakefulness and they increase proportionally with the duration and intensity of arousal . the accumulation of metabolites up to a critical level would be involved in the initiation of sleep specifically promoting nrem sleep . during the course of sleep metabolite levels would be reduced from the critical concentration to basal levels ( datta and maclean , 2007 ) . many molecules have been implicated in the processes of sleep regulation , but only a few meet the criteria for being considered nrem sleep - regulatory substances . among these substances are : adenosine , nitric oxide , prostaglandin d2 , interleukin-1 , tumor necrosis factor , and growth hormone - releasing hormone ( krueger et al . , 2008 ) . these molecules are involved in regulating the intensity and duration of nrem sleep and they act in biochemical cascades . neuronal activity during wakefulness increases the synthesis of nrem sleep regulating molecules ; this increase , as occurs with the effects of adenosine on the a1-receptor , leads to the inhibition of neuronal activity , thereby increasing sleep pressure , and finally nrem begins ( vassalli and dijk , 2009 ) . although nrem - promoting substances act on basal forebrain and hypothalamic nrem sleep regulating circuits ( gvilia et al . , 2006a , b ) , they can also act locally on the cerebral cortex and thalamus to change the electrical properties of thalamic and cortical neurons thus altering their input output relationships ( krueger et al . , these observations help explain the capacity of the isolated forebrain to exhibit nrem sleep rebound ( corpas and de andrs , 1991 ) . currently , nrem and rem sleep are considered behavioral states involving the whole organism and governed by central control mechanisms . although accepting this point of view , there is increasing evidence that nrem sleep ( and thus sleep ) might be a fundamental property of local neuronal networks since it can be initiated at small brain assemblies in response to their use , and only later on be consolidated by general central mechanisms ( krueger et al . , 2008 ; rector et al . , 2009 ) . one of the smallest sleep units could be the individual cortical columns in which sensory stimulation produces fluctuating high and low amplitude evoked responses depending on the behavioral state . in sleeping animals , the sleep - like response in cortical columns is characterized by evoked response potentials with a greater amplitude that the evoked responses during wakefulness ( rector et al . , 2005 ) . however , the sleep-/wake - like response also seems to be an intrinsic property of the individual cortical columns since , independently of the behavioral state of the whole - animal , the probability of finding a column in a sleep - like state depends on the length of time that column has previously spent in the wake - like state ( rector et al . , 2005 ) , suggesting that in different cortical regions cortical columns can be sleeping while others are awake during both whole - animal wakefulness and sleep . thus , these findings suggest that nrem sleep may be regulated at local neuronal assembly levels and this regulation appears to be a fundamental property of the neural networks and it depends on prior activity in each network ( krueger et al . , 2008 ) . the nrem sleep of each local cortical column can be initiated by metabolically driven changes derived from the manufacture of molecules of sleep - regulatory substances produced by neuronal activity during wakefulness - like activity . the process may involve increased electrical activity , blood flow , extracellular levels of atp and extra - and intracellular levels of adenosine , all of which would decrease the intracellular atp level , and this would drive individual columns to enter a sleep - like activity state . once a final sleep state was reached this process would be reversed : reduced electrical activity , blood flow , extracellular levels of atp and extra - and intracellular levels of adenosine would raise the intracellular atp level in individual local cortical columns , and thus , the cortical column would be prepared for wakefulness . the sleep or wakefulness states of individual cortical columns could be synchronized through humoral and electrical connections and therefore the sleep or wakefulness of the whole organism would occur as an emergent property of the interaction of individual networks ( krueger et al . , 2008 ) . nowadays there is an emerging understanding of the role of astrocytes as potential mediators of the known effect of adenosine in sleep regulation and this mediation would involve both neuroenergetic and synaptic plasticity roles ( jones , 2009 ) . the power of delta swa in the eeg is considered to be an indicator of nrem sleep intensity , and delta swa is homeostatically regulated since its power increases after wakefulness and returns to baseline during sleep . indeed , experiments have shown that the power of swa reflects the duration of prior wakefulness regardless of the circadian time . delta swa seems to reflect a form of restorative process for cortical local circuits : swa delta power is increased in cortical areas that have been most active during the day . also , imaging techniques have shown that there is a localized decrease in metabolic activity at night in areas that have been activated during the day ( mignot and huguenard , 2009 ) . 2004 ) have indicated that sleep homeostasis has a local component , one that can be triggered in specific human brain regions by a learning task ; these authors also showed that the local increase in swa after learning correlates with improved performance of the task after sleep . thus , sleep homeostasis can be induced on a local level and can improve performance . as we have indicated above delta eeg waves are synchronized in the different cortical areas in correlation with the neuronal events that express cyclic potential membrane fluctuations ( < 1 hz ) between hyperpolarized ( down ) and depolarized ( up ) periods in which neurons are respectively silent or active ( dossi et al . , 1992 ; contreras and steriade , 1995 ) . 2009 ) have shown that during nrem , barrel cortex multiunit activity expresses cyclic ( < 1 hz ) changes with silent periods ( off ) alternating with others ( on ) in which there is a sustained firing activity ; these on / off cortical multiunit activities closely resemble the neuronal membrane potential events of up / down states . ( 2009 ) also showed that the multiunit off periods in barrel cortical neurons are temporally correlated with the negative component of the eeg delta waves as are the down states that occur at cellular level ( contreras and steriade , 1995 ) . during the first nrem sleep after an uninterrupted waking period , neuronal barrel cortex population on periods are short and frequent , while neuronal silence periods ( down states ) are long and frequent . down states ( accompanied by an overall decrease in neuronal activity ) are a phenomenon that is thought to be associated with energy savings . after a sustained sleep period , which reduces the sleep debt , barrel cortex off periods decrease and the duration of the on periods increases . in addition , the changes in firing patterns in nrem sleep correlate positively with changes in swa intensity ( vyazovskiy et al . , 2009 ) . recent data ( jones et al . , 2008 ) indicate that sleep reduces the number of synaptic connections to a basic level that is augmented during waking experience that is , nrem sleep seems to ensure cell homeostasis ; simultaneously during sleep the expression of molecules involved in synaptic reorganization is increased . based on simple energy demands and since the cerebral cortex thalamus unit is a large part of the human and mammal brain , being awake for longer and longer periods of time seems to be very expensive , hence energy economy by the brain during nrem sleep is one of the prevalent hypotheses to explain nrem sleep pressure ( mignot and huguenard , 2009 ) . however , during nrem sleep neurons must down - fire in synchrony rather than simply stay silent ; for some authors this circumstance supports the need to transfer memory to different cortical areas , as has been demonstrated to occur between the hippocampus and visual cortex . to vyazovskiy et al . ( 2009 ) , wakefulness is associated with learning , and enhancing long - term potentiation strengthens glutamatergic synapses , a process with an unsustainable energy requirement , whereas during sleep only robust connections remain intact , reducing the energy requirements to the levels necessary for maintaining critical learned circuits . this reduction of synapses during sleep increases the signal to noise ratio for the remaining connections , improving performance ( mignot and huguenard , 2009 ; vyazovskiy et al . , 2009 ) . the close reciprocal connections between the thalamus and the cerebral cortex would force nrem sleep homeostatic processes to take place simultaneously in the two structures . the thalamus cerebral cortex is a strictly necessary unit in the expression of the most significant bioelectric and behavioral events of nrem sleep and the thalamus cerebral cortex unit is connected to subcortical nrem sleep - promoting structures and with all the subcortical structures involved in the organization of the other phases of swc ( reinoso - surez et al . , 2011 ) . the former connections would act on the thalamus cerebral cortex unit to facilitate the nrem sleep neurophysiological and homeostatic processes and also through neurophysiological and homeostatic mechanisms , the latter subcortical connections would inhibit the other phases of swc : rem sleep and wakefulness . 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 .

the state of non - rem sleep ( nrem ) , or slow wave sleep , is associated with a synchronized eeg pattern in which sleep spindles and/or k complexes and high - voltage slow wave activity ( swa ) can be recorded over the entire cortical surface . in humans , nrem is subdivided into stages 2 and 34 ( presently named n3 ) depending on the proportions of each of these polygraphic events . nrem is necessary for normal physical and intellectual performance and behavior . an overview of the brain structures involved in nrem generation shows that the thalamus and the cerebral cortex are absolutely necessary for the most significant bioelectric and behavioral events of nrem to be expressed ; other structures like the basal forebrain , anterior hypothalamus , cerebellum , caudal brain stem , spinal cord and peripheral nerves contribute to nrem regulation and modulation . in nrem stage 2 , sustained hyperpolarized membrane potential levels resulting from interaction between thalamic reticular and projection neurons gives rise to spindle oscillations in the membrane potential ; the initiation and termination of individual spindle sequences depends on corticothalamic activities . cortical and thalamic mechanisms are also involved in the generation of eeg delta swa that appears in deep stage 34 ( n3 ) nrem ; the cortex has classically been considered to be the structure that generates this activity , but delta oscillations can also be generated in thalamocortical neurons . nrem is probably necessary to normalize synapses to a sustainable basal condition that can ensure cellular homeostasis . sleep homeostasis depends not only on the duration of prior wakefulness but also on its intensity , and sleep need increases when wakefulness is associated with learning . nrem seems to ensure cell homeostasis by reducing the number of synaptic connections to a basic level ; based on simple energy demands , cerebral energy economizing during nrem sleep is one of the prevalent hypotheses to explain nrem homeostasis .

Introduction Overview of the Brain Structures Involved in NREM Generation The Thalamus and the Cerebral Cortex Basal Forebrain Anterior Hypothalamic Structures Brainstem and Cerebellar Structures NREM Sleep Homeostasis Conflict of Interest Statement

the state of non - rem sleep ( nrem ) , or slow wave sleep , is associated with a synchronized eeg pattern in which specific electrographic events take place . these events are sleep spindles and/or k complexes and high - voltage slow wave activity ( swa ) within the delta frequency band ( between 0.5 and 4.0 hz ) that can be recorded over the entire cortical surface . in humans , nrem is subdivided into stages 2 and 34 depending on the proportions of each of these polygraphic events . stage 2 , with sleep spindles and k complexes and less than 20% of swa in the recording , corresponds , from the behavioral point of view , to a light sleep with a low - threshold to awakening . in young adults stage 34 ( n3 ) is especially abundant in the first third of the night ( figure 1 ) . its proportions show notable stability in long and short sleepers , and it is the first stage to recover after sleep deprivation ; it is considered to be the main component of the core sleep necessary for normal physical and intellectual performance and behavior . conversely , decrease of swa in the eeg ( and therefore enhancement of desynchronized eeg ) takes place after suppression of trigeminal nerve afferents ( vies - morros , 1959 ) , and after lesion or blockage of the spinal cord , thus indicating that ascending synchronizing impulses can also be generated in the spinal cord ( hodes , 1964 ; de andrs et al . the main connections between these structures and the structures responsible of the organization of wakefulness ( encompassed in red line ) or rem sleep ( blue lines ) with the exception of the efferent hypothalamic and midbrain connections and the brainstem connections from the basal forebrain are shown . ac , anterior commissure ; cc , corpus callosum ; dcn , deep cerebellar nuclei ; fo , fornix ; g7 , genu of the facial nerve ; ic , inferior colliculus ; io , inferior olive ; lv , lateral ventricle ; mrf ; midbrain reticular formation ; mt , medullary tegmentum ; och , optic chiasm ; pgs , periaqueductal gray substance ; rpc , caudal pontine reticular nucleus ; rpo , oral pontine reticular nucleus ; sc , superior colliculus ; sc and pn , spinal cord and peripheral nerves ; sn , solitary nucleus ; tb , trapezoid body . in the case of lesion to the brachium conjunctivum tract , the suppression of the synchronizing impulses was associated to a bilateral increase of desynchronized eeg but also to the generation of bursts of intermediate- to high - voltage theta band eeg activity recorded in cortical areas ( motor and parietal cortices ) that are targets of the thalamic nuclei where cerebellar projections end ( camacho - evangelista and reinoso - surez , 1965 ; reinoso - surez , 1992 , 1993 ) . also , lesion , stimulation , and clinical studies demonstrate that the thalamus is necessary to generate the electrophysiological and behavioral manifestations that characterize nrem sleep ( hess , 1931 , 1968 ; morrison and dempsey , 1942 ; villablanca , 1974 ; steriade et al . also villablanca ( 1972 , 2004 ) affirmed that true nrem sleep is absent in chronic cats without telencephalon ( diencephalic cats ) because neither delta swa nor sleep spindles could be recorded in the thalamus . in summary , it appears that the thalamus and the cerebral cortex are absolutely necessary for the expression of the most significant bioelectric and behavioral events of nrem sleep . other structures like the basal forebrain , cerebellum and caudal brain stem , and impulses from the spinal cord and peripheral nerves modulate nrem sleep but are not required for the actual generation of the bioelectric and behavioral signs accompanying the expression of nrem sleep ( figure 2 ) . these authors showed that the combination of the intrinsic electrophysiological properties of the thalamic and cortical neurons , together with their synaptic connections , is responsible for the generation of sleep spindles and delta swa oscillations in the eeg during nrem sleep ( for review see nez - molina and amzica , 2004 ; fuentealba and steriade , 2005 ) . thus , the eeg activities , which appear during stages 2 and 34 ( n3 ) of nrem sleep are essentially generated by the activity of neurons in the thalamic reticular nucleus , in the thalamocortical projecting nuclei , and in the cerebral cortex . 1985 ) proposed , early on , that the neurons of the thalamic reticular nucleus are pacemakers for sleep spindles , based on the absence of the sleep spindle oscillation in thalamocortical systems when they are disconnected from the thalamic reticular nucleus and the presence of spindle rhythmicity in the deafferentized reticular thalamic nucleus . simultaneous intracellular recordings in reticular , thalamocortical , and cortical neurons ( steriade and deschenes , 1988 ) indicated that sleep spindle generation was produced by a chain of events with three main consecutive steps : ( 1 ) repetitive spike - bursts at spindle frequency generated by gabaergic thalamic reticular nucleus neurons ; ( 2 ) transfer of this activity to relay thalamocortical nuclei producing rhythmic ( at spindle frequency ) inhibitory postsynaptic potentials ( ipsps ) in thalamocortical neurons ; and ( 3 ) transfer to the cerebral cortex of postinhibitory rebound spike - bursts of the thalamocortical cells producing excitatory postsynaptic potentials ( epsps ) in cortical cells at spindle frequency . thalamocortical relay cells are able to generate ca spikes at membrane hyperpolarizing levels ( jahnsen and llins , 1984 ) , these ca spike makes it possible to generate na potentials in the re - polarizing phase of the ipsps that can reach the cerebral cortex and produce rhythmic epsps in cortical neurons at the sleep spindle frequencies ( steriade and deschenes , 1988 ) since the thalamic afferents to the cerebral cortex are glutamatergic ( figures 3 and 5 ) . finally , in relation with the cellular basis of the sleep spindles , it is noteworthy that spindle oscillations in the membrane potential of the reticular and thalamocortical neurons arise from sustained hyperpolarized membrane potential levels ( llins and steriade , 2006 ) , and that thalamocortical or corticothalamic collaterals to the thalamic reticular nucleus can reinforce the generation of sleep spindles . schematic representation of the anatomical bases for the sleep spindles of nrem sleep and the intermediate- to high - voltage theta eeg activity of drowsiness . the recruiting responses are the neurophysiological activity that can be experimentally elicited in the cat cortex after low - frequency stimulation of the intralaminar and related nuclei ( morrison and dempsey , 1942 ) ; these responses resemble the eeg activity recorded in superficial layer i of the cortex during sleep spindles ( hess , 1968 ) . for this to occur , as happens in stage 2 of nrem sleep , it is necessary to decrease the brainstem activating system action on both the thalamic reticular nucleus neurons , which consequently fire at their intrinsic burst activity , and the paralaminar neurons , which then transfer the slow burst activity to the cortex . altogether this permits the thalamic paralaminar m - type neurons projecting to layer i of the cerebral cortex , in their turn , impose this activity on the cortex , which has also been released from the influence of the ascending activating system ( figure 5 ; reinoso - surez , 1992 , 1993 ; reinoso - surez et al . consequently corticothalamic activity is involved not only in the long - range synchronization of spindles but also in the initiation and termination of individual spindle sequences , and this controls spindle duration . cortical and thalamic mechanisms are also involved in the generation of eeg delta swa that appear in deep nrem sleep stage 34 . eeg with swa can be registered in the cortex of cats without thalamus ( villablanca , 1974 ) ; therefore , the cortex has classically been considered to be the structure that generates this activity . ( 1991 , 1993 ) showed that delta oscillations can also be generated in thalamocortical neurons . action potentials at delta frequency that can be transferred to the cortex are generated in the depolarizing phase of the ca low - threshold spikes . therefore , there is a degree of incompatibility in simultaneously generating thalamic spindles and delta rhythms ; this may explain the distinct prevalences of these two types of oscillations during the different stages of nrem [ stages 2 and 34 ( n3 ) respectively ] with synchronization of the electroencephalogram ( nuez et al . in fact , the thalamus and the cerebral cortex are close structures and abundantly interconnected , and they form a non - divisible unit in brain functions . during nrem sleep oscillating cortical activities can be transferred to specific thalamic structures via cortical efferents from layer vi and v pyramidal neurons ; in turn , thalamic oscillating activities can reach cortical neurons principally through m - type , but also c - type , thalamocortical neurons , since the former innervate extensive zones of layer i of the cerebral cortex and can convey oscillatory information activity to the distal apical tufts of pyramidal neurons from layers ii , iii , and v , neurons which are source for corticocortical and subcortical connections . the firing rate of the cholinergic laterodorsal and pedunculopontine mesopontine nuclei neurons is increased during wakefulness and rem sleep in comparison with firing rate during nrem sleep ( steriade et al . , 1982 ) ; therefore , when the activity of the cholinergic mesopontine , hypothalamic and basal forebrain , and glutamatergic and aminergic mesopontine structures decreases during nrem , the membrane potential of thalamic and cortical cells is hyperpolarized . interestingly , the generation of k complexes during stage 2 of nrem sleep seems to be related with the < 1 hz rhythm of the cortical pyramidal cells as the k complexes can be generated by stimulation of a cortical area other than the one in which they are recorded . k complexes , during nrem stage 2 , are usually followed by a sleep spindle ; in these circumstances , it is possible that the < 1 hz cortical activity would be sufficient to induce thalamic reticular nucleus neuron oscillation at the sleep spindle frequencies , since , as we have indicated above , stimulation of corticothalamic afferents to the reticular nucleus is one the most efficient stimuli in evoking rhythmic activity in reticular thalamic cells at sleep spindle frequencies . beside thalamus and cerebral cortex , the preoptic region and the anterior hypothalamus are other prosencephalic regions involved in nrem mechanisms ( figure 2 ) . , 2011 ) ; later on in the sixties , the importance of this region in the mechanisms for nrem generation was confirmed after behavioral sleep associated with synchronized eeg was obtained with electrical stimulation of the preoptic region and of the basal forebrain ( sterman and clemente , 1962 ) and by the insomnia that followed electrolytic or diathermocoagulation lesions in these areas ( madoz and reinoso - surez , 1968 ; mcginty and sterman , 1968 ) . , 1961 ) , but lesion and stimulation studies in the peripheral nerves , spinal cord , caudal pontine tegmentum , and deep cerebellar nuclei can also induce hypnogenic synchronizing impulses ( figure 2 ) . the solitary tract nucleus does not directly project to the cerebral cortex ( saper , 1995 ) , although it does project to several brainstem , thalamic , and hypothalamic areas that innervate the cortex and can mediate eeg and sleep responses . lesions in these areas decreased swa in the cerebral cortex and the bioelectrical and behavioral signals of nrem and rem sleep ( figure 7 ; camacho - evangelista and reinoso - surez , 1964 ; zarranz and reinoso - surez , 1971 ; reinoso - surez and de andrs , 1976 ) . more recent experiments microinjecting small amounts of a carbachol solution in rostral and intermediate parts of the nucleus reticularis pontis caudalis produced a synchronized eeg pattern associated with some rem sleep signs such as isolated and/or clustered pgo waves ; the most rostral microinjections were also associated with muscular atonia ( garzn , 1996 ; reinoso - surez et al . these results indicate that not only is the pontine tegmentum the neural substrate for rem sleep and desynchronized eeg mechanisms , but that its caudal region especially the rostral and intermediate areas of the nucleus reticularis pontis caudalis ; together with the dorsal part of the oral region seems to be involved in generating the pattern of bioelectrical activity that announces rem onset from nrem sleep . suppression of the impulses from the deep cerebellar nuclei through lesion of the brachium conjunctivum tract produce , together with a bilateral activation of the eeg , the appearance of synchronized bursts of intermediate- to high - voltage eeg activity in the theta band ( 713 hz , bigger of 250 mv ) that can be recorded in frontal and parietal cortical areas that are targets of the projections from the thalamic nuclei that receive cerebellar afferents ( camacho - evangelista and reinoso - surez , 1965 ; reinoso - surez , 1992 , 1993 ) . this intermediate- to high - voltage swa in the theta band can also be recorded in the thalamic paralaminar nuclei that receive afferents from the cerebellum ; it looks as if suppression of the cerebellar afferents releases these nuclei and allows them to fire at a possible intrinsic theta frequency , modulated by thalamic and other extrathalamic connections that , projecting to layer i , impose their bioelectrical activity on the frontal and parietal cortices ( figure 5 ; reinoso - surez , 1992 , 1993 ; reinoso - surez et al . , the two bioelectric phenomena after the lesion on the superior cerebellar peduncle ( increased eeg activation and synchronized bursts of eeg activity in the theta band ) are coherent with the swc modifications after lesion of the superior cerebellar peduncle that significantly increase wakefulness and drowsiness while decreasing rem and nrem sleep ( de andrs and reinoso - surez , 1979 ) . all these studies point to an important participation by the cerebellum in swc mechanisms through a functional antagonism in the regulation of the proportions of sleep and wakefulness between cerebellar cortex and deep cerebellar nuclei ; that is , like the changes occurring after the brachium conjunctivum tract lesions , increased wakefulness , and decreased nrem and rem sleep were reported after bilateral lesions of the fastigial nuclei ( giannazzo et al . these results confirm cerebellar involvement in mechanisms of synchronized eeg and support the hypothesis that the cerebellum , through dual opposite cortical and subcortical effects , would have an important regulatory action in the fine adjustments in the proportions of swc states for maintaining the sleep modifications in the total duration of wakefulness , drowsiness , nrem ( slow sleep ) , and rem ( paradoxical ) sleep , as well as their episode mean duration and number per recording after different cerebellar lesions . , 2008 ) with functional magnetic resonance imaging has reported that the slow oscillation < 1 hz rhythm of nrem sleep is associated with significantly increased local activity in specific cerebral regions : the parahippocampal gyrus and cerebellum ( in both cerebellar hemispheres and vermis ) . as yet no further studies have examined cerebellar participation at the cellular level in the generation of the slow < 1 hz nrem sleep rhythm , but the dang - vu et al . , 2006 ) the current dominant theory in sleep research considers that the connections between structures putatively responsible for each of the three phases of the swc ( wakefulness , rem , and nrem sleep ) would provide the anatomical basis for the central organization of reciprocal interactions among these structures and result in the alternation of the different phases of the swc under the circadian control of the suprachiasmatic nucleus ( reinoso - surez et al . in addition , we would note that , in humans and other mammals , sleep is not only regulated by the circadian clock , but also by sleep homeostasis . sleep would be necessary to normalize the synapses to a basal condition that is sustainable and ensures cellular homeostasis ( cirelli and tononi , 2008 ) . nrem sleep homeostasis depends not only on the duration of prior wakefulness but also on intensity , and sleep intensity increases when wakefulness is associated with learning ( cirelli , 2009 ) . one of the smallest sleep units could be the individual cortical columns in which sensory stimulation produces fluctuating high and low amplitude evoked responses depending on the behavioral state . however , the sleep-/wake - like response also seems to be an intrinsic property of the individual cortical columns since , independently of the behavioral state of the whole - animal , the probability of finding a column in a sleep - like state depends on the length of time that column has previously spent in the wake - like state ( rector et al . the power of delta swa in the eeg is considered to be an indicator of nrem sleep intensity , and delta swa is homeostatically regulated since its power increases after wakefulness and returns to baseline during sleep . indeed , experiments have shown that the power of swa reflects the duration of prior wakefulness regardless of the circadian time . , 2008 ) indicate that sleep reduces the number of synaptic connections to a basic level that is augmented during waking experience that is , nrem sleep seems to ensure cell homeostasis ; simultaneously during sleep the expression of molecules involved in synaptic reorganization is increased . based on simple energy demands and since the cerebral cortex thalamus unit is a large part of the human and mammal brain , being awake for longer and longer periods of time seems to be very expensive , hence energy economy by the brain during nrem sleep is one of the prevalent hypotheses to explain nrem sleep pressure ( mignot and huguenard , 2009 ) . ( 2009 ) , wakefulness is associated with learning , and enhancing long - term potentiation strengthens glutamatergic synapses , a process with an unsustainable energy requirement , whereas during sleep only robust connections remain intact , reducing the energy requirements to the levels necessary for maintaining critical learned circuits . the close reciprocal connections between the thalamus and the cerebral cortex would force nrem sleep homeostatic processes to take place simultaneously in the two structures . the thalamus cerebral cortex is a strictly necessary unit in the expression of the most significant bioelectric and behavioral events of nrem sleep and the thalamus cerebral cortex unit is connected to subcortical nrem sleep - promoting structures and with all the subcortical structures involved in the organization of the other phases of swc ( reinoso - surez et al .

copd is a chronic , progressive disease of the lung characterized by poor airflow , shortness of breath , and cough , and leads to long - term decline in lung function.1 approximately 5% of the world population ( ~329 million people ) are affected by copd,2 which is correlated with tobacco use,3 older age,4 pollution,5,6 and genetics.1,7,8 this disease is a major cause of morbidity and mortality , and was responsible for 3 million deaths globally as estimated by the world health organization in 2012.1,6,9,10 the world health organization also estimates that over one - third of premature deaths attributable to copd in low- and middle - income countries are due to indoor exposure to smoke.11 in addition , copd patients may experience frequent exacerbations , respiratory infections , and copd - related hospitalizations that contribute to a substantial social and economic burden,12 accounting for an estimated $ 18 billion in direct medical costs in the usa and ~38.7 billion in the european union.10 there is no cure for copd , but appropriate pharmacologic therapy is critical in reducing the frequency and severity of symptoms . bronchodilators , which alter airway smooth muscle tone , are central to the management of copd symptoms . long - acting 2-agonists ( labas ) and long - acting muscarinic antagonists ( lamas ) are two commonly used bronchodilators.1 laba monotherapy is contraindicated for patients with asthma , due to an increased risk of the exacerbation of asthma symptoms.13 a variety of labas with different durations of action , routes of administration , delivery devices , and associated rates of exacerbation , breathlessness , and bronchodilator effects are currently available.1,14 commonly used labas include twice - daily ( bid ) salmeterol 50 g and formoterol 12 g , which have a duration of action of 12 hours . newer agents such as indacaterol 75/150/300 g,15 olodaterol 5/10 g,16 and vilanterol 25 g17 are given once daily ( od ) with a duration of action of 24 hours . ( indacaterol 150 and 300 g , olodaterol 10 g , and vilanterol 25 g are not commercially available in the usa . ) given the variety of available labas for the treatment of copd , physicians are faced with the difficulty of choosing the laba with optimal efficacy . randomized controlled trials ( rcts ) have evaluated the efficacy of long - acting laba monotherapies against placebo and/or short - acting labas . in rcts , indacaterol was found to have a significantly greater bronchodilator effect than placebo , formoterol 12 g bid , and salmeterol 50 g bid.15,18,19 in addition , olodaterol ( 5/10 g ) was superior to placebo and formoterol 12 g bid,16,20 and vilanterol 25 g od was superior to placebo.17,21,22 in addition , several earlier network meta - analyses ( nmas ) have indirectly compared the efficacy among a limited number of labas / lamas.23,24 in 2013 , cope et al compared 40 rcts in a bayesian meta - analysis and found that indacaterol ( 150/300 g ) , glycopyrronium 50 g , and tiotropium 5 g were superior to other labas , with indacaterol dominant in forced expiratory volume in 1 second ( fev1 ) and st george s respiratory questionnaire ( sgrq ) score improvement.23 in 2014 , roskell et al compared olodaterol 5 g and indacaterol ( 75/150 g ) in a meta - analysis of 18 rcts , and found no significant differences in their primary analysis.24 however , studies evaluating the comparative efficacy of all currently available labas , including the newer agents in different doses , have not been conducted . thus , this study aimed to evaluate the comparative efficacy of all available laba monotherapy inhalers trialed in patients with moderate to severe copd using an nma . labas included in the network were salmeterol 50 g bid ( inhalation powder ) , formoterol 12 g bid ( inhalation powder ) , indacaterol 75/150/300 g od ( inhalation powder ) , olodaterol 5 and 10 g od ( inhalation spray ) , and vilanterol 25 g od ( inhalation powder ) . ( indacaterol 150 and 300 g , olodaterol 10 g , and vilanterol 25 g are not commercially available in the usa . ) the efficacy of these labas was evaluated using the following outcomes : 1 ) trough fev1 at 12 and 24 weeks ; 2 ) transition dyspnea index ( tdi ) focal score at 12 and 24 weeks ; 3 ) sgrq total score at 12 and 24 weeks ; and 4 ) rate of exacerbation . a systematic literature review was conducted to update an earlier systematic review completed in 2013.23 the updated search was performed in medline and medline - in - process , embase , and cochrane databases through ovid for rcts evaluating the efficacy of laba monotherapies ( indacaterol [ indacaterol inhalation powder ] , salmeterol [ salmeterol xinafoate inhalation powder ] , olodaterol [ olodaterol inhalation spray ] , vilanterol [ vilanterol inhalation powder ] , and formoterol [ formoterol inhalation powder ] ) trialed in patients with moderate to severe copd . the studies identified from the updated search spanned from january 1 , 2013 to march 24 , 2015 , while the earlier search had extended back to 1989.23 full - text terms and common abbreviations , listed in the supplementary material , were used for the search strategy . eligible studies from both the earlier and updated systematic literature reviews were included in the current meta - analysis . all articles identified in the initial database search were screened for relevance based on title , abstract , and full - text articles . rcts that reported at least one of the outcomes of interest for the targeted interventions among adults with moderate to severe copd were selected . to be included into the network , trials were further required to include a comparison of at least two of the interventions of interest or one of the above interventions against placebo . the selection criteria for the study population , interventions , comparators , and outcomes are detailed in table 1 . the screening process was independently conducted by and reconciled between two researchers , and in the event of a discrepancy , a third researcher was consulted . trials were excluded if they were duplications , conference abstracts only , < 12 weeks in duration , or if the patient population , trial design , intervention , comparator , or outcomes did not meet the inclusion criteria ( table 1 ) . continuous outcomes included trough fev1 , tdi focal score , and sgrq total score at 12 and 24 weeks . in the absence of 12- and 24-week data , data within a 2-week range for each time point of interest were allowed ( ie , between 10 and 14 weeks for the 12-week time point and between 22 and 26 weeks for the 6-month time point ) . differences between the least square mean at follow - up or the change from baseline for the active treatment versus the comparator were used for the network analysis . to be included in the network , outcomes had to be reported for each treatment group in a clear manner to allow reliable estimation of the treatment differences and their associated standard errors . rates of exacerbation were compared between the treatment groups at the end of trial follow - up . to be included in the network , this outcome had to be reported as the number of events of exacerbation with the total patient years of follow - up . if such event rates were not available , the rates were then calculated as the number of total events divided by the total patient - years which allowed the rates of exacerbations accumulated over differing periods of follow - up to be compared ( assuming the risk of exacerbations remained constant over time ) . severity of the exacerbation could not be incorporated into the analysis due to lack of granular severity reporting within the trials . nma combines data from several different randomized comparisons of different treatments to deliver an internally consistent set of estimates while respecting the randomization within each trial . this nma was carried out within a generalized linear model framework with a link function which specified the relationship between the outcome and the model coefficients to be estimated . when an outcome was continuous , such as trough fev1 , the likelihood was modeled as normal . when the outcome was an event rate , such as the per patient - year event rate of exacerbation , the likelihood was modeled as poisson.25,26 random effect models were utilized for this analysis . the estimation was performed under a bayesian context , using noninformative prior distributions for parameters . the model was evaluated using the deviance information criterion , a measure which combines model fit and complexity . all analyses were implemented using the statistical software r ( v3.2.2 ; ross ihaka and robert gentleman , open source ) and openbugs ( v3.2.3 ; openbugs foundation ) . because trough fev1 was the primary efficacy outcome of the majority of the rcts , sensitivity analyses were conducted for the fev1 12- and 24-week outcomes to test the robustness of the nma results . specifically , because concomitant medications and copd severity are potential treatment effect modifiers , the sensitivity analyses included : 1 ) a subset of trials with no concomitant lama usage ( all trials which permitted concomitant usage were excluded ) ; 2 ) a meta - regression adjusting for disease severity ( adjusting for the percent of patients with severe / very severe copd ) ; and 3 ) a meta - regression adjusting for inhaled corticosteroid ( ics ) use ( adjusting for the percent of patients with ics use within each trial ) . a systematic literature review was conducted to update an earlier systematic review completed in 2013.23 the updated search was performed in medline and medline - in - process , embase , and cochrane databases through ovid for rcts evaluating the efficacy of laba monotherapies ( indacaterol [ indacaterol inhalation powder ] , salmeterol [ salmeterol xinafoate inhalation powder ] , olodaterol [ olodaterol inhalation spray ] , vilanterol [ vilanterol inhalation powder ] , and formoterol [ formoterol inhalation powder ] ) trialed in patients with moderate to severe copd . the studies identified from the updated search spanned from january 1 , 2013 to march 24 , 2015 , while the earlier search had extended back to 1989.23 full - text terms and common abbreviations , listed in the supplementary material , were used for the search strategy . eligible studies from both the earlier and updated systematic literature reviews were included in the current meta - analysis . all articles identified in the initial database search were screened for relevance based on title , abstract , and full - text articles . rcts that reported at least one of the outcomes of interest for the targeted interventions among adults with moderate to severe copd were selected . to be included into the network , trials were further required to include a comparison of at least two of the interventions of interest or one of the above interventions against placebo . the selection criteria for the study population , interventions , comparators , and outcomes are detailed in table 1 . the screening process was independently conducted by and reconciled between two researchers , and in the event of a discrepancy , a third researcher was consulted . trials were excluded if they were duplications , conference abstracts only , < 12 weeks in duration , or if the patient population , trial design , intervention , comparator , or outcomes did not meet the inclusion criteria ( table 1 ) . continuous outcomes included trough fev1 , tdi focal score , and sgrq total score at 12 and 24 weeks . in the absence of 12- and 24-week data , data within a 2-week range for each time point of interest were allowed ( ie , between 10 and 14 weeks for the 12-week time point and between 22 and 26 weeks for the 6-month time point ) . differences between the least square mean at follow - up or the change from baseline for the active treatment versus the comparator were used for the network analysis . to be included in the network , outcomes had to be reported for each treatment group in a clear manner to allow reliable estimation of the treatment differences and their associated standard errors . rates of exacerbation were compared between the treatment groups at the end of trial follow - up . to be included in the network , this outcome had to be reported as the number of events of exacerbation with the total patient years of follow - up . if such event rates were not available , the rates were then calculated as the number of total events divided by the total patient - years which allowed the rates of exacerbations accumulated over differing periods of follow - up to be compared ( assuming the risk of exacerbations remained constant over time ) . severity of the exacerbation could not be incorporated into the analysis due to lack of granular severity reporting within the trials . nma combines data from several different randomized comparisons of different treatments to deliver an internally consistent set of estimates while respecting the randomization within each trial . this nma was carried out within a generalized linear model framework with a link function which specified the relationship between the outcome and the model coefficients to be estimated . when an outcome was continuous , such as trough fev1 , the likelihood was modeled as normal . when the outcome was an event rate , such as the per patient - year event rate of exacerbation , the likelihood was modeled as poisson.25,26 random effect models were utilized for this analysis . the estimation was performed under a bayesian context , using noninformative prior distributions for parameters . the model was evaluated using the deviance information criterion , a measure which combines model fit and complexity . all analyses were implemented using the statistical software r ( v3.2.2 ; ross ihaka and robert gentleman , open source ) and openbugs ( v3.2.3 ; openbugs foundation ) . because trough fev1 was the primary efficacy outcome of the majority of the rcts , sensitivity analyses were conducted for the fev1 12- and 24-week outcomes to test the robustness of the nma results . specifically , because concomitant medications and copd severity are potential treatment effect modifiers , the sensitivity analyses included : 1 ) a subset of trials with no concomitant lama usage ( all trials which permitted concomitant usage were excluded ) ; 2 ) a meta - regression adjusting for disease severity ( adjusting for the percent of patients with severe / very severe copd ) ; and 3 ) a meta - regression adjusting for inhaled corticosteroid ( ics ) use ( adjusting for the percent of patients with ics use within each trial ) . the updated systematic review identified 916,17,2022,2730 full - text articles detailing 12 rcts that met the inclusion criteria ( figure 1 ) . these were pooled with 21 laba monotherapy rcts identified in the previous search,15,18,19,3148 resulting in a total of 33 rcts included in the nma . a list of included studies and details of the systematic literature search can be found in figure 1 , and the details of each study s own inclusion criteria are listed in table s1 . all studies were double - blind , multicenter rcts ( figure 2 ) , ranging from 12 weeks to 3 years in duration . all studies were placebo controlled , with the exception of one head - to - head study which compared the efficacy of indacaterol 150 g od to salmeterol 50 g bid.19 the studies were predominantly conducted in multiple countries simultaneously , although four were limited to the usa32,35,36,44 and one was limited to the netherlands.40 the majority of the studies were conducted in patients over the age of 40 with a smoking history of 10 pack - years and predicted fev1 of 80% . each trial predominantly enrolled male patients , and the mean age was > 60 years in all trials . the percentage of ics use , current smokers , and patients with severe or very severe copd varied among studies . the 33 rcts were synthesized in the nma ; a network diagram of the included studies is detailed in figure 2 . the network for each outcome measure consisted of a subset of the presented network based on the availability of different outcomes within the 33 rcts . changes in baseline trough fev1 at 12 and 24 weeks were reported in a total of 24 and 19 trials , respectively . all interventions were found to be significantly better than placebo in terms of fev1 , at both 12 and 24 weeks . relative to placebo at 12 weeks , indacaterol 300 g ( difference : 0.167 l , 95% credible interval : [ 0.151 , 0.183 ] ) had the largest difference in change in baseline trough fev1 , followed by indacaterol 150 g ( 0.163 l [ 0.148 , 0.177 ] ) , indacaterol 75 g ( 0.129 l [ 0.099 , 0.157 ] ) , salmeterol 50 g bid ( 0.105 l [ 0.085 , 0.125 ] ) , vilanterol 25 g od ( 0.098 l [ 0.076 , 0.120 ] ) , olodaterol 10 g od ( 0.083 [ 0.063 , 0.103 ] ) , olodaterol 5 g od ( 0.073 [ 0.053 , 0.092 ] ) , and formoterol 12 g bid ( 0.071 l [ 0.057 , 0.085 ] ; figure 3a ) . indacaterol 300 g od and indacaterol 150 g od were associated with significantly better trough fev1 compared to indacaterol 75 g od , salmeterol 50 g bid , vilanterol 25 g od , olodaterol 10 g od , olodaterol 5 g od , and formoterol 12 g bid , and were not statistically different from each other . indacaterol 75 g was associated with significantly better trough fev1 compared to olodaterol 10 g od , olodaterol 5 g od , and formoterol 12 g bid . salmeterol 50 g bid was associated with significantly better trough fev1 compared to olodaterol 5 g od and formoterol 12 g bid . there were no significant differences between indacaterol 75 g od , salmeterol 50 g bid , and vilanterol 25 g od . relative to placebo at 24 weeks , indacaterol 300 g ( 0.162 l [ 0.143 , 0.181 ] ) had the largest difference in change in baseline trough fev1 , followed by indacaterol 150 g ( 0.147 l [ 0.129 , 0.164 ] ) , vilanterol 25 g od ( 0.094 l [ 0.065 , 0.124 ] ) , salmeterol 50 g bid ( 0.082 l [ 0.066 , 0.098 ] ) , olodaterol 10 g od ( 0.079 l [ 0.059 , 0.099 ] ) , olodaterol 5 g od ( 0.074 [ 0.055 , 0.094 ] ) , and formoterol 12 g bid ( 0.061 [ 0.046 , 0.076 ] ; figure 3b ) . indacaterol 75 g od was not included in the 24-week analysis . as in the 12-week analysis , indacaterol 300 g od and indacaterol 150 g od were associated with significantly better trough fev1 compared to vilanterol 25 g od , salmeterol 50 g bid , olodaterol 10 g od , olodaterol 5 g od , and formoterol 12 g bid , and were not statistically different from each other ( table 2 ) . in addition , vilanterol 25 g od had significantly higher mean trough fev1 than formoterol 12 g bid . changes in baseline mean tdi focal scores at 12 and 24 weeks were reported in 14 and 15 trials , respectively . at 12 and 24 relative to placebo at 12 weeks , indacaterol 300 , 150 , and 75 g od ( 1.171 [ 0.906 , 1.401 ] , 1.051 [ 0.826 , 1.291 ] , and 0.831 [ 0.330 , 1.336 ] , respectively ) had the highest difference in tdi focal scores at 12 weeks , followed by olodaterol 10 g od ( 0.734 [ 0.278 , 1.166 ] ) , vilanterol 25 g od ( 0.665 [ 0.284 , 1.054 ] ) , olodaterol 5 g od ( 0.629 [ 0.187 , 1.058 ] ) , formoterol 12 g bid ( 0.618 [ 0.281 , 0.925 ] ) , and salmeterol 50 g bid ( 0.555 [ 0.246 , 0.887 ] ; figure 4a ) . indacaterol 300 g od and indacaterol 150 g od were associated with significantly higher mean tdi focal score compared to salmeterol 50 g bid and formoterol 12 g bid . indacaterol 300 g od was also associated with significantly higher mean tdi focal score compared to olodaterol 5 g od and vilanterol 25 g od ( table 3 ) . relative to placebo at 24 weeks , indacaterol 300 g od ( 1.184 [ 0.942 , 1.433 ] ) had the highest difference in tdi focal scores at 24 weeks , followed by indacaterol 150 g od ( 0.894 [ 0.653 , 1.139 ] ) , salmeterol 50 g bid ( 0.696 [ 0.423 , 0.965 ] ) , vilanterol 25 g od ( 0.693 [ 0.297 , 1.093 ] ) , formoterol 12 g bid ( 0.594 [ 0.359 , 0.838 ] ) , olodaterol 5 g od ( 0.556 [ 0.143 , 0.975 ) , and olodaterol 10 g od ( 0.501 [ 0.097 , 0.920 ] ; figure 4b ) . indacaterol 300 g od was associated with a significantly higher mean tdi focal score compared to all other labas ( including indacaterol 150 g od ; table 3 ) . changes from baseline in sgrq total score at weeks 12 and 24 were reported in a total of 14 and 16 trials , respectively . at 12 and 24 weeks , all interventions were found to be significantly better than placebo . no significant differences were noted at 12 or 24 weeks between the different labas , except that indacaterol 150 g was significantly better than salmeterol 50 g bid ( 1.776 [ 3.430 , 0.023 ] ) at week 24 ( table 4 ) . relative to placebo , the numerically best sgrq scores at 12 weeks belonged to ( in order ) olodaterol 10 g od ( 4.144 [ 6.089 , 2.161 ] ) , indacaterol 150 g ( 4.022 [ 5.096 , 2.962 ] ) , indacaterol 300 g ( 3.704 [ 4.922 , 2.501 ] ) , and indacaterol 75 g od ( 3.691 [ 5.825 , 1.509 ] ) , followed by formoterol 12 g bid ( 3.150 [ 4.464 , 1.890 ] ) , olodaterol 5 g od ( 3.047 [ 5.014 , 1.107 ] ) , and salmeterol 50 g bid ( 2.710 [ 4.463 , 0.935 ] ) . for 24 weeks , the best scores belonged to olodaterol 10 g od ( 3.589 [ 5.704 , 1.429 ] ) , indacaterol 150 g od ( 3.155 [ 4.504 , 1.752 ] ) , and vilanterol 25 g od ( 2.906 [ 5.042 , 0.769 ] ) , followed by indacaterol 300 g od ( 2.843 [ 4.321 , 1.407 ] ) , formoterol 12 g bid ( 1.401 [ 2.694 , 0.113 ] ) , and salmeterol 50 g bid ( 1.379 [ 2.559 , 0.286 ] ; figure 5 ) . vilanterol 25 g od was not included in the 12-week analysis , and indacaterol 75 g od was not included in the 24-week analysis . a total of 14 trials that reported the exacerbation rate were included in the evidence network including salmeterol 50 g bid , formoterol 12 g bid , indacaterol 150 g od , indacaterol 300 g od , olodaterol 5 g od , olodaterol 10 g od , and placebo . the exacerbation rates were significantly lower for salmeterol 50 g bid , indacaterol 150 g od , and indacaterol 300 g od , compared with placebo . in addition , indacaterol 150 g od was significantly better than olodaterol 5 g od ( 0.773 [ 0.590 , 0.991 ] ) and olodaterol 10 g od ( 0.737 [ 0.565 , 0.939 ] ; table 5 and figure 6 ) . a summary of the nma results for trough fev1 after adjusting for female percentage , disease severity , ics use , and the subgroup with no concomitant lama use at 12 and 24 weeks is presented in figure 7 . all the changes were minimal , ranging from 0.005 to 0.004 l for week 12 outcomes and from 0.006 to 0.012 l for week 24 outcomes , illustrating that the nma results were robust . the updated systematic review identified 916,17,2022,2730 full - text articles detailing 12 rcts that met the inclusion criteria ( figure 1 ) . these were pooled with 21 laba monotherapy rcts identified in the previous search,15,18,19,3148 resulting in a total of 33 rcts included in the nma . a list of included studies and details of the systematic literature search can be found in figure 1 , and the details of each study s own inclusion criteria are listed in table s1 . all studies were double - blind , multicenter rcts ( figure 2 ) , ranging from 12 weeks to 3 years in duration . all studies were placebo controlled , with the exception of one head - to - head study which compared the efficacy of indacaterol 150 g od to salmeterol 50 g bid.19 the studies were predominantly conducted in multiple countries simultaneously , although four were limited to the usa32,35,36,44 and one was limited to the netherlands.40 the majority of the studies were conducted in patients over the age of 40 with a smoking history of 10 pack - years and predicted fev1 of 80% . each trial predominantly enrolled male patients , and the mean age was > 60 years in all trials . the percentage of ics use , current smokers , and patients with severe or very severe copd varied among studies . the 33 rcts were synthesized in the nma ; a network diagram of the included studies is detailed in figure 2 . the network for each outcome measure consisted of a subset of the presented network based on the availability of different outcomes within the 33 rcts . changes in baseline trough fev1 at 12 and 24 weeks were reported in a total of 24 and 19 trials , respectively . all interventions were found to be significantly better than placebo in terms of fev1 , at both 12 and 24 weeks . relative to placebo at 12 weeks , indacaterol 300 g ( difference : 0.167 l , 95% credible interval : [ 0.151 , 0.183 ] ) had the largest difference in change in baseline trough fev1 , followed by indacaterol 150 g ( 0.163 l [ 0.148 , 0.177 ] ) , indacaterol 75 g ( 0.129 l [ 0.099 , 0.157 ] ) , salmeterol 50 g bid ( 0.105 0.098 l [ 0.076 , 0.120 ] ) , olodaterol 10 g od ( 0.083 [ 0.063 , 0.103 ] ) , olodaterol 5 g od ( 0.073 [ 0.053 , 0.092 ] ) , and formoterol 12 g bid ( 0.071 l [ 0.057 , 0.085 ] ; figure 3a ) . indacaterol 300 g od and indacaterol 150 g od were associated with significantly better trough fev1 compared to indacaterol 75 g od , salmeterol 50 g bid , vilanterol 25 g od , olodaterol 10 g od , olodaterol 5 g od , and formoterol 12 g bid , and were not statistically different from each other . indacaterol 75 g was associated with significantly better trough fev1 compared to olodaterol 10 g od , olodaterol 5 g od , and formoterol 12 g bid . salmeterol 50 g bid was associated with significantly better trough fev1 compared to olodaterol 5 g od and formoterol 12 g bid . there were no significant differences between indacaterol 75 g od , salmeterol 50 g bid , and vilanterol 25 g od . relative to placebo at 24 weeks , indacaterol 300 g ( 0.162 l [ 0.143 , 0.181 ] ) had the largest difference in change in baseline trough fev1 , followed by indacaterol 150 g ( 0.147 l [ 0.129 , 0.164 ] ) , vilanterol 25 g od ( 0.094 l [ 0.065 , 0.124 ] ) , salmeterol 50 g bid ( 0.082 l [ 0.066 , 0.098 ] ) , olodaterol 10 g od ( 0.079 l [ 0.059 , 0.099 ] ) , olodaterol 5 g od ( 0.074 [ 0.055 , 0.094 ] ) , and formoterol 12 g bid ( 0.061 [ 0.046 , 0.076 ] ; figure 3b ) . indacaterol 75 g od was not included in the 24-week analysis . as in the 12-week analysis , indacaterol 300 g od and indacaterol 150 g od were associated with significantly better trough fev1 compared to vilanterol 25 g od , salmeterol 50 g bid , olodaterol 10 g od , olodaterol 5 g od , and formoterol 12 g bid , and were not statistically different from each other ( table 2 ) . in addition , vilanterol 25 g od had significantly higher mean trough fev1 than formoterol 12 g bid . changes in baseline mean tdi focal scores at 12 and 24 weeks were reported in 14 and 15 trials , respectively . at 12 and 24 relative to placebo at 12 weeks , indacaterol 300 , 150 , and 75 g od ( 1.171 [ 0.906 , 1.401 ] , 1.051 [ 0.826 , 1.291 ] , and 0.831 [ 0.330 , 1.336 ] , respectively ) had the highest difference in tdi focal scores at 12 weeks , followed by olodaterol 10 g od ( 0.734 [ 0.278 , 1.166 ] ) , vilanterol 25 g od ( 0.665 [ 0.284 , 1.054 ] ) , olodaterol 5 g od ( 0.629 [ 0.187 , 1.058 ] ) , formoterol 12 g bid ( 0.618 [ 0.281 , 0.925 ] ) , and salmeterol 50 g bid ( 0.555 [ 0.246 , 0.887 ] ; figure 4a ) . indacaterol 300 g od and indacaterol 150 g od were associated with significantly higher mean tdi focal score compared to salmeterol 50 g bid and formoterol 12 g bid . indacaterol 300 g od was also associated with significantly higher mean tdi focal score compared to olodaterol 5 g od and vilanterol 25 g od ( table 3 ) . relative to placebo at 24 weeks , indacaterol 300 g od ( 1.184 [ 0.942 , 1.433 ] ) had the highest difference in tdi focal scores at 24 weeks , followed by indacaterol 150 g od ( 0.894 [ 0.653 , 1.139 ] ) , salmeterol 50 g bid ( 0.696 [ 0.423 , 0.965 ] ) , vilanterol 25 g od ( 0.693 [ 0.297 , 1.093 ] ) , formoterol 12 g bid ( 0.594 [ 0.359 , 0.838 ] ) , olodaterol 5 g od ( 0.556 [ 0.143 , 0.975 ) , and olodaterol 10 g od ( 0.501 [ 0.097 , 0.920 ] ; figure 4b ) . indacaterol 300 g od was associated with a significantly higher mean tdi focal score compared to all other labas ( including indacaterol 150 g od ; table 3 ) . changes from baseline in sgrq total score at weeks 12 and 24 were reported in a total of 14 and 16 trials , respectively . at 12 and 24 weeks , all interventions were found to be significantly better than placebo . no significant differences were noted at 12 or 24 weeks between the different labas , except that indacaterol 150 g was significantly better than salmeterol 50 g bid ( 1.776 [ 3.430 , 0.023 ] ) at week 24 ( table 4 ) . relative to placebo , the numerically best sgrq scores at 12 weeks belonged to ( in order ) olodaterol 10 g od ( 4.144 [ 6.089 , 2.161 ] ) , indacaterol 150 g ( 4.022 [ 5.096 , 2.962 ] ) , indacaterol 300 g ( 3.704 [ 4.922 , 2.501 ] ) , and indacaterol 75 g od ( 3.691 [ 5.825 , 1.509 ] ) , followed by formoterol 12 g bid ( 3.150 [ 4.464 , 1.890 ] ) , olodaterol 5 g od ( 3.047 [ 5.014 , 1.107 ] ) , and salmeterol 50 g bid ( 2.710 [ 4.463 , 0.935 ] ) . for 24 weeks , the best scores belonged to olodaterol 10 g od ( 3.589 [ 5.704 , 1.429 ] ) , indacaterol 150 g od ( 3.155 [ 4.504 , 1.752 ] ) , and vilanterol 25 g od ( 2.906 [ 5.042 , 0.769 ] ) , followed by indacaterol 300 g od ( 2.843 [ 4.321 , 1.407 ] ) , formoterol 12 g bid ( 1.401 [ 2.694 , 0.113 ] ) , and salmeterol 50 g bid ( 1.379 [ 2.559 , 0.286 ] ; figure 5 ) . vilanterol 25 g od was not included in the 12-week analysis , and indacaterol 75 g od was not included in the 24-week analysis . a total of 14 trials that reported the exacerbation rate were included in the evidence network including salmeterol 50 g bid , formoterol 12 g bid , indacaterol 150 g od , indacaterol 300 g od , olodaterol 5 g od , olodaterol 10 g od , and placebo . the exacerbation rates were significantly lower for salmeterol 50 g bid , indacaterol 150 g od , and indacaterol 300 g od , compared with placebo . in addition , indacaterol 150 g od was significantly better than olodaterol 5 g od ( 0.773 [ 0.590 , 0.991 ] ) and olodaterol 10 g od ( 0.737 [ 0.565 , 0.939 ] ; table 5 and figure 6 ) . a summary of the nma results for trough fev1 after adjusting for female percentage , disease severity , ics use , and the subgroup with no concomitant lama use at 12 and 24 weeks is presented in figure 7 . all the changes were minimal , ranging from 0.005 to 0.004 l for week 12 outcomes and from 0.006 to 0.012 l for week 24 outcomes , illustrating that the nma results were robust . this study is the first nma to analyze the comparative efficacy of all currently available labas , including the newer agents for the treatment of moderate to severe copd . thus , this study provides the most up - to - date understanding of the treatment landscape for copd in terms of laba monotherapies as well as the most complete comparative analysis of effective treatment options and dosages in terms of efficacy outcomes : fev1 , tdi focal score , sgrq , and exacerbation rate . the results indicate that indacaterol was the most effective laba monotherapy for the treatment of copd , similar to the findings of earlier studies comparing laba efficacy.23,24 specifically , indacaterol 150 g od and indacaterol 300 g od were associated with significant improvement in 12- and 24-week trough fev1 compared to all other labas , and indacaterol 75 g od was associated with significant improvement in trough fev1 at week 12 compared to formoterol and olodaterol ( both 5 and 10 g doses ) . in addition , indacaterol , 300 g in particular , showed statistical superiority over other labas in tdi score , and indacaterol 150 g showed statistical superiority over olodaterol 5 g and olodaterol 10 g od in exacerbation rates . olodaterol 10 g od showed numerical superiority in sgrq scores at 12 and 24 weeks , although the results were not statistically different from the other labas . the outcomes compared in this meta - analysis each have valid thresholds for clinically relevant differences versus placebo . for example , for fev1 , a widely accepted threshold , is a change of 100 ml from baseline ; for tdi focal score , a 1 unit score reduction ; for sgrq total score , a reduction of 4 units ; and for exacerbation rate , an annual rate reduction of 20%.49 these outcomes were used for the determination of efficacy against placebo in respective clinical trials ; however , the validity of using these thresholds for post hoc active - arm comparisons has not been empirically evaluated . thus , nmas such as the current analysis are useful for comparing active treatments by testing for statistically significant differences between treatment outcomes after showing clinically meaningful efficacy against placebo . future prospective studies evaluating the thresholds for clinically meaningful differences between active treatments in copd therapy trials are needed . the results of this study add to and update the pre - existing literature on the comparative efficacy of labas in the treatment of copd , while coming to similar conclusions as previous studies about the efficacy of indacaterol . for example , a 2013 nma on the comparative efficacy of long - acting bronchodilators for copd found that indacaterol was associated with higher trough fev1 and superior improvement in sgrq score over comparative labas.23 in addition , a 2012 comparative effectiveness study evaluated indacaterol for copd versus placebo , formoterol , and salmeterol in rcts using the outcomes trough fev1 , sgrq , and tdi total scores . it found that indacaterol was as good as or superior to these bronchodilators in all the outcomes measured , and that indacaterol 300 g resulted in the best overall efficacy.50 a 2014 systematic review compared efficacy outcomes ( fev1 , sgrq and tdi scores , exacerbations , and use of rescue medication at 12 weeks ) for olodaterol and indacaterol and determined that these drugs had similar efficacy.24 however , a comment published later in 2014 noted that the study suffered from several limitations including a restricted search date resulting in exclusion of relevant clinical trials , study design heterogeneity , and reliance on data from other nmas rather than primary data within rcts.51 in the current analysis , only trials of the inhalation powder form of formoterol were included , in order to maintain consistency with the delivery device of the other comparators . however , nebulized formoterol may be beneficial for patients who are unable to use inhalation powder for reasons including frailty , arthritis , visual impairment , compromised mental capacity , exacerbation , difficulty using an inhaler , or inadequate hand / breath coordination . important differences may exist between real - world practice and clinical trial populations , such as training for the use of inhalers , adherence to treatment , and routine medical care , all of which may limit the applicability of the current results . some limitations inherent to nmas apply to the results of this study . for example , although the trials included in the nma were of good caliber , the validity of the current findings depends on the quality , biases , and study and patient characteristic reporting consistency of the included rcts . some variation existed in their inclusion criteria regarding the concomitant use of laba and icss , smoking history , age , the severity of copd , and exacerbation history . though sensitivity analyses have been conducted for our main outcome fev1 , meta - regression analyses of study - level data can be prone to ecological bias ( ie , the association between the study - level effect patient characteristics and treatment effects may not reflect the individual - level effect modification of a covariate ) . since there was only a single head - to - head trial , the ability to check the consistency of the direct and indirect evidence was limited . however , in this head - to - head trial , indacaterol 150 g was associated with a 0.06 l higher fev1 compared to salmeterol 50 g,19 which is consistent with the 0.057 l estimated in this analysis . in addition , other outcomes , which are important in the measurement of copd treatment efficacy and safety and played an essential role in treatment decisions , such as the use of rescue treatments and the severity of exacerbations , were not assessed in this study . future studies are warranted to further evaluate these outcomes among different treatment options for copd patients . lastly , exacerbations were expected to be defined differently among the included studies , and the nma results might be subject to those inconsistencies , if any . because the number of studies that contained that study outcome ( exacerbations ) was small , no subgroup analysis was conducted in this study . future research may be needed to conduct subgroup analysis among studies with consistent criteria and definitions , when there are sufficient studies . in conclusion , indacaterol 300 g od , followed by 150 g od , and 75 g od , were the most effective laba monotherapies for copd in terms of trough fev1 and tdi focal scores .

purposelong - acting 2-agonists ( labas ) have demonstrated efficacy in patients with copd in clinical trials . the purpose of this study was to assess the comparative efficacy of all available dosages of all laba monotherapies using a network meta-analysis.methodsa systematic literature review identified 33 randomized controlled trials of laba monotherapies ( salmeterol 50 g twice daily [ bid ] ; formoterol 12 g bid ; indacaterol 75 , 150 , and 300 g once daily [ od ] ; olodaterol 5 and 10 g od , and vilanterol 25 g od ) . clinical efficacy was evaluated at 12 and 24 weeks in terms of trough forced expiratory volume in 1 second ( fev1 ) , transition dyspnea index focal score , st george s respiratory questionnaire total score , and rate of copd exacerbations . the relative effectiveness of all laba monotherapies was estimated by bayesian network meta-analysis.resultsat 12 and 24 weeks , indacaterol 300 and 150 g od were associated with statistically significant improvement in trough fev1 compared to all other laba monotherapies ; vilanterol 25 g od was superior to formoterol 12 g bid . at 12 weeks , indacaterol 75 g od was associated with significant improvement in trough fev1 compared to formoterol 12 g bid and olodaterol ( 5 and 10 g od ) ; salmeterol 50 g bid was superior to formoterol 12 g bid and olodaterol 5 g od . indacaterol 300 g od was also associated with significant improvement in transition dyspnea index focal score compared to all other labas at 12 or 24 weeks . indacaterol 150 g od had significantly better results in exacerbation rates than olodaterol 5 g and olodaterol 10 g od.conclusionindacaterol 300 g , followed by 150 and 75 g , were the most effective laba monotherapies for moderate to severe copd .

Introduction Methods Study identification and selection Outcome measures Network meta-analyses Sensitivity analyses Results Evidence base Network meta-analysis Trough FEV TDI focal score at 12 and 24 weeks SGRQ total score at 12 and 24 weeks Exacerbation rate Sensitivity analysis for trough FEV Discussion Conclusion

long - acting 2-agonists ( labas ) and long - acting muscarinic antagonists ( lamas ) are two commonly used bronchodilators.1 laba monotherapy is contraindicated for patients with asthma , due to an increased risk of the exacerbation of asthma symptoms.13 a variety of labas with different durations of action , routes of administration , delivery devices , and associated rates of exacerbation , breathlessness , and bronchodilator effects are currently available.1,14 commonly used labas include twice - daily ( bid ) salmeterol 50 g and formoterol 12 g , which have a duration of action of 12 hours . in rcts , indacaterol was found to have a significantly greater bronchodilator effect than placebo , formoterol 12 g bid , and salmeterol 50 g bid.15,18,19 in addition , olodaterol ( 5/10 g ) was superior to placebo and formoterol 12 g bid,16,20 and vilanterol 25 g od was superior to placebo.17,21,22 in addition , several earlier network meta - analyses ( nmas ) have indirectly compared the efficacy among a limited number of labas / lamas.23,24 in 2013 , cope et al compared 40 rcts in a bayesian meta - analysis and found that indacaterol ( 150/300 g ) , glycopyrronium 50 g , and tiotropium 5 g were superior to other labas , with indacaterol dominant in forced expiratory volume in 1 second ( fev1 ) and st george s respiratory questionnaire ( sgrq ) score improvement.23 in 2014 , roskell et al compared olodaterol 5 g and indacaterol ( 75/150 g ) in a meta - analysis of 18 rcts , and found no significant differences in their primary analysis.24 however , studies evaluating the comparative efficacy of all currently available labas , including the newer agents in different doses , have not been conducted . labas included in the network were salmeterol 50 g bid ( inhalation powder ) , formoterol 12 g bid ( inhalation powder ) , indacaterol 75/150/300 g od ( inhalation powder ) , olodaterol 5 and 10 g od ( inhalation spray ) , and vilanterol 25 g od ( inhalation powder ) . a systematic literature review was conducted to update an earlier systematic review completed in 2013.23 the updated search was performed in medline and medline - in - process , embase , and cochrane databases through ovid for rcts evaluating the efficacy of laba monotherapies ( indacaterol [ indacaterol inhalation powder ] , salmeterol [ salmeterol xinafoate inhalation powder ] , olodaterol [ olodaterol inhalation spray ] , vilanterol [ vilanterol inhalation powder ] , and formoterol [ formoterol inhalation powder ] ) trialed in patients with moderate to severe copd . relative to placebo at 12 weeks , indacaterol 300 g ( difference : 0.167 l , 95% credible interval : [ 0.151 , 0.183 ] ) had the largest difference in change in baseline trough fev1 , followed by indacaterol 150 g ( 0.163 l [ 0.148 , 0.177 ] ) , indacaterol 75 g ( 0.129 l [ 0.099 , 0.157 ] ) , salmeterol 50 g bid ( 0.105 l [ 0.085 , 0.125 ] ) , vilanterol 25 g od ( 0.098 l [ 0.076 , 0.120 ] ) , olodaterol 10 g od ( 0.083 [ 0.063 , 0.103 ] ) , olodaterol 5 g od ( 0.073 [ 0.053 , 0.092 ] ) , and formoterol 12 g bid ( 0.071 l [ 0.057 , 0.085 ] ; figure 3a ) . indacaterol 300 g od and indacaterol 150 g od were associated with significantly better trough fev1 compared to indacaterol 75 g od , salmeterol 50 g bid , vilanterol 25 g od , olodaterol 10 g od , olodaterol 5 g od , and formoterol 12 g bid , and were not statistically different from each other . indacaterol 75 g was associated with significantly better trough fev1 compared to olodaterol 10 g od , olodaterol 5 g od , and formoterol 12 g bid . salmeterol 50 g bid was associated with significantly better trough fev1 compared to olodaterol 5 g od and formoterol 12 g bid . relative to placebo at 24 weeks , indacaterol 300 g ( 0.162 l [ 0.143 , 0.181 ] ) had the largest difference in change in baseline trough fev1 , followed by indacaterol 150 g ( 0.147 l [ 0.129 , 0.164 ] ) , vilanterol 25 g od ( 0.094 l [ 0.065 , 0.124 ] ) , salmeterol 50 g bid ( 0.082 l [ 0.066 , 0.098 ] ) , olodaterol 10 g od ( 0.079 l [ 0.059 , 0.099 ] ) , olodaterol 5 g od ( 0.074 [ 0.055 , 0.094 ] ) , and formoterol 12 g bid ( 0.061 [ 0.046 , 0.076 ] ; figure 3b ) . as in the 12-week analysis , indacaterol 300 g od and indacaterol 150 g od were associated with significantly better trough fev1 compared to vilanterol 25 g od , salmeterol 50 g bid , olodaterol 10 g od , olodaterol 5 g od , and formoterol 12 g bid , and were not statistically different from each other ( table 2 ) . at 12 and 24 relative to placebo at 12 weeks , indacaterol 300 , 150 , and 75 g od ( 1.171 [ 0.906 , 1.401 ] , 1.051 [ 0.826 , 1.291 ] , and 0.831 [ 0.330 , 1.336 ] , respectively ) had the highest difference in tdi focal scores at 12 weeks , followed by olodaterol 10 g od ( 0.734 [ 0.278 , 1.166 ] ) , vilanterol 25 g od ( 0.665 [ 0.284 , 1.054 ] ) , olodaterol 5 g od ( 0.629 [ 0.187 , 1.058 ] ) , formoterol 12 g bid ( 0.618 [ 0.281 , 0.925 ] ) , and salmeterol 50 g bid ( 0.555 [ 0.246 , 0.887 ] ; figure 4a ) . indacaterol 300 g od and indacaterol 150 g od were associated with significantly higher mean tdi focal score compared to salmeterol 50 g bid and formoterol 12 g bid . indacaterol 300 g od was also associated with significantly higher mean tdi focal score compared to olodaterol 5 g od and vilanterol 25 g od ( table 3 ) . relative to placebo at 24 weeks , indacaterol 300 g od ( 1.184 [ 0.942 , 1.433 ] ) had the highest difference in tdi focal scores at 24 weeks , followed by indacaterol 150 g od ( 0.894 [ 0.653 , 1.139 ] ) , salmeterol 50 g bid ( 0.696 [ 0.423 , 0.965 ] ) , vilanterol 25 g od ( 0.693 [ 0.297 , 1.093 ] ) , formoterol 12 g bid ( 0.594 [ 0.359 , 0.838 ] ) , olodaterol 5 g od ( 0.556 [ 0.143 , 0.975 ) , and olodaterol 10 g od ( 0.501 [ 0.097 , 0.920 ] ; figure 4b ) . indacaterol 300 g od was associated with a significantly higher mean tdi focal score compared to all other labas ( including indacaterol 150 g od ; table 3 ) . relative to placebo , the numerically best sgrq scores at 12 weeks belonged to ( in order ) olodaterol 10 g od ( 4.144 [ 6.089 , 2.161 ] ) , indacaterol 150 g ( 4.022 [ 5.096 , 2.962 ] ) , indacaterol 300 g ( 3.704 [ 4.922 , 2.501 ] ) , and indacaterol 75 g od ( 3.691 [ 5.825 , 1.509 ] ) , followed by formoterol 12 g bid ( 3.150 [ 4.464 , 1.890 ] ) , olodaterol 5 g od ( 3.047 [ 5.014 , 1.107 ] ) , and salmeterol 50 g bid ( 2.710 [ 4.463 , 0.935 ] ) . for 24 weeks , the best scores belonged to olodaterol 10 g od ( 3.589 [ 5.704 , 1.429 ] ) , indacaterol 150 g od ( 3.155 [ 4.504 , 1.752 ] ) , and vilanterol 25 g od ( 2.906 [ 5.042 , 0.769 ] ) , followed by indacaterol 300 g od ( 2.843 [ 4.321 , 1.407 ] ) , formoterol 12 g bid ( 1.401 [ 2.694 , 0.113 ] ) , and salmeterol 50 g bid ( 1.379 [ 2.559 , 0.286 ] ; figure 5 ) . a total of 14 trials that reported the exacerbation rate were included in the evidence network including salmeterol 50 g bid , formoterol 12 g bid , indacaterol 150 g od , indacaterol 300 g od , olodaterol 5 g od , olodaterol 10 g od , and placebo . in addition , indacaterol 150 g od was significantly better than olodaterol 5 g od ( 0.773 [ 0.590 , 0.991 ] ) and olodaterol 10 g od ( 0.737 [ 0.565 , 0.939 ] ; table 5 and figure 6 ) . relative to placebo at 12 weeks , indacaterol 300 g ( difference : 0.167 l , 95% credible interval : [ 0.151 , 0.183 ] ) had the largest difference in change in baseline trough fev1 , followed by indacaterol 150 g ( 0.163 l [ 0.148 , 0.177 ] ) , indacaterol 75 g ( 0.129 l [ 0.099 , 0.157 ] ) , salmeterol 50 g bid ( 0.105 0.098 l [ 0.076 , 0.120 ] ) , olodaterol 10 g od ( 0.083 [ 0.063 , 0.103 ] ) , olodaterol 5 g od ( 0.073 [ 0.053 , 0.092 ] ) , and formoterol 12 g bid ( 0.071 l [ 0.057 , 0.085 ] ; figure 3a ) . indacaterol 300 g od and indacaterol 150 g od were associated with significantly better trough fev1 compared to indacaterol 75 g od , salmeterol 50 g bid , vilanterol 25 g od , olodaterol 10 g od , olodaterol 5 g od , and formoterol 12 g bid , and were not statistically different from each other . indacaterol 75 g was associated with significantly better trough fev1 compared to olodaterol 10 g od , olodaterol 5 g od , and formoterol 12 g bid . salmeterol 50 g bid was associated with significantly better trough fev1 compared to olodaterol 5 g od and formoterol 12 g bid . relative to placebo at 24 weeks , indacaterol 300 g ( 0.162 l [ 0.143 , 0.181 ] ) had the largest difference in change in baseline trough fev1 , followed by indacaterol 150 g ( 0.147 l [ 0.129 , 0.164 ] ) , vilanterol 25 g od ( 0.094 l [ 0.065 , 0.124 ] ) , salmeterol 50 g bid ( 0.082 l [ 0.066 , 0.098 ] ) , olodaterol 10 g od ( 0.079 l [ 0.059 , 0.099 ] ) , olodaterol 5 g od ( 0.074 [ 0.055 , 0.094 ] ) , and formoterol 12 g bid ( 0.061 [ 0.046 , 0.076 ] ; figure 3b ) . as in the 12-week analysis , indacaterol 300 g od and indacaterol 150 g od were associated with significantly better trough fev1 compared to vilanterol 25 g od , salmeterol 50 g bid , olodaterol 10 g od , olodaterol 5 g od , and formoterol 12 g bid , and were not statistically different from each other ( table 2 ) . at 12 and 24 relative to placebo at 12 weeks , indacaterol 300 , 150 , and 75 g od ( 1.171 [ 0.906 , 1.401 ] , 1.051 [ 0.826 , 1.291 ] , and 0.831 [ 0.330 , 1.336 ] , respectively ) had the highest difference in tdi focal scores at 12 weeks , followed by olodaterol 10 g od ( 0.734 [ 0.278 , 1.166 ] ) , vilanterol 25 g od ( 0.665 [ 0.284 , 1.054 ] ) , olodaterol 5 g od ( 0.629 [ 0.187 , 1.058 ] ) , formoterol 12 g bid ( 0.618 [ 0.281 , 0.925 ] ) , and salmeterol 50 g bid ( 0.555 [ 0.246 , 0.887 ] ; figure 4a ) . indacaterol 300 g od and indacaterol 150 g od were associated with significantly higher mean tdi focal score compared to salmeterol 50 g bid and formoterol 12 g bid . indacaterol 300 g od was also associated with significantly higher mean tdi focal score compared to olodaterol 5 g od and vilanterol 25 g od ( table 3 ) . relative to placebo at 24 weeks , indacaterol 300 g od ( 1.184 [ 0.942 , 1.433 ] ) had the highest difference in tdi focal scores at 24 weeks , followed by indacaterol 150 g od ( 0.894 [ 0.653 , 1.139 ] ) , salmeterol 50 g bid ( 0.696 [ 0.423 , 0.965 ] ) , vilanterol 25 g od ( 0.693 [ 0.297 , 1.093 ] ) , formoterol 12 g bid ( 0.594 [ 0.359 , 0.838 ] ) , olodaterol 5 g od ( 0.556 [ 0.143 , 0.975 ) , and olodaterol 10 g od ( 0.501 [ 0.097 , 0.920 ] ; figure 4b ) . indacaterol 300 g od was associated with a significantly higher mean tdi focal score compared to all other labas ( including indacaterol 150 g od ; table 3 ) . relative to placebo , the numerically best sgrq scores at 12 weeks belonged to ( in order ) olodaterol 10 g od ( 4.144 [ 6.089 , 2.161 ] ) , indacaterol 150 g ( 4.022 [ 5.096 , 2.962 ] ) , indacaterol 300 g ( 3.704 [ 4.922 , 2.501 ] ) , and indacaterol 75 g od ( 3.691 [ 5.825 , 1.509 ] ) , followed by formoterol 12 g bid ( 3.150 [ 4.464 , 1.890 ] ) , olodaterol 5 g od ( 3.047 [ 5.014 , 1.107 ] ) , and salmeterol 50 g bid ( 2.710 [ 4.463 , 0.935 ] ) . for 24 weeks , the best scores belonged to olodaterol 10 g od ( 3.589 [ 5.704 , 1.429 ] ) , indacaterol 150 g od ( 3.155 [ 4.504 , 1.752 ] ) , and vilanterol 25 g od ( 2.906 [ 5.042 , 0.769 ] ) , followed by indacaterol 300 g od ( 2.843 [ 4.321 , 1.407 ] ) , formoterol 12 g bid ( 1.401 [ 2.694 , 0.113 ] ) , and salmeterol 50 g bid ( 1.379 [ 2.559 , 0.286 ] ; figure 5 ) . a total of 14 trials that reported the exacerbation rate were included in the evidence network including salmeterol 50 g bid , formoterol 12 g bid , indacaterol 150 g od , indacaterol 300 g od , olodaterol 5 g od , olodaterol 10 g od , and placebo . the exacerbation rates were significantly lower for salmeterol 50 g bid , indacaterol 150 g od , and indacaterol 300 g od , compared with placebo . in addition , indacaterol 150 g od was significantly better than olodaterol 5 g od ( 0.773 [ 0.590 , 0.991 ] ) and olodaterol 10 g od ( 0.737 [ 0.565 , 0.939 ] ; table 5 and figure 6 ) . the results indicate that indacaterol was the most effective laba monotherapy for the treatment of copd , similar to the findings of earlier studies comparing laba efficacy.23,24 specifically , indacaterol 150 g od and indacaterol 300 g od were associated with significant improvement in 12- and 24-week trough fev1 compared to all other labas , and indacaterol 75 g od was associated with significant improvement in trough fev1 at week 12 compared to formoterol and olodaterol ( both 5 and 10 g doses ) . in addition , indacaterol , 300 g in particular , showed statistical superiority over other labas in tdi score , and indacaterol 150 g showed statistical superiority over olodaterol 5 g and olodaterol 10 g od in exacerbation rates . in conclusion , indacaterol 300 g od , followed by 150 g od , and 75 g od , were the most effective laba monotherapies for copd in terms of trough fev1 and tdi focal scores .

previously , these large - scale genome - changing events were studied through genetic or cytological studies . with the availability of many complete genome sequences the publication of the yeast genome has led to much better insight into the duplication events that have occurred in fungal and eukaryotic evolution ( for example , see ) . large chromosomal duplications have also been found from analysis of completed chromosomes of arabidopsis thaliana . the ability to detect large - scale genomic changes is dependent in large part on which genomes are available . such studies in bacteria , for example , have been limited by the availability of genomes only from distantly related sets of species . recently , however , the genomes of sets of closely related bacterial species have become available . we have compared these closely related bacterial genomes and have discovered an unusual phenomenon - alignments of whole genomes that show an x - shaped pattern ( which we refer to as x - alignments ) . here we present the evidence for these x - alignments and discuss mechanisms that might have produced them . we compared the dna sequences of the two chromosomes of vibrio cholerae with the sequence of the escherichia coli chromosome using a suffix tree alignment algorithm . the analysis revealed a significant alignment at the dna level between the v. cholerae large chromosome ( chri ) and the e. coli chromosome spanning the entire length of these chromosomes ( figure 1a ) . analysis of the reverse complement of v. cholerae chri with e. coli also produced a significant alignment ( figure 1b ) . when superimposed , the two alignments produce a clear ' x ' shape ( figure 1c ) that is symmetric about the origin of replication of both genomes . this symmetry indicates that matching sequences tend to occur at the same distance from the origin but not necessarily on the same side of the origin . the x - alignment between v. cholerae and e. coli was found to be statistically significant using a test based on the number of matches found in diagonal strips in the alignment ( see the materials and methods section ) . specifically , when v. cholerae chri is aligned in the forward direction against e. coli , there are 459 maximal unique matching subsequences ( mums ; see the materials and methods section ) , of which 177 occurred in a diagonal strip covering 10% of the total area ( compared to the expected value of 46 ) . the probability of observing this high a number of mums by chance is 4.7 10 . the alignment of v. cholerae chri in the reverse direction against e. coli ( which corresponds to the mums on the anti - diagonal ) has a probability of 1.8 10 . as a control , we compared the genomes of distantly related species , such as e. coli and mycobacterium tuberculosis . we have found that x - alignments of whole genomes are not limited to the v. cholerae versus e. coli comparison . for example , a whole - genome comparison of two bacteria in the genus streptococcus - s. pyogenes and s. pneumoniae ( h. tettelin , personal communication ) - reveals a global x - alignment similar to that of v. cholerae versus e. coli ( figure 1d ) which is also statistically significant ( table 1 ) . in addition , an x - alignment is found between two species in the genus mycobacterium - m. tuberculosis and m. leprae ( figure 1e ) - as well as between two strains of helicobacter pylori ( data not shown ) . the x - alignments observed between any two pairs of genomes are not identical in every aspect . for example , in the alignment between the two mycobacterium species , each conserved region is much longer than in the other genome pairs . we believe this is due to different numbers of evolutionary events between the species ( see below ) . whole - genome x - alignments were not found between any other pairs of species , although a related pattern was seen between some of the chlamydial species ( see below ) . to test whether the x - alignments found in the dna analysis could also be found at the level of whole proteomes , we conducted comparisons of homologous proteins between species ( see the materials and methods section ) . figure 2a shows a scatterplot of chromosome positions of all proteins homologous between v. cholerae chri and e. coli . the presence of many large gene families causes a great deal of noise in this comparison . this noise can be reduced by considering only the best matching homolog for each open reading frame ( orf ) , rather than all protein homologs ( figure 2b ) . this filtered protein comparison results in an x - alignment that is statistically significant ( table 2 ) . the finding of the x - alignment pattern between species led us to search for similar patterns within species ; that is , global alignments of a genome with its own reverse complement . of the genomes for which we found between - species x - alignments ( m. tuberculosis , m. leprae , s. pyogenes , s. pneumoniae , e. coli and v. cholerae ) , statistically significant self - alignments are detected for all except m. tuberculosis ( figure 3 ; probabilities shown in table 1 ) . proteome analysis also shows an x - alignment within species ( shown for v. cholerae chri in figure 2d ; probabilities shown in table 2 ) . the x - alignment of proteins within v. cholerae chri is statistically significant only for recently duplicated - genes , but disappears when all paralogs are included . one possible explanation for an x - alignment within and between species is an ancestral inverted duplication of the whole genome , as has been suggested for e. coli . the weak or missing x - alignment within species could be explained by gene loss of one of the two duplicates of many of the pairs of genes in the different lineages . this gene loss is thought to stabilize large duplications by preventing recombination events between duplicate genes . if gene loss is responsible for the weak x - alignment within species , then to maintain the x - alignments between species , the member of the gene pair lost in a particular lineage should be essentially random . if an ancient inverted duplication followed by differential gene loss is the correct explanation for the observed x - alignments , one would expect the genes along one diagonal to be orthologous between species ( related to each other by the speciation event ) , while the genes along the other diagonal should be paralogous ( related to each other by the genome duplication event before the speciation of the two lineages ) . however , the evidence appears to contradict this model : likely orthologous gene pairs are equally distributed on each diagonal ( data not shown ) . a second possible explanation for the x - alignments is that an underlying mechanism allows sections of dna to move within the genome but maintains the distance of these sections from the origin and/or terminus . there are a variety of possible mechanisms for such movement , but we believe the most likely explanation is the occurrence of large chromosomal inversions that pivot around the replication origin and/or terminus . large chromosomal inversions , including those that occur around the replication origin and terminus , have been shown to occur in e. coli and salmonella typhimurium in the laboratory ( see , for example , ) . the occurrence of such inversions over evolutionary time scales was first suggested by comparative analysis of the complete genomes of four strains in the genus chlamydia . in that study , we found that the major chromosomal differences between c. pneumoniae and c. trachomatis ( shown in figure 2c ) were consistent with the occurrence of large inversions that pivoted around the origin and terminus ( including multiple inversions of different sizes ) . in figure 4 we present a hypothetical model showing how a small number of inversions centered around the origin or terminus could produce patterns very similar to those seen in the chlamydia , mycobacterium and helicobacter comparisons . the continued occurrence of such inversion over longer time scales would result in an x - alignment similar to that seen in the v. cholerae versus e. coli and s. pneumoniae versus s. pyogenes comparisons . thus the different between - species x - alignments could be the result of different numbers of inversions between particular pairs of species . inversions about the origin and terminus could also produce an x - alignment within species , through the splitting of tandemly duplicated sequence . many sets of tandemly duplicated genes are found in most bacterial genomes ( also see figure 3a , c ) . as tandem duplications are inherently unstable ( one of the duplicates can be rapidly eliminated by slippage and/or recombination events ) , the fact that many tandem pairs are present within each genome suggests that tandem duplications occur frequently . thus , it is reasonable to assume that occasionally a large inversion will split a pair of tandemly duplicated genes . an inversion that pivots about the origin and also splits a tandem duplication will result in a pair of paralogous genes spaced symmetrically on opposite sides of the origin . if our inversion model is correct , then the genes along both diagonals in the between - species alignments should be orthologous , which is the case ( see above ) . in contrast , genes along the anti - diagonal in the within - species x - alignments should be recent tandem duplicates that have been separated by inversions . this also appears to be the case - in the within - species analysis of v. cholerae chri orfs , the x - alignment shows up best when only recent duplicates are analyzed ( figure 2d ) . the splitting of tandem duplicates by inversions may be a general mechanism to stabilize the coexistence of duplicated genes , as it will prevent their elimination by unequal crossing - over or replication slippage events . what could cause inversions that pivot around the origin and terminus of the genome to occur more frequently than other inversions ? one possibility is that many inversions occur , but there is selection against those that change the distance of a gene from the origin or terminus . additional studies have , however , suggested that there is little selective difference between inversions and that instead there may be certain regions that are more prone to inversion than others . alternatively , the inversion events could be linked to replication , as has been suggested for small local inversion events . whatever the mechanisms , the fact that we find evidence for such inversions between many pairs of species suggests that they are a common feature of bacterial evolution . for example , to split a tandem duplication , an inversion must fall precisely on the boundary between two duplicated genes . this would appear to be unlikely , requiring a large number of inversions in order to generate a sufficient number of split gene pairs . if the mechanisms of gene duplication are somehow related to the mechanisms of inversion , however , then this model is more plausible . the process of duplicating a gene , if it occurs during replication , might promote a recombination event within the bacterial chromosome that inverts the sequence from the origin up to that point . as with inversion events , recombination and replication we compared the dna sequences of the two chromosomes of vibrio cholerae with the sequence of the escherichia coli chromosome using a suffix tree alignment algorithm . the analysis revealed a significant alignment at the dna level between the v. cholerae large chromosome ( chri ) and the e. coli chromosome spanning the entire length of these chromosomes ( figure 1a ) . analysis of the reverse complement of v. cholerae chri with e. coli also produced a significant alignment ( figure 1b ) . when superimposed , the two alignments produce a clear ' x ' shape ( figure 1c ) that is symmetric about the origin of replication of both genomes . this symmetry indicates that matching sequences tend to occur at the same distance from the origin but not necessarily on the same side of the origin . the x - alignment between v. cholerae and e. coli was found to be statistically significant using a test based on the number of matches found in diagonal strips in the alignment ( see the materials and methods section ) . specifically , when v. cholerae chri is aligned in the forward direction against e. coli , there are 459 maximal unique matching subsequences ( mums ; see the materials and methods section ) , of which 177 occurred in a diagonal strip covering 10% of the total area ( compared to the expected value of 46 ) . the probability of observing this high a number of mums by chance is 4.7 10 . the alignment of v. cholerae chri in the reverse direction against e. coli ( which corresponds to the mums on the anti - diagonal ) has a probability of 1.8 10 . as a control , we compared the genomes of distantly related species , such as e. coli and mycobacterium tuberculosis . we have found that x - alignments of whole genomes are not limited to the v. cholerae versus e. coli comparison . for example , a whole - genome comparison of two bacteria in the genus streptococcus - s. pyogenes and s. pneumoniae ( h. tettelin , personal communication ) - reveals a global x - alignment similar to that of v. cholerae versus e. coli ( figure 1d ) which is also statistically significant ( table 1 ) . in addition , an x - alignment is found between two species in the genus mycobacterium - m. tuberculosis and m. leprae ( figure 1e ) - as well as between two strains of helicobacter pylori ( data not shown ) . the x - alignments observed between any two pairs of genomes are not identical in every aspect . for example , in the alignment between the two mycobacterium species , each conserved region is much longer than in the other genome pairs . we believe this is due to different numbers of evolutionary events between the species ( see below ) . whole - genome x - alignments were not found between any other pairs of species , although a related pattern was seen between some of the chlamydial species ( see below ) . to test whether the x - alignments found in the dna analysis could also be found at the level of whole proteomes , we conducted comparisons of homologous proteins between species ( see the materials and methods section ) . figure 2a shows a scatterplot of chromosome positions of all proteins homologous between v. cholerae chri and e. coli . the presence of many large gene families causes a great deal of noise in this comparison . this noise can be reduced by considering only the best matching homolog for each open reading frame ( orf ) , rather than all protein homologs ( figure 2b ) . this filtered protein comparison results in an x - alignment that is statistically significant ( table 2 ) . the finding of the x - alignment pattern between species led us to search for similar patterns within species ; that is , global alignments of a genome with its own reverse complement . of the genomes for which we found between - species x - alignments ( m. tuberculosis , m. leprae , s. pyogenes , s. pneumoniae , e. coli and v. cholerae ) , statistically significant self - alignments are detected for all except m. tuberculosis ( figure 3 ; probabilities shown in table 1 ) . proteome analysis also shows an x - alignment within species ( shown for v. cholerae chri in figure 2d ; probabilities shown in table 2 ) . the x - alignment of proteins within v. cholerae chri is statistically significant only for recently duplicated - genes , but disappears when all paralogs are included . one possible explanation for an x - alignment within and between species is an ancestral inverted duplication of the whole genome , as has been suggested for e. coli . the weak or missing x - alignment within species could be explained by gene loss of one of the two duplicates of many of the pairs of genes in the different lineages . this gene loss is thought to stabilize large duplications by preventing recombination events between duplicate genes . if gene loss is responsible for the weak x - alignment within species , then to maintain the x - alignments between species , the member of the gene pair lost in a particular lineage should be essentially random . if an ancient inverted duplication followed by differential gene loss is the correct explanation for the observed x - alignments , one would expect the genes along one diagonal to be orthologous between species ( related to each other by the speciation event ) , while the genes along the other diagonal should be paralogous ( related to each other by the genome duplication event before the speciation of the two lineages ) . however , the evidence appears to contradict this model : likely orthologous gene pairs are equally distributed on each diagonal ( data not shown ) . a second possible explanation for the x - alignments is that an underlying mechanism allows sections of dna to move within the genome but maintains the distance of these sections from the origin and/or terminus . there are a variety of possible mechanisms for such movement , but we believe the most likely explanation is the occurrence of large chromosomal inversions that pivot around the replication origin and/or terminus . large chromosomal inversions , including those that occur around the replication origin and terminus , have been shown to occur in e. coli and salmonella typhimurium in the laboratory ( see , for example , ) . the occurrence of such inversions over evolutionary time scales was first suggested by comparative analysis of the complete genomes of four strains in the genus chlamydia . in that study , we found that the major chromosomal differences between c. pneumoniae and c. trachomatis ( shown in figure 2c ) were consistent with the occurrence of large inversions that pivoted around the origin and terminus ( including multiple inversions of different sizes ) . in figure 4 we present a hypothetical model showing how a small number of inversions centered around the origin or terminus could produce patterns very similar to those seen in the chlamydia , mycobacterium and helicobacter comparisons . the continued occurrence of such inversion over longer time scales would result in an x - alignment similar to that seen in the v. cholerae versus e. coli and s. pneumoniae versus s. pyogenes comparisons . thus the different between - species x - alignments could be the result of different numbers of inversions between particular pairs of species . inversions about the origin and terminus could also produce an x - alignment within species , through the splitting of tandemly duplicated sequence . many sets of tandemly duplicated genes are found in most bacterial genomes ( also see figure 3a , c ) . as tandem duplications are inherently unstable ( one of the duplicates can be rapidly eliminated by slippage and/or recombination events ) , the fact that many tandem pairs are present within each genome suggests that tandem duplications occur frequently . thus , it is reasonable to assume that occasionally a large inversion will split a pair of tandemly duplicated genes . an inversion that pivots about the origin and also splits a tandem duplication will result in a pair of paralogous genes spaced symmetrically on opposite sides of the origin . if our inversion model is correct , then the genes along both diagonals in the between - species alignments should be orthologous , which is the case ( see above ) . in contrast , genes along the anti - diagonal in the within - species x - alignments should be recent tandem duplicates that have been separated by inversions . this also appears to be the case - in the within - species analysis of v. cholerae chri orfs , the x - alignment shows up best when only recent duplicates are analyzed ( figure 2d ) . the splitting of tandem duplicates by inversions may be a general mechanism to stabilize the coexistence of duplicated genes , as it will prevent their elimination by unequal crossing - over or replication slippage events . what could cause inversions that pivot around the origin and terminus of the genome to occur more frequently than other inversions ? one possibility is that many inversions occur , but there is selection against those that change the distance of a gene from the origin or terminus . additional studies have , however , suggested that there is little selective difference between inversions and that instead there may be certain regions that are more prone to inversion than others . alternatively , the inversion events could be linked to replication , as has been suggested for small local inversion events . whatever the mechanisms , the fact that we find evidence for such inversions between many pairs of species suggests that they are a common feature of bacterial evolution . for example , to split a tandem duplication , an inversion must fall precisely on the boundary between two duplicated genes . this would appear to be unlikely , requiring a large number of inversions in order to generate a sufficient number of split gene pairs . if the mechanisms of gene duplication are somehow related to the mechanisms of inversion , however , then this model is more plausible . the process of duplicating a gene , if it occurs during replication , might promote a recombination event within the bacterial chromosome that inverts the sequence from the origin up to that point . as with inversion events , recombination and replication we present here a novel observation regarding the conservation between bacterial species of the distance of particular genes from the replication origin or terminus . the initial observation was only possible due to the availability of complete genome sequences from pairs of moderately closely related species ( for example , v. cholerae and e. coli ) . comparisons of distantly related species enable the determination of universal features of life as well as of events that occur very rarely . comparison of very closely related species allows the identification of frequent events such as transitional changes at third codon positions or tandem duplications . to elucidate all other events in the history of life complete published genome sequences were obtained from the national center for biotechnology information website or from the tigr comprehensive microbial resource . these included aeropyrum pernix , aquifex aeolicus , archaeoglobus fulgidus , bacillus subtilis , borrelia burgdorferi , campylobacter jejuni , chlamydia pneumoniae ar39 , chlamydia pneumoniae cwl029 , chlamydia trachomatis ( d / uw-3/cx ) , chlamydia trachomatis mopn , deinococcus radiodurans , escherichia coli , haemophilus influenzae , helicobacter pylori , helicobacter pylori j99 , methanobacterium thermoautotrophicum , methanococcus jannaschii , mycobacterium tuberculosis , mycoplasma genitalium , mycoplasma pneumoniae , neisseria meningitidis mc58 , neisseria meningitidis serogroup a strain z2491 , pyrococcus horikoshii , rickettsia prowazekii , synechocystis sp . , thermotoga maritima , treponema pallidum , and vibrio cholerae . in addition , a few unpublished genomes were analyzed : streptococcus pyogenes ( obtained from the oklahoma university genome center website ) , streptococcus pneumoniae ( h. tettelin , personal communication ) , and mycobacterium leprae ( obtained from the sanger centre pathogen sequencing group website ) . dna alignments of the complete genomic sequences of all bacteria used in this study were accomplished with the mummer program . this program uses an efficient suffix tree construction algorithm to rapidly compute alignments of entire genomes . the algorithm identifies all exact matches of nucleotide subsequences that are contained in both input sequences ; these exact matches must be longer than a specified minimum length , which was set to 20 base pairs for this comparison . to search for genome - scale alignments within species , complete bacterial and archaeal genomes ( 25 in total including all published genomes ) were aligned with their own reverse complements . to search for between - species alignments , the predicted proteome of each complete genome sequence ( all predicted proteins in the genome ) was compared to the proteomes of all complete genome sequences ( including itself ) using the fasta3 program . matches with an expected score ( e - value ) of 10 or less were considered significant . to calculate the statistical significance of the x - alignments , the maximal unique matching subsequences ( mums ) for unrelated genomes were examined and found to be uniformly distributed . with a uniform background , the expected density of mums in any region of an alignment plot is a simple proportion of the area of that region to the entire plot . in particular , in an alignment with n total mums , the probability ( pr ) of observing at least m matches in a region with area p can be computed using the binomial distribution in equation 1 : the alignment of v. cholerae chri ( both forward and reverse strands ) versus e. coli contains 926 mums . the mums forming x - alignments appear along the diagonal ( y = x ) and the anti - diagonal ( y = l -x , where l is the genome length ) . to estimate the significance of the alignments in both directions , diagonal strips were sampled along each of the diagonals . the width of each strip was set at 10% of the plot area and significance values were calculated ( table 1 ) . the origins of replication for the bacterial genomes have been characterized by a variety of methods . for e. coli , m. tuberculosis and m. leprae , the origins have been well - characterized by laboratory studies . the origins and termini of c. trachomatis , c. pneumoniae and v. cholerae were identified by gc - skew and by characteristic genes in the region of the origin . gc - skew uses the function ( g - c)/(g+c ) computed on 2,000 bp windows across the genome , which exhibits a clear tendency in many bacterial genomes to be positive for the leading strand and negative for the lagging strand . the origin of h. pylori was determined by oligomer skew and confirmed by gc - skew . the origins and termini of s. pneumoniae and s. pyogenes were determined by the authors of the present study using gc - skew analysis and the locations of characteristic genes , particularly the chromosome replication initiator gene dnaa . complete published genome sequences were obtained from the national center for biotechnology information website or from the tigr comprehensive microbial resource . these included aeropyrum pernix , aquifex aeolicus , archaeoglobus fulgidus , bacillus subtilis , borrelia burgdorferi , campylobacter jejuni , chlamydia pneumoniae ar39 , chlamydia pneumoniae cwl029 , chlamydia trachomatis ( d / uw-3/cx ) , chlamydia trachomatis mopn , deinococcus radiodurans , escherichia coli , haemophilus influenzae , helicobacter pylori , helicobacter pylori j99 , methanobacterium thermoautotrophicum , methanococcus jannaschii , mycobacterium tuberculosis , mycoplasma genitalium , mycoplasma pneumoniae , neisseria meningitidis mc58 , neisseria meningitidis serogroup a strain z2491 , pyrococcus horikoshii , rickettsia prowazekii , synechocystis sp . , thermotoga maritima , treponema pallidum , and vibrio cholerae . in addition , a few unpublished genomes were analyzed : streptococcus pyogenes ( obtained from the oklahoma university genome center website ) , streptococcus pneumoniae ( h. tettelin , personal communication ) , and mycobacterium leprae ( obtained from the sanger centre pathogen sequencing group website ) . dna alignments of the complete genomic sequences of all bacteria used in this study were accomplished with the mummer program . this program uses an efficient suffix tree construction algorithm to rapidly compute alignments of entire genomes . the algorithm identifies all exact matches of nucleotide subsequences that are contained in both input sequences ; these exact matches must be longer than a specified minimum length , which was set to 20 base pairs for this comparison . to search for genome - scale alignments within species , complete bacterial and archaeal genomes ( 25 in total including all published genomes ) were aligned with their own reverse complements . to search for between - species alignments , the predicted proteome of each complete genome sequence ( all predicted proteins in the genome ) was compared to the proteomes of all complete genome sequences ( including itself ) using the fasta3 program . matches with an expected score ( e - value ) of 10 or less were considered significant . to calculate the statistical significance of the x - alignments , the maximal unique matching subsequences ( mums ) for unrelated genomes were examined and found to be uniformly distributed . with a uniform background , the expected density of mums in any region of an alignment plot is a simple proportion of the area of that region to the entire plot . in particular , in an alignment with n total mums , the probability ( pr ) of observing at least m matches in a region with area p can be computed using the binomial distribution in equation 1 : the alignment of v. cholerae chri ( both forward and reverse strands ) versus e. coli contains 926 mums . the mums forming x - alignments appear along the diagonal ( y = x ) and the anti - diagonal ( y = l -x , where l is the genome length ) . to estimate the significance of the alignments in both directions , diagonal strips were sampled along each of the diagonals . the width of each strip was set at 10% of the plot area and significance values were calculated ( table 1 ) . the origins of replication for the bacterial genomes have been characterized by a variety of methods . for e. coli , m. tuberculosis and m. leprae , the origins and termini of c. trachomatis , c. pneumoniae and v. cholerae were identified by gc - skew and by characteristic genes in the region of the origin . gc - skew uses the function ( g - c)/(g+c ) computed on 2,000 bp windows across the genome , which exhibits a clear tendency in many bacterial genomes to be positive for the leading strand and negative for the lagging strand . the origin of h. pylori was determined by oligomer skew and confirmed by gc - skew . the origins and termini of s. pneumoniae and s. pyogenes were determined by the authors of the present study using gc - skew analysis and the locations of characteristic genes , particularly the chromosome replication initiator gene dnaa . riley , t. read , a. stoltzfus , m - i benito and i. paulsen for helpful comments , suggestions and discussions . s.l.s . was supported in part by nsf grant iis-9902923 and nih grant r01 lm06845 . data for all published complete genome sequences were obtained from the ncbi genomes database or from the institute for genomic research ( tigr ) microbial genome database . the sequences of v. cholerae , s. pneumoniae , and m. tuberculosis ( cdc 1551 ) were determined at tigr with support from nih and the niaid . the m. leprae sequence data were produced by the pathogen sequencing group at the sanger centre . sequencing of m. leprae is funded by the heiser program for research in leprosy and tuberculosis of the new york community trust and by l'association raoul follereau . the source of the s. pyogenes genome sequence was the streptococcal genome sequencing project funded by usphs / nih grant ai38406 , and was kindly made available by b. a. roe , s.p . mclaughlin , m. mcshan and j. ferretti , and can be obtained from the website of the oklahoma university genome center . plots of maximally unique matching subsequences ( mums ) between genomes as identified by the mummer program . tuberculosis forward versus m. leprae forward and reverse overlaid . a point ( x , y ) indicates a dna sequence that occurs once within each genome , at location x in one genome and at location y in the other genome . the matching sequences may occur on either the forward or the reverse strand ; in either case , the locations indicate the 5 ' end of the sequences . plots show the chromosome locations of pairs of predicted proteins that have significant similarity ( on the basis of fasta3 comparisons ) . the filtering for top matches for each orf removes noise due to the presence of many large multigene families . the position of the origins ( r ) and termini ( t ) of replication are slightly shifted to see the inversions better . this pattern is consistent with the occurrence a small number of inversions around the origin and terminus in the two lineages since their divergence from a common ancestor ( see figure 4 ) . recent duplications were operationally defined as those genes that were more similar to another gene in v. cholerae than to any gene in any other complete genome sequence . no significant x - alignment was detected when all pairs of paralogs were included . within - genome dna alignments . plots of exactly matching sequences within four genomes as identified by the mummer program : ( a)v . cholerae ; ( b)e . . pneumoniae . a point ( x , y ) indicates a dna sequence that is repeated within the genome , occurring once at location x and again at location y. points near the diagonal ( y = x ) correspond to tandem repeats . points near the anti - diagonal ( y = l - x , where l is genome length ) correspond to repeats that occur at symmetric locations about the origin of location . the model shows an initial speciation event , followed by a series of inversions in the different lineages ( a and b ) . numbers on the chromosome refer to hypothetical genes 1 - 32 . at time point 1 , the genomes of the two species are still co - linear ( as indicated in the scatterplot of a1 versus b1 ) . between time point 1 and time point 2 , each species ( a and b ) undergoes a large inversion about the terminus ( as indicated in the scatterplots of a1 versus a2 and b1 versus b2 ) . this results in the between - species scatterplot looking as if there have been two nested inversions ( a2 versus b2 ) , similar to that seen for c. trachomatis versus c. pneumoniae ( see figure 2 ) . between time point 2 and time point 3 each species undergoes an additional inversion ( as indicated in the scatterplots of b2 versus b3 and a2 versus a3 ) . this results in the between - species scatterplots beginning to resemble an x - alignment , similar to that seen in m. tuberculosis versus m. leprae ( see figure 2 ) . whole - genome dna alignments using mummer statistical significance was estimated as described in the text ; rev , reverse complement sequence . whole - genome protein - level comparisons statistical significance was estimated as described in the text .

background : whole - genome comparisons can provide great insight into many aspects of biology . until recently , however , comparisons were mainly possible only between distantly related species . complete genome sequences are now becoming available from multiple sets of closely related strains or species.results:by comparing the recently completed genome sequences of vibrio cholerae , streptococcus pneumoniae and mycobacterium tuberculosis to those of closely related species - escherichia coli , streptococcus pyogenes and mycobacterium leprae , respectively - we have identified an unusual and previously unobserved feature of bacterial genome structure . scatterplots of the conserved sequences ( both dna and protein ) between each pair of species produce a distinct x - shaped pattern , which we call an x - alignment . the key feature of these alignments is that they have symmetry around the replication origin and terminus ; that is , the distance of a particular conserved feature ( dna or protein ) from the replication origin ( or terminus ) is conserved between closely related pairs of species . statistically significant x - alignments are also found within some genomes , indicating that there is symmetry about the replication origin for paralogous features as well.conclusions:the most likely mechanism of generation of x - alignments involves large chromosomal inversions that reverse the genomic sequence symmetrically around the origin of replication . the finding of these x - alignments between many pairs of species suggests that chromosomal inversions around the origin are a common feature of bacterial genome evolution .

Background Results and discussion Whole-genome X-alignments between species at the DNA level Whole-genome X-alignments between species are also found at the proteome level Whole-genome X-alignments within species Model I: whole-genome inverted duplications Model II: chromosomal inversions about the origin and/or terminus Conclusions Materials and methods Genomes analyzed Whole-genome DNA alignments Whole-genome protein comparisons Statistical significance of X-alignments Identification of origins of replication Acknowledgements Figures and Tables

with the availability of many complete genome sequences the publication of the yeast genome has led to much better insight into the duplication events that have occurred in fungal and eukaryotic evolution ( for example , see ) . such studies in bacteria , for example , have been limited by the availability of genomes only from distantly related sets of species . recently , however , the genomes of sets of closely related bacterial species have become available . we have compared these closely related bacterial genomes and have discovered an unusual phenomenon - alignments of whole genomes that show an x - shaped pattern ( which we refer to as x - alignments ) . here we present the evidence for these x - alignments and discuss mechanisms that might have produced them . we compared the dna sequences of the two chromosomes of vibrio cholerae with the sequence of the escherichia coli chromosome using a suffix tree alignment algorithm . when superimposed , the two alignments produce a clear ' x ' shape ( figure 1c ) that is symmetric about the origin of replication of both genomes . this symmetry indicates that matching sequences tend to occur at the same distance from the origin but not necessarily on the same side of the origin . the x - alignment between v. cholerae and e. coli was found to be statistically significant using a test based on the number of matches found in diagonal strips in the alignment ( see the materials and methods section ) . as a control , we compared the genomes of distantly related species , such as e. coli and mycobacterium tuberculosis . we have found that x - alignments of whole genomes are not limited to the v. cholerae versus e. coli comparison . for example , a whole - genome comparison of two bacteria in the genus streptococcus - s. pyogenes and s. pneumoniae ( h. tettelin , personal communication ) - reveals a global x - alignment similar to that of v. cholerae versus e. coli ( figure 1d ) which is also statistically significant ( table 1 ) . in addition , an x - alignment is found between two species in the genus mycobacterium - m. tuberculosis and m. leprae ( figure 1e ) - as well as between two strains of helicobacter pylori ( data not shown ) . the x - alignments observed between any two pairs of genomes are not identical in every aspect . whole - genome x - alignments were not found between any other pairs of species , although a related pattern was seen between some of the chlamydial species ( see below ) . this filtered protein comparison results in an x - alignment that is statistically significant ( table 2 ) . the finding of the x - alignment pattern between species led us to search for similar patterns within species ; that is , global alignments of a genome with its own reverse complement . of the genomes for which we found between - species x - alignments ( m. tuberculosis , m. leprae , s. pyogenes , s. pneumoniae , e. coli and v. cholerae ) , statistically significant self - alignments are detected for all except m. tuberculosis ( figure 3 ; probabilities shown in table 1 ) . proteome analysis also shows an x - alignment within species ( shown for v. cholerae chri in figure 2d ; probabilities shown in table 2 ) . the x - alignment of proteins within v. cholerae chri is statistically significant only for recently duplicated - genes , but disappears when all paralogs are included . one possible explanation for an x - alignment within and between species is an ancestral inverted duplication of the whole genome , as has been suggested for e. coli . the weak or missing x - alignment within species could be explained by gene loss of one of the two duplicates of many of the pairs of genes in the different lineages . if gene loss is responsible for the weak x - alignment within species , then to maintain the x - alignments between species , the member of the gene pair lost in a particular lineage should be essentially random . if an ancient inverted duplication followed by differential gene loss is the correct explanation for the observed x - alignments , one would expect the genes along one diagonal to be orthologous between species ( related to each other by the speciation event ) , while the genes along the other diagonal should be paralogous ( related to each other by the genome duplication event before the speciation of the two lineages ) . a second possible explanation for the x - alignments is that an underlying mechanism allows sections of dna to move within the genome but maintains the distance of these sections from the origin and/or terminus . there are a variety of possible mechanisms for such movement , but we believe the most likely explanation is the occurrence of large chromosomal inversions that pivot around the replication origin and/or terminus . large chromosomal inversions , including those that occur around the replication origin and terminus , have been shown to occur in e. coli and salmonella typhimurium in the laboratory ( see , for example , ) . in that study , we found that the major chromosomal differences between c. pneumoniae and c. trachomatis ( shown in figure 2c ) were consistent with the occurrence of large inversions that pivoted around the origin and terminus ( including multiple inversions of different sizes ) . in figure 4 we present a hypothetical model showing how a small number of inversions centered around the origin or terminus could produce patterns very similar to those seen in the chlamydia , mycobacterium and helicobacter comparisons . the continued occurrence of such inversion over longer time scales would result in an x - alignment similar to that seen in the v. cholerae versus e. coli and s. pneumoniae versus s. pyogenes comparisons . thus the different between - species x - alignments could be the result of different numbers of inversions between particular pairs of species . inversions about the origin and terminus could also produce an x - alignment within species , through the splitting of tandemly duplicated sequence . as tandem duplications are inherently unstable ( one of the duplicates can be rapidly eliminated by slippage and/or recombination events ) , the fact that many tandem pairs are present within each genome suggests that tandem duplications occur frequently . an inversion that pivots about the origin and also splits a tandem duplication will result in a pair of paralogous genes spaced symmetrically on opposite sides of the origin . this also appears to be the case - in the within - species analysis of v. cholerae chri orfs , the x - alignment shows up best when only recent duplicates are analyzed ( figure 2d ) . what could cause inversions that pivot around the origin and terminus of the genome to occur more frequently than other inversions ? one possibility is that many inversions occur , but there is selection against those that change the distance of a gene from the origin or terminus . additional studies have , however , suggested that there is little selective difference between inversions and that instead there may be certain regions that are more prone to inversion than others . whatever the mechanisms , the fact that we find evidence for such inversions between many pairs of species suggests that they are a common feature of bacterial evolution . as with inversion events , recombination and replication we compared the dna sequences of the two chromosomes of vibrio cholerae with the sequence of the escherichia coli chromosome using a suffix tree alignment algorithm . when superimposed , the two alignments produce a clear ' x ' shape ( figure 1c ) that is symmetric about the origin of replication of both genomes . this symmetry indicates that matching sequences tend to occur at the same distance from the origin but not necessarily on the same side of the origin . the x - alignment between v. cholerae and e. coli was found to be statistically significant using a test based on the number of matches found in diagonal strips in the alignment ( see the materials and methods section ) . as a control , we compared the genomes of distantly related species , such as e. coli and mycobacterium tuberculosis . we have found that x - alignments of whole genomes are not limited to the v. cholerae versus e. coli comparison . for example , a whole - genome comparison of two bacteria in the genus streptococcus - s. pyogenes and s. pneumoniae ( h. tettelin , personal communication ) - reveals a global x - alignment similar to that of v. cholerae versus e. coli ( figure 1d ) which is also statistically significant ( table 1 ) . in addition , an x - alignment is found between two species in the genus mycobacterium - m. tuberculosis and m. leprae ( figure 1e ) - as well as between two strains of helicobacter pylori ( data not shown ) . the x - alignments observed between any two pairs of genomes are not identical in every aspect . whole - genome x - alignments were not found between any other pairs of species , although a related pattern was seen between some of the chlamydial species ( see below ) . this filtered protein comparison results in an x - alignment that is statistically significant ( table 2 ) . the finding of the x - alignment pattern between species led us to search for similar patterns within species ; that is , global alignments of a genome with its own reverse complement . of the genomes for which we found between - species x - alignments ( m. tuberculosis , m. leprae , s. pyogenes , s. pneumoniae , e. coli and v. cholerae ) , statistically significant self - alignments are detected for all except m. tuberculosis ( figure 3 ; probabilities shown in table 1 ) . proteome analysis also shows an x - alignment within species ( shown for v. cholerae chri in figure 2d ; probabilities shown in table 2 ) . the x - alignment of proteins within v. cholerae chri is statistically significant only for recently duplicated - genes , but disappears when all paralogs are included . one possible explanation for an x - alignment within and between species is an ancestral inverted duplication of the whole genome , as has been suggested for e. coli . the weak or missing x - alignment within species could be explained by gene loss of one of the two duplicates of many of the pairs of genes in the different lineages . if gene loss is responsible for the weak x - alignment within species , then to maintain the x - alignments between species , the member of the gene pair lost in a particular lineage should be essentially random . if an ancient inverted duplication followed by differential gene loss is the correct explanation for the observed x - alignments , one would expect the genes along one diagonal to be orthologous between species ( related to each other by the speciation event ) , while the genes along the other diagonal should be paralogous ( related to each other by the genome duplication event before the speciation of the two lineages ) . a second possible explanation for the x - alignments is that an underlying mechanism allows sections of dna to move within the genome but maintains the distance of these sections from the origin and/or terminus . there are a variety of possible mechanisms for such movement , but we believe the most likely explanation is the occurrence of large chromosomal inversions that pivot around the replication origin and/or terminus . large chromosomal inversions , including those that occur around the replication origin and terminus , have been shown to occur in e. coli and salmonella typhimurium in the laboratory ( see , for example , ) . in that study , we found that the major chromosomal differences between c. pneumoniae and c. trachomatis ( shown in figure 2c ) were consistent with the occurrence of large inversions that pivoted around the origin and terminus ( including multiple inversions of different sizes ) . in figure 4 we present a hypothetical model showing how a small number of inversions centered around the origin or terminus could produce patterns very similar to those seen in the chlamydia , mycobacterium and helicobacter comparisons . the continued occurrence of such inversion over longer time scales would result in an x - alignment similar to that seen in the v. cholerae versus e. coli and s. pneumoniae versus s. pyogenes comparisons . thus the different between - species x - alignments could be the result of different numbers of inversions between particular pairs of species . inversions about the origin and terminus could also produce an x - alignment within species , through the splitting of tandemly duplicated sequence . as tandem duplications are inherently unstable ( one of the duplicates can be rapidly eliminated by slippage and/or recombination events ) , the fact that many tandem pairs are present within each genome suggests that tandem duplications occur frequently . an inversion that pivots about the origin and also splits a tandem duplication will result in a pair of paralogous genes spaced symmetrically on opposite sides of the origin . this also appears to be the case - in the within - species analysis of v. cholerae chri orfs , the x - alignment shows up best when only recent duplicates are analyzed ( figure 2d ) . what could cause inversions that pivot around the origin and terminus of the genome to occur more frequently than other inversions ? one possibility is that many inversions occur , but there is selection against those that change the distance of a gene from the origin or terminus . additional studies have , however , suggested that there is little selective difference between inversions and that instead there may be certain regions that are more prone to inversion than others . whatever the mechanisms , the fact that we find evidence for such inversions between many pairs of species suggests that they are a common feature of bacterial evolution . as with inversion events , recombination and replication we present here a novel observation regarding the conservation between bacterial species of the distance of particular genes from the replication origin or terminus . the initial observation was only possible due to the availability of complete genome sequences from pairs of moderately closely related species ( for example , v. cholerae and e. coli ) . comparisons of distantly related species enable the determination of universal features of life as well as of events that occur very rarely . these included aeropyrum pernix , aquifex aeolicus , archaeoglobus fulgidus , bacillus subtilis , borrelia burgdorferi , campylobacter jejuni , chlamydia pneumoniae ar39 , chlamydia pneumoniae cwl029 , chlamydia trachomatis ( d / uw-3/cx ) , chlamydia trachomatis mopn , deinococcus radiodurans , escherichia coli , haemophilus influenzae , helicobacter pylori , helicobacter pylori j99 , methanobacterium thermoautotrophicum , methanococcus jannaschii , mycobacterium tuberculosis , mycoplasma genitalium , mycoplasma pneumoniae , neisseria meningitidis mc58 , neisseria meningitidis serogroup a strain z2491 , pyrococcus horikoshii , rickettsia prowazekii , synechocystis sp . in addition , a few unpublished genomes were analyzed : streptococcus pyogenes ( obtained from the oklahoma university genome center website ) , streptococcus pneumoniae ( h. tettelin , personal communication ) , and mycobacterium leprae ( obtained from the sanger centre pathogen sequencing group website ) . to search for between - species alignments , the predicted proteome of each complete genome sequence ( all predicted proteins in the genome ) was compared to the proteomes of all complete genome sequences ( including itself ) using the fasta3 program . to calculate the statistical significance of the x - alignments , the maximal unique matching subsequences ( mums ) for unrelated genomes were examined and found to be uniformly distributed . for e. coli , m. tuberculosis and m. leprae , the origins have been well - characterized by laboratory studies . the origins and termini of c. trachomatis , c. pneumoniae and v. cholerae were identified by gc - skew and by characteristic genes in the region of the origin . the origin of h. pylori was determined by oligomer skew and confirmed by gc - skew . these included aeropyrum pernix , aquifex aeolicus , archaeoglobus fulgidus , bacillus subtilis , borrelia burgdorferi , campylobacter jejuni , chlamydia pneumoniae ar39 , chlamydia pneumoniae cwl029 , chlamydia trachomatis ( d / uw-3/cx ) , chlamydia trachomatis mopn , deinococcus radiodurans , escherichia coli , haemophilus influenzae , helicobacter pylori , helicobacter pylori j99 , methanobacterium thermoautotrophicum , methanococcus jannaschii , mycobacterium tuberculosis , mycoplasma genitalium , mycoplasma pneumoniae , neisseria meningitidis mc58 , neisseria meningitidis serogroup a strain z2491 , pyrococcus horikoshii , rickettsia prowazekii , synechocystis sp . in addition , a few unpublished genomes were analyzed : streptococcus pyogenes ( obtained from the oklahoma university genome center website ) , streptococcus pneumoniae ( h. tettelin , personal communication ) , and mycobacterium leprae ( obtained from the sanger centre pathogen sequencing group website ) . to search for between - species alignments , the predicted proteome of each complete genome sequence ( all predicted proteins in the genome ) was compared to the proteomes of all complete genome sequences ( including itself ) using the fasta3 program . to calculate the statistical significance of the x - alignments , the maximal unique matching subsequences ( mums ) for unrelated genomes were examined and found to be uniformly distributed . for e. coli , m. tuberculosis and m. leprae , the origins and termini of c. trachomatis , c. pneumoniae and v. cholerae were identified by gc - skew and by characteristic genes in the region of the origin . the origins and termini of s. pneumoniae and s. pyogenes were determined by the authors of the present study using gc - skew analysis and the locations of characteristic genes , particularly the chromosome replication initiator gene dnaa . data for all published complete genome sequences were obtained from the ncbi genomes database or from the institute for genomic research ( tigr ) microbial genome database . this pattern is consistent with the occurrence a small number of inversions around the origin and terminus in the two lineages since their divergence from a common ancestor ( see figure 4 ) . no significant x - alignment was detected when all pairs of paralogs were included . points near the anti - diagonal ( y = l - x , where l is genome length ) correspond to repeats that occur at symmetric locations about the origin of location . this results in the between - species scatterplots beginning to resemble an x - alignment , similar to that seen in m. tuberculosis versus m. leprae ( see figure 2 ) .

brazil is among the 25 countries in the world with the fastest aging rates [ 1 , 2 ] . in 1950 , 2.6 million ( 4.9% ) brazilians were older than 60 years of age , and this number has increased to 20.6 million people ( 10.8% ) according to the 2010 census . improvements in medical care and living standards have been shown to translate into higher life expectancy . in 1950 , for example , life expectancy at birth in brazil was 50.9 years , but the figure increased to 72.2 years in 2010 . however , the number of disabled people is expected to increase in the coming years , given the rapid growth rate of the elderly population and the rise in the prevalence of obesity and chronic diseases . fast changes in the population 's nutritional intake that have occurred in brazil in recent decades have resulted in an increase in the prevalence of obesity [ 6 , 7 ] . in the past three decades , obesity rates in brazil tripled among men and almost doubled among women . with the exception of higher - income urban women , few studies focus on the impact of bmi on mortality and disability in the latin american and the caribbean ( lac ) region . based on the baseline for the health , well - being , and aging in latin america and the caribbean study ( sabe ) , al snih and colleagues showed that obese individuals were 1.6 times more likely to face difficulties performing activities of daily living ( adl ) than those with normal bmi . corona and colleagues found that older adults who were underweight ( bmi 23 kg / m ) and obese ( bmi 30 kg / m ) were more likely to develop limitations on instrumental activity of daily living ( iadl ) , but their analysis did not explore bmi associations with mortality or recovery from disability . monteverde and colleagues found that , based on relative cutoffs ( quintiles ) , heavier older adults in mexico face higher mortality risks than those in the united states . however , when bmi was categorized following the traditional world health organization cutoffs , no excess mortality was found among overweight and obese subjects . in fact , coefficients for overweight and obesity were not in the expected direction . however , none of these studies jointly examined the association between bmi , disability transitions , and mortality . we examine the association between body mass index and weight changes on disability transitions and mortality . obesity has been associated with higher prevalence of disability in cross - sectional and longitudinal studies [ 4 , 1113 ] . this positive association has been found among middle- and older - aged adults [ 1315 ] , and it appears that this association has not changed over time [ 16 , 17 ] . additionally , the association between body mass index ( bmi ) and disability is strongest among those who are underweight ( bmi < 18.5 kg / m ) and among obese subjects ( bmi > 30 obese women face higher prevalence of mobility impairment than men . in the united states , severe functional limitations are higher among older adults who gain or lose weight after age 50 compared to those with stable weight . emerging evidence supports the proposition that bmi is an important predictor of the onset of mobility limitations . older adults in the united states who gain weight over time have higher incidence of mobility limitations than those who maintain their weight . large systematic reviews have shown that the relationship between bmi and mortality seems to follow a j - shaped ( sometimes u - shaped ) curve [ 17 , 2022 ] . all - cause mortality appears to be lowest at bmi levels between 20 and 24.9 kg / m [ 20 , 22 ] , with obese individuals facing higher mortality risks than their normal - weight counterparts [ 20 , 22 , 23 ] . recent studies , however , show that overweight individuals have higher life expectancy than individuals of normal weight or those who are obese , whereas other studies have an l - shape ( see , for a short review ) . higher mortality at low levels of bmi has been associated primarily with lung cancer and respiratory diseases . at older ages , results from a systematic review and meta - analysis indicated that bmi in the overweight range of 2529.9 kg / m is not associated with increased mortality , whereas other studies have shown that the burden of obesity on mortality seems to be reduced or eliminated among older adults [ 11 , 12 , 2630 ] . the use of bmi categories has been criticized for not reproducing well the complexities of the bmi and mortality relationship [ 3133 ] . gronniger used semiparametric models and found that men in the mild - obese category ( bmis of 3035 kg / m ) had similar mortality than those of normal weight , but among women bmi levels above 27 kg / m were associated with higher mortality in the u.s . wong and colleagues used multivariable fractional polynomials to explore the association between bmi and mortality in a sample of adults in the ua they found that the best fitting model contained the powers 1 and 2 for bmi . their results indicated that the nadir of the bmi mortality curve was in the normal range for women but overweight range for men . zajacova and burgard used generalized additive models and found that the nadir was at bmis 23 to 26 , which is also in the normal overweight range . however , they point out that there are important differences depending on the cause of death . for example , the association between bmi and diabetes mortality increases monotonically , but , for all - cause mortality , it followed more a v - shape . however , even though these alternative models often provide better fit to the data , the results of these studies are often interpreted making references for traditional cutoffs as they are more easily understood by the general audience and health practitioners . the use of bmi and bmi categories have also been criticized because it can be related to underlying health status . for example , individuals may be underweight based on their bmi because they have health conditions such as cancer , thyroid problems , infectious , or digestive diseases that lead to low body weight . one approach to address this limitation has been to take into account body weight changes . our study uses data a large cohort study conducted in so paulo , brazil , to examine the association between bmi and body weight changes on disability transitions and mortality , while controlling for a series of demographic , socioeconomic , and health determinants . we investigate these associations on three types of disability ( activities of daily living , instrumental activities of daily living , and nagi 's limitations ) transitions . data from the two waves ( 2000 and 2006 ) of the sabe cohort study conducted in so paulo , brazil , were used in this study . sabe is a multicenter survey with respondents in seven major cities throughout lac countries that have been investigating the health and well - being of older adults ( age 60 and over ) . the study was approved by the institutional review boards at the collaborating institutions [ 34 , 35 ] , and the participants provided consent to have their data used for research purposes . the baseline sample was obtained using a two - stage stratified sampling based on the 1995 national household survey master sampling frame . the first stage was a household interview conducted by a single interviewer using a standardized questionnaire that included several questions about the living conditions and health status of the subjects . the second stage of data collection consisted of a household visit by a pair of interviewers who completed anthropometric and physical - performance measurements . at baseline , additional characteristics of the baseline data collection process have been described elsewhere [ 3638 ] . in 2006 , to reestablish contact , trained interviewers visited the addresses and neighborhoods of surviving participants from the 2000 survey . for those not found during these visits , interviewers used the additional contact information collected at baseline ( e.g. , telephone numbers of children or other relatives ) to obtain further information about the subjects ' current location . in 2006 , researchers collected data via face - to - face interviews using a standardized questionnaire . the 2006 questionnaire was very similar to the one used in 2000 but included additional questions that complemented the previous study . vital statistics records were used to identify subjects who had died between 2000 and 2006 . the search was based on the names , sex , dates of birth , and addresses listed in the 2000 database . of the 2,143 participants in the first wave of sabe so paulo , 355 ( 16.6% ) had missing data on selected variables . those with missing data were older ( 75.1 years ) than those with complete data ( 72.9 years ) ( p = 0.0001 ) , but there were no sex differences . the prevalence of all measures of disability was higher among those with missing data ( p < 0.001 ) . the final sample is composed of 1,788 individuals , with a subset of 961 with weight change included in the analyses . self - reported disability in six adl measures ( dressing , bathing , eating , getting in and out of a bed , toileting , and getting across a room ) were used to measure disability . please tell me if you have any difficulty with these because of a health problem . exclude any difficulties you expect to last less than three months . after this introduction , they were asked do you have difficulty ? and does not know , and no response for each one of the six adl measures . participants who answered does not know and no response were classified as missing . the iadl items included were preparing a hot meal , managing money , shopping , using of transportation within the community , ability to use the telephone , and responsibility for one 's own medications . can not do it , does not know , and no response . those who answered can not do it were classified as having difficulty performing the activity , whereas those answering does not know and no response were classified as missing . the nagi physical performance measure included lifting or carrying objects that weighed five kilograms or more ; lifting a coin ; pulling or pushing a large object , such as a living - room chair ; stooping , kneeling , or crouching ; and reaching or extending the arms above shoulder level . each of the three disability measures was converted into binary form , in which respondents scored 0 if they did not indicate any limitations and 1 if they reported having difficulty performing one or more activity in the scale . body weight and height were measured without shoes and with light clothing by trained examiners . four bmi categories were defined according to the criteria adopted by the pan american health organization for the sabe study : underweight ( bmi 23.0 ) , normal ( bmi > 23 and < 28 ) , overweight ( bmi 28.0 and < 30 ) , and obese ( bmi 30 ) . change in bmi was calculated as bmi in 2006 minus the bmi at baseline . this difference was divided by the baseline bmi and then recoded into three categories : ( a ) an increase of 5% or more ; ( b ) a decrease of 5% or more ; and ( c ) changes within 5% of the baseline weight ( reference category ) [ 19 , 39 ] . the following sociodemographic characteristics were included in the analysis : age ( in years ) , gender , smoking status ( never , former , or current smoker ) , schooling ( in years of formal education ) , and household arrangement ( living alone or accompanied ) . all regression analysis also included a measure of number of chronic conditions at the baseline . health status based on the number of self - reported chronic conditions included diabetes , hypertension , cardiovascular disease , stroke , cancer , arthritis , and osteoporosis . 12.1 for windows ( statacorp , college station , tx ) was used for all the statistical analyses . weighted logistic regressions were then used to assess the influence of bmi on disability prevalence . multinomial logistic regressions were used to assess the influence of bmi on disability transitions and mortality . for those free of disability , four outcomes were considered in the multinomial logistic regressions : remained free of disability ( reference category ) , became disabled ( incidence ) , died , or were lost to followup . for those who were disabled at the baseline , four outcomes were included in the multinomial logistic regressions : remained disabled ( reference category ) , recovered from disability , died , or were lost to followup . multinomial logistic regressions were used to analyze the role of weight change on health transitions as discussed above , excluding mortality , as we do not have information on weight change prior to death in between waves . in the baseline , there were 1,420 individuals free of adl and 368 individuals with adl . in 2006 , among those free of adl , 606 individuals had remained free of adl , 226 had developed adl , 329 had died , and 259 were lost in the followup or had missing data on adl status . among those who had adl in the baseline , 99 remained with adl , 75 recovered from adl , 144 died and 50 were lost in the followup or had missing data on adl in 2006 . for iadl limitations , 1,207 were free of iadl , and 581 had iadl in the baseline . among those who were free of iadl in the baseline , 491 remained free of limitations , 257 developed iadl , 230 had died , and 229 were lost in the followup or had missing data on iadl in the second wave . among those with iadl in the baseline , 220 remained with iadl , 36 recovered from iadl , 243 had died , and 82 were lost in the followup or had missing data on iadl status in 2006 . regarding the nagi , 654 participants were free of it in 2000 , and 1,134 had at least one nagi limitation . among those free of nagi , 192 remained free of it , 210 developed nagi , 129 died , and 123 were lost in the followup or had missing data on the nagi variable in 2006 . among those who had at least one nagi limitation in 2000 , 539 remained with nagi 's limitations , 70 recovered from nagi 's limitations , 344 died , and 181 were lost in the followup or had missing data on nagi 's limitations in the second wave . in the final sample , 23.4% were underweight , 43.3% had normal weight , 12.4% were overweight , and 21.1% of the participants were classified as obese . table 1 presents the prevalence estimates of disability according to measures of adl , iadl , and nagi 's limitations by sex and bmi category at baseline . prevalence of adl and nagi 's limitations was highest among obese individuals , whereas prevalence of iadl was highest among underweight older adults . weighted estimates indicated that 16.7% of the sample in so paulo had difficulty performing at least one adl . prevalence of iadl reached 24.4% , and most ( 57.8% ) of the older brazilian adults reported nagi 's limitations . in logistic regressions , after adjusting for age and sex ( not shown ) , individuals who were underweight did not differ from those of normal weight on their likelihood of reporting having adl , iadl , or nagi in the baseline . obese individuals were more likely than normal weight participants to report having at least one adl ( or = 1.8 , 95% ci = 1.2 , 2.6 ) and nagi 's limitations ( or = 2.5 , 95% ci = 1.8 , 3.6 ) . there were no statistical differences between normal weight and obese participants regarding iadl prevalence at baseline . there were no statistical differences between normal and overweight subjects regarding adl and iadl prevalence at baseline , but overweight individuals were more likely than those of normal weight to have nagi 's limitations ( or = 1.6 , 95% ci = 1.1 , 2.2 ) . women were more likely than men to report having adl , iadl , and nagi 's limitations at baseline ( p < 0.05 ) . table 2 shows the multinomial logistic regression results of the disability transitions and mortality between 2000 and 2006 among those who were free of disability in the baseline . compared to normal weight individuals , obese individuals were more likely to develop adl ( rrr = 2.1 ) and iadl ( rrr = 2.4 ) , whereas individuals who were underweight were more likely to develop iadl ( rrr = 1.9 ) . mortality risks were higher among those who were overweight ( rrr = 2.5 ) compared to those of normal weight in the nagi model in which the reference category was remaining free of nagi 's limitations . for all measures of disability , the risk of becoming disabled increased with age . as expected , women were more likely to develop adl and nagi 's limitations , but not iadl , between waves . higher number of chronic conditions was associated with higher mortality and higher incidence of adl and iadl . table 3 shows the multinomial logistic regression results of the disability transitions and mortality between 2000 and 2006 among those who had disability in the baseline . being obese was also associated with lower recovery from nagi ( rrr = 0.46 ) versus remaining with at least one nagi limitation . older age and higher number of chronic conditions were negatively associated with recovery . in the last set of analyses , we focus on the role of weight gain between waves on disability transitions ( table 4 ) . the results presented in table 4 indicate that those who gained weight between waves were more likely to develop adl ( rrr = 2.3 ) and nagi 's limitations ( rrr = 2.2 ) than those who maintained their weight , even after controlling for initial bmi categories and other covariates . women faced higher incidence of adl ( rrr = 1.8 ) and nagi ( rrr = 2.4 ) than men . when the analyses focused on those who had disability in the baseline , we found that weight gain was associated with lower recovery from adl ( rrr = 0.18 ) . . a higher number of chronic conditions were associated with lower recovery of adl and nagi . most previous studies have focused on the association between bmi and disability [ 4 , 9 , 13 , 15 , 16 ] or bmi and mortality [ 10 , 2029 , 3133 ] , but few have analyzed the effect of bmi on both disability and mortality [ 11 , 12 , 14 , 30 ] . using three disability measures and data from a large cohort study , this study contributes to the literature by exploring the impact of bmi and weight changes on disability status transitions and on mortality . this study confirmed the negative effects of obesity on disability in so paulo , brazil . higher levels of nagi 's limitations were also found among those who were overweight at baseline . most longitudinal studies have found that obese older adults are more likely to have experienced incidence of disability in the followup than those of normal weight [ 13 , 14 , 40 ] , and our study confirmed these findings . older adult brazilians who were obese at baseline faced higher risks of becoming disabled with adl or iadl limitations compared to those of normal weight . however , being overweight was not associated with higher incidence of disability after controls were included in the analysis , which is consistent with previous findings . in terms of recovery , we also found that obese individuals were less likely to recover ( versus remaining disabled in the followup ) , as other studies have also found . there is growing interest in the role of weight changes on health transitions [ 13 , 18 , 19 , 42 ] . studies have shown that weight gain in older adults is associated with decreased physical function and role limitations [ 18 , 19 ] . we found similar findings in which older adults who gained weight between waves were more likely to develop adl and nagi 's limitations than those who maintained their weight , even after controlling for initial bmi categories . al snih and colleagues also reported higher adl incidence among individuals who had weight gain of more than 5% between waves . some studies have indicated that weight loss is associated with improvements in mobility and functioning , whereas others have reported increased adl disability . weight - loss therapy among obese older adults seems to be beneficial for improving quality of life and physical functioning . ritchie and colleagues found that intentional weight loss was not associated with functional decline ; however , those who unintentionally lost weight faced higher rates of functional decline , regardless of the initial bmi . in our study , we found no differences between those who lost weight and those who maintained weight on disability transitions after controlling for initial bmi . given the lack of data on intent related to weight changes , further studies are necessary to explore the impact of weight change on mortality and disability in latin american countries . in additional analyses ( not shown and available upon request ) , we have explored additional models to test whether bmi and weight changes influence changes in the number of disabilities over time . we found that obesity was associated with the increases in the number of nagi 's limitations . weight loss and weight gain were associated with an increase in the number of adl and nagi 's limitations over time . changes in the number of iadl limitations were not statistically associated with bmi categories or weight changes . as expected , older age was associated with the increases in the number of adl , iadl , and nagi 's limitations over time . a higher number of chronic conditions were also associated with an increase in the number of adl , iadl and nagi 's limitations over time . we also tested fractional polynomial models following the approach suggested by wong and colleagues to examine the relationship between bmi , disability and mortality ( results available upon request ) , and our main conclusions remain the same , which indicates that findings are robust to different model specifications . the only mortality differential by bmi categories was found among overweight participants who were more likely to die than to remain free of nagi 's limitations . in further analyses ( not shown ) , results from a logistic regression that controlled for the same covariates included in this study , revealed no differences in mortality among underweight , normal weight , overweight , and obese participants . this is consistent with previous studies suggesting that the association between bmi and mortality becomes less u - shaped at older ages , and others that suggest that higher bmi may not be detrimental for mortality at older ages . monteverde and colleagues also did not find statistical differences in mortality among older adults between higher bmi categories ( overweight and obese ) and normal subjects when using traditional bmi cutoffs , though they reported statistical differences when bmi was categorized in relative terms . however , some studies allude to the fact that the association between bmi and mortality is differential between individuals who are healthy versus those with chronic conditions . the obesity paradox literature indicates that excess weight is actually protective among patients with chronic disease . in our sample , there were no mortality differentials by bmi categories among those with chronic conditions , but overweight individuals free of chronic conditions had lower mortality than those of normal weight ( results available upon request ) . our findings also contribute to a growing debate in the field about whether greater life expectancy implies better health for the expanding surviving elderly female population in latin america [ 37 , 4651 ] . we found that brazilian women experience higher levels of disability than men , which is consistent with previous studies [ 52 , 53 ] . previous studies have indicated that brazilian women face lower mortality than their male counterparts [ 37 , 54 ] , and this study confirms these findings . aging is related to the increase of fat mass , and there is growing evidence of the detrimental impact of obesity on disability at older ages . there is evidence as well that changes in lifestyle , such as walking , have positive effects on preventing mobility limitations . a large proportion of older adults , however , do not engage in physical activity . in a study based on an urban sample in brazil , for example , 71% of older adults reported living sedentary lives . when asked about neighborhood characteristics related to concerns of leaving home to go out , most ( 78% ) reported fear of being robbed , while almost half ( 48.2% ) said that they were afraid of falling because of sidewalk defects . fear of falling due to poor sidewalk conditions was associated with a 62% increase in the expected number of adl conditions . therefore , investments aimed at improving urban infrastructure and safety may be effective in addressing the health conditions of older adults in brazil . this study advances the literature on the impact of body weight and body weight changes on disability and mortality . first , the data used in the study on disability measures were self - reported . although this could be a possible source of bias , methodological studies have shown that self - reported data on functional disability are consistent with medical diagnoses . second , the use of bmi as a measure for body weight composition among older adults is very controversial as it does not take into account body fat distribution . in addition , bmi at baseline can be associated with health status [ 16 , 17 ] . therefore , it is important to control for weight changes , which we accomplished in this study . some authors have argued that waist circumference or waist - to - hip ratio could be better predictors of disability and mortality ; however , most studies to date have focused on the use of bmi and the categories used here . other scholars have indicated that , at least for developed countries , information on bmi , waist circumference , or waist - to - hip ratio do not necessarily improve prediction of mortality due to cardiovascular disease ; instead , they suggest using information on systolic blood pressure , diabetes status , and lipids when those are available . waist circumference and waist - to - hip ratio can be useful in better understanding mortality risks [ 59 , 60 ] . in brazil , as in other developing countries , data on blood pressure and lipids are often lacking , so the use of anthropometric measures , such as waist circumference , may improve our understanding of the impact of body composition changes on mortality and disability . third , the first wave of sabe focuses on the civilian population not residing in institutions . as a result , estimates may be biased if one expects institutionalized individuals , particularly those residing in nursing homes , to be more likely to have a higher prevalence of disability than the noninstitutionalized population . however , because the institutionalized population in brazil is relatively small , this possible bias is likely not to be very significant . this study confirms previous studies that have found obesity to be associated with increased disability in brazilian older adults . historically , brazil has mainly been concerned with curbing malnutrition ; however , in recent years , new policies have targeted the marketing of highly processed and unhealthy foods . owing to the fact that obesity rates in brazil have been increasing drastically for the past three decades , our findings have important implications for policy makers in brazil with regard to curbing disability risk by promoting the use of effective preventive measures to reduce body weight , thereby making healthy aging a reality .

the aim of this study was to examine the association between body mass index and weight changes on disability transitions and mortality among brazilian older adults . longitudinal data from the health , well - being , and aging in latin america and the caribbean study conducted in so paulo , brazil ( 2000 and 2006 ) , were used to examine the impact of obesity on disability and mortality and of weight changes on health transitions related to disability . logistic and multinomial regression models were used in the analyses . individuals who were obese were more likely than those of normal weight to have limitations on activities of daily living ( adl ) , instrumental activity of daily living ( iadl ) , and nagi 's limitations . obesity was associated with higher incidence of adl and iadl limitations and with lower recovery from nagi 's limitations . compared to those who maintained their weight , those who gained weight experienced higher incidence of adl and nagi 's limitations , even after controlling for initial body mass index . higher mortality among overweight individuals was only found when the reference category was remaining free of nagi limitations . the findings of the study underline the importance of maintaining normal weight for preventing disability at older ages .

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

however , the number of disabled people is expected to increase in the coming years , given the rapid growth rate of the elderly population and the rise in the prevalence of obesity and chronic diseases . fast changes in the population 's nutritional intake that have occurred in brazil in recent decades have resulted in an increase in the prevalence of obesity [ 6 , 7 ] . with the exception of higher - income urban women , few studies focus on the impact of bmi on mortality and disability in the latin american and the caribbean ( lac ) region . based on the baseline for the health , well - being , and aging in latin america and the caribbean study ( sabe ) , al snih and colleagues showed that obese individuals were 1.6 times more likely to face difficulties performing activities of daily living ( adl ) than those with normal bmi . corona and colleagues found that older adults who were underweight ( bmi 23 kg / m ) and obese ( bmi 30 kg / m ) were more likely to develop limitations on instrumental activity of daily living ( iadl ) , but their analysis did not explore bmi associations with mortality or recovery from disability . monteverde and colleagues found that , based on relative cutoffs ( quintiles ) , heavier older adults in mexico face higher mortality risks than those in the united states . however , none of these studies jointly examined the association between bmi , disability transitions , and mortality . we examine the association between body mass index and weight changes on disability transitions and mortality . obesity has been associated with higher prevalence of disability in cross - sectional and longitudinal studies [ 4 , 1113 ] . additionally , the association between body mass index ( bmi ) and disability is strongest among those who are underweight ( bmi < 18.5 kg / m ) and among obese subjects ( bmi > 30 obese women face higher prevalence of mobility impairment than men . in the united states , severe functional limitations are higher among older adults who gain or lose weight after age 50 compared to those with stable weight . older adults in the united states who gain weight over time have higher incidence of mobility limitations than those who maintain their weight . recent studies , however , show that overweight individuals have higher life expectancy than individuals of normal weight or those who are obese , whereas other studies have an l - shape ( see , for a short review ) . higher mortality at low levels of bmi has been associated primarily with lung cancer and respiratory diseases . at older ages , results from a systematic review and meta - analysis indicated that bmi in the overweight range of 2529.9 kg / m is not associated with increased mortality , whereas other studies have shown that the burden of obesity on mortality seems to be reduced or eliminated among older adults [ 11 , 12 , 2630 ] . the use of bmi categories has been criticized for not reproducing well the complexities of the bmi and mortality relationship [ 3133 ] . gronniger used semiparametric models and found that men in the mild - obese category ( bmis of 3035 kg / m ) had similar mortality than those of normal weight , but among women bmi levels above 27 kg / m were associated with higher mortality in the u.s . wong and colleagues used multivariable fractional polynomials to explore the association between bmi and mortality in a sample of adults in the ua they found that the best fitting model contained the powers 1 and 2 for bmi . their results indicated that the nadir of the bmi mortality curve was in the normal range for women but overweight range for men . zajacova and burgard used generalized additive models and found that the nadir was at bmis 23 to 26 , which is also in the normal overweight range . for example , the association between bmi and diabetes mortality increases monotonically , but , for all - cause mortality , it followed more a v - shape . our study uses data a large cohort study conducted in so paulo , brazil , to examine the association between bmi and body weight changes on disability transitions and mortality , while controlling for a series of demographic , socioeconomic , and health determinants . we investigate these associations on three types of disability ( activities of daily living , instrumental activities of daily living , and nagi 's limitations ) transitions . data from the two waves ( 2000 and 2006 ) of the sabe cohort study conducted in so paulo , brazil , were used in this study . sabe is a multicenter survey with respondents in seven major cities throughout lac countries that have been investigating the health and well - being of older adults ( age 60 and over ) . the study was approved by the institutional review boards at the collaborating institutions [ 34 , 35 ] , and the participants provided consent to have their data used for research purposes . the first stage was a household interview conducted by a single interviewer using a standardized questionnaire that included several questions about the living conditions and health status of the subjects . vital statistics records were used to identify subjects who had died between 2000 and 2006 . the search was based on the names , sex , dates of birth , and addresses listed in the 2000 database . of the 2,143 participants in the first wave of sabe so paulo , 355 ( 16.6% ) had missing data on selected variables . those with missing data were older ( 75.1 years ) than those with complete data ( 72.9 years ) ( p = 0.0001 ) , but there were no sex differences . the final sample is composed of 1,788 individuals , with a subset of 961 with weight change included in the analyses . self - reported disability in six adl measures ( dressing , bathing , eating , getting in and out of a bed , toileting , and getting across a room ) were used to measure disability . and does not know , and no response for each one of the six adl measures . each of the three disability measures was converted into binary form , in which respondents scored 0 if they did not indicate any limitations and 1 if they reported having difficulty performing one or more activity in the scale . four bmi categories were defined according to the criteria adopted by the pan american health organization for the sabe study : underweight ( bmi 23.0 ) , normal ( bmi > 23 and < 28 ) , overweight ( bmi 28.0 and < 30 ) , and obese ( bmi 30 ) . this difference was divided by the baseline bmi and then recoded into three categories : ( a ) an increase of 5% or more ; ( b ) a decrease of 5% or more ; and ( c ) changes within 5% of the baseline weight ( reference category ) [ 19 , 39 ] . the following sociodemographic characteristics were included in the analysis : age ( in years ) , gender , smoking status ( never , former , or current smoker ) , schooling ( in years of formal education ) , and household arrangement ( living alone or accompanied ) . weighted logistic regressions were then used to assess the influence of bmi on disability prevalence . multinomial logistic regressions were used to assess the influence of bmi on disability transitions and mortality . for those free of disability , four outcomes were considered in the multinomial logistic regressions : remained free of disability ( reference category ) , became disabled ( incidence ) , died , or were lost to followup . for those who were disabled at the baseline , four outcomes were included in the multinomial logistic regressions : remained disabled ( reference category ) , recovered from disability , died , or were lost to followup . multinomial logistic regressions were used to analyze the role of weight change on health transitions as discussed above , excluding mortality , as we do not have information on weight change prior to death in between waves . in the baseline , there were 1,420 individuals free of adl and 368 individuals with adl . in 2006 , among those free of adl , 606 individuals had remained free of adl , 226 had developed adl , 329 had died , and 259 were lost in the followup or had missing data on adl status . among those who had adl in the baseline , 99 remained with adl , 75 recovered from adl , 144 died and 50 were lost in the followup or had missing data on adl in 2006 . for iadl limitations , 1,207 were free of iadl , and 581 had iadl in the baseline . among those who were free of iadl in the baseline , 491 remained free of limitations , 257 developed iadl , 230 had died , and 229 were lost in the followup or had missing data on iadl in the second wave . among those with iadl in the baseline , 220 remained with iadl , 36 recovered from iadl , 243 had died , and 82 were lost in the followup or had missing data on iadl status in 2006 . regarding the nagi , 654 participants were free of it in 2000 , and 1,134 had at least one nagi limitation . among those free of nagi , 192 remained free of it , 210 developed nagi , 129 died , and 123 were lost in the followup or had missing data on the nagi variable in 2006 . among those who had at least one nagi limitation in 2000 , 539 remained with nagi 's limitations , 70 recovered from nagi 's limitations , 344 died , and 181 were lost in the followup or had missing data on nagi 's limitations in the second wave . in the final sample , 23.4% were underweight , 43.3% had normal weight , 12.4% were overweight , and 21.1% of the participants were classified as obese . table 1 presents the prevalence estimates of disability according to measures of adl , iadl , and nagi 's limitations by sex and bmi category at baseline . prevalence of adl and nagi 's limitations was highest among obese individuals , whereas prevalence of iadl was highest among underweight older adults . weighted estimates indicated that 16.7% of the sample in so paulo had difficulty performing at least one adl . prevalence of iadl reached 24.4% , and most ( 57.8% ) of the older brazilian adults reported nagi 's limitations . in logistic regressions , after adjusting for age and sex ( not shown ) , individuals who were underweight did not differ from those of normal weight on their likelihood of reporting having adl , iadl , or nagi in the baseline . obese individuals were more likely than normal weight participants to report having at least one adl ( or = 1.8 , 95% ci = 1.2 , 2.6 ) and nagi 's limitations ( or = 2.5 , 95% ci = 1.8 , 3.6 ) . there were no statistical differences between normal and overweight subjects regarding adl and iadl prevalence at baseline , but overweight individuals were more likely than those of normal weight to have nagi 's limitations ( or = 1.6 , 95% ci = 1.1 , 2.2 ) . women were more likely than men to report having adl , iadl , and nagi 's limitations at baseline ( p < 0.05 ) . table 2 shows the multinomial logistic regression results of the disability transitions and mortality between 2000 and 2006 among those who were free of disability in the baseline . compared to normal weight individuals , obese individuals were more likely to develop adl ( rrr = 2.1 ) and iadl ( rrr = 2.4 ) , whereas individuals who were underweight were more likely to develop iadl ( rrr = 1.9 ) . mortality risks were higher among those who were overweight ( rrr = 2.5 ) compared to those of normal weight in the nagi model in which the reference category was remaining free of nagi 's limitations . as expected , women were more likely to develop adl and nagi 's limitations , but not iadl , between waves . higher number of chronic conditions was associated with higher mortality and higher incidence of adl and iadl . table 3 shows the multinomial logistic regression results of the disability transitions and mortality between 2000 and 2006 among those who had disability in the baseline . being obese was also associated with lower recovery from nagi ( rrr = 0.46 ) versus remaining with at least one nagi limitation . in the last set of analyses , we focus on the role of weight gain between waves on disability transitions ( table 4 ) . the results presented in table 4 indicate that those who gained weight between waves were more likely to develop adl ( rrr = 2.3 ) and nagi 's limitations ( rrr = 2.2 ) than those who maintained their weight , even after controlling for initial bmi categories and other covariates . women faced higher incidence of adl ( rrr = 1.8 ) and nagi ( rrr = 2.4 ) than men . when the analyses focused on those who had disability in the baseline , we found that weight gain was associated with lower recovery from adl ( rrr = 0.18 ) . a higher number of chronic conditions were associated with lower recovery of adl and nagi . most previous studies have focused on the association between bmi and disability [ 4 , 9 , 13 , 15 , 16 ] or bmi and mortality [ 10 , 2029 , 3133 ] , but few have analyzed the effect of bmi on both disability and mortality [ 11 , 12 , 14 , 30 ] . using three disability measures and data from a large cohort study , this study contributes to the literature by exploring the impact of bmi and weight changes on disability status transitions and on mortality . this study confirmed the negative effects of obesity on disability in so paulo , brazil . higher levels of nagi 's limitations were also found among those who were overweight at baseline . most longitudinal studies have found that obese older adults are more likely to have experienced incidence of disability in the followup than those of normal weight [ 13 , 14 , 40 ] , and our study confirmed these findings . older adult brazilians who were obese at baseline faced higher risks of becoming disabled with adl or iadl limitations compared to those of normal weight . however , being overweight was not associated with higher incidence of disability after controls were included in the analysis , which is consistent with previous findings . in terms of recovery , we also found that obese individuals were less likely to recover ( versus remaining disabled in the followup ) , as other studies have also found . there is growing interest in the role of weight changes on health transitions [ 13 , 18 , 19 , 42 ] . studies have shown that weight gain in older adults is associated with decreased physical function and role limitations [ 18 , 19 ] . we found similar findings in which older adults who gained weight between waves were more likely to develop adl and nagi 's limitations than those who maintained their weight , even after controlling for initial bmi categories . ritchie and colleagues found that intentional weight loss was not associated with functional decline ; however , those who unintentionally lost weight faced higher rates of functional decline , regardless of the initial bmi . in our study , we found no differences between those who lost weight and those who maintained weight on disability transitions after controlling for initial bmi . given the lack of data on intent related to weight changes , further studies are necessary to explore the impact of weight change on mortality and disability in latin american countries . in additional analyses ( not shown and available upon request ) , we have explored additional models to test whether bmi and weight changes influence changes in the number of disabilities over time . we found that obesity was associated with the increases in the number of nagi 's limitations . weight loss and weight gain were associated with an increase in the number of adl and nagi 's limitations over time . changes in the number of iadl limitations were not statistically associated with bmi categories or weight changes . as expected , older age was associated with the increases in the number of adl , iadl , and nagi 's limitations over time . a higher number of chronic conditions were also associated with an increase in the number of adl , iadl and nagi 's limitations over time . we also tested fractional polynomial models following the approach suggested by wong and colleagues to examine the relationship between bmi , disability and mortality ( results available upon request ) , and our main conclusions remain the same , which indicates that findings are robust to different model specifications . the only mortality differential by bmi categories was found among overweight participants who were more likely to die than to remain free of nagi 's limitations . in further analyses ( not shown ) , results from a logistic regression that controlled for the same covariates included in this study , revealed no differences in mortality among underweight , normal weight , overweight , and obese participants . this is consistent with previous studies suggesting that the association between bmi and mortality becomes less u - shaped at older ages , and others that suggest that higher bmi may not be detrimental for mortality at older ages . monteverde and colleagues also did not find statistical differences in mortality among older adults between higher bmi categories ( overweight and obese ) and normal subjects when using traditional bmi cutoffs , though they reported statistical differences when bmi was categorized in relative terms . however , some studies allude to the fact that the association between bmi and mortality is differential between individuals who are healthy versus those with chronic conditions . in our sample , there were no mortality differentials by bmi categories among those with chronic conditions , but overweight individuals free of chronic conditions had lower mortality than those of normal weight ( results available upon request ) . our findings also contribute to a growing debate in the field about whether greater life expectancy implies better health for the expanding surviving elderly female population in latin america [ 37 , 4651 ] . previous studies have indicated that brazilian women face lower mortality than their male counterparts [ 37 , 54 ] , and this study confirms these findings . aging is related to the increase of fat mass , and there is growing evidence of the detrimental impact of obesity on disability at older ages . fear of falling due to poor sidewalk conditions was associated with a 62% increase in the expected number of adl conditions . therefore , investments aimed at improving urban infrastructure and safety may be effective in addressing the health conditions of older adults in brazil . this study advances the literature on the impact of body weight and body weight changes on disability and mortality . first , the data used in the study on disability measures were self - reported . therefore , it is important to control for weight changes , which we accomplished in this study . some authors have argued that waist circumference or waist - to - hip ratio could be better predictors of disability and mortality ; however , most studies to date have focused on the use of bmi and the categories used here . in brazil , as in other developing countries , data on blood pressure and lipids are often lacking , so the use of anthropometric measures , such as waist circumference , may improve our understanding of the impact of body composition changes on mortality and disability . as a result , estimates may be biased if one expects institutionalized individuals , particularly those residing in nursing homes , to be more likely to have a higher prevalence of disability than the noninstitutionalized population . this study confirms previous studies that have found obesity to be associated with increased disability in brazilian older adults .

despite efforts to eradicate tuberculosis , it remains one of the most successful and deadly human diseases . the tubercle bacilli resides within dendritic cells and macrophages , and in an immunocompetent host , the infection is walled off within a granuloma where it can remain dormant for years . while many factors contribute to its success , it is the thick , waxy cell wall of the bacillus that prevents dehydration , affords protection against varying levels of acidity and the detrimental affects of free radicals . in many respects , it is the unique architecture of the cell wall itself that makes mycobacterium tuberculosis ( mtb ) infection relatively difficult to treat with antibiotics . since it is also a reservoir for many proteins and nonproteinaceous antigens , which are secreted into the extracellular milieu to stimulate and/or suppress the host immune response , its definition can be exploited for vaccine development . for decades , the macromolecular features of the mycobacterial cell wall , including the mycolic acid and arabinogalactan core , have been studied in detail.(3 ) structurally , the cell wall of mtb is composed of a distinct inner core of mycolic acid - arabinogalactan - peptidoglycan ( magp ) . in addition to the covalently attached lipids and carbohydrates , it is well - known that the free lipids , lipoglycans , and phosphotidyl inositols that reside in the outer core of the cell wall play key roles in modulation of the host immune response.(4 ) specifically , the molecules , lipoarabinomannan ( lam ) , lipomannan ( lm ) , and phosphatidyl inositol mannoside ( pim ) are known to aid the process of host immune evasion.(2 ) in addition , virulence lipids such as trehalose dimycolate / monomycolate ( tdm / tmm ) , phthiocerol dimycocerosate ( pdim ) , and sulfolipids ( sl ) and the protein machinery , such as mmpls , required for their export are intercalated in the cell wall.(5 ) numerous cell wall associated proteins , including many lipoproteins and lipoglycoproteins , have also been described . for example , the tlr2 agonists , lpqh ( 19 kda ) , psts1 ( 38 kda ) , and lprg ( rv14llc ) , are all found in the cell wall , where they function to regulate the action of macrophages and dendritic cells.(12 ) psts1 also plays a role in bacterial escape from the host macrophage through apoptosis.(13 ) many other lipoproteins of unknown function are identified in the secreted proteome of mtb culture filtrate . the study of the secreted proteome of mtb was driven by the search for novel immunodominant antigens , drug targets , and biomarkers for disease . to build upon this work , a number of studies employed advances in proteomic technologies such as two - dimensional gel electrophoresis ( 2dge ) and liquid chromatography mass spectrometry ( lcms / ms ) to further mine additional subcellular compartments of mtb cytosol , membrane , and cell wall . historically , the proteins within the cell wall have been difficult to resolve and identify by traditional 2dge methods . mawuenyega et al . , employing two - dimensional liquid chromatography coupled with mass spectrometry ( 2dlc / ms ) were the most successful at defining the cell wall proteome with the identification of 306 proteins.(24 ) in this study , we set out to comprehensively describe the mtb cell wall proteome in an effort to exploit additional proteins that may play a role in host - pathogen interactions and define new potential drug targets via discovery of unique biosynthetic or metabolic processes . we used a combination of detergent extraction , 2dge , multidimensional liquid chromatography , and mass spectrometry to achieve our goal . mtb strain h37rv was cultured in 2 l of glycerol alanine salts ( gas ) medium(28 ) in roller bottles for 14 d at 37 c with gentle agitation . cells were harvested,(24 ) washed with phosphate - buffered saline ( pbs ) , ph 7.4 , and inactivated by -irradiation . cells were disrupted in a french press , free lipids were removed , and the cell wall was obtained as previously described.(29 ) briefly , 1 g of lyophilized cell wall was subjected to two extractions of 2 h each followed by one 18 h extraction with chloroform / methanol ( 2:1 , v / v ) at a ratio of 30 ml / g of cell wall . centrifugation at 27 000 g for 30 min was performed to collect cell wall material . the 2:1 extracted cell wall was dried under n2 and further extracted twice for 2 h and one 18 h extraction with chloroform / methanol / water ( 10:10:3 , v / v / v ) each at 22 c . the fully delipidated cell wall was dried under n2 and resuspended in pbs , ph 7.4 . cell wall protein was quantified by bicinchoninic acid ( bca ) assay ( thermo pierce ) . cell wall protein ( cwp ) was solubilized by one of three methods using ( i ) 6 m guanidine hcl ( guhcl ) , ( ii ) 2% sodium dodecyl sulfate ( sds ) , or ( iii ) 4% tritionx-114 ( tx-114 ) . for method ( i ) , 75 mg of cwp was incubated at 22 c for 4 h with agitation . solubilized proteins were exchanged into 0.01 m nh4hco3 , and the protein amount was determined by bca assay . the average protein recovery following guhcl extraction of cwp was 40% . for method ( ii ) , sds soluble proteins were generated by extraction of 100 mg of cwp with 2% sds in pbs ( w / v ) as described previously.(29 ) briefly , the sample was bath sonicated for 3 h at 90 c . the sample was centrifuged at 27 000 g at 22 c for 30 min , collected , and centrifuged again . the fully cleared supernatant was then subjected to paired - ion extraction for removal of sds.(30 ) proteins were concentrated by centrifugation as above and pellets washed with acetone at 20 c for 4 h. the final pellet was resuspended in 0.01 m nh4hco3 and protein amount was determined by bca . the average protein recovery following sds extraction of cwp was 60% . in method ( iii ) , a stock solution of 32% tx-114 in pbs was added to 300 mg of cwp to a final concentration of 4% detergent . primary extraction occurred at 4 c for 16 h. the extract was allowed to biphase at 37 c for 30 min and was fully separated by centrifugation at 27 000 g for 30 min . the tx-114 detergent phases were pooled , and proteins collected by cold acetone precipitation.(31 ) final tx-114 proteins were resuspended as above and protein amount was determined by bca assay . samples of 200 g of each cwp preparation were solubilized for 914 h in rehydration buffer ( 7 m urea/2 m thiourea , 1% amidosulfobetaine-14 ( asb-14 ) , 1% 3-[(3-cholamidopropyl ) dimethylammonio]-1-propanesulfonate ( chaps ) , 1 mm dithiothreitol ( dtt ) , 0.25% np-40 , 0.625% zoom carrier ampholytes 310 , and 1.9% cwp was applied on zoom precast immobilized ph 47 linear gradient strips ( 7.0 cm ; invitrogen ) according to manufacturer s instructions . focusing was achieved using a stepwise voltage gradient of 200 , 450 , 700 , and 1000 v , for 10 min each followed by focusing at 2000 v for 2 h. sds - page of the isoelectric focusing ( ief ) strips was performed using zoom 412% bis - tris sds - page gradient gels ( invitrogen ) . images were captured using a gel - doc system ( bio - rad , hercules , ca ) , and spot detection was performed using delta 2d software ( greifswald , germany ) . a total of 5.0 mg of tx-114 cwp was digested with modified trypsin ( roche diagnostics ) at a ratio of 1:20 ( e / s ) , in 0.1 m urea and 20 mm methylamine . the digest was desalted using sep - pak light c18 cartridge ( waters , inc . ) and concentrated under vacuum . the resultant peptides were separated by strong cation - exchange ( scx ) chromatography using a polysulfylethyl a column ( 460 m 200 mm , 300 ; poly lc , inc . ) the digest was applied in buffer a ( 5 mm k2po4 , 20% acetonitrile ( acn ) , ph 3.0 ) using a gradient of 080% buffer b ( a with 0.5 m kcl ) over 75 min with a flow rate of 1 ml / min . the elution of peptides was monitored at 214 nm , and fractions ( 3 ml each ) were pooled based on uv absorbance . each fraction ( 6 total ) was concentrated under vacuum and resuspended into reverse phase buffer a ( 0.1% tfa in h2o ) . individual scx pools were subjected to further separation by reversed - phase hplc ( rp - hplc ) using a monomeric c18 column ( 4.6 mm 150 mm , vydac ) . peptides were eluted using a gradient of 050% reverse - phase buffer b ( 90% acn in a ) over 40 min at a flow rate of 1 ml / min . a total of 139 rp - hplc peptide fractions were collected and concentrated under vacuum . coomassie blue stained spots were excised from 2-de gels and subjected to in - gel digestion with modified trypsin ( roche diagnostics ) . digests and 2dlc peptide fractions were resolved by liquid chromatographymass spectrometry ( lcms ) using either an lcq ( 2dge ) or ltq ( 2dlc ) as described previously . tandem mass spectra were extracted , charge state deconvoluted , and deisotoped by bioworks version 3.2 ( thermo finnigan , san jose , ca ) . all ms / ms samples were analyzed using sequest ( thermo finnigan ; version 27 , rev . 12 ) sequest was set up to search the tb genome database ( version 2.0 , genbank accession no . al123456 , 3912 entries ) assuming the digestion enzyme trypsin , a fragment ion mass tolerance of 1.0 da , a parent ion tolerance of 2.5 da , and 3 allowable missed cleavages . oxidation of methionine and acrylamide adduct of cysteine ( 2dge spots ) were specified in sequest as variable modifications . sequest identifications required at least deltacn scores of greater than 0.3 and xcorr scores of greater than 1.5 , 2.2 , 2.5 , and 2.5 for singly , doubly , triply , and quadruply charged peptides ( supplementary table s1 ) . scaffold ( version scaffold-01_05_21 , proteome software , inc . , portland , or ) was used to validate ms / ms based peptide and protein identifications . peptide identifications were accepted if they could be established at greater than 90% probability as specified by the peptide prophet algorithm.(35 ) protein identifications were accepted if they could be established at greater than 90% probability as assigned by the protein prophet algorithm(35 ) and contained at least two unique peptides . the protein probability false discovery rate for the cwp identifications was 3.5%.(36 ) proteins that contained similar peptides and could not be differentiated based on ms / ms analysis alone were grouped to satisfy the principles of parsimony ( supplementary table s1 ) . fasta files for each annotated mtb protein were collected and organized into small groups of 50 . each group was then submitted to the signalp server ( http://www.cbs.dtu.dk/services/signalp/ ) using the gram - positive option . the output was saved in html format and a perl script written to combine the results from all html files into one file and then converted to tab delimited format . this generated a complete list of results that were sorted in a spreadsheet to create the subsets of neural network positive , hidden markov model positive , and the union of the nn and hmm sets . these lists were input into a java application along with the list of proteins isolated from the cell wall fractions . this application simply compared the predicted list to those isolated and identified the proteins found in the cell wall that were also predicted in each subset as having a signal peptide . to predict lipoproteins , the mtb peptide fasta file from above was broken down into 1000 sequences per file . the output files were combined in a spreadsheet , and from the sorted data , a list was extracted with every signal peptidase ii cleavage site generating a predicted lipoprotein list . this list was input into a java application that would compare the cell wall proteins , identifying the predicted lipoproteins from each cell wall fraction . protein preparations from the three cwp fractions resolved by 2dge or sds - page were transferred to pvdf membranes and incubated with mouse monoclonal antibodies ( -glcb [ rv1837c ] , -psts1 [ rv0934 ; it-23 ] , -lprg [ rv1411c ] , -ald [ rv2780 ] , -lpqh [ rv3763 ; it-19 ] ; all available through the nih , niaid contract , tuberculosis vaccine testing and research materials ) and polyclonal antibody against fructose bisphosphate aldolase ( -fba [ rv0363c ] , kindly provided by dr . mary jackson , colorado state university ) in tbs and 0.25% tween 80 ( tbst ) . after incubation , membranes were rinsed with tbst and developed using amersham ecl advance western blotting detection kit ( ge healthcare life science , piscataway , nj ) as per manufacturers instructions . protein immunoblots were visualized with the typhoon 9400 multiplex fluorescent imager ( ge healthcare life science , piscataway , nj ) . to maximize the resolution and protein identification of cell wall proteins , the delipidated cell wall of mtb was subjected to extraction based on solubility within three reagents an anionic ( sds ) , a chaotrophic ( guhcl ) , and a nonionic ( tx-114 ) detergent . 2dge of all cwp fractions demonstrated the majority of proteins resolving within a range of mw 2575 kda ( supplementary figure 1 ) . on average , 210 spots were resolved for guhcl and sds cwp fractions and 170 from tx-114 . from the 2dge analysis , 290 proteins were identified with 122 , 131 , and 37 proteins identified in the guhcl , sds , and tx-114 fractions , respectively . for all fractions , over half were unique to each subset ( figure 1a ) . analysis of the data demonstrated 80% of proteins had known functions ( figure 2 ) . in contrast , the tx-114 extracted fraction was less amenable to gel separation most likely due to its increased hydrophobic content . a multidimensional chromatography approach was employed to better resolve proteins in this fraction , in order to exploit any unique or uncharacterized protein families within the tx-114 subset . while 2dge was insufficient in identifying a significant number of proteins in the tx-114 detergent extract ( figure 1a ) , scx chromatography combined with reverse phase chromatography of a digest of this cwp preparation allowed the resolution of 364 proteins , including an additional 294 proteins not found in any of the 2dge preparations ( figure 1b ) . ( a ) identification of cell wall proteins by two - dimensional gel electrophoresis and two - dimensional liquid chromatography . detergent extraction and separation of cell wall protein subsets via 2dge demonstrated the majority of proteins to be resolved using sds and guhcl . ( b ) cumulatively , separation of cell wall proteins by 2dge resulted in the identification of 234 proteins . separating the tx-114 protein subset by multidimensional chromatography ( 2dlcms / ms ) resolved an additional 294 proteins . proteins identified for each extraction method ( guhcl , sds , tx114 ) were sorted by functional category as a percentage of total proteins per sample . representation of all protein groups was identified with the exception of groups 4 and 8 ( data not shown ) . seventy percent of the proteins identified in the cell wall are associated with lipid metabolism ( group 1 ) , cell wall processes ( group 3 ) , and intermediary metabolism ( group 7 ) . black , sds ; white , guhcl ; gray , tx114 ; hash , total . validation of a few selected proteins , by western blot analysis of samples resolved by 2dge ( supplementary figure 1 ) and sds - page ( supplementary figure 2 ) , corroborated the identification of these proteins by mass spectrometry . all identified proteins were grouped by functional category as defined by institute pasteur , which demonstrated mtb protein families present in each extract . proteins in categories 3 ( cell wall and cell wall processes ) and 7 ( intermediary metabolism ) were consistently overrepresented among the cwp preparations ( figure 2 ) . combining the data sets of both gel and non - gel based protein identifications led to the detection of 528 proteins ( supplementary table s2 ) . these were further classified into functional groups as defined by the sanger institute ( figure 3ac ) . one hundred and five of the 528 cell wall proteins identified were not reported in previous mtb proteome publications , including a comprehensive proteomic database http://web.mpiib-berlin.mpg.de/cgi-bin/pdbs/2d-page/extern/index.cgi,(19 ) and http://web.mpiib-berlin.mpg.de/cgi-bin/pdbs/2d-page/extern/index.cgi(42 ) ( table 1 ) . the distributions are among the major functional groups and the subgroups within functional groups i and ii . the percentage for each subgroup indicates the percentage of the total number of identified proteins in its major functional group . i.x is defined by proteins involved in central intermediary metabolism , amino acid biosynthesis , polyamine synthesis , biosynthesis of cofactors , prosthetic groups , and carriers , lipid biosynthesis , polyketide and non - ribosomal peptide synthesis , and broad regulatory functions . cell wall protein fractions : a , 2% sds ; b , 6 m guhcl ; c , 4% tx-114 ; d , 2dlc tx-114 . a majority of proteins were classified in either category i , small molecule metabolism ( 35% ) , or category ii , macromolecule synthesis and degradation ( 25% ) ( figure 3 ) . subclasses of category i showed 19% classified in small molecule metabolism - other ( i.x ) , which included the classes : central intermediary metabolism and amino acid biosynthesis ( figure 3 ; upper right ) ; and subclasses of category ii demonstrated an even distribution of proteins between synthesis / degradation of macromolecules ( ii.a , ii.b , 12.5% ) and cell envelope proteins ( ii.c , 12.1% ) ( figure 3 ; lower right ) . the secreted proteins of mtb have traditionally been characterized as important antigens and immune - modulators . prior to being exported , many of these proteins are resident within the cell wall where their function remains largely unknown . to find the putative secreted proteins identified within the cell wall proteome , all identified proteins were subject to interrogation against neural network ( nn ) and hidden markov model ( hmm ) algorithms ( signalp , http://www.cbs.dtu.dk/services/signalp/ ) which revealed 18% , 19% , 27% putative secreted proteins in guhcl , sds , tx-114 cwp samples , respectively , and 13% in the 2dlc resolved tx-114 cwp fraction . cumulatively , of the 528 proteins identified in this study , 87 proteins were predicted to contain secretion signals and included the identification of 23 proteins uniquely found in this study ( table 2 ) . a majority ( 60% ) of the cwp associated with secretion are indeed within the category of the cell wall and its processes ( figure 4 ) . further , many of these cwp are also membrane associated , either by description or functional annotation . of the 23 secreted cwp unique to this study , 11 have been described to be involved with small molecule and peptide binding . additionally , 23 of the 87 secreted proteins are classified as hypothetical or unknown ( categories v and vi of sanger institute ) illustrating that to a large extent , the functions of these exported proteins are poorly understood . eighty - seven cell wall proteins were predicted to contain secretion signals and putative lipoprotein motifs . sixty percent of these proteins are classified within the cell wall and cell wall processes functional category . next , we interrogated the cwp to identify those with putative lipoprotein motifs . here , a total of 16 proteins were predicted by lipop ( lipop , http://www.cbs.dtu.dk/services/lipop/)(43 ) to contain a signal peptidase ii cleavage motif , and an additional 8 proteins were identified when compared to the 99 putative lipoproteins ( 2.5% of the genome ) that reportedly exist within the mtb proteome.(7 ) a majority of the lipoproteins contain no additional functional classification ; however , 4 proteins ( rv0928 , rv0934 , rv2864c , rv3666c ) are involved with substrate binding and transport within the periplasm . one of two superoxide dismutases in mtb , sodc ( rv0432 ) , was also identified within the lipoprotein - enriched tx-114 cwp fraction . this protein has recently been defined as a highly glycosylated putative lipoprotein and is a defined b - cell antigen . other putative lipoproteins identified in this study contain o - glycosylation motifs as predicted by netoglyc and were found within the mtb glycoproteome ( table 2).(46 ) the function of these dual - modified lipo - glycoproteins , as well as the nature of their modification remain largely undefined . in this study , we focused on the gel - based two - dimensional separation of the cell wall and complemented that separation by liquid chromatography of digested tx-114 cwp . identified protein families included known antigens , fbp a , b , and c ( rv 3804c , rv1886c , and rv0129c ) , cfp10 ( rv3874 ) and esat-6 like proteins esxj and esxl ( rv1038c , rv1198 ) along with antigens lpqh ( rv3763 ) , lprg ( rv1411c ) , and lpra ( rv1270c ) . in concordance with previous work , a number of ribosomal proteins are reported here , which must indicate a high level of protein synthesis occurring at the cytosolcell wall interface , most likely to facilitate the entry of proteins into the cellular envelope . for over a decade , the advancement of techniques in 2dge , mass spectrometry , and increased access to bioinformatic tools greatly enhanced proteomic studies of mtb . largely descriptive , these studies were undertaken to identify novel virulence factors and drug targets . subsequent studies set out to understand functional relationships between proteins and discover antigens responsible for adaptive t - cell responses . the quantitative techniques of mass spectrometric profiling , isotope coded affinity tag ( icat ) and isobaric tagging for relative and absolute quantification ( itraq ) , have afforded the accurate monitoring of up- and down - regulation of proteins on a global scale . previous work mining subcellular fractions of mtb , focused on the cytosol and culture filtrate fractions , resulted in thorough characterization and the creation of 2d gel databases . efforts have been made more recently to resolve the insoluble cell wall fraction . as mentioned previously , mawuenyega et al . was able to identify 300 proteins within the cell wall using 2dlc as a separation method . their study demonstrated functional relationships among key protein families within the fatty - acid synthesis pathway . using these studies as experimental platforms , we were able to focus on the mtb cell wall and were successful in reporting over 100 additional proteins that had not been identified previously . the elucidation of the cell wall proteome can facilitate subsequent studies of a more targeted nature where specific proteins can be monitored in response to various environmental , nutritional , or drug pressures . one of the most interesting yet poorly understood protein families residing within the cell wall of mtb are the triacylated lipoproteins . as stated above , sutcliffe et al . predicted 2.5% of the genome to encode for lipoproteins . defining these proteins beyond their antigenic and immune modulatory potential remains quite elusive with only a few studies predicting functional roles for mtb lipoproteins . in this study , a vast majority of proteins found were involved in small molecule and macromolecule metabolism . this builds upon the evidence that clearly defines two mtb cw lipoproteins , lppx and lprg , as aiding and/or facilitating the transport of lipids(57 ) and small molecules,(58 ) respectively . the presence of these lipoproteins and perhaps other secreted , cw resident proteins provides a conduit between various nutrient transportation pathways that are required for cellular survival and growth while allowing the mycobacterial cell wall to maintain its rigid hydrophobic integrity . while bacterial cell walls have historically been thought of as structural scaffolding with little active interplay between the extracellular milieu and inner cytosolic environment , this perception of a static bacterial structure is fading and we are beginning to understand the true complexity and dynamic nature of the biological processes being facilitated within the cell wall . for instance , these proteins may contribute to bacterial cell wall remodeling events in response to environmental stressors such as nutrient depletion , antibiotic pressures , the host immune response , and tissue remodeling events throughout the course of infection . for mtb , the highly complex framework of the magpouter lipid architecture can now be definitively complemented with a highly diverse protein population . this new perspective will provide further insight into cell wall remodeling processes during mycobacterial infection as well as give a more comprehensive list of potential drug targets and diagnostic candidates . several classes of proteins were identified , including putative secreted proteins , lipoproteins , and known t and b cell antigens . many proteins have unknown function ; however , their presence within the cell wall may be of biological relevance .

the cell envelope of mycobacterium tuberculosis ( mtb ) is complex and diverse ; composed of proteins intermingled in a matrix of peptidoglycan , mycolic acids , lipids , and carbohydrates . proteomic studies of the mtb cell wall have been limited ; nonetheless , the characterization of resident and secreted proteins associated with the cell wall are critical to understanding bacterial survival and immune modulation in the host . in this study , the cell wall proteome was defined in order to better understand its unique biosynthetic and secretion processes . mtb cell wall was subjected to extraction with organic solvents to remove noncovalently bound lipids and lipoglycans and remaining proteins were solubilized with either sds , guanidine - hcl , or tx-114 . these extracts were analyzed by two - dimensional gel electrophoresis and mass - spectrometry and resulted in the identification of 234 total proteins . the lipoproteome of mtb , enriched in the tx-114 extract , was further resolved by multidimensional chromatography and mass spectrometry to identify an additional 294 proteins . a query of the 528 total protein identifications against neural network or hidden markov model algorithms predicted secretion signals in 87 proteins . classification of these 528 proteins also demonstrated that 35% are involved in small molecule metabolism and 25% are involved in macromolecule synthesis and degradation building upon evidence that the mtb cell wall is actively engaged in mycobacterial survival and remodeling .

Introduction Methods Results Discussion Conclusion

the tubercle bacilli resides within dendritic cells and macrophages , and in an immunocompetent host , the infection is walled off within a granuloma where it can remain dormant for years . while many factors contribute to its success , it is the thick , waxy cell wall of the bacillus that prevents dehydration , affords protection against varying levels of acidity and the detrimental affects of free radicals . in many respects , it is the unique architecture of the cell wall itself that makes mycobacterium tuberculosis ( mtb ) infection relatively difficult to treat with antibiotics . for decades , the macromolecular features of the mycobacterial cell wall , including the mycolic acid and arabinogalactan core , have been studied in detail. (3 ) structurally , the cell wall of mtb is composed of a distinct inner core of mycolic acid - arabinogalactan - peptidoglycan ( magp ) . in addition to the covalently attached lipids and carbohydrates , it is well - known that the free lipids , lipoglycans , and phosphotidyl inositols that reside in the outer core of the cell wall play key roles in modulation of the host immune response. (4 ) specifically , the molecules , lipoarabinomannan ( lam ) , lipomannan ( lm ) , and phosphatidyl inositol mannoside ( pim ) are known to aid the process of host immune evasion. (2 ) in addition , virulence lipids such as trehalose dimycolate / monomycolate ( tdm / tmm ) , phthiocerol dimycocerosate ( pdim ) , and sulfolipids ( sl ) and the protein machinery , such as mmpls , required for their export are intercalated in the cell wall. for example , the tlr2 agonists , lpqh ( 19 kda ) , psts1 ( 38 kda ) , and lprg ( rv14llc ) , are all found in the cell wall , where they function to regulate the action of macrophages and dendritic cells. (12 ) psts1 also plays a role in bacterial escape from the host macrophage through apoptosis. (13 ) many other lipoproteins of unknown function are identified in the secreted proteome of mtb culture filtrate . the study of the secreted proteome of mtb was driven by the search for novel immunodominant antigens , drug targets , and biomarkers for disease . to build upon this work , a number of studies employed advances in proteomic technologies such as two - dimensional gel electrophoresis ( 2dge ) and liquid chromatography mass spectrometry ( lcms / ms ) to further mine additional subcellular compartments of mtb cytosol , membrane , and cell wall . historically , the proteins within the cell wall have been difficult to resolve and identify by traditional 2dge methods . , employing two - dimensional liquid chromatography coupled with mass spectrometry ( 2dlc / ms ) were the most successful at defining the cell wall proteome with the identification of 306 proteins. (24 ) in this study , we set out to comprehensively describe the mtb cell wall proteome in an effort to exploit additional proteins that may play a role in host - pathogen interactions and define new potential drug targets via discovery of unique biosynthetic or metabolic processes . we used a combination of detergent extraction , 2dge , multidimensional liquid chromatography , and mass spectrometry to achieve our goal . cells were harvested,(24 ) washed with phosphate - buffered saline ( pbs ) , ph 7.4 , and inactivated by -irradiation . cells were disrupted in a french press , free lipids were removed , and the cell wall was obtained as previously described. (29 ) briefly , 1 g of lyophilized cell wall was subjected to two extractions of 2 h each followed by one 18 h extraction with chloroform / methanol ( 2:1 , v / v ) at a ratio of 30 ml / g of cell wall . centrifugation at 27 000 g for 30 min was performed to collect cell wall material . the 2:1 extracted cell wall was dried under n2 and further extracted twice for 2 h and one 18 h extraction with chloroform / methanol / water ( 10:10:3 , v / v / v ) each at 22 c . the fully delipidated cell wall was dried under n2 and resuspended in pbs , ph 7.4 . cell wall protein ( cwp ) was solubilized by one of three methods using ( i ) 6 m guanidine hcl ( guhcl ) , ( ii ) 2% sodium dodecyl sulfate ( sds ) , or ( iii ) 4% tritionx-114 ( tx-114 ) . solubilized proteins were exchanged into 0.01 m nh4hco3 , and the protein amount was determined by bca assay . for method ( ii ) , sds soluble proteins were generated by extraction of 100 mg of cwp with 2% sds in pbs ( w / v ) as described previously. (29 ) briefly , the sample was bath sonicated for 3 h at 90 c . the sample was centrifuged at 27 000 g at 22 c for 30 min , collected , and centrifuged again . the fully cleared supernatant was then subjected to paired - ion extraction for removal of sds. (30 ) proteins were concentrated by centrifugation as above and pellets washed with acetone at 20 c for 4 h. the final pellet was resuspended in 0.01 m nh4hco3 and protein amount was determined by bca . the tx-114 detergent phases were pooled , and proteins collected by cold acetone precipitation. (31 ) final tx-114 proteins were resuspended as above and protein amount was determined by bca assay . samples of 200 g of each cwp preparation were solubilized for 914 h in rehydration buffer ( 7 m urea/2 m thiourea , 1% amidosulfobetaine-14 ( asb-14 ) , 1% 3-[(3-cholamidopropyl ) dimethylammonio]-1-propanesulfonate ( chaps ) , 1 mm dithiothreitol ( dtt ) , 0.25% np-40 , 0.625% zoom carrier ampholytes 310 , and 1.9% cwp was applied on zoom precast immobilized ph 47 linear gradient strips ( 7.0 cm ; invitrogen ) according to manufacturer s instructions . focusing was achieved using a stepwise voltage gradient of 200 , 450 , 700 , and 1000 v , for 10 min each followed by focusing at 2000 v for 2 h. sds - page of the isoelectric focusing ( ief ) strips was performed using zoom 412% bis - tris sds - page gradient gels ( invitrogen ) . images were captured using a gel - doc system ( bio - rad , hercules , ca ) , and spot detection was performed using delta 2d software ( greifswald , germany ) . individual scx pools were subjected to further separation by reversed - phase hplc ( rp - hplc ) using a monomeric c18 column ( 4.6 mm 150 mm , vydac ) . al123456 , 3912 entries ) assuming the digestion enzyme trypsin , a fragment ion mass tolerance of 1.0 da , a parent ion tolerance of 2.5 da , and 3 allowable missed cleavages . sequest identifications required at least deltacn scores of greater than 0.3 and xcorr scores of greater than 1.5 , 2.2 , 2.5 , and 2.5 for singly , doubly , triply , and quadruply charged peptides ( supplementary table s1 ) . , portland , or ) was used to validate ms / ms based peptide and protein identifications . this generated a complete list of results that were sorted in a spreadsheet to create the subsets of neural network positive , hidden markov model positive , and the union of the nn and hmm sets . these lists were input into a java application along with the list of proteins isolated from the cell wall fractions . this application simply compared the predicted list to those isolated and identified the proteins found in the cell wall that were also predicted in each subset as having a signal peptide . to predict lipoproteins , the mtb peptide fasta file from above was broken down into 1000 sequences per file . the output files were combined in a spreadsheet , and from the sorted data , a list was extracted with every signal peptidase ii cleavage site generating a predicted lipoprotein list . this list was input into a java application that would compare the cell wall proteins , identifying the predicted lipoproteins from each cell wall fraction . to maximize the resolution and protein identification of cell wall proteins , the delipidated cell wall of mtb was subjected to extraction based on solubility within three reagents an anionic ( sds ) , a chaotrophic ( guhcl ) , and a nonionic ( tx-114 ) detergent . from the 2dge analysis , 290 proteins were identified with 122 , 131 , and 37 proteins identified in the guhcl , sds , and tx-114 fractions , respectively . analysis of the data demonstrated 80% of proteins had known functions ( figure 2 ) . in contrast , the tx-114 extracted fraction was less amenable to gel separation most likely due to its increased hydrophobic content . a multidimensional chromatography approach was employed to better resolve proteins in this fraction , in order to exploit any unique or uncharacterized protein families within the tx-114 subset . while 2dge was insufficient in identifying a significant number of proteins in the tx-114 detergent extract ( figure 1a ) , scx chromatography combined with reverse phase chromatography of a digest of this cwp preparation allowed the resolution of 364 proteins , including an additional 294 proteins not found in any of the 2dge preparations ( figure 1b ) . ( a ) identification of cell wall proteins by two - dimensional gel electrophoresis and two - dimensional liquid chromatography . detergent extraction and separation of cell wall protein subsets via 2dge demonstrated the majority of proteins to be resolved using sds and guhcl . ( b ) cumulatively , separation of cell wall proteins by 2dge resulted in the identification of 234 proteins . separating the tx-114 protein subset by multidimensional chromatography ( 2dlcms / ms ) resolved an additional 294 proteins . proteins identified for each extraction method ( guhcl , sds , tx114 ) were sorted by functional category as a percentage of total proteins per sample . representation of all protein groups was identified with the exception of groups 4 and 8 ( data not shown ) . seventy percent of the proteins identified in the cell wall are associated with lipid metabolism ( group 1 ) , cell wall processes ( group 3 ) , and intermediary metabolism ( group 7 ) . validation of a few selected proteins , by western blot analysis of samples resolved by 2dge ( supplementary figure 1 ) and sds - page ( supplementary figure 2 ) , corroborated the identification of these proteins by mass spectrometry . all identified proteins were grouped by functional category as defined by institute pasteur , which demonstrated mtb protein families present in each extract . combining the data sets of both gel and non - gel based protein identifications led to the detection of 528 proteins ( supplementary table s2 ) . one hundred and five of the 528 cell wall proteins identified were not reported in previous mtb proteome publications , including a comprehensive proteomic database http://web.mpiib-berlin.mpg.de/cgi-bin/pdbs/2d-page/extern/index.cgi,(19 ) and http://web.mpiib-berlin.mpg.de/cgi-bin/pdbs/2d-page/extern/index.cgi(42 ) ( table 1 ) . i.x is defined by proteins involved in central intermediary metabolism , amino acid biosynthesis , polyamine synthesis , biosynthesis of cofactors , prosthetic groups , and carriers , lipid biosynthesis , polyketide and non - ribosomal peptide synthesis , and broad regulatory functions . a majority of proteins were classified in either category i , small molecule metabolism ( 35% ) , or category ii , macromolecule synthesis and degradation ( 25% ) ( figure 3 ) . subclasses of category i showed 19% classified in small molecule metabolism - other ( i.x ) , which included the classes : central intermediary metabolism and amino acid biosynthesis ( figure 3 ; upper right ) ; and subclasses of category ii demonstrated an even distribution of proteins between synthesis / degradation of macromolecules ( ii.a , ii.b , 12.5% ) and cell envelope proteins ( ii.c , 12.1% ) ( figure 3 ; lower right ) . the secreted proteins of mtb have traditionally been characterized as important antigens and immune - modulators . prior to being exported , many of these proteins are resident within the cell wall where their function remains largely unknown . to find the putative secreted proteins identified within the cell wall proteome , all identified proteins were subject to interrogation against neural network ( nn ) and hidden markov model ( hmm ) algorithms ( signalp , http://www.cbs.dtu.dk/services/signalp/ ) which revealed 18% , 19% , 27% putative secreted proteins in guhcl , sds , tx-114 cwp samples , respectively , and 13% in the 2dlc resolved tx-114 cwp fraction . cumulatively , of the 528 proteins identified in this study , 87 proteins were predicted to contain secretion signals and included the identification of 23 proteins uniquely found in this study ( table 2 ) . a majority ( 60% ) of the cwp associated with secretion are indeed within the category of the cell wall and its processes ( figure 4 ) . of the 23 secreted cwp unique to this study , 11 have been described to be involved with small molecule and peptide binding . additionally , 23 of the 87 secreted proteins are classified as hypothetical or unknown ( categories v and vi of sanger institute ) illustrating that to a large extent , the functions of these exported proteins are poorly understood . eighty - seven cell wall proteins were predicted to contain secretion signals and putative lipoprotein motifs . sixty percent of these proteins are classified within the cell wall and cell wall processes functional category . next , we interrogated the cwp to identify those with putative lipoprotein motifs . here , a total of 16 proteins were predicted by lipop ( lipop , http://www.cbs.dtu.dk/services/lipop/)(43 ) to contain a signal peptidase ii cleavage motif , and an additional 8 proteins were identified when compared to the 99 putative lipoproteins ( 2.5% of the genome ) that reportedly exist within the mtb proteome. (7 ) a majority of the lipoproteins contain no additional functional classification ; however , 4 proteins ( rv0928 , rv0934 , rv2864c , rv3666c ) are involved with substrate binding and transport within the periplasm . one of two superoxide dismutases in mtb , sodc ( rv0432 ) , was also identified within the lipoprotein - enriched tx-114 cwp fraction . other putative lipoproteins identified in this study contain o - glycosylation motifs as predicted by netoglyc and were found within the mtb glycoproteome ( table 2). in this study , we focused on the gel - based two - dimensional separation of the cell wall and complemented that separation by liquid chromatography of digested tx-114 cwp . identified protein families included known antigens , fbp a , b , and c ( rv 3804c , rv1886c , and rv0129c ) , cfp10 ( rv3874 ) and esat-6 like proteins esxj and esxl ( rv1038c , rv1198 ) along with antigens lpqh ( rv3763 ) , lprg ( rv1411c ) , and lpra ( rv1270c ) . for over a decade , the advancement of techniques in 2dge , mass spectrometry , and increased access to bioinformatic tools greatly enhanced proteomic studies of mtb . largely descriptive , these studies were undertaken to identify novel virulence factors and drug targets . the quantitative techniques of mass spectrometric profiling , isotope coded affinity tag ( icat ) and isobaric tagging for relative and absolute quantification ( itraq ) , have afforded the accurate monitoring of up- and down - regulation of proteins on a global scale . previous work mining subcellular fractions of mtb , focused on the cytosol and culture filtrate fractions , resulted in thorough characterization and the creation of 2d gel databases . efforts have been made more recently to resolve the insoluble cell wall fraction . was able to identify 300 proteins within the cell wall using 2dlc as a separation method . using these studies as experimental platforms , we were able to focus on the mtb cell wall and were successful in reporting over 100 additional proteins that had not been identified previously . the elucidation of the cell wall proteome can facilitate subsequent studies of a more targeted nature where specific proteins can be monitored in response to various environmental , nutritional , or drug pressures . one of the most interesting yet poorly understood protein families residing within the cell wall of mtb are the triacylated lipoproteins . predicted 2.5% of the genome to encode for lipoproteins . defining these proteins beyond their antigenic and immune modulatory potential remains quite elusive with only a few studies predicting functional roles for mtb lipoproteins . in this study , a vast majority of proteins found were involved in small molecule and macromolecule metabolism . the presence of these lipoproteins and perhaps other secreted , cw resident proteins provides a conduit between various nutrient transportation pathways that are required for cellular survival and growth while allowing the mycobacterial cell wall to maintain its rigid hydrophobic integrity . while bacterial cell walls have historically been thought of as structural scaffolding with little active interplay between the extracellular milieu and inner cytosolic environment , this perception of a static bacterial structure is fading and we are beginning to understand the true complexity and dynamic nature of the biological processes being facilitated within the cell wall . for instance , these proteins may contribute to bacterial cell wall remodeling events in response to environmental stressors such as nutrient depletion , antibiotic pressures , the host immune response , and tissue remodeling events throughout the course of infection . for mtb , the highly complex framework of the magpouter lipid architecture can now be definitively complemented with a highly diverse protein population . several classes of proteins were identified , including putative secreted proteins , lipoproteins , and known t and b cell antigens . many proteins have unknown function ; however , their presence within the cell wall may be of biological relevance .

postpartum depression is a significant public health issue with rates reported between 10% and 15% in the general population . mothers of preterm infants are at particular risk for poor postpartum functioning with rates of depression from 14% to 27% . indeed , rates of clinically elevated depressive symptoms can be as high as 63% during the preterm infant s hospitalization . more recently , increased levels of anxiety and acute stress among mothers of infants cared for in the neonatal intensive care unit ( nicu ) have been reported . maternal postpartum depression and anxiety affect child development with detrimental effects on cognitive and social - emotional development as well as parent - child interactions . preterm infants appear more sensitive to the impact of poor maternal mental health as compared to full term infants . the risk factors for postpartum depression in the general population include a prior history of depression , life stress , poor social support , marital discord , single marital status and low socioeconomic status . another important factor , maternal smoking , is a risk factor for preterm delivery , as well as depressive symptoms during pregnancy and the postpartum period in mothers of both preterm and full - term infants . , prior research has identified risk factors for anxiety in mothers of very low birth weight ( < 1000 g ) infants including marital status and social support , though this study measured trait anxiety reflecting mothers underlying propensity for anxiety rather than their current anxiety levels . additional risk factors for postpartum depression and anxiety specific to the nicu setting include perception of the infant s illness severity , poor coping skills , increased parenting stress , perceived parental role alteration , length of ventilation and length of stay in the nicu . in contrast to the general population , demographic risk factors , such as maternal education and age , have not been consistently associated with depressive symptoms among mothers of preterm infants though marital status was still an important risk factor . ethnicity is another demographic factor that has not been consistently related to maternal postpartum functioning . it is well - established that african americans are at the highest risk of preterm birth with rates at least twice that of caucasians . it is unclear , however , if they are at higher risk for postpartum depression and anxiety . among the general population , african americans are at increased risk of depression including during the postpartum period compared to non - hispanic caucasians . among mothers of preterm infants , some report increased rates of postpartum depressive symptoms for african americans while others do not . identification of the risk factors predictive for poor postpartum functioning would assist in the recognition and referral of mothers at greatest risk prior to taking home their vulnerable infants . one study in mothers of infants born less than 35 weeks gestation reported that the interaction between maternal demographic characteristics conferred greater risk than the infant s medical severity for mothers with elevated demographic risk , low social support and infants with very low birthweight . to date , however , the risk factors for poor postpartum functioning have not been well - studied in a sample that has both elevated demographic risk and medically fragile very preterm infants , representing the most vulnerable mother - infant dyads . to address this gap , the current study aims to assess which factors are most useful for identifying mothers at - risk for postpartum depression or anxiety at the time of discharge from the nicu by investigating the impact of demographic , maternal psychosocial and infant factors in a cohort of caucasian and african american mothers of very preterm infants at a level iii nicu . this study was a prospective cohort design recruiting preterm infants born at less than 30 weeks gestation within the first 3 days of life over a three - year period . exclusion criteria included having a known congenital anomaly or being moribund with severe sepsis or respiratory failure in the first days of life . all infants whose parents gave informed consent for magnetic resonance imaging ( mri ) were scanned during the nicu hospitalization . in addition , clinical information on the infant and mother were collected from the medical record , and each primary caregiver completed a comprehensive questionnaire at discharge from the nicu . the epds is a 10-item self - report measure with good validity that assesses symptoms of postpartum depression among mothers over the last 7 days . a score of 13 ( range 030 ) or greater indicates a mother with clinically significant depression . the state - trait anxiety inventory ( stai ) is a widely used and reliable assessment of anxiety in adults and consists of two 20-item scales that differentiate between anxiety as a personality trait and conditional anxiety . state anxiety was used as the outcome measure to reflect the anxiety that resulted from having a very preterm infant in the nicu . moderate anxiety was defined by a score between 40 and 59 with severe anxiety between 60 and 80 , as per published guidelines . information on maternal age , race , education , and marital status were collected from the questionnaire by caregiver report . insurance status , used as a proxy measure for socioeconomic status ( ses ) , was obtained from the clinical record and dichotomized into public insurance or private insurance . maternal education was dichotomized as those who attained the equivalent or less than high school degree or a college education or higher . data on prior history of depression or anxiety , smoking , stressful life events , and social support satisfaction were collected . histories of depression and/or anxiety were obtained both by self - report in the discharge questionnaire as well as from the maternal clinical records . maternal smoking was obtained by self - report and dichotomized into those who smoked at least 1 cigarette / day and non - smokers . stressful life events were measured via the life stress subscale of the parenting stress index ( psi ) , assessing stressful situational circumstances outside of parenting ( e.g. , death of a relative , loss of a job ) . social support satisfaction was measured with the social support questionnaire ( ssq ) which rates satisfaction with self - identified sources of support . parental role alteration was assessed using the parental role alteration subscale of the parental stressor scale : neonatal intensive care unit ( pss ) , which has good reliability and internal consistency . the following infant factors were hypothesized a priori to impact maternal depression or anxiety : number of days of ventilation , length of stay , and extent of brain injury . the extent of brain injury was assessed from cranial ultrasound conducted at clinical discretion and from a study - related mri conducted without sedation . mri results were interpreted by a single , trained neurologist to determine the presence of focal white matter lesions , cystic periventricular leukomalacia ( pvl ) , and cerebellar hemorrhage . brain injury was dichotomized into no to mild abnormality or moderate to severe abnormality ( ivh grade iii or iv ; and/or moderate - severe white matter injury including multiple focal lesions and/or cystic pvl ; and cerebellar hemorrhage ) . the results of the neuroimaging were communicated to the primary caregivers prior to completion of the discharge questionnaire , including estimated prognosis for motor and cognitive development for the infant , by a trained neonatologist and neurologist ( ti ) . for mothers of multiples , the infant with the most severe clinical presentation based on length of stay , length of ventilation , and degree of brain injury was chosen for the analysis in order to best assess the association between having a medically ill infant and maternal functioning . to assess the differential impact of the individual demographic ( maternal age , race , education , marital status and insurance status ) , maternal psychosocial ( prior history of depression or anxiety , smoking , stressful life events , parental role alteration , and social support satisfaction ) , and infant risk factors ( number of days of ventilation , length of stay , and extent of brain injury ) on depression and anxiety , each set of factors was entered into hierarchical linear regression models with epds and stai - s scores as the outcome variables . the maternal psychosocial factors were entered in the first step , demographic factors entered in the second step and infant factors added in the final step . maternal psychosocial factors were entered first based on the larger impact of these factors over demographic factors on depressive symptoms in the general population . infant factors were added last as the literature has been mixed on the impact of infant health status on outcome . within each step , the factors were entered with a stepwise procedure which retained variables with a p value < 0.1 . possible disparities in the rates of risk factors between caucasian and african american mothers were calculated using t - tests for continuous variables and chi - square analyses for categorical variables . significant factors from the hierarchical regression model and factors more prevalent in one race ( age , insurance status , and marital status ) were evaluated for interactions with race by analysis of covariance . this study was a prospective cohort design recruiting preterm infants born at less than 30 weeks gestation within the first 3 days of life over a three - year period . exclusion criteria included having a known congenital anomaly or being moribund with severe sepsis or respiratory failure in the first days of life . all infants whose parents gave informed consent for magnetic resonance imaging ( mri ) were scanned during the nicu hospitalization . in addition , clinical information on the infant and mother were collected from the medical record , and each primary caregiver completed a comprehensive questionnaire at discharge from the nicu . the epds is a 10-item self - report measure with good validity that assesses symptoms of postpartum depression among mothers over the last 7 days . a score of 13 ( range 030 ) or greater indicates a mother with clinically significant depression . the state - trait anxiety inventory ( stai ) is a widely used and reliable assessment of anxiety in adults and consists of two 20-item scales that differentiate between anxiety as a personality trait and conditional anxiety . state anxiety was used as the outcome measure to reflect the anxiety that resulted from having a very preterm infant in the nicu . moderate anxiety was defined by a score between 40 and 59 with severe anxiety between 60 and 80 , as per published guidelines . information on maternal age , race , education , and marital status were collected from the questionnaire by caregiver report . insurance status , used as a proxy measure for socioeconomic status ( ses ) , was obtained from the clinical record and dichotomized into public insurance or private insurance . maternal education was dichotomized as those who attained the equivalent or less than high school degree or a college education or higher . data on prior history of depression or anxiety , smoking , stressful life events , and social support satisfaction were collected . histories of depression and/or anxiety were obtained both by self - report in the discharge questionnaire as well as from the maternal clinical records . maternal smoking was obtained by self - report and dichotomized into those who smoked at least 1 cigarette / day and non - smokers . stressful life events were measured via the life stress subscale of the parenting stress index ( psi ) , assessing stressful situational circumstances outside of parenting ( e.g. , death of a relative , loss of a job ) . social support satisfaction was measured with the social support questionnaire ( ssq ) which rates satisfaction with self - identified sources of support . parental role alteration was assessed using the parental role alteration subscale of the parental stressor scale : neonatal intensive care unit ( pss ) , which has good reliability and internal consistency . the following infant factors were hypothesized a priori to impact maternal depression or anxiety : number of days of ventilation , length of stay , and extent of brain injury . the extent of brain injury was assessed from cranial ultrasound conducted at clinical discretion and from a study - related mri conducted without sedation . mri results were interpreted by a single , trained neurologist to determine the presence of focal white matter lesions , cystic periventricular leukomalacia ( pvl ) , and cerebellar hemorrhage . brain injury was dichotomized into no to mild abnormality or moderate to severe abnormality ( ivh grade iii or iv ; and/or moderate - severe white matter injury including multiple focal lesions and/or cystic pvl ; and cerebellar hemorrhage ) . the results of the neuroimaging were communicated to the primary caregivers prior to completion of the discharge questionnaire , including estimated prognosis for motor and cognitive development for the infant , by a trained neonatologist and neurologist ( ti ) . for mothers of multiples , the infant with the most severe clinical presentation based on length of stay , length of ventilation , and degree of brain injury was chosen for the analysis in order to best assess the association between having a medically ill infant and maternal functioning . to assess the differential impact of the individual demographic ( maternal age , race , education , marital status and insurance status ) , maternal psychosocial ( prior history of depression or anxiety , smoking , stressful life events , parental role alteration , and social support satisfaction ) , and infant risk factors ( number of days of ventilation , length of stay , and extent of brain injury ) on depression and anxiety , each set of factors was entered into hierarchical linear regression models with epds and stai - s scores as the outcome variables . the maternal psychosocial factors were entered in the first step , demographic factors entered in the second step and infant factors added in the final step . maternal psychosocial factors were entered first based on the larger impact of these factors over demographic factors on depressive symptoms in the general population . infant factors were added last as the literature has been mixed on the impact of infant health status on outcome . within each step , the factors were entered with a stepwise procedure which retained variables with a p value < 0.1 . possible disparities in the rates of risk factors between caucasian and african american mothers were calculated using t - tests for continuous variables and chi - square analyses for categorical variables . significant factors from the hierarchical regression model and factors more prevalent in one race ( age , insurance status , and marital status ) were evaluated for interactions with race by analysis of covariance . there were a total of 83 caucasian and african - american mothers of surviving infants enrolled in the study . those with missing data had shorter lengths of stay ( t=2.23 , p=.03 ) compared to those included in the analysis . there were no other significant differences between those with missing data and those included in the analysis . significant differences in demographic factors were as follows : african american mothers were more likely to be unmarried ( p<.001 ) , be on public aid ( p<.001 ) , and be younger ( p=.009 ) . there was a trend for african american mothers to have less education ( p=.09 ) . the results of the hierarchical regressions with the individual risk factors that were significant for each block for the epds are displayed in table 2 . the only psychosocial risk factor that remained in the model was parental role alteration . among demographic risks , being married was the only significant factor in the second step . in the final step , the final model with all of three of these factors was significant ( r = 0.31 and p<.001 ) . interactions between race and significant factors ( marital status , ventilation days , and parental role alteration ) and between race and factors that differed between caucasian and african american mothers ( age , insurance status ) were tested and were not significant ( data not shown ) . given the unexpected finding that being married was associated with higher levels of depressive symptoms , we conducted exploratory analyses of this relationship . comparing those that were married to those that were unmarried , married mothers had higher mean epds scores ( t=2.4 , p=.02 ) . twenty - six percent of married mothers had clinical levels of depression compared to 15% of unmarried mothers ( = 1.2 , p=.28 ) . married mothers had higher epds scores compared to unmarried mothers cohabitating ( mean epds 8.9 vs. 4.3 respectively , f=2.9 , p=.03 ) but not from single mothers ( mean epds 6.7 , figure 1 . ) . other factors reported to be associated with depressive symptoms , notably receiving fertility treatments and marital separation used as a proxy for marital discord were evaluated . use of fertility treatment was reported in only five participants , all of whom were married . separation from their spouse was reported in three participants . being married continued to be a predictor after adjusting for fertility treatment ( = .46 , p=.002 ) or marital separation ( = .6 , p<.001 ) . another potential confound , multiple births , noted to be associated with postpartum depression was also assessed . there was no difference in the rate of multiple births between married and unmarried mothers ( = 0.77 p=.38 ) . additionally , there were no differences between the infant factors with reported impact on postpartum depression for married and unmarried mothers ( ventilation days t=.83 , p=.41 ; length of stay , t=.58 , p=.57 ) . none of the psychosocial , demographic , or infant risk factors significantly predicted stai state ( stai - s ) scores . post hoc analyses found no correlation between the number of children and stai - s scores ( r=.11 p=.23 ) nor any difference in mean stai - s scores between those who did or did not have children previously ( t = .10 , p=.92 ) . the mean stai trait score was 33.8 with 27% of mothers having moderate trait anxiety and none having severe trait anxiety . there was a modest correlation between the stai state and stai trait scores ( r=.4 , p=.001 ) . the results of the hierarchical regressions with the individual risk factors that were significant for each block for the epds are displayed in table 2 . the only psychosocial risk factor that remained in the model was parental role alteration . among demographic risks , being married was the only significant factor in the second step . in the final step , the final model with all of three of these factors was significant ( r = 0.31 and p<.001 ) . interactions between race and significant factors ( marital status , ventilation days , and parental role alteration ) and between race and factors that differed between caucasian and african american mothers ( age , insurance status ) were tested and were not significant ( data not shown ) . given the unexpected finding that being married was associated with higher levels of depressive symptoms , we conducted exploratory analyses of this relationship . comparing those that were married to those that were unmarried , married mothers had higher mean epds scores ( t=2.4 , p=.02 ) . twenty - six percent of married mothers had clinical levels of depression compared to 15% of unmarried mothers ( = 1.2 , p=.28 ) . married mothers had higher epds scores compared to unmarried mothers cohabitating ( mean epds 8.9 vs. 4.3 respectively , f=2.9 , p=.03 ) but not from single mothers ( mean epds 6.7 , figure 1 . ) . other factors reported to be associated with depressive symptoms , notably receiving fertility treatments and marital separation used as a proxy for marital discord were evaluated . use of fertility treatment was reported in only five participants , all of whom were married . being married continued to be a predictor after adjusting for fertility treatment ( = .46 , p=.002 ) or marital separation ( = .6 , p<.001 ) . another potential confound , multiple births , noted to be associated with postpartum depression was also assessed . there was no difference in the rate of multiple births between married and unmarried mothers ( = 0.77 p=.38 ) . additionally , there were no differences between the infant factors with reported impact on postpartum depression for married and unmarried mothers ( ventilation days t=.83 , p=.41 ; length of stay , t=.58 , p=.57 ) . none of the psychosocial , demographic , or infant risk factors significantly predicted stai state ( stai - s ) scores . post hoc analyses found no correlation between the number of children and stai - s scores ( r=.11 p=.23 ) nor any difference in mean stai - s scores between those who did or did not have children previously ( t = .10 , p=.92 ) . the mean stai trait score was 33.8 with 27% of mothers having moderate trait anxiety and none having severe trait anxiety . there was a modest correlation between the stai state and stai trait scores ( r=.4 , p=.001 ) . in this study of caucasian and african american mothers of very preterm infants , there were specific factors that were associated with postpartum depressive symptoms at the time of discharge . in contrast , none of the risk factors analyzed were associated with anxiety at nicu discharge . there was no difference in the rate of postpartum depression between caucasian and african american mothers . while modest levels of anxiety are reasonably anticipated upon discharge from the nicu , rates of moderate to severe state anxiety were found in 43% of mothers in this cohort , present at the time of discharge often many months after the initial admission . indeed , the cohorts mean anxiety score was similar to means reported at much earlier time points in the nicu hospitalization . the modest association between trait and state anxiety suggests that the elevated state anxiety was not due to an over - representation of temperamentally anxious mothers . the rates of moderate - severe anxiety did not differ between caucasian and african american mothers . we found that being married , experiencing parental role alteration , and increased length of ventilation were the only risk factors associated with elevated postpartum depressive symptoms . it was a surprising finding that being married was associated with postpartum depression , given unmarried status has been previously associated with postpartum depression . this discrepancy was accounted for by unmarried , cohabitating mothers having the lowest epds scores . evidence has shown that it is poor relationship quality , and not marital status per se , that is related to increased postpartum depression . the infants in this study had a much longer nicu hospitalization than similar research that found marriage protective which may have increased the stress on married couples . it is also possible that more married couples compared to cohabiting couples had planned or intended pregnancies and thus , preterm delivery and this prolonged nicu stay may have had a greater impact on married mothers emotional well - being . information on relationship quality and pregnancy intention were not captured in the current study . experiencing parental role alteration and the length of ventilation both factors relate to the disruption in the typical maternal - infant connectedness that occurs when an infant is in the nicu . thus , both of these factors may identify dyads at greatest risk for poor attachment which is detrimental to both maternal functioning and infant development . another unexpected finding was that a prior history of depression and anxiety was not related to current symptoms of depression and anxiety . one possible explanation is that mothers underreported their prior histories of symptoms and this may have been better assessed with a clinical interview rather than by self - report . nevertheless , while a history of depression / anxiety has been noted to be predictive in other studies in the general population , this has not been confirmed in prior cohorts of preterm infants . unfortunately , prior research with mothers of preterm infants with racially diverse samples did not evaluate the impact of a history of prior depression on postpartum depression symptoms . prior research had found risk factors for trait anxiety , a different construct , with a sample that was less racially diverse , had less social disadvantage and had infants with an older mean gestational age . it is particularly notable that there was no relationship between the severity of the medical course or the presence of brain injury with maternal anxiety . while there may be other unmeasured risk factors for anxiety , this finding suggests that a large proportion of mothers of very preterm infants are at elevated risk for significant and impairing levels of anxiety even after the infant s medical condition has stabilized . this highlights the importance of surveillance of mothers of very preterm infants for impairing levels of anxiety at discharge from the nicu , at postpartum visits and well baby check - ups . it also highlights how challenging it may be for the clinical team to identify the mother at greatest risk as the majority of clinical teams would assign risk based on demographic , psychosocial history and/or severity of infant illness . this study has several strengths including the racial and socioeconomic diversity of the cohort as well as a more comprehensive set of risk factors including a prior history of depression / anxiety and severity of brain injury . limitations include that we report on findings at only one time point so it is unclear if these factors continue to confer risk over time . this cohort is part of a longitudinal study and subsequent investigation of the persistence of maternal postpartum symptoms during infancy and childhood as well the impact of these early risk factors will be undertaken . nevertheless , early attachment with infants is important for promoting development and elevated depressive or anxiety symptoms at discharge may increase the risk of attachment difficulties and impact the care of these infants when they are still medically fragile . another possible limitation is that this cohort was recruited from an urban level iii nicu , and thus , these results may not be generalizable to all mothers of vpt infants , nor to all caucasian and african american mothers . the findings from this study confirm the common nature of maternal postpartum depression and anxiety disorders in mothers of very preterm infants at the time of discharge from the nicu . identification of risk factors ( marital state / satisfaction , parental role alteration , and length of ventilation ) may assist physician and other team members to screen for this disorder . universal screening for anxiety in the nicu would be required to identify mothers with moderate - severe anxiety . future research is needed to study the relationship between the identified risk factors and the duration of depression and anxiety in this population and to determine whether targeting interventions early in the nicu hospitalization to mothers identified as at - risk reduces symptoms prior to discharge .

objectivewe investigated whether particular demographic , maternal psychosocial , and infant factors identified mothers of very preterm infants at risk for postpartum depression or anxiety at the time of discharge from a level iii urban neonatal intensive care unit ( nicu).study designa racially diverse cohort of mothers ( n=73 ) of preterm infants ( gestational age < 30 weeks ) completed a comprehensive questionnaire at discharge from the nicu assessing postpartum depression , anxiety , and psychosocial and demographic factors . additionally , infants underwent brain magnetic resonance imaging prior to discharge.resulttwenty percent of mothers had clinically significant levels of depression while 43% had moderate - severe anxiety . being married ( p<.01 ) , parental role alteration ( p<.01 ) and prolonged ventilation ( p<.05 ) were associated with increased depressive symptoms . no psychosocial , demographic , or infant factors , including severity of brain injury , were associated with state anxiety levels.conclusionmaternal factors , such as marital status , stress from parental role alteration , and infant factors , such as prolonged ventilation , are associated with increased depression . however , clinically significant levels of anxiety are common in mothers of very preterm infants with few identifiable risk factors . these findings support the need for universal screening within the nicu .

Introduction Patients and Methods Participants Measures Demographic Factors Maternal Psychosocial History Infant Factors Data Analysis Results Depressive Symptoms Anxiety symptoms Discussion Conclusion

postpartum depression is a significant public health issue with rates reported between 10% and 15% in the general population . mothers of preterm infants are at particular risk for poor postpartum functioning with rates of depression from 14% to 27% . indeed , rates of clinically elevated depressive symptoms can be as high as 63% during the preterm infant s hospitalization . more recently , increased levels of anxiety and acute stress among mothers of infants cared for in the neonatal intensive care unit ( nicu ) have been reported . maternal postpartum depression and anxiety affect child development with detrimental effects on cognitive and social - emotional development as well as parent - child interactions . preterm infants appear more sensitive to the impact of poor maternal mental health as compared to full term infants . the risk factors for postpartum depression in the general population include a prior history of depression , life stress , poor social support , marital discord , single marital status and low socioeconomic status . another important factor , maternal smoking , is a risk factor for preterm delivery , as well as depressive symptoms during pregnancy and the postpartum period in mothers of both preterm and full - term infants . , prior research has identified risk factors for anxiety in mothers of very low birth weight ( < 1000 g ) infants including marital status and social support , though this study measured trait anxiety reflecting mothers underlying propensity for anxiety rather than their current anxiety levels . additional risk factors for postpartum depression and anxiety specific to the nicu setting include perception of the infant s illness severity , poor coping skills , increased parenting stress , perceived parental role alteration , length of ventilation and length of stay in the nicu . in contrast to the general population , demographic risk factors , such as maternal education and age , have not been consistently associated with depressive symptoms among mothers of preterm infants though marital status was still an important risk factor . it is well - established that african americans are at the highest risk of preterm birth with rates at least twice that of caucasians . it is unclear , however , if they are at higher risk for postpartum depression and anxiety . among mothers of preterm infants , some report increased rates of postpartum depressive symptoms for african americans while others do not . identification of the risk factors predictive for poor postpartum functioning would assist in the recognition and referral of mothers at greatest risk prior to taking home their vulnerable infants . one study in mothers of infants born less than 35 weeks gestation reported that the interaction between maternal demographic characteristics conferred greater risk than the infant s medical severity for mothers with elevated demographic risk , low social support and infants with very low birthweight . to date , however , the risk factors for poor postpartum functioning have not been well - studied in a sample that has both elevated demographic risk and medically fragile very preterm infants , representing the most vulnerable mother - infant dyads . to address this gap , the current study aims to assess which factors are most useful for identifying mothers at - risk for postpartum depression or anxiety at the time of discharge from the nicu by investigating the impact of demographic , maternal psychosocial and infant factors in a cohort of caucasian and african american mothers of very preterm infants at a level iii nicu . this study was a prospective cohort design recruiting preterm infants born at less than 30 weeks gestation within the first 3 days of life over a three - year period . all infants whose parents gave informed consent for magnetic resonance imaging ( mri ) were scanned during the nicu hospitalization . in addition , clinical information on the infant and mother were collected from the medical record , and each primary caregiver completed a comprehensive questionnaire at discharge from the nicu . the epds is a 10-item self - report measure with good validity that assesses symptoms of postpartum depression among mothers over the last 7 days . the state - trait anxiety inventory ( stai ) is a widely used and reliable assessment of anxiety in adults and consists of two 20-item scales that differentiate between anxiety as a personality trait and conditional anxiety . state anxiety was used as the outcome measure to reflect the anxiety that resulted from having a very preterm infant in the nicu . moderate anxiety was defined by a score between 40 and 59 with severe anxiety between 60 and 80 , as per published guidelines . information on maternal age , race , education , and marital status were collected from the questionnaire by caregiver report . insurance status , used as a proxy measure for socioeconomic status ( ses ) , was obtained from the clinical record and dichotomized into public insurance or private insurance . data on prior history of depression or anxiety , smoking , stressful life events , and social support satisfaction were collected . histories of depression and/or anxiety were obtained both by self - report in the discharge questionnaire as well as from the maternal clinical records . stressful life events were measured via the life stress subscale of the parenting stress index ( psi ) , assessing stressful situational circumstances outside of parenting ( e.g. parental role alteration was assessed using the parental role alteration subscale of the parental stressor scale : neonatal intensive care unit ( pss ) , which has good reliability and internal consistency . the following infant factors were hypothesized a priori to impact maternal depression or anxiety : number of days of ventilation , length of stay , and extent of brain injury . the extent of brain injury was assessed from cranial ultrasound conducted at clinical discretion and from a study - related mri conducted without sedation . mri results were interpreted by a single , trained neurologist to determine the presence of focal white matter lesions , cystic periventricular leukomalacia ( pvl ) , and cerebellar hemorrhage . brain injury was dichotomized into no to mild abnormality or moderate to severe abnormality ( ivh grade iii or iv ; and/or moderate - severe white matter injury including multiple focal lesions and/or cystic pvl ; and cerebellar hemorrhage ) . the results of the neuroimaging were communicated to the primary caregivers prior to completion of the discharge questionnaire , including estimated prognosis for motor and cognitive development for the infant , by a trained neonatologist and neurologist ( ti ) . for mothers of multiples , the infant with the most severe clinical presentation based on length of stay , length of ventilation , and degree of brain injury was chosen for the analysis in order to best assess the association between having a medically ill infant and maternal functioning . to assess the differential impact of the individual demographic ( maternal age , race , education , marital status and insurance status ) , maternal psychosocial ( prior history of depression or anxiety , smoking , stressful life events , parental role alteration , and social support satisfaction ) , and infant risk factors ( number of days of ventilation , length of stay , and extent of brain injury ) on depression and anxiety , each set of factors was entered into hierarchical linear regression models with epds and stai - s scores as the outcome variables . the maternal psychosocial factors were entered in the first step , demographic factors entered in the second step and infant factors added in the final step . maternal psychosocial factors were entered first based on the larger impact of these factors over demographic factors on depressive symptoms in the general population . possible disparities in the rates of risk factors between caucasian and african american mothers were calculated using t - tests for continuous variables and chi - square analyses for categorical variables . significant factors from the hierarchical regression model and factors more prevalent in one race ( age , insurance status , and marital status ) were evaluated for interactions with race by analysis of covariance . this study was a prospective cohort design recruiting preterm infants born at less than 30 weeks gestation within the first 3 days of life over a three - year period . all infants whose parents gave informed consent for magnetic resonance imaging ( mri ) were scanned during the nicu hospitalization . in addition , clinical information on the infant and mother were collected from the medical record , and each primary caregiver completed a comprehensive questionnaire at discharge from the nicu . the epds is a 10-item self - report measure with good validity that assesses symptoms of postpartum depression among mothers over the last 7 days . a score of 13 ( range 030 ) or greater indicates a mother with clinically significant depression . state anxiety was used as the outcome measure to reflect the anxiety that resulted from having a very preterm infant in the nicu . information on maternal age , race , education , and marital status were collected from the questionnaire by caregiver report . insurance status , used as a proxy measure for socioeconomic status ( ses ) , was obtained from the clinical record and dichotomized into public insurance or private insurance . data on prior history of depression or anxiety , smoking , stressful life events , and social support satisfaction were collected . histories of depression and/or anxiety were obtained both by self - report in the discharge questionnaire as well as from the maternal clinical records . parental role alteration was assessed using the parental role alteration subscale of the parental stressor scale : neonatal intensive care unit ( pss ) , which has good reliability and internal consistency . the following infant factors were hypothesized a priori to impact maternal depression or anxiety : number of days of ventilation , length of stay , and extent of brain injury . the extent of brain injury was assessed from cranial ultrasound conducted at clinical discretion and from a study - related mri conducted without sedation . mri results were interpreted by a single , trained neurologist to determine the presence of focal white matter lesions , cystic periventricular leukomalacia ( pvl ) , and cerebellar hemorrhage . brain injury was dichotomized into no to mild abnormality or moderate to severe abnormality ( ivh grade iii or iv ; and/or moderate - severe white matter injury including multiple focal lesions and/or cystic pvl ; and cerebellar hemorrhage ) . the results of the neuroimaging were communicated to the primary caregivers prior to completion of the discharge questionnaire , including estimated prognosis for motor and cognitive development for the infant , by a trained neonatologist and neurologist ( ti ) . for mothers of multiples , the infant with the most severe clinical presentation based on length of stay , length of ventilation , and degree of brain injury was chosen for the analysis in order to best assess the association between having a medically ill infant and maternal functioning . to assess the differential impact of the individual demographic ( maternal age , race , education , marital status and insurance status ) , maternal psychosocial ( prior history of depression or anxiety , smoking , stressful life events , parental role alteration , and social support satisfaction ) , and infant risk factors ( number of days of ventilation , length of stay , and extent of brain injury ) on depression and anxiety , each set of factors was entered into hierarchical linear regression models with epds and stai - s scores as the outcome variables . the maternal psychosocial factors were entered in the first step , demographic factors entered in the second step and infant factors added in the final step . maternal psychosocial factors were entered first based on the larger impact of these factors over demographic factors on depressive symptoms in the general population . infant factors were added last as the literature has been mixed on the impact of infant health status on outcome . possible disparities in the rates of risk factors between caucasian and african american mothers were calculated using t - tests for continuous variables and chi - square analyses for categorical variables . significant factors from the hierarchical regression model and factors more prevalent in one race ( age , insurance status , and marital status ) were evaluated for interactions with race by analysis of covariance . significant differences in demographic factors were as follows : african american mothers were more likely to be unmarried ( p<.001 ) , be on public aid ( p<.001 ) , and be younger ( p=.009 ) . the only psychosocial risk factor that remained in the model was parental role alteration . among demographic risks , being married was the only significant factor in the second step . interactions between race and significant factors ( marital status , ventilation days , and parental role alteration ) and between race and factors that differed between caucasian and african american mothers ( age , insurance status ) were tested and were not significant ( data not shown ) . given the unexpected finding that being married was associated with higher levels of depressive symptoms , we conducted exploratory analyses of this relationship . comparing those that were married to those that were unmarried , married mothers had higher mean epds scores ( t=2.4 , p=.02 ) . twenty - six percent of married mothers had clinical levels of depression compared to 15% of unmarried mothers ( = 1.2 , p=.28 ) . married mothers had higher epds scores compared to unmarried mothers cohabitating ( mean epds 8.9 vs. 4.3 respectively , f=2.9 , p=.03 ) but not from single mothers ( mean epds 6.7 , figure 1 . ) other factors reported to be associated with depressive symptoms , notably receiving fertility treatments and marital separation used as a proxy for marital discord were evaluated . being married continued to be a predictor after adjusting for fertility treatment ( = .46 , p=.002 ) or marital separation ( = .6 , p<.001 ) . another potential confound , multiple births , noted to be associated with postpartum depression was also assessed . there was no difference in the rate of multiple births between married and unmarried mothers ( = 0.77 p=.38 ) . additionally , there were no differences between the infant factors with reported impact on postpartum depression for married and unmarried mothers ( ventilation days t=.83 , p=.41 ; length of stay , t=.58 , p=.57 ) . none of the psychosocial , demographic , or infant risk factors significantly predicted stai state ( stai - s ) scores . the results of the hierarchical regressions with the individual risk factors that were significant for each block for the epds are displayed in table 2 . the only psychosocial risk factor that remained in the model was parental role alteration . interactions between race and significant factors ( marital status , ventilation days , and parental role alteration ) and between race and factors that differed between caucasian and african american mothers ( age , insurance status ) were tested and were not significant ( data not shown ) . given the unexpected finding that being married was associated with higher levels of depressive symptoms , we conducted exploratory analyses of this relationship . twenty - six percent of married mothers had clinical levels of depression compared to 15% of unmarried mothers ( = 1.2 , p=.28 ) . married mothers had higher epds scores compared to unmarried mothers cohabitating ( mean epds 8.9 vs. 4.3 respectively , f=2.9 , p=.03 ) but not from single mothers ( mean epds 6.7 , figure 1 . ) other factors reported to be associated with depressive symptoms , notably receiving fertility treatments and marital separation used as a proxy for marital discord were evaluated . being married continued to be a predictor after adjusting for fertility treatment ( = .46 , p=.002 ) or marital separation ( = .6 , p<.001 ) . another potential confound , multiple births , noted to be associated with postpartum depression was also assessed . there was no difference in the rate of multiple births between married and unmarried mothers ( = 0.77 p=.38 ) . additionally , there were no differences between the infant factors with reported impact on postpartum depression for married and unmarried mothers ( ventilation days t=.83 , p=.41 ; length of stay , t=.58 , p=.57 ) . none of the psychosocial , demographic , or infant risk factors significantly predicted stai state ( stai - s ) scores . in this study of caucasian and african american mothers of very preterm infants , there were specific factors that were associated with postpartum depressive symptoms at the time of discharge . in contrast , none of the risk factors analyzed were associated with anxiety at nicu discharge . while modest levels of anxiety are reasonably anticipated upon discharge from the nicu , rates of moderate to severe state anxiety were found in 43% of mothers in this cohort , present at the time of discharge often many months after the initial admission . the modest association between trait and state anxiety suggests that the elevated state anxiety was not due to an over - representation of temperamentally anxious mothers . the rates of moderate - severe anxiety did not differ between caucasian and african american mothers . we found that being married , experiencing parental role alteration , and increased length of ventilation were the only risk factors associated with elevated postpartum depressive symptoms . it was a surprising finding that being married was associated with postpartum depression , given unmarried status has been previously associated with postpartum depression . evidence has shown that it is poor relationship quality , and not marital status per se , that is related to increased postpartum depression . experiencing parental role alteration and the length of ventilation both factors relate to the disruption in the typical maternal - infant connectedness that occurs when an infant is in the nicu . thus , both of these factors may identify dyads at greatest risk for poor attachment which is detrimental to both maternal functioning and infant development . nevertheless , while a history of depression / anxiety has been noted to be predictive in other studies in the general population , this has not been confirmed in prior cohorts of preterm infants . unfortunately , prior research with mothers of preterm infants with racially diverse samples did not evaluate the impact of a history of prior depression on postpartum depression symptoms . prior research had found risk factors for trait anxiety , a different construct , with a sample that was less racially diverse , had less social disadvantage and had infants with an older mean gestational age . it is particularly notable that there was no relationship between the severity of the medical course or the presence of brain injury with maternal anxiety . while there may be other unmeasured risk factors for anxiety , this finding suggests that a large proportion of mothers of very preterm infants are at elevated risk for significant and impairing levels of anxiety even after the infant s medical condition has stabilized . this highlights the importance of surveillance of mothers of very preterm infants for impairing levels of anxiety at discharge from the nicu , at postpartum visits and well baby check - ups . it also highlights how challenging it may be for the clinical team to identify the mother at greatest risk as the majority of clinical teams would assign risk based on demographic , psychosocial history and/or severity of infant illness . this study has several strengths including the racial and socioeconomic diversity of the cohort as well as a more comprehensive set of risk factors including a prior history of depression / anxiety and severity of brain injury . this cohort is part of a longitudinal study and subsequent investigation of the persistence of maternal postpartum symptoms during infancy and childhood as well the impact of these early risk factors will be undertaken . nevertheless , early attachment with infants is important for promoting development and elevated depressive or anxiety symptoms at discharge may increase the risk of attachment difficulties and impact the care of these infants when they are still medically fragile . another possible limitation is that this cohort was recruited from an urban level iii nicu , and thus , these results may not be generalizable to all mothers of vpt infants , nor to all caucasian and african american mothers . the findings from this study confirm the common nature of maternal postpartum depression and anxiety disorders in mothers of very preterm infants at the time of discharge from the nicu . identification of risk factors ( marital state / satisfaction , parental role alteration , and length of ventilation ) may assist physician and other team members to screen for this disorder . universal screening for anxiety in the nicu would be required to identify mothers with moderate - severe anxiety . future research is needed to study the relationship between the identified risk factors and the duration of depression and anxiety in this population and to determine whether targeting interventions early in the nicu hospitalization to mothers identified as at - risk reduces symptoms prior to discharge .

we used data from the inter99 study , a population - based randomized controlled trial ( ct00289237 , clinicaltrials.gov ) on residents ( n = 61,301 ) in the southern part of former copenhagen county , which is investigating the effects of lifestyle intervention ( smoking cessation , increased physical activity , and healthier dietary habits ) on cvd ( 12 ) . the study population was drawn from the danish civil registration system and prerandomized into four groups : 1 ) high - intensity intervention group ( n = 11,708 invited ) , 2 ) low - intensity intervention group ( n = 1,308 invited ) , 3 ) control group receiving questionnaires ( n = 5,264 invited ) , and 4 ) control group followed only in central registries ( n = 43,021 ) . subjects in groups 1 and 2 were invited to participate in a health examination in 19992001 and included in the current observational study . the health examination included self - administered questionnaires , a physical examination , a 2-h oral glucose tolerance test ( ogtt ) , and various blood tests , as well as a lifestyle consultation with personal health advice . furthermore , participants at high risk of ischemic heart disease were included for lifestyle intervention ( 60% ) . group 1 was offered lifestyle counseling in groups on smoking cessation and physical activity / diet during 6-month periods , and group 2 was referred to their general practitioner ( 12 ) . more specifically , groups 1 and 2 consisted of an age- and sex - stratified random sample of 13,016 men and women aged 3065 years with 12,934 eligible for invitation . participation at baseline was associated with obesity , nonsmoking , and fewer admissions for ischemic heart disease , cvd , and diabetes ( 12 ) . after 5 years ( median : 5.5 ; range : 5.06.0 ) of follow - up , all participants were invited in 20042006 to a reexamination with essentially the same questionnaires and examinations , with 4,513 participating . nonparticipation at follow - up was associated with the following baseline characteristics : female sex , younger age , a less healthful lifestyle , and a less favorable risk factor profile , including lower 25(oh)d levels and higher diabetes prevalence . only participants with a northern european origin ( denmark , norway , sweden , iceland , and faroe islands ) were included ( n = 6,405 at baseline ; n = 4,296 at follow - up ) . the study was approved by the ethical committees of copenhagen ( ka 98155 ) and was in accordance with helsinki declaration ii principles . both at baseline and follow - up , all participants without a history of diabetes underwent a 2-h standardized 75-g ogtt in the morning after an overnight fast . plasma glucose and serum insulin were measured at 0 , 30 , and 120 min . glucose concentrations were analyzed by the hexokinase / glucose-6-phosphate dehydrogenase assay ( boehringer , mannheim , germany ) . insulin levels were measured by a fluoroimmunoassay technique ( dako diagnostics ltd . , cambridgeshire , u.k . ) . diabetes was diagnosed based on ogtt results as fasting plasma glucose 7.0 or 2-h plasma glucose 11.1 mmol / l ( 13 ) . hemoglobin a1c ( hba1c ) also was measured for all participants for a definition of diabetes as hba1c 6.5% ( 14 ) . prevalent diabetes at baseline was defined as having diabetes according to ogtt criteria , hba1c criteria , a known history of diabetes , and/or use of diabetes medication . incident diabetes was defined as diabetes at follow - up among those without diabetes at baseline according to the criteria given above . insulin resistance and pancreatic -cell function were estimated using both the widely used homeostasis model assessment ( homa ) , which is based on fasting glucose and insulin levels only ( 15 ) , and the bigtt ( -cell function , insulin sensitivity , and glucose tolerance test ) ( 16 ) , which is based on serum insulin and plasma glucose for the entire ogtt at 0 , 30 , and 120 min , as well as bmi and sex . thus , homa gives estimates of basal insulin secretion ( homa-%b ) and hepatic insulin resistance ( homa - ir ) . the bigtt model , on the other hand , gives estimates of the acute glucose - stimulated insulin response ( bigtt - air ) and stimulated insulin resistance ( bigtt - ir ) that reflect both hepatic and muscle insulin resistance . fasting blood was collected beginning with initial examinations in 1999 , and serum samples were stored at 20c until analysis in 2009 . vitamin d status was measured at baseline as serum 25(oh)d by high - performance liquid chromatography . stability of 25(oh)d in serum samples under different conditions has been demonstrated , so we assumed our measurements were unaffected by storage time ( 17 ) . nmol / l , and observations below this limit were assigned 10 nmol / l ( n = 74 ) . 25(oh)d was categorized into < 25 , 2550 , 5075 , and 75 nmol / l . for physical activity during leisure time , participants scored themselves as mostly sedentary , moderate activity , regular exercise , or heavy training . smoking status was never , ex - smoker , or current smoker at < 15 g / day , 1525 g / day , or 25 g / day . on the basis of responses to qualitative questions about intake of fruit , vegetables , fish , and the average amount and type of alcoholic beverages per week during the past 12 months was used to estimate a total alcohol consumption of 0 , 17 , > 714 , > 1421 , or > 21 standard drinks ( 1.5 cl or 12 g ethanol ) per week . social class was defined based on questions regarding years of vocational training and employment status and was categorized into five classes . family history of diabetes was defined as having a first - degree relative with diabetes . an extensive food frequency questionnaire provided information on total energy intake and specific nutrients ( 19 ) . self - reported changes in dietary habits , physical activity , smoking status , and alcohol consumption were recorded at follow - up . bmi was calculated as weight divided by height squared and categorized as underweight ( < 18.5 kg / m ) , normal ( 18.525 kg / m ) , overweight ( 2530 kg / m ) , or obese ( 30 kg / m ) . changes in weight during follow - up were calculated by subtraction and categorized as increased , unchanged , or decreased . statistics were performed with sas , version 9.1 ( sas institute inc , cary , nc ) . all reported p values are two - tailed , and statistical significance was defined as p < 0.05 . crude and adjusted associations were evaluated in a series of linear and logistic regression models . effects from logistic regression models were reported as odds ratios ( or ) with 95% cis . -coefficients from linear regression models with log - transformed outcomes were back transformed and reported as percent with 95% ci . to adjust for regression to the mean and allow effect estimates to be interpreted as outcome changes from baseline to follow - up , linear regression models with outcomes measured at follow - up were adjusted for the baseline value of the outcome . continuous 25(oh)d was tested for linear associations by including the squared term of 25(oh)d in the models . statistical interaction effects were evaluated by including a product term between 25(oh)d and relevant covariates . f tests and wald tests for single parameters were used to determine significance in regression analyses . the number of participants included in the analyses differed since complete case analyses were performed and not all participants had complete information on all variables examined . we used data from the inter99 study , a population - based randomized controlled trial ( ct00289237 , clinicaltrials.gov ) on residents ( n = 61,301 ) in the southern part of former copenhagen county , which is investigating the effects of lifestyle intervention ( smoking cessation , increased physical activity , and healthier dietary habits ) on cvd ( 12 ) . the study population was drawn from the danish civil registration system and prerandomized into four groups : 1 ) high - intensity intervention group ( n = 11,708 invited ) , 2 ) low - intensity intervention group ( n = 1,308 invited ) , 3 ) control group receiving questionnaires ( n = 5,264 invited ) , and 4 ) control group followed only in central registries ( n = 43,021 ) . subjects in groups 1 and 2 were invited to participate in a health examination in 19992001 and included in the current observational study . the health examination included self - administered questionnaires , a physical examination , a 2-h oral glucose tolerance test ( ogtt ) , and various blood tests , as well as a lifestyle consultation with personal health advice . furthermore , participants at high risk of ischemic heart disease were included for lifestyle intervention ( 60% ) . group 1 was offered lifestyle counseling in groups on smoking cessation and physical activity / diet during 6-month periods , and group 2 was referred to their general practitioner ( 12 ) . more specifically , groups 1 and 2 consisted of an age- and sex - stratified random sample of 13,016 men and women aged 3065 years with 12,934 eligible for invitation . participation at baseline was associated with obesity , nonsmoking , and fewer admissions for ischemic heart disease , cvd , and diabetes ( 12 ) . after 5 years ( median : 5.5 ; range : 5.06.0 ) of follow - up , all participants were invited in 20042006 to a reexamination with essentially the same questionnaires and examinations , with 4,513 participating . nonparticipation at follow - up was associated with the following baseline characteristics : female sex , younger age , a less healthful lifestyle , and a less favorable risk factor profile , including lower 25(oh)d levels and higher diabetes prevalence . only participants with a northern european origin ( denmark , norway , sweden , iceland , and faroe islands ) were included ( n = 6,405 at baseline ; n = 4,296 at follow - up ) . the study was approved by the ethical committees of copenhagen ( ka 98155 ) and was in accordance with helsinki declaration ii principles . both at baseline and follow - up , all participants without a history of diabetes underwent a 2-h standardized 75-g ogtt in the morning after an overnight fast . plasma glucose and serum insulin were measured at 0 , 30 , and 120 min . glucose concentrations were analyzed by the hexokinase / glucose-6-phosphate dehydrogenase assay ( boehringer , mannheim , germany ) . insulin levels were measured by a fluoroimmunoassay technique ( dako diagnostics ltd . , cambridgeshire , u.k . ) . diabetes was diagnosed based on ogtt results as fasting plasma glucose 7.0 or 2-h plasma glucose 11.1 mmol / l ( 13 ) . hemoglobin a1c ( hba1c ) also was measured for all participants for a definition of diabetes as hba1c 6.5% ( 14 ) . prevalent diabetes at baseline was defined as having diabetes according to ogtt criteria , hba1c criteria , a known history of diabetes , and/or use of diabetes medication . incident diabetes was defined as diabetes at follow - up among those without diabetes at baseline according to the criteria given above . insulin resistance and pancreatic -cell function were estimated using both the widely used homeostasis model assessment ( homa ) , which is based on fasting glucose and insulin levels only ( 15 ) , and the bigtt ( -cell function , insulin sensitivity , and glucose tolerance test ) ( 16 ) , which is based on serum insulin and plasma glucose for the entire ogtt at 0 , 30 , and 120 min , as well as bmi and sex . thus , homa gives estimates of basal insulin secretion ( homa-%b ) and hepatic insulin resistance ( homa - ir ) . the bigtt model , on the other hand , gives estimates of the acute glucose - stimulated insulin response ( bigtt - air ) and stimulated insulin resistance ( bigtt - ir ) that reflect both hepatic and muscle insulin resistance . fasting blood was collected beginning with initial examinations in 1999 , and serum samples were stored at 20c until analysis in 2009 . vitamin d status was measured at baseline as serum 25(oh)d by high - performance liquid chromatography . stability of 25(oh)d in serum samples under different conditions has been demonstrated , so we assumed our measurements were unaffected by storage time ( 17 ) . the detection limit was 10 nmol / l , and observations below this limit were assigned 10 nmol / l ( n = 74 ) . 25(oh)d was categorized into < 25 , 2550 , 5075 , and 75 nmol / l . for physical activity during leisure time , participants scored themselves as mostly sedentary , moderate activity , regular exercise , or heavy training . smoking status was never , ex - smoker , or current smoker at < 15 g / day , 1525 g / day , or 25 g / day . on the basis of responses to qualitative questions about intake of fruit , vegetables , fish , and the average amount and type of alcoholic beverages per week during the past 12 months was used to estimate a total alcohol consumption of 0 , 17 , > 714 , > 1421 , or > 21 standard drinks ( 1.5 cl or 12 g ethanol ) per week . social class was defined based on questions regarding years of vocational training and employment status and was categorized into five classes . family history of diabetes was defined as having a first - degree relative with diabetes . an extensive food frequency questionnaire provided information on total energy intake and specific nutrients ( 19 ) . self - reported changes in dietary habits , physical activity , smoking status , and alcohol consumption were recorded at follow - up . bmi was calculated as weight divided by height squared and categorized as underweight ( < 18.5 kg / m ) , normal ( 18.525 kg / m ) , overweight ( 2530 kg / m ) , or obese ( 30 kg / m ) . changes in weight during follow - up were calculated by subtraction and categorized as increased , unchanged , or decreased . data were considered observational . statistics were performed with sas , version 9.1 ( sas institute inc , cary , nc ) . all reported p values are two - tailed , and statistical significance was defined as p < 0.05 . crude and adjusted associations were evaluated in a series of linear and logistic regression models . effects from logistic regression models were reported as odds ratios ( or ) with 95% cis . -coefficients from linear regression models with log - transformed outcomes were back transformed and reported as percent with 95% ci . to adjust for regression to the mean and allow effect estimates to be interpreted as outcome changes from baseline to follow - up , linear regression models with outcomes measured at follow - up were adjusted for the baseline value of the outcome . continuous 25(oh)d was tested for linear associations by including the squared term of 25(oh)d in the models . statistical interaction effects were evaluated by including a product term between 25(oh)d and relevant covariates . f tests and wald tests for single parameters were used to determine significance in regression analyses . the number of participants included in the analyses differed since complete case analyses were performed and not all participants had complete information on all variables examined . the baseline study population was 48.4% ( n = 3,099 ) men and 51.6% ( n = 3,306 ) women with a mean age of 46.3 years ( range : 29.761.3 ) . 25(oh)d levels strongly correlated to the season in which blood was collected , whereas dietary vitamin d intake showed only a weak and nonsignificant association with circulating 25(oh)d ( table 1 ) . furthermore , male sex , current smoking , low physical activity , less healthful dietary habits , obesity , low social class , and prevalent diabetes were associated with lower 25(oh)d concentrations in crude analyses ( table 1 ) . no association with 25(oh)d concentration was observed for age , alcohol intake , total energy intake , or family history of diabetes . baseline characteristics of the study population by 25(oh)d status the prevalence of diabetes at baseline was 10.6% ( n = 677 ) . during follow - up , 3.6% ( n = 141 ) of participants without diabetes at baseline developed diabetes . low 25(oh)d status was significantly associated with a higher prevalence of diabetes at baseline both in crude analyses and after adjustment for confounders ( supplementary table 1 ) . low 25(oh)d status also was significantly associated with a higher risk of incident diabetes in crude analyses ( table 2 ) . supplemental analyses of incident diabetes defined according to either ogtt or hba1c criteria showed a borderline significant association between 25(oh)d and ogtt - based diabetes ( or per 10 nmol / l was 0.92 [ 95% ci 0.841.01 ] ; p = 0.080 ) , whereas no association was observed with hba1c - based diabetes ( supplementary table 3 ) . table 3 shows the associations between 25(oh)d status and longitudinal changes in continuous outcome variables adjusted for confounders . the degree of insulin resistance as assessed by the homa - ir and bigtt - ir indexes increased significantly more during follow - up among those with low 25(oh)d levels compared with those with higher levels . in addition , low 25(oh)d status was significantly associated with an increase in fasting and 2-h glucose as well as in fasting and 2-h insulin during follow - up . no association was observed with hba1c or with surrogate measures of pancreatic -cell function ( table 3 ) . multiple logistic regression analyses of the prospective association between baseline 25(oh)d status and 5-year diabetes incidence multiple linear regression analyses of association between baseline serum 25(oh)d and longitudinal changes in continuous markers of glucose homeostasis during 5 years of follow - up low 25(oh)d status was not significantly associated with incident diabetes after adjustment for confounders . however , it was significantly associated with unfavorable longitudinal changes in continuous markers of glucose homeostasis . our results indicate that low vitamin d status could be related to deterioration of glucose homeostasis . the major strengths of this study include its prospective study design , which allowed for assessment of temporal relationships , and the use of a large random sample of the general population of denmark , a country with a high prevalence of low vitamin d status . the use of serum 25(oh)d as a measure of vitamin d status is an important strength because 25(oh)d measures total , rather than solely dietary , vitamin d. the objective definitions of diabetes by ogtt and hba1c are also important strengths . in addition , the availability of detailed information on important covariates reduced potential confounding . on the other hand , the many covariates , together with the relatively low number of incident cases , may have diminished the power and contributed to the nonsignificant results in the fully adjusted models with incident diabetes . the major limitation of the study is its observational design , which despite the detailed covariate information , can not exclude residual confounding . another weakness was the single serum 25(oh)d measurement , which did not account for individual seasonal variation . this potential random misclassification could contribute to the low power and cause an underestimation of the true association between 25(oh)d and diabetes risk . the offer of intervention may also have influenced the results , and again , any beneficial effects of the lifestyle intervention program would cause an underestimation of the true association . finally , loss to follow - up may have introduced bias , and the fact that participants were only of northern european origin limits the generalization of the results . previous cross - sectional and case - control studies find an inverse association between serum 25(oh)d and type 2 diabetes as well as markers of adverse glucose homeostasis in many , although not all , studies ( 4,20 ) . the major drawback of these studies is the potential for reverse causation ( i.e. , diabetes or its early determinants causing low vitamin d status rather than vice versa ) . previous prospective studies are inconsistent . the nurses health study and women s health study both reported association of low supplemental and dietary vitamin d intake with increased incident diabetes risk ( 5,11 ) , whereas no association was observed in a japanese cohort ( 8) . however , those studies are limited by self - reports of dietary vitamin d intake , which do not consider the large proportion of vitamin d produced in the skin . a prospective study based on the framingham offspring cohort associates a predicted 25(oh)d four studies previously examined the prospective association of serum 25(oh)d and diabetes incidence ( 6,7,9,10 ) . one used pooled data from two finnish cohorts , the finnish mobile clinic health examination survey and the mini - finland health survey , and finds an inverse association in men , but not in women ( 6 ) . a major limitation of the study is the ascertainment of diabetes from registry data of patients receiving reimbursements for type 2 diabetes medication , resulting in misclassification of undiagnosed individuals or those treated with dietary changes only . the second study also used registry data and finds a significant inverse association between 25(oh)d and incidence of diabetes ( 9 ) . that study had a relatively short follow - up time ( average 1.3 years ) and was restricted to patients with measured 25(oh)d . the third study was nested within the nurses health study and , thus , restricted to women ; it finds that higher levels of plasma 25(oh)d were associated with lower risk of self - reported incident type 2 diabetes ( 7 ) . in contrast , a fourth study restricted to older women does not find an association of 25(oh)d with self - reported diabetes in a post hoc analysis of three nested case - control studies within the women s health initiative clinical trials and observational study ( 10 ) . finally , in agreement with our results , a previous study reports that baseline serum 25(oh)d was predictive of future continuous distribution of glycemic status and insulin resistance in a nondiabetic population after 10 years of follow - up . that study lacked statistical power to examine clinical outcomes and was restricted by sample size to quantitative traits ( 22 ) . results from randomized trials of the effect of vitamin d supplementation on glucose homeostasis are inconsistent , but several studies had other primary end points and , therefore , were underpowered for our outcomes of interest ( 2325 ) . as yet , no randomized intervention trials specifically designed to test the effect of vitamin d supplementation on incident diabetes in healthy individuals have been published . however , one study reports no effect of a relatively low dose ( 400 iu ) of vitamin d supplementation on incident diabetes after 7 years of follow - up , in post hoc analysis ( 24 ) . the mechanism by which vitamin d deficiency and type 2 diabetes may be related is not well understood . experimental evidence from animal and in vitro studies suggests that vitamin d may preserve glucose tolerance through effects on insulin secretion and sensitivity . these effects may be caused by effects on intracellular calcium , regulation of insulin receptor expression in peripheral tissues , or increased resilience of -cells to the systemic inflammation seen in type 2 diabetes ( 23 ) . in conclusion , however , it was significantly associated with unfavorable longitudinal changes in continuous markers of glucose homeostasis , indicating that low vitamin d status could be related to deterioration of glucose homeostasis . this study emphasizes the need for randomized intervention trials specifically designed to assess whether vitamin d supplementation effectively decreases the risk of developing diabetes in high - risk individuals .

objectivethis study aimed to examine vitamin d status as a determinant for development of type 2 diabetes and deterioration of glucose homeostasis.research design and methodsa random sample of the general population of copenhagen , denmark , was taken as part of the inter99 study . included were 6,405 men and women aged 3065 years at baseline ( 19992001 ) , with 4,296 participating in the follow - up examination 5 years later ( 20042006 ) . vitamin d was determined at baseline as serum 25-hydroxyvitamin d [ 25(oh)d ] . diabetes was defined based on an oral glucose tolerance test and a glycosylated hemoglobin ( hba1c ) test . secondary outcomes included continuous markers of glucose homeostasis.resultsthe risk of incident diabetes associated with a 10 nmol / l increase in 25(oh)d was odds ratio ( or ) 0.91 ( 95% ci 0.840.97 ) in crude analyses . the association became statistically nonsignificant after adjustment for confounders , with an or per 10 nmol / l of 0.94 ( 0.861.03 ) . low 25(oh)d status was significantly associated with unfavorable longitudinal changes in continuous markers of glucose homeostasis after adjustment for confounders . fasting and 2-h glucose and insulin as well as the degree of insulin resistance increased significantly more during follow - up among those with low 25(oh)d levels compared with those with higher levels.conclusionslow 25(oh)d status was not significantly associated with incident diabetes after adjustment for confounders . however , it was significantly associated with unfavorable longitudinal changes in continuous markers of glucose homeostasis , indicating that low vitamin d status could be related to deterioration of glucose homeostasis .

RESEARCH DESIGN AND METHODS Study population Diabetes and markers of glucose homeostasis Vitamin D Self-administered questionnaire Physical examinations Statistical analyses RESULTS CONCLUSIONS Supplementary Material

we used data from the inter99 study , a population - based randomized controlled trial ( ct00289237 , clinicaltrials.gov ) on residents ( n = 61,301 ) in the southern part of former copenhagen county , which is investigating the effects of lifestyle intervention ( smoking cessation , increased physical activity , and healthier dietary habits ) on cvd ( 12 ) . the study population was drawn from the danish civil registration system and prerandomized into four groups : 1 ) high - intensity intervention group ( n = 11,708 invited ) , 2 ) low - intensity intervention group ( n = 1,308 invited ) , 3 ) control group receiving questionnaires ( n = 5,264 invited ) , and 4 ) control group followed only in central registries ( n = 43,021 ) . subjects in groups 1 and 2 were invited to participate in a health examination in 19992001 and included in the current observational study . the health examination included self - administered questionnaires , a physical examination , a 2-h oral glucose tolerance test ( ogtt ) , and various blood tests , as well as a lifestyle consultation with personal health advice . furthermore , participants at high risk of ischemic heart disease were included for lifestyle intervention ( 60% ) . more specifically , groups 1 and 2 consisted of an age- and sex - stratified random sample of 13,016 men and women aged 3065 years with 12,934 eligible for invitation . participation at baseline was associated with obesity , nonsmoking , and fewer admissions for ischemic heart disease , cvd , and diabetes ( 12 ) . after 5 years ( median : 5.5 ; range : 5.06.0 ) of follow - up , all participants were invited in 20042006 to a reexamination with essentially the same questionnaires and examinations , with 4,513 participating . nonparticipation at follow - up was associated with the following baseline characteristics : female sex , younger age , a less healthful lifestyle , and a less favorable risk factor profile , including lower 25(oh)d levels and higher diabetes prevalence . only participants with a northern european origin ( denmark , norway , sweden , iceland , and faroe islands ) were included ( n = 6,405 at baseline ; n = 4,296 at follow - up ) . the study was approved by the ethical committees of copenhagen ( ka 98155 ) and was in accordance with helsinki declaration ii principles . both at baseline and follow - up , all participants without a history of diabetes underwent a 2-h standardized 75-g ogtt in the morning after an overnight fast . plasma glucose and serum insulin were measured at 0 , 30 , and 120 min . diabetes was diagnosed based on ogtt results as fasting plasma glucose 7.0 or 2-h plasma glucose 11.1 mmol / l ( 13 ) . hemoglobin a1c ( hba1c ) also was measured for all participants for a definition of diabetes as hba1c 6.5% ( 14 ) . prevalent diabetes at baseline was defined as having diabetes according to ogtt criteria , hba1c criteria , a known history of diabetes , and/or use of diabetes medication . incident diabetes was defined as diabetes at follow - up among those without diabetes at baseline according to the criteria given above . insulin resistance and pancreatic -cell function were estimated using both the widely used homeostasis model assessment ( homa ) , which is based on fasting glucose and insulin levels only ( 15 ) , and the bigtt ( -cell function , insulin sensitivity , and glucose tolerance test ) ( 16 ) , which is based on serum insulin and plasma glucose for the entire ogtt at 0 , 30 , and 120 min , as well as bmi and sex . thus , homa gives estimates of basal insulin secretion ( homa-%b ) and hepatic insulin resistance ( homa - ir ) . the bigtt model , on the other hand , gives estimates of the acute glucose - stimulated insulin response ( bigtt - air ) and stimulated insulin resistance ( bigtt - ir ) that reflect both hepatic and muscle insulin resistance . vitamin d status was measured at baseline as serum 25(oh)d by high - performance liquid chromatography . nmol / l , and observations below this limit were assigned 10 nmol / l ( n = 74 ) . 25(oh)d was categorized into < 25 , 2550 , 5075 , and 75 nmol / l . smoking status was never , ex - smoker , or current smoker at < 15 g / day , 1525 g / day , or 25 g / day . on the basis of responses to qualitative questions about intake of fruit , vegetables , fish , and the average amount and type of alcoholic beverages per week during the past 12 months was used to estimate a total alcohol consumption of 0 , 17 , > 714 , > 1421 , or > 21 standard drinks ( 1.5 cl or 12 g ethanol ) per week . social class was defined based on questions regarding years of vocational training and employment status and was categorized into five classes . family history of diabetes was defined as having a first - degree relative with diabetes . self - reported changes in dietary habits , physical activity , smoking status , and alcohol consumption were recorded at follow - up . bmi was calculated as weight divided by height squared and categorized as underweight ( < 18.5 kg / m ) , normal ( 18.525 kg / m ) , overweight ( 2530 kg / m ) , or obese ( 30 kg / m ) . changes in weight during follow - up were calculated by subtraction and categorized as increased , unchanged , or decreased . all reported p values are two - tailed , and statistical significance was defined as p < 0.05 . effects from logistic regression models were reported as odds ratios ( or ) with 95% cis . -coefficients from linear regression models with log - transformed outcomes were back transformed and reported as percent with 95% ci . to adjust for regression to the mean and allow effect estimates to be interpreted as outcome changes from baseline to follow - up , linear regression models with outcomes measured at follow - up were adjusted for the baseline value of the outcome . continuous 25(oh)d was tested for linear associations by including the squared term of 25(oh)d in the models . the number of participants included in the analyses differed since complete case analyses were performed and not all participants had complete information on all variables examined . we used data from the inter99 study , a population - based randomized controlled trial ( ct00289237 , clinicaltrials.gov ) on residents ( n = 61,301 ) in the southern part of former copenhagen county , which is investigating the effects of lifestyle intervention ( smoking cessation , increased physical activity , and healthier dietary habits ) on cvd ( 12 ) . the study population was drawn from the danish civil registration system and prerandomized into four groups : 1 ) high - intensity intervention group ( n = 11,708 invited ) , 2 ) low - intensity intervention group ( n = 1,308 invited ) , 3 ) control group receiving questionnaires ( n = 5,264 invited ) , and 4 ) control group followed only in central registries ( n = 43,021 ) . the health examination included self - administered questionnaires , a physical examination , a 2-h oral glucose tolerance test ( ogtt ) , and various blood tests , as well as a lifestyle consultation with personal health advice . more specifically , groups 1 and 2 consisted of an age- and sex - stratified random sample of 13,016 men and women aged 3065 years with 12,934 eligible for invitation . participation at baseline was associated with obesity , nonsmoking , and fewer admissions for ischemic heart disease , cvd , and diabetes ( 12 ) . after 5 years ( median : 5.5 ; range : 5.06.0 ) of follow - up , all participants were invited in 20042006 to a reexamination with essentially the same questionnaires and examinations , with 4,513 participating . nonparticipation at follow - up was associated with the following baseline characteristics : female sex , younger age , a less healthful lifestyle , and a less favorable risk factor profile , including lower 25(oh)d levels and higher diabetes prevalence . only participants with a northern european origin ( denmark , norway , sweden , iceland , and faroe islands ) were included ( n = 6,405 at baseline ; n = 4,296 at follow - up ) . the study was approved by the ethical committees of copenhagen ( ka 98155 ) and was in accordance with helsinki declaration ii principles . both at baseline and follow - up , all participants without a history of diabetes underwent a 2-h standardized 75-g ogtt in the morning after an overnight fast . plasma glucose and serum insulin were measured at 0 , 30 , and 120 min . diabetes was diagnosed based on ogtt results as fasting plasma glucose 7.0 or 2-h plasma glucose 11.1 mmol / l ( 13 ) . hemoglobin a1c ( hba1c ) also was measured for all participants for a definition of diabetes as hba1c 6.5% ( 14 ) . prevalent diabetes at baseline was defined as having diabetes according to ogtt criteria , hba1c criteria , a known history of diabetes , and/or use of diabetes medication . incident diabetes was defined as diabetes at follow - up among those without diabetes at baseline according to the criteria given above . insulin resistance and pancreatic -cell function were estimated using both the widely used homeostasis model assessment ( homa ) , which is based on fasting glucose and insulin levels only ( 15 ) , and the bigtt ( -cell function , insulin sensitivity , and glucose tolerance test ) ( 16 ) , which is based on serum insulin and plasma glucose for the entire ogtt at 0 , 30 , and 120 min , as well as bmi and sex . thus , homa gives estimates of basal insulin secretion ( homa-%b ) and hepatic insulin resistance ( homa - ir ) . the bigtt model , on the other hand , gives estimates of the acute glucose - stimulated insulin response ( bigtt - air ) and stimulated insulin resistance ( bigtt - ir ) that reflect both hepatic and muscle insulin resistance . vitamin d status was measured at baseline as serum 25(oh)d by high - performance liquid chromatography . the detection limit was 10 nmol / l , and observations below this limit were assigned 10 nmol / l ( n = 74 ) . 25(oh)d was categorized into < 25 , 2550 , 5075 , and 75 nmol / l . smoking status was never , ex - smoker , or current smoker at < 15 g / day , 1525 g / day , or 25 g / day . on the basis of responses to qualitative questions about intake of fruit , vegetables , fish , and the average amount and type of alcoholic beverages per week during the past 12 months was used to estimate a total alcohol consumption of 0 , 17 , > 714 , > 1421 , or > 21 standard drinks ( 1.5 cl or 12 g ethanol ) per week . social class was defined based on questions regarding years of vocational training and employment status and was categorized into five classes . family history of diabetes was defined as having a first - degree relative with diabetes . self - reported changes in dietary habits , physical activity , smoking status , and alcohol consumption were recorded at follow - up . bmi was calculated as weight divided by height squared and categorized as underweight ( < 18.5 kg / m ) , normal ( 18.525 kg / m ) , overweight ( 2530 kg / m ) , or obese ( 30 kg / m ) . changes in weight during follow - up were calculated by subtraction and categorized as increased , unchanged , or decreased . all reported p values are two - tailed , and statistical significance was defined as p < 0.05 . effects from logistic regression models were reported as odds ratios ( or ) with 95% cis . -coefficients from linear regression models with log - transformed outcomes were back transformed and reported as percent with 95% ci . to adjust for regression to the mean and allow effect estimates to be interpreted as outcome changes from baseline to follow - up , linear regression models with outcomes measured at follow - up were adjusted for the baseline value of the outcome . continuous 25(oh)d was tested for linear associations by including the squared term of 25(oh)d in the models . the number of participants included in the analyses differed since complete case analyses were performed and not all participants had complete information on all variables examined . the baseline study population was 48.4% ( n = 3,099 ) men and 51.6% ( n = 3,306 ) women with a mean age of 46.3 years ( range : 29.761.3 ) . 25(oh)d levels strongly correlated to the season in which blood was collected , whereas dietary vitamin d intake showed only a weak and nonsignificant association with circulating 25(oh)d ( table 1 ) . furthermore , male sex , current smoking , low physical activity , less healthful dietary habits , obesity , low social class , and prevalent diabetes were associated with lower 25(oh)d concentrations in crude analyses ( table 1 ) . baseline characteristics of the study population by 25(oh)d status the prevalence of diabetes at baseline was 10.6% ( n = 677 ) . during follow - up , 3.6% ( n = 141 ) of participants without diabetes at baseline developed diabetes . low 25(oh)d status was significantly associated with a higher prevalence of diabetes at baseline both in crude analyses and after adjustment for confounders ( supplementary table 1 ) . low 25(oh)d status also was significantly associated with a higher risk of incident diabetes in crude analyses ( table 2 ) . supplemental analyses of incident diabetes defined according to either ogtt or hba1c criteria showed a borderline significant association between 25(oh)d and ogtt - based diabetes ( or per 10 nmol / l was 0.92 [ 95% ci 0.841.01 ] ; p = 0.080 ) , whereas no association was observed with hba1c - based diabetes ( supplementary table 3 ) . table 3 shows the associations between 25(oh)d status and longitudinal changes in continuous outcome variables adjusted for confounders . the degree of insulin resistance as assessed by the homa - ir and bigtt - ir indexes increased significantly more during follow - up among those with low 25(oh)d levels compared with those with higher levels . in addition , low 25(oh)d status was significantly associated with an increase in fasting and 2-h glucose as well as in fasting and 2-h insulin during follow - up . multiple logistic regression analyses of the prospective association between baseline 25(oh)d status and 5-year diabetes incidence multiple linear regression analyses of association between baseline serum 25(oh)d and longitudinal changes in continuous markers of glucose homeostasis during 5 years of follow - up low 25(oh)d status was not significantly associated with incident diabetes after adjustment for confounders . however , it was significantly associated with unfavorable longitudinal changes in continuous markers of glucose homeostasis . our results indicate that low vitamin d status could be related to deterioration of glucose homeostasis . the major strengths of this study include its prospective study design , which allowed for assessment of temporal relationships , and the use of a large random sample of the general population of denmark , a country with a high prevalence of low vitamin d status . the use of serum 25(oh)d as a measure of vitamin d status is an important strength because 25(oh)d measures total , rather than solely dietary , vitamin d. the objective definitions of diabetes by ogtt and hba1c are also important strengths . on the other hand , the many covariates , together with the relatively low number of incident cases , may have diminished the power and contributed to the nonsignificant results in the fully adjusted models with incident diabetes . the major limitation of the study is its observational design , which despite the detailed covariate information , can not exclude residual confounding . this potential random misclassification could contribute to the low power and cause an underestimation of the true association between 25(oh)d and diabetes risk . the offer of intervention may also have influenced the results , and again , any beneficial effects of the lifestyle intervention program would cause an underestimation of the true association . finally , loss to follow - up may have introduced bias , and the fact that participants were only of northern european origin limits the generalization of the results . previous cross - sectional and case - control studies find an inverse association between serum 25(oh)d and type 2 diabetes as well as markers of adverse glucose homeostasis in many , although not all , studies ( 4,20 ) . , diabetes or its early determinants causing low vitamin d status rather than vice versa ) . the nurses health study and women s health study both reported association of low supplemental and dietary vitamin d intake with increased incident diabetes risk ( 5,11 ) , whereas no association was observed in a japanese cohort ( 8) . however , those studies are limited by self - reports of dietary vitamin d intake , which do not consider the large proportion of vitamin d produced in the skin . a prospective study based on the framingham offspring cohort associates a predicted 25(oh)d four studies previously examined the prospective association of serum 25(oh)d and diabetes incidence ( 6,7,9,10 ) . a major limitation of the study is the ascertainment of diabetes from registry data of patients receiving reimbursements for type 2 diabetes medication , resulting in misclassification of undiagnosed individuals or those treated with dietary changes only . that study had a relatively short follow - up time ( average 1.3 years ) and was restricted to patients with measured 25(oh)d . the third study was nested within the nurses health study and , thus , restricted to women ; it finds that higher levels of plasma 25(oh)d were associated with lower risk of self - reported incident type 2 diabetes ( 7 ) . finally , in agreement with our results , a previous study reports that baseline serum 25(oh)d was predictive of future continuous distribution of glycemic status and insulin resistance in a nondiabetic population after 10 years of follow - up . that study lacked statistical power to examine clinical outcomes and was restricted by sample size to quantitative traits ( 22 ) . results from randomized trials of the effect of vitamin d supplementation on glucose homeostasis are inconsistent , but several studies had other primary end points and , therefore , were underpowered for our outcomes of interest ( 2325 ) . as yet , no randomized intervention trials specifically designed to test the effect of vitamin d supplementation on incident diabetes in healthy individuals have been published . however , one study reports no effect of a relatively low dose ( 400 iu ) of vitamin d supplementation on incident diabetes after 7 years of follow - up , in post hoc analysis ( 24 ) . the mechanism by which vitamin d deficiency and type 2 diabetes may be related is not well understood . experimental evidence from animal and in vitro studies suggests that vitamin d may preserve glucose tolerance through effects on insulin secretion and sensitivity . these effects may be caused by effects on intracellular calcium , regulation of insulin receptor expression in peripheral tissues , or increased resilience of -cells to the systemic inflammation seen in type 2 diabetes ( 23 ) . in conclusion , however , it was significantly associated with unfavorable longitudinal changes in continuous markers of glucose homeostasis , indicating that low vitamin d status could be related to deterioration of glucose homeostasis . this study emphasizes the need for randomized intervention trials specifically designed to assess whether vitamin d supplementation effectively decreases the risk of developing diabetes in high - risk individuals .

specific fibril aggregation of more than 20 proteins has been linked to a pathogenesis of many amyloid - associated diseases such as alzheimer s disease , parkinson s disease , huntington s diseases , and prion diseases . moreover , a formation of amyloid - like fibrils takes place in other maladies associated with protein misfolding , including diabetes type ii , prolactoma , primary amyloidosis , etc . striking evidence has recently accumulated suggesting that the ability to form amyloid fibrils is not a peculiarity of this small group of disease - related proteins but , rather , the ability to form amyloid is a generic property of the polypeptide chain . amyloid fibrils are -sheet rich protein aggregates with different morphologies that constitute a phenomenon known as fibril polymorphism.in vitro aggregation of various amyloid associated proteins , such as calcitonin , amylin , insulin , transthyretin , and mouse prion , commonly leads to the formation of two distinct fibril morphologies : tape - like and helical - twisted . microscopic examination of post - mortem extracted amyloids from brains of patients who were diagnosed with alzheimer s disease also revealed both of these fibril polymorphs . thus , a controllable formation of one fibril polymorph versus another could possibly slow down fibril development and progression of various amyloid associated disorders . at the same time , the factors leading to one fibril polymorph or another are not currently known . it has also been observed that , with protein aggregation , many physical and chemical factors , such as ionic strength , temperature , or pressure , determine the fibril structure and morphology . for example , makarava and baskakov found that hamster cellular isoform of mammalian prion protein forms variable fibril polymorphs under different aggregation conditions . separately , petkova et al . observed that agitation of a peptide solution results in the formation of structurally and morphologically different fibril polymorphs compared to the fibrils formed without agitation . a few years ago , dzwolak demonstrated that vortexing of insulin solutions during protein aggregation at 60 c causes the formation of fibril superstructures exhibiting negative induced circular dichroism ( icd ) upon binding the achiral dye , thioflavin t ( tht ) . if , however , the same fibrillation was carried out at a temperature , below 50 c , randomly two differing supramolecular structures were obtained that showed opposite icd ( icd and + icd polymorphs ) . insulin fibrils that were not agitated , while being formed , did not exhibit any icd signal . interestingly , the morphologies of the two observed chiral polymorphs although similar showed a tendency for more lateral alignment of filaments for the + icd polymorph compared to the icd polymorph . this discovery emphasized the importance of utilizing alternative methods beyond classical microscopy , such as afm ( atomic force microscopy ) and sem ( scanning electron microscopy ) , that are capable of distinguishing chiral fibril polymorphs that otherwise appear identical . recently , it was discovered that vcd is a unique tool that can directly probe supramolecular fibril chirality in solution as well as in dried films . studies of insulin fibrils indicated that enhanced vcd sensitivity arises directly from the long - range supramolecular chirality of fibril structures at all hierarchical levels . it was shown that vcd probes deeper levels of fibril supramolecular chiral organization that are not apparent using existing forms of microscopy . in particular , we found that insulin aggregation at ph 2.4 and higher results in the formation of fibrils that show a strong vcd spectrum with peaks near 1554 , 1593 , 1627 , 1647 , 1670 cm , which have a sign pattern ( + + microscopic examination of these fibrils indicated that a majority of them have a left - handed helical twist . however , if the ph of the aggregation solution is lower than 2.4 , the distribution of fibrils shifts to increasingly flat , tape - like , or binary fibrils as the incubation ph continues to be lowered that under microscopic examination show no noticeable chirality or twist on their surface . nevertheless , these fibrils show a strong , but often somewhat smaller , vcd with a sign pattern ( + ) that is nearly the mirror - image of normal vcd fibril spectrum and is referred to as the reversed polymorph . the fact that flat tape - like fibrils show a strong reversed vcd signal indicates that they must be composed of right - handed filaments , the chirality of which lies below the limit of afm or sem detection . our combined vcd - microscopic studies showed that ph determines not only the net handedness of the filaments , precursors of mature fibrils , but also controls their association pathways . left - handed filaments intertwine , forming left - handed proto - fibrils and mature fibrils that have normal vcd . on the other hand , right - handed filaments associate side - by - side , forming flat , tape - like , or binary fibrils . thus , ph most likely alters protein solvent interactions or causes protonation of some amino acid side chains , which are lying on the surface of the filaments . these changes cause variations in the way these filaments bind together to form mature fibrils that either twist together to form braids or align side - by - side without braiding . in addition , it was discovered that that ph not only determines insulin fibril morphology and net chirality at the stage of the protein aggregation , but also may dramatically change the morphology of mature fibrils and overturn their initial chirality . the key question we answer in this paper is whether the supramolecular chirality of the filaments is the underlying cause of morphological differences only for insulin fibrils , or whether it is a more general phenomenon that determines the polymorphism of other amyloidogenic proteins and peptides . a related question is whether the supramolecular chirality of all fibril filaments can be controlled by ph . to address these questions we studied aggregation as a function of incubation ph for hen - egg lysozyme , bovine apo--lactalbumin , het - s ( 218289 ) prion - forming domain from fungus podospora anserina , and a short 11-residue peptide fragment of human transthyretin ( ttr ( 105115 ) ) . the intrinsic supramolecular chirality of these fibrils was measured by vcd , and in parallel , fibril morphology was characterized by atomic force microscopy ( afm ) , scanning electron microscopy ( sem ) , and cryo - sem . in addition the secondary structure of each of these amyloid fibrils was determined by fourier transform infrared ( ftir ) and deep uv resonance raman ( duvrr ) spectroscopy . lysozyme , like insulin , forms opposite chiral polymorph depending on the incubation ph . hen - egg lysozyme is a widely used model protein for amyloidogenic studies that aggregates at low ph and high temperatures , forming rod - like fibrils . hen - egg lysozyme ( 60 mg / ml ) was incubated at 65 c for 3 days at different ph values . the solution was centrifuged at 10 000 rpm for 20 min , and the resulting gelatinous solution , dominated by mature fibrils , was analyzed by ir and vcd ( figure 1 ) . vcd ( a ) and ir ( b ) spectra of lysozyme fibrils grown at ph 1.0 ( blue ) , 1.5 ( green ) , 2.3 ( black ) , and 2.7 ( red ) for 3 days at 65 c . the strong peak at 1623 cm in the ir spectra of all studied samples indicates a dominance of -sheet structure ( figure 1 , bottom ) . the peak at 1663 cm that increases in intensity with increasing ph is associated with nonordered or loop structure , and may be related to the rate of fibril formation during the specified incubation conditions . we found that relatively small variations in the ph of the aggregation solution cause dramatic changes in the vcd spectra of lysozyme fibrils , the largest of which is almost 10 times more intense than the corresponding intense vcd spectra observed for insulin fibrils grown under the same ph values . as is evident from the vcd spectra , lysozyme fibrils show normal vcd at ph 2.7 ( figure 1 ) and higher ( data not shown ) . fibrils that were grown at low ph ( 1.01.5 ) , on the other hand , show reversed vcd with intensity comparable in magnitude but roughly opposite in sign compared to normal vcd . fibrils that were grown at ph 2.3 have very weak vcd intensity , with small residual vcd peaks for both normal ( negative , 1610 cm ) and reversed ( positive , 1624 cm ) fibrils present . one therefore concludes that ph 2.3 is close to an equilibrium balance point in the distribution of normal and reversed lysozyme fibrils . similar vcd spectral patterns were obtained for insulin fibrils grown at the same ph values . we also investigated whether prolonged aggregation at elevated temperature causes any changes in lysozyme fibrils . solutions with ph from 1.0 to 2.7 ( 60 mg / ml ) were incubated at 65 c for 5 days , and their vcd and ir spectra were recorded ( supporting information figure s1 ) . we found that vcd intensity of all fibril solutions , except the very small vcd intensity from fibrils incubated at ph 2.3 , decreased compared to fibrils grown for 3 days at 65 c . in addition to the decrease of vcd intensity we found that the position of a negative peak changed : lysozyme fibrils that were grown at ph 2.7 had a negative peak at 1610 cm ( 3 days of incubation ) and at 1616 cm ( 5 days of incubation ) . the same band minimum shifted from 1610 cm to 1623 cm for lysozyme fibrils grown at ph 2.3 . fluid - cell afm and sem were utilized to investigate morphological differences between normal and reversed lysozyme fibrils . both afm and sem ( but not tem ) microscopy can provide information about the sense , or handedness , of fibril helical chirality afm and sem are complementary microscopic methods for the determination of accurate species dimensions : afm provides accurate height data , while width dimensions may be larger due to a finite tip - width convolution error . sem , on the other hand , may not be utilized for height determination , but does provides accurate width data . we found that lysozyme fibrils that exhibit reversed vcd have a flat tape - like morphology without a noticeable twist . they have a height between 2.5 and 3.0 nm and a width in the range of 25 nm . some of these fibrils are composed of two side - to - side associated fibrils ( figure 2 , b ) . reported that these tape - like lysozyme fibrils consist of as many as 17 laterally assembled filaments . according to our data , binary lysozyme fibrils have the same height as the discussed tape - like fibrils , while their width is significantly larger ( 45 nm ) . some of these fibrils exhibit a well - defined left - handed twist ( figure 2 , c ; identified by yellow arrows ) . they have a height between 6 and 8 nm and a width between 18 and 20 nm . as with insulin , lysozyme fibrils exhibiting normal and reversed vcd have cylindrical left - twisted and flat tape - like fibril morphologies , respectively . sem ( a , c ) and fluid - cell afm ( b , d ) images of reversed vcd ( a , b ) and normal vcd ( c and d ) lysozyme fibrils grown at 65 c for 3 days . helical twisted fibrils ( all left - handed ) are indicated by yellow arrows . scale bars are 100 nm . it is interesting that neither for insulin nor lysozyme fibrils exhibiting reversed vcd is there any evidence of right - handed twist on their surface . one might suspect that fibril dehydration after fixation on copper grids for sem imaging could result in significant deformations of fibril topology . as a result , it is possible that a right - twist of reversed vcd fibrils may become distorted and therefore not apparent on sem images . in order to show that reversed vcd fibrils do not have a right - handed twist on their surface we utilized one of the most preservative microscopic tools : cryo - sem . cryo - sem shows that reversed vcd lysozyme fibrils have flat - like topology without a right - handed twist . in fact applications of cryo - sem for studies of insulin reversed vcd fibrils showed similar results , while at the same time this method confirmed the left - handed twist of normal vcd insulin fibrils ( supporting information figure s2 ) . deep uv resonance raman ( duvrr ) spectroscopy has proven to be an efficient technique for characterizing protein structural rearrangements at all stages of fibrillation . in particular , duvrr spectroscopy , together with hydrogen deuterium exchange , has been found to be a powerful method for the determination of fibril cross--core structure . previously , using duvrr spectroscopy we found that both normal and reversed insulin fibrils have the same spectrum and hence the same cross--core structure , while they have different environments for tyrosine ( tyr ) amino acid residues . in the present study we used duvrr spectroscopy to demonstrate the same similarity of cross--sheet structures of normal and reversed vcd lysozyme fibrils with small differences for the aromatic side - chains between normal and reversed fibrils ( supporting information figure s3 ) . apo--lactalbumin aggregation results in the formation of only reversed vcd fibrils at 37 c . the protein -lactalbumin is a small ( 14 kda ) metal binding protein present in the whey milk of most mammal species as a regulatory subunit of lactose synthase . -lactalbumin consists of 123 amino acid residues arranged into two domains , designated and . the two domains are divided by a cleft , where a calcium ion is located . reduction of two of four disulfide bonds in -lactalbumin results in its adopting a molten globule conformation . the molten globule state of -lactalbumin is a well - studied structure that has native - like secondary structure with an absence of specific tertiary packing interactions of amino acid side chains . apo--lactalbumin is of particular interest since it is homologous to lysozyme , albeit with different aggregating and denaturing properties . aggregates of apo--lactalbumin form long unbranched fibrils under low ph and high ionic strength ( supporting information figure s4 ) . previously the secondary structure of apo--lactalbumin fibrils has been extensively characterized in our group . in the current study we investigate the effect of ph on the formation of different vcd chiral polymorphs . apo--lactalbumin ( 5 mg / ml ) was incubated at different values of ph in the presence of 150 mm nacl at 37 c for 3 days . after fibril formation was terminated by centrifugation and temperature reduction , the vcd and ir spectra of the sample were measured . we found that fibrils grown at different ph values show reversed vcd , with a positive peak near 1624 cm and a negative peak near 1639 cm ( figure 3 ) . vcd ( top ) and ir ( bottom ) spectra of apo--lactalbumin fibrils grown at ph 1.5 ( blue ) , 2.5 ( green ) , 3.0 ( black ) , and 4.0 ( red ) for 3 days at 37 c . as depicted , apo--lactalbumin aggregation at this concentration over the ph range from 1.5 to 4.0 at 37 c results in an excess of only one chiral polymorph that has reversed vcd supramolecular chirality . fibrils that were grown at ph ( 2.5 ) have the highest intensity of a peak at 1617 cm . fibrils that were grown at ph 1.5 have relatively smaller intensity at 1617 cm , while the intensity of a negative peak at 1666 cm is almost the same as for ph 2.5 fibrils . fibrils that were grown at ph 3.0 have smaller intensity for all vcd bands relative to ph 1.5 and 2.5 fibrils centered at 1624 cm . finally , fibrils grown at ph 4.0 have the smallest vcd intensity , and the peak previously observed at 1617 cm for ph 1.5 and 2.5 is shifted to 1612 cm . afm was used to image fibrils of apo--lactalbumin that were deposited onto a freshly cleaved mica surface . the afm images revealed both tape - like and left - twisted fibrils at all ph values . tape - like fibrils had 26 nm width and 5 nm height , while left - twisted fibrils had the same width but were over twice as high ( 12 nm ) . fluid - cell afm images of left - twisted ( a ) and tape - like ( b ) fibrils of apo--lactalbumin grown for 3 days at 37 c . the amount of left - twisted fibrils decreased with an increase of aggregation ph ( supporting information figure s5 ) . specifically , they were abundant in a fibril solution that was grown at ph 1.5 , while less frequently observed at ph 2.5 . finally , in the fibril solutions grown at ph 3.0 and 4.0 left - twisted fibrils were extremely rare ( supporting information figure s5 ) . at the same time , the tape - like morphology was commonly observed for fibrils grown at all ph values . het - s ( 218289 ) peptide fragment of prion protein forms opposite chiral polymorphs at different ph values . it was previously shown that at low ph its c - terminal fragment ( het - s 218289 ) slowly aggregates forming amyloid - like fibrils . several independent studies have shown that ( het - s 218289 ) aggregation at ph 2.0 leads to a formation of thick left - twisted cables composed of 36 filaments . at the same time , its aggregation at ph above 3.0 causes formation of thin fibrils without a noticeable twist . mizuno et al . proposed that these thin fibrils ought to have a left - handed twist , while no repeatable twist on the cryo - em provided pictures was evident . these thin fibrils were proposed to form a solenoid ( -roll ) with a triangular hydrophobic core . it is also of interest to note that these two ( het - s 218289 ) fibril polymorphs have different infectivity : fibrils at ph 2.0 showed very little if any infectivity , while fibrils formed at ph above 3.0 were found to be highly infectious . on the basis of electronic circular dichroism ( ecd ) and ir spectroscopy , thick left - twisted cables and thin fibrils were found to have different secondary structure . it was also shown that these two polymorphs have different assembly kinetics , resistance to denaturants , and intrinsic fluorescence . in our study we investigated supramolecular chirality of het - s ( 218289 ) prion fibrils grown at ph 2.0 , 3.3 , and 3.9 ( figure 6 ) . it appears that ph 2.0 fibrils , which have a left - handed twist ( figure 6 ) , exhibited very intense normal vcd . thin fibrils grown at ph 3.3 and 3.9 ( figure 5 ) showed vcd spectra of the opposite sign with 12 orders of magnitude lower intensity . the normal sign patterns for ph 2.0 fibrils are 1557 ( + ) , 1578 ( + ) , 1614 ( ) , 1640 ( + ) , and 1666 ( + ) cm , while the reversed sign pattern for ph 3.3 is 1560 ( ) , 1611 ( + ) , 1636 ( ) cm . het - s ( 218289 ) prion fibrils grown at ph 3.9 exhibit the same reversed sign pattern but have some differing vcd band frequencies : 1557 ( + ) , 1614 ( + ) , and 1660 ( ) cm . vcd ( a ) and ir ( b ) spectra of het - s ( 218289 ) fibrils grown at ph 2.0 ( blue ) , 3.3 ( green ) , and 3.9 ( black ) for 2 months at room temperature . for better visualization of band positions and intensities the insert shows vcd spectra of only ph 3.3 ( green ) and ph 3.9 ( black ) fibrils . afm images of het - s ( 218289 ) prion fibrils grown in ph 2.0 ( a ) and 3.9 ( b ) . the ir spectra of all studied samples exhibit a peak around 1620 cm , indicating a predominance of -sheet structure ( figure 5b ) . it was found that that the position of this peak is slightly shifted in the spectrum of fibrils grown at ph 2.0 ( 1624 cm ) compared to the corresponding ir band for fibrils grown at ph 3.3 and 3.9 fibrils ( 1620 cm ) . moreover , the ir at ph 2.0 has higher intensity for peaks at 1725 and 1671 cmcompared to those of fibrils grown at ph 3.3 and 3.9 . these differences indicate structural deviations of normal and reversed vcd chiral polymorphs of het - s fibrils . for more detailed structural characterization of their secondary structure we applied duvrr ( supporting information figure s6 ) . the positions of amide i , ii bands and c-h band are nearly identical in the spectra of both chiral polymorphs , which indicates a high level of similarity of secondary structure for all three fibril ph values . at the same time , we found that het - s fibrils that were grown at ph 3.9 have substantially higher amount of unordered structure ( a peak 1268 cm ) compared to the ph 2.0 fibrils . afm was utilized to reveal morphological organization of the analyzed het - s ( 218289 ) prion fibrils . we found that fibrils grown at ph 2.0 have a well - defined left - handed twist , 40 nm in width and around 3 nm in height ( figure 7 ) . at the same time , fibrils that were grown at ph 3.9 ( reversed vcd ) exhibited no twist on their surface . they had a shape of flat tapes that have 25 nm in width and 2 nm in height . vcd ( top ) and ir ( bottom ) spectra of ttr ( 105115 ) fibrils grown at ph 1.0 ( blue ) , 1.5 ( green ) , 2.0 ( black ) , 2.5 ( red ) , and 3.0 ( violet ) for 2 days at 37 c followed by 14 days at room temperature . opposite chiral polymorphs are observed for fibrils formed from a small peptide of human transthyretin . ttr ( 105115 ) is a peptide fragment of transthyretin , a 55 kda protein involved in the transport pathway of thyroxine and retinol in plasma . although this peptide is itself not implicated in any human disease , fibrillization of the full - length protein has been linked to familial amyloid polyneuropathy ( fap ) and senile systemic amyloidosis ( ssa ) . transthyretin has a -sheet rich structure and can form amyloid - like aggregates in vivo . previously , it was demonstrated that ttr ( 105115 ) is able to aggregate in vitro , forming amyloid - like fibrils . we grew ttr ( 105115 ) fibrils as described previously , at different ph values for 2 days at 37 c followed by 14 days at room temperature . as shown in figure 7 , the ir spectra of ttr ( 105115 ) feature a strong band at 1623 cm that is characteristic of -sheet structure . as with insulin , lysozyme , and het - s prion fibrils described above , the vcd sign of ttr ( 105115 ) reverses as a function of small changes in ph during protein aggregation . ttr ( 105115 ) fibrils formed at ph 2.0 and above show strong reversed vcd , and the intensity of the positive 1624 cm vcd band increases within increase of aggregation ph . similar to het - s fibrils , and opposite insulin and lysozyme , higher ph favors formation of the reversed vcd for ttr ( 105115 ) fibrils . the magnitude of reversed vcd intensities observed for these fibrils is nearly the same as those previously observed for reversed vcd insulin fibrils ( formed at low ph ) . by contrast , ttr ( 105115 ) fibrils grown at ph values 1.5 and 1.0 show normal vcd with their vcd intensities in the range 26 times smaller than the vcd intensities of reversed ttr ( 105115 ) fibrils . afm was applied to characterize morphologies of normal and reversed vcd ttr ( 105115 ) fibrils ( figure 8) . we found that normal and reversed vcd afm images of ttr ( 105115 ) display different morphologies . ttr ( 105115 ) fibrils that were grown at ph 1.5 are long and have cylindrical shapes . together with long rod - like fibrils we observed large amounts of small spherical species , which may be fibril precursors ( figure 8a ) . ttr ( 105115 ) fibrils grown at higher ph ( ph 2.5 ) are significantly shorter than fibrils grown at ph 1.5 . in addition to the diverse morphologies fibrils with normal and reversed vcd , ttr ( 105115 ) fibril polymorphs have different heights : normal ( ph 1.5 ) vcd fibrils are around 5 nm , while reversed vcd ( ph 2.0 ) fibrils are around 9 nm . afm images of ttr ( 105115 ) fibrils grown at ( a ) ph 1.5 and ( b ) ph 2.5 . more detailed surface analysis of ttr ( 105115 ) fibrils grown at ph 2.5 ( figure 8b ) indicates that some of them are composed of several side - by - side associated proto - fibrils . we did not observe any twist either on the surface of this chiral polymorph or on the surface of fibrils grown at ph 1.5 . flat - like topology of ttr ( 105115 ) fibrils grown at ph 2.0 , 37 c ( here reversed vcd ) was previously confirmed by jaroniec et al . it was shown that these fibrils are composed of several side - by - side associated proto - fibrils . the application of duvrr spectroscopy allowed us to elucidate the secondary structure organization of both normal and reversed vcd of ttr ( 105115 ) fibrils ( supporting information figure s7 ) . both chiral polymorphs of ttr ( 105115 ) fibrils have narrow and intense amide i , ii , and iii bands , which indicate that they share the same cross- core structure . we found that the intensity of the tyr band is also identical in both normal and reversed vcd ttr ( 105115 ) fibrils , indicating high similarities in tyr local environment of both polymorphs . with the results presented here , vcd intensity , enhanced by more than 1 order of magnitude over isolated protein levels , has been observed for three additional amyloid fibrils , namely apo--lactalbumin , the het - s ( 218289 ) prion - forming domain , and ttr ( 105115 ) peptide fragment from transthyretin . in a separate publication , similarly enhanced vcd has also been reported for a number of polyglutamine ( polyq ) fibrils from q15 to q45 . also with this paper , control of the sense of amyloid fibril chirality , normal versus reversed , by incubation under relatively small variations in ph , previously reported only for insulin , has been extended to lysozyme as well as apo--lactalbumin , het - s ( 218289 ) , and ttr ( 105115 ) . as with insulin , the sign pattern , defined above , for enhanced vcd intensity is ( + + + + ) for normal fibrils , and the nearly mirror - image relative intensity pattern with signs ( + ) for reversed fibrils reflects an underlying level of fibril chirality . of these signed bands , the band near 1620 cm , corresponding to the principal ir marker band for fibril protein structure , is the most intense and is the primary marker band for vcd fibril chirality . the principal focus of this paper is to correlate fibril supramolecular chirality , based on the sign pattern of enhanced vcd , to the two major classes of fibril morphology , either left - twisted or flat tape - like , as revealed by afm or sem imaging . the details of this correlation from results described in the previous section , as well as the earlier reported results for insulin , are summarized in table 1 for ease of comparison . for the proteins and peptide fragments reported here , each has a different sensitivity to ph although the sensitivity of lysozyme is similar to that of insulin : normal vcd is observed above approximately ph 2.2 and reversed vcd below that ph . though , no point of vcd sign reversal is reached between ph 1.5 and 4.0 . if it were to reverse sign pattern , it is predicted to do so only below ph 1.5 when the population of left - twisted fibrils becomes sufficiently dominant relative to flat tape - like , as reasoned further in the next paragraph . we have found that , at ph 1.5 , the relative populations of reversed tape - like fibrils and normal left - twisted fibrils marginally favor the left - twisted fibrils ( see table 1 and supporting information figure s5 ) ; however , the vcd spectrum indicates in figure 3 that reversed tape - like fibrils are more influential , perhaps because reversed fibrils in this region have intrinsically higher vcd intensity . at ph 2.5 , the reversed vcd increases , as the population of normal left - twisted fibrils decreases , in agreement with the idea that these two morphologies have opposite vcd and reduction of left - twisted fibrils permits an overall increase in the observed , net - reversed vcd spectrum . as the ph is increased to 3.0 and then to 4.0 , the population of left - twisted fibrils becomes close to zero , but unexpectedly the observed reversed vcd decreases dramatically from ph 2.5 to 3.0 and then to a relative small value for ph 4.0 . this reduction must solely be due to a reduction in the long - range chirality of the flat tape - like fibrils . from supporting information figure s5 the observed height distribution of the flat tape - like fibrils narrows considerably to near 6 nm with increasing ph . one possible explanation for these observations is that as the ph increases beyond ph 2.5 , the stability of the aggregation of right - handed ( reversed ) filaments that form multifilament tape - like fibrils decreases until at ph 4 only the lowest level of fibril structure remains , possibly only individual filaments having no lateral associations of filaments . beyond ph 4 , not even the simplest fibril filament structures are stable , and fibrillation of any kind no longer occurs . thus , although the ph dependence of apo--lactalbumin differs substantially from that of insulin and lysozyme , our understanding of the vcd and afm imaging for this protein still supports the evidence that the sign pattern for vcd correlates without exception to the morphology of the observed microscopic images of the fibrils grown at various ph values . the two peptide fragments , het - s ( 218289 ) and ttr ( 105115 ) , show the opposite vcd sign behavior compared with insulin and lysozyme , with normal fibrils appearing at low ph and reversed fibrils at high ph , although in these two cases the level of vcd enhancement differs dramatically . for the het - s ( 218289 ) peptide , the low - ph normal vcd is extremely large with high - ph reversed vcd significantly smaller , whereas for the ttr ( 105115 ) peptide the low - ph normal vcd is small and the high - ph reversed vcd is much larger . one possible influence beyond the value of ph was explored by introducing differing amounts of sodium chloride and thereby changing the ionic strength at a given ph value . experiments to date have shown no such influence of ionic strength ( supporting information figure s8 ) . another variable partially explored is the effect of incubation temperature , duration of incubation , and time after incubation . here variations in vcd intensity have been observed from which one concludes , as is well - known from many previous studies , that fibril formation , development , and stability can depend significantly on these variables . while studying the dependence of lysozyme fibril supramolecular chirality on incubation conditions we found that prolonged fibril exposure at the elevated temperature results in a decrease of vcd intensity . as previously demonstrated , fibril formation and development is associated with the continuous increase in the vcd intensity of the analyzed solution . since fibril fragmentation is the opposite process , it should be associated with a decrease in vcd intensity . therefore , one would expect that the observed decrease of fibril vcd intensity for lysozyme is indicative of fibril fragmentation during extended development . in fact , this propensity of fibrils has been extensively studied and proposed to be an intrinsic mechanism of fibril self - propagation , since each individual fibril fragment may act as a seed and give a birth to a new full - length fibril . in the results section , we noted on several occasions that the sign of the major vcd peak appears to shift from the region of 1620 cm to near 1610 cm . however , there is no corresponding shift in the peak maxima of the corresponding ir bands near 1620 cm . a highly likely explanation for the large frequency shift away from the vicinity of 1620 cm is that two opposite - signed vcd bands , with peak maxima only a few wavenumbers apart , interfere and give rise to a couplet of opposite signed vcd bands reduced in intensity but separated by a frequency difference related with the widths of the individual opposite signed vcd bands . such interference and splitting for peak maxima do not occur for the parent ir bands since their intensity contributions reinforce rather than partially cancel . combining these vcd observations with correlated afm images as summarized in table 1 reveals a correlation , without exception , of the normal vcd sign pattern with fibrils displaying a predominantly rounded left - twisted helical morphology and the reversed vcd sign pattern with fibrils having a flat tape - like morphology . for the flat tape - like fibrils there is no indication of fibril chirality in their afm or sem images . in the case of insulin , this correlation led us to propose that the enhanced , opposite - signed , mirror - image - like intensity pattern of vcd must have its origins at a deeper level of fibril structure than that visible for fibril morphology using afm or sem imaging . further , we proposed that opposite vcd sign patterns for normal and reversed fibrils originated at the level of individual fibril filaments , the simplest level of fibril cross- core structure , with filament axial helical twists of opposite sense , left - handed for normal fibrils and right - handed for reversed fibrils . it follows from this proposition that left - twisted filaments form braids of multiple - filament left - twisted fibrils with observable left - twisted morphology , while right - twisted fibrils do not braid and instead adopt a flat tape - like morphology consisting of parallel side - by - side filaments . to our knowledge , imaging the chirality of individual filaments has not been conclusively observed , and consequently , vcd to date is the only known structure probe of fibril chirality at the level of individual filaments . this point is reinforced by the fact that vcd can follow fibril formation as intensity enhancement from the earliest stages of filament formation , and that the enhanced vcd intensity grows without a substantial change in the relative intensity pattern as filaments first form , then associate and develop into mature fibrils . further in the case of insulin , on the basis of the observation that reversed fibrils spontaneously convert to normal fibrils upon raising the ph into the normal fibril ph range whereas normal fibrils do not convert to reversed fibrils when the ph is correspondingly reduced to the reversed fibril ph range , we have concluded that normal left - twisted mature fibrils are more thermodynamically stable , at least for insulin , than flat tape - like fibrils . this correlates with the idea that left - twisted filament - braided mature fibrils are in deeper thermodynamic sinks from which reversal or decomposition requires extremes of temperature or ph . we have also considered whether there is any correlation between the ph dependence of filament chirality and pi values for the protein / peptide and found that there appears to be little or no correlation between protein pi and ph dependence of filament chirality . for example , the pi for insulin is 5.305.35 , while for lysozyme the pi is 10.610.9 . nevertheless , both of these proteins exhibit nearly the same ph dependent fibril chirality . at the same time , the pi of het - s ( 218289 ) is 9.19 , while it exhibits the opposite ph chirality dependence of lysozyme ( pi 10.610.9 ) . for apo--lactalbumin the pi ( 4.24.5 ) is very close to the pi of insulin ( 5.305.35 ) ; however , we observed formation of only one chiral polymorph for apo--lactalbumin . finally , het - s ( 218289 ) and ttr ( 105115 ) exhibit similar ph dependence of fibril chirality ( normal at low ph and reversed at high ph ) . nevertheless , their pis are substantially different : 9.19 for het - s ( 218289 ) and 6.09 for ttr ( 105115 ) . the only correlation might be that the substantial difference in pi s of apo--lactalbumin ( 4.24.5 ) and lysozyme ( pi 10.610.9 ) might be related to the difference in ph chirality dependence for these two isostructural proteins in their native states . although the focus here is to relate the sign pattern of the enhanced vcd to the major classes of fibril morphology , inspection of the ir absorption spectra for fibrils described in figures 1 , 3 , 5 , and 7 reveals variations in a band in the 16501670 region that also shows intensity differences that correlate with vcd sign and fibril morphology . not as dramatic as the vcd sign pattern , there is likely interesting information about the differences between left - twisted and flat tape - like fibrils associated with these ir bands . in the results section for lysozyme we attributed this band to nonordered or loop structure . an open question to be pursued in future studies is whether this band is associated with a fully formed fibril filament ( cross--core ) or with partially unfolded proteins yet to be incorporated into the filament or fibril structure . at least for lysozyme and insulin ( not shown here ) , this band may be associated with parts of the protein structure not in the cross--core for which there would be more residues than for the shorter peptides from het - s and ttr . finally , examination of the structural organization of the opposite chiral polymorphs using duvrr and ir spectroscopy shows that insulin , lysozyme , and ttr ( 105115 ) fibril polymorphs have the same secondary structure . at the same time , we have found that normal and reversed vcd chiral polymorphs of het - s ( 218289 ) fibrils have small differences secondary structure distributions with the same cross--core structure . although the primary findings of this paper are drawn from new experimental results , namely vcd spectra and afm / sem images as a function of incubation ph for four protein molecules beyond our previously published findings for insulin , we offer in this section our views of the theoretical , molecular - level implications for what the experimental evidence suggests . the theoretical model of schweitzer - stenner is the first and simplest computational model of enhanced vcd in fibrils , first observed by ma et al . the model is based on exciton coupling among amide i transition dipoles arrayed as dual , stacked -sheet ribbons corresponding to the simplest representation of the structure of a single cross - beta core filament . the strands in this model , only two peptide units long , run perpendicular to the filament axis direction . in order to generate enhanced vcd , a twist of 2 was introduced between the strands yielding a long - range gradual helical twist to the filament structure with a one full - turn distance of 180 strands . it was found that such a structure yields enhanced vcd that corresponds to what is referred to as normal vcd ( negative vcd near 1620 cm with positive vcd to higher wavenumber frequencies ) for a left - hand helical fibril twist . a corresponding 2 intrastrand helical twist in the opposite direction , corresponding to a right - hand helical filament , reversed the sign of the enhanced vcd couplet and corresponds to our reversed vcd . filament chirality is therefore a very delicate , subtle level of protein chirality that is apparently sensitive to incubation ph . most likely , the sensitivity of amino - acid side chains to the aqueous solvent is responsible for this ph sensitivity , and apparently , differing arrays of constituent side - chains , exposed or not to the solvent , results in different ph sensitivity ( high versus low ) for the sense of filament chirality observed . this is a long - range fibril property that likely can not be predicted , even qualitatively , without a realistic model of protein side chain influence on the sense of helical chirality as a function of ph . as far as we are aware , the chirality of individual fibril filaments lies below the sensitivity of afm or sem imaging , but can be observed with vcd at the initial and subsequent stages of fibril formation . it is known from afm and sem images that fibril filaments form higher levels of structure subsequent to the formation of individual filaments . it has been demonstrated , for example by kinetic studies , that the degree of enhancement of vcd is sensitive to the degree of filament association as fibrils mature and develop . this is particularly true for the left - twisted morphologies composed of multifilament chiral braids of left - helical filaments and to a lesser degree for the flat morphologies of side - by - side associations of right - helical filaments . an important unanswered question is why only left - helical filaments form braids corresponding to the left - twisted fibril morphology as illustrated diagrammatically in figure 9 . the answer most likely is that left- and right - helical filaments are not mirror - image structures as both consist of l - amino acid residues . a model calculation of this property , the appearance of left - twisted braids and nonappearance of right - twisted braids of helical filaments , again involves consideration of the structure and influence of amino acid side chains . the model of schweitzer - stenner applies only to the filament level of structure and does not include any side chain chirality ( l- or d - amino acid residues ) or filament filament interactions or braiding . answering the question of why only left - twisted fibril images and vcd are observed is two levels beyond the schweitzer - stenner ( s - s ) model of enhanced vcd . one first needs a more realistic model of cross--core filament structure that includes side chain detail , and second , one needs to extend such a model to a new , higher level of fibril chirality , namely the chiral braiding of multiple - filament fibril structure . diagram illustrating the progression from folded protein to individual straight filament with chirality below the senstivity of afm / sem and on to mature multifilament fibrils with right helical filaments forming flat tape - like fibrils of straight filaments and left helical filaments forming , possibly more stable , left twisted braids of filaments . the ph dependence may vary between proteins / peptides but not the vcd intensity patterns ( normal left helical filaments versus reversed for right helical filaments ) . a step in this direction has just been published by welch , kubelka , and keiderling ( wkk ) . they employ modeling at the level of a full dft calculation with transfer of parameters from smaller peptides to build large -sheet structures . their model is quite different in dimension from the ( s - s ) model although both use stacked -sheets to simulate the cross--core structure . the wkk model involves strands that are 10 residues instead of 2 residues wide , but the fibril axis is only 5 strands long rather than in the 100-strand range . to induce enhanced vcd , they introduce a twist along the strand direction , not the fibril axis direction , of 15 between residues . they also explore a rotation of the stacked sheets relative to each of 45 , again a chiral departure from an initial achiral cross- core structure . these two degrees of chiral freedom may be important as filaments aggregate and braid about a common axis . the point needs to be explored as the models move beyond individual filament models and on to multifilament simulations of more mature fibrils . nevertheless , it seems reasonable that chirality along the filament axis , as emphasized in this paper ( see figure 9 ) , as opposed to the more local strand or intersheet twist chirality , is the form of filament chirality that can be extended most easily to the supramolecular level of filament structure and hence to vcd enhanced by up to 2 orders of magnitude relative to the isolated - protein vcd intensity level . at this stage , even with the recent work of wkk , there are no structural models of interfilament association leading to the formation of different fibril morphologies . it is our view that the realization of a model , even at the course grain level , that is capable of rationalizing the correlation of left - helical filaments to left - twisted fibril morphology and right - helical filaments to flat fibril morphology , is beyond the reach of near - term possibilities and must await further advances in our understanding of the unusual sensitivity of vcd to filament and mature fibril chirality as well as the many chiral degrees of freedom that may influence fibril morphology . two further observations relevant to this discussion have been established previously for insulin and remain to be confirmed more systematically for the new fibril results presented here . as fibrils grow by adding filaments and becoming thicker , the vcd grows from initially observed levels , whereas the ir intensity does not grow in overall magnitude . this is more dramatically true for the braided left - twisted morphology where an additional order of magnitude of vcd may be observed compared to the initial enhanced vcd of newly formed filaments . this strongly implies a second level of chirality beyond the filament level that is ir intensity neutral . second , the left twisted morphology is more stable than the flat tape - like morphology . thus , the interfilament interactions of the left - twisted braided fibril consisting of left - helical filaments are stronger than the corresponding side - by - side interactions of the flat tape - like fibrils composed of right - helical filaments . a successful model of fibril morphology and associated vcd intensity should reflect this difference in thermodynamic stability . the results of the present study , and the absence of any observable chirality by afm for flat tape - like fibrils , extends , beyond the case of insulin , the correlation of the sign of vcd intensity pattern with the two major classes of amyloid fibril morphology , round left - twisted and flat tape - like . also extended by this study ( to fibrils of four new protein or peptide fragments ) is the hypothesis that the near mirror - like opposite - signed vcd patterns associated with these two morphologies arise from the opposite sense of helical twist of individual fibril filaments that comprise observed multifilament , more - fully developed fibrils . further , for all five analyzed amyloid fibrils documented in table 1 , filament chirality appears to lie below the level of direct detection of afm or sem imaging . finally , there is wide variability in the ph dependence of filament chirality , being one sense ( reversed , right - handed at lower ph and normal , left - handed at higher ph ) for the proteins insulin , lysozyme , and apo--lactalbumin , and the opposite sense ( normal , left - handed at lower ph and reversed right - handed at higher ph ) for the peptide fragments het - s ( 218289 ) from prion protein , and ttr ( 105115 ) from transthyretin . we have investigated whether there is any correlation between ph dependence of filament chirality and the pi of the protein / peptide and found no direct correlation . thus , we have shown extensive new evidence that supports an affirmative answer to the question posed in the title of this paper . namely , yes , the chirality of individual amyloid fibril filaments does indeed appear to be the underlying cause of the major morphological differences ( round twisted versus flat tape - like ) in all amyloid fibrils explored to date by vcd . nevertheless , the protein / peptide fibrils sampled for morphology and vcd is so far limited to five , albeit somewhat diverse , cases , and there well could be surprises and exceptions in future studies . a final understanding of the correlations reported here must await sufficiently realistic and accurate modeling of filament and fibril chirality together with the calculation of the associated vcd spectra .

the unique enhanced sensitivity of vibrational circular dichroism ( vcd ) to the formation and development of amyloid fibrils in solution is extended to four additional fibril - forming proteins or peptides where it is shown that the sign of the fibril vcd pattern correlates with the sense of supramolecular filament chirality and , without exception , to the dominant fibril morphology as observed in afm or sem images . previously for insulin , it has been demonstrated that the sign of the vcd band pattern from filament chirality can be controlled by adjusting the ph of the incubating solution , above ph 2 for normal left - hand - helical filaments and below ph 2 for reversed right - hand - helical filaments . from afm or sem images , left - helical filaments form multifilament braids of left - twisted fibrils while the right - helical filaments form parallel filament rows of fibrils with a flat tape - like morphology , the two major classes of fibril morphology that from deep uv resonance raman scattering exhibit the same cross--core secondary structure . here we investigate whether fibril supramolecular chirality is the underlying cause of the major morphology differences in all amyloid fibrils by showing that the morphology ( twisted versus flat ) of fibrils of lysozyme , apo--lactalbumin , het - s ( 218289 ) prion , and a short polypeptide fragment of transthyretin , ttr ( 105115 ) , directly correlates to their supramolecular chirality as revealed by vcd . the result is strong evidence that the chiral supramolecular organization of filaments is the principal underlying cause of the morphological heterogeneity of amyloid fibrils . because fibril morphology is linked to cell toxicity , the chirality of amyloid aggregates should be explored in the widely used in vitro models of amyloid - associated diseases .

Introduction Results Discussion Theoretical Considerations Conclusions

specific fibril aggregation of more than 20 proteins has been linked to a pathogenesis of many amyloid - associated diseases such as alzheimer s disease , parkinson s disease , huntington s diseases , and prion diseases . amyloid fibrils are -sheet rich protein aggregates with different morphologies that constitute a phenomenon known as fibril polymorphism.in vitro aggregation of various amyloid associated proteins , such as calcitonin , amylin , insulin , transthyretin , and mouse prion , commonly leads to the formation of two distinct fibril morphologies : tape - like and helical - twisted . however , if the ph of the aggregation solution is lower than 2.4 , the distribution of fibrils shifts to increasingly flat , tape - like , or binary fibrils as the incubation ph continues to be lowered that under microscopic examination show no noticeable chirality or twist on their surface . the fact that flat tape - like fibrils show a strong reversed vcd signal indicates that they must be composed of right - handed filaments , the chirality of which lies below the limit of afm or sem detection . the key question we answer in this paper is whether the supramolecular chirality of the filaments is the underlying cause of morphological differences only for insulin fibrils , or whether it is a more general phenomenon that determines the polymorphism of other amyloidogenic proteins and peptides . to address these questions we studied aggregation as a function of incubation ph for hen - egg lysozyme , bovine apo--lactalbumin , het - s ( 218289 ) prion - forming domain from fungus podospora anserina , and a short 11-residue peptide fragment of human transthyretin ( ttr ( 105115 ) ) . we found that relatively small variations in the ph of the aggregation solution cause dramatic changes in the vcd spectra of lysozyme fibrils , the largest of which is almost 10 times more intense than the corresponding intense vcd spectra observed for insulin fibrils grown under the same ph values . tape - like fibrils had 26 nm width and 5 nm height , while left - twisted fibrils had the same width but were over twice as high ( 12 nm ) . fluid - cell afm images of left - twisted ( a ) and tape - like ( b ) fibrils of apo--lactalbumin grown for 3 days at 37 c . het - s ( 218289 ) peptide fragment of prion protein forms opposite chiral polymorphs at different ph values . several independent studies have shown that ( het - s 218289 ) aggregation at ph 2.0 leads to a formation of thick left - twisted cables composed of 36 filaments . in our study we investigated supramolecular chirality of het - s ( 218289 ) prion fibrils grown at ph 2.0 , 3.3 , and 3.9 ( figure 6 ) . het - s ( 218289 ) prion fibrils grown at ph 3.9 exhibit the same reversed sign pattern but have some differing vcd band frequencies : 1557 ( + ) , 1614 ( + ) , and 1660 ( ) cm . vcd ( a ) and ir ( b ) spectra of het - s ( 218289 ) fibrils grown at ph 2.0 ( blue ) , 3.3 ( green ) , and 3.9 ( black ) for 2 months at room temperature . afm images of het - s ( 218289 ) prion fibrils grown in ph 2.0 ( a ) and 3.9 ( b ) . at the same time , we found that het - s fibrils that were grown at ph 3.9 have substantially higher amount of unordered structure ( a peak 1268 cm ) compared to the ph 2.0 fibrils . afm was utilized to reveal morphological organization of the analyzed het - s ( 218289 ) prion fibrils . ttr ( 105115 ) is a peptide fragment of transthyretin , a 55 kda protein involved in the transport pathway of thyroxine and retinol in plasma . previously , it was demonstrated that ttr ( 105115 ) is able to aggregate in vitro , forming amyloid - like fibrils . as with insulin , lysozyme , and het - s prion fibrils described above , the vcd sign of ttr ( 105115 ) reverses as a function of small changes in ph during protein aggregation . ttr ( 105115 ) fibrils formed at ph 2.0 and above show strong reversed vcd , and the intensity of the positive 1624 cm vcd band increases within increase of aggregation ph . similar to het - s fibrils , and opposite insulin and lysozyme , higher ph favors formation of the reversed vcd for ttr ( 105115 ) fibrils . by contrast , ttr ( 105115 ) fibrils grown at ph values 1.5 and 1.0 show normal vcd with their vcd intensities in the range 26 times smaller than the vcd intensities of reversed ttr ( 105115 ) fibrils . in addition to the diverse morphologies fibrils with normal and reversed vcd , ttr ( 105115 ) fibril polymorphs have different heights : normal ( ph 1.5 ) vcd fibrils are around 5 nm , while reversed vcd ( ph 2.0 ) fibrils are around 9 nm . with the results presented here , vcd intensity , enhanced by more than 1 order of magnitude over isolated protein levels , has been observed for three additional amyloid fibrils , namely apo--lactalbumin , the het - s ( 218289 ) prion - forming domain , and ttr ( 105115 ) peptide fragment from transthyretin . also with this paper , control of the sense of amyloid fibril chirality , normal versus reversed , by incubation under relatively small variations in ph , previously reported only for insulin , has been extended to lysozyme as well as apo--lactalbumin , het - s ( 218289 ) , and ttr ( 105115 ) . as with insulin , the sign pattern , defined above , for enhanced vcd intensity is ( + + + + ) for normal fibrils , and the nearly mirror - image relative intensity pattern with signs ( + ) for reversed fibrils reflects an underlying level of fibril chirality . the principal focus of this paper is to correlate fibril supramolecular chirality , based on the sign pattern of enhanced vcd , to the two major classes of fibril morphology , either left - twisted or flat tape - like , as revealed by afm or sem imaging . if it were to reverse sign pattern , it is predicted to do so only below ph 1.5 when the population of left - twisted fibrils becomes sufficiently dominant relative to flat tape - like , as reasoned further in the next paragraph . we have found that , at ph 1.5 , the relative populations of reversed tape - like fibrils and normal left - twisted fibrils marginally favor the left - twisted fibrils ( see table 1 and supporting information figure s5 ) ; however , the vcd spectrum indicates in figure 3 that reversed tape - like fibrils are more influential , perhaps because reversed fibrils in this region have intrinsically higher vcd intensity . at ph 2.5 , the reversed vcd increases , as the population of normal left - twisted fibrils decreases , in agreement with the idea that these two morphologies have opposite vcd and reduction of left - twisted fibrils permits an overall increase in the observed , net - reversed vcd spectrum . as the ph is increased to 3.0 and then to 4.0 , the population of left - twisted fibrils becomes close to zero , but unexpectedly the observed reversed vcd decreases dramatically from ph 2.5 to 3.0 and then to a relative small value for ph 4.0 . this reduction must solely be due to a reduction in the long - range chirality of the flat tape - like fibrils . one possible explanation for these observations is that as the ph increases beyond ph 2.5 , the stability of the aggregation of right - handed ( reversed ) filaments that form multifilament tape - like fibrils decreases until at ph 4 only the lowest level of fibril structure remains , possibly only individual filaments having no lateral associations of filaments . thus , although the ph dependence of apo--lactalbumin differs substantially from that of insulin and lysozyme , our understanding of the vcd and afm imaging for this protein still supports the evidence that the sign pattern for vcd correlates without exception to the morphology of the observed microscopic images of the fibrils grown at various ph values . the two peptide fragments , het - s ( 218289 ) and ttr ( 105115 ) , show the opposite vcd sign behavior compared with insulin and lysozyme , with normal fibrils appearing at low ph and reversed fibrils at high ph , although in these two cases the level of vcd enhancement differs dramatically . for the het - s ( 218289 ) peptide , the low - ph normal vcd is extremely large with high - ph reversed vcd significantly smaller , whereas for the ttr ( 105115 ) peptide the low - ph normal vcd is small and the high - ph reversed vcd is much larger . as previously demonstrated , fibril formation and development is associated with the continuous increase in the vcd intensity of the analyzed solution . in the results section , we noted on several occasions that the sign of the major vcd peak appears to shift from the region of 1620 cm to near 1610 cm . combining these vcd observations with correlated afm images as summarized in table 1 reveals a correlation , without exception , of the normal vcd sign pattern with fibrils displaying a predominantly rounded left - twisted helical morphology and the reversed vcd sign pattern with fibrils having a flat tape - like morphology . for the flat tape - like fibrils there is no indication of fibril chirality in their afm or sem images . in the case of insulin , this correlation led us to propose that the enhanced , opposite - signed , mirror - image - like intensity pattern of vcd must have its origins at a deeper level of fibril structure than that visible for fibril morphology using afm or sem imaging . further , we proposed that opposite vcd sign patterns for normal and reversed fibrils originated at the level of individual fibril filaments , the simplest level of fibril cross- core structure , with filament axial helical twists of opposite sense , left - handed for normal fibrils and right - handed for reversed fibrils . it follows from this proposition that left - twisted filaments form braids of multiple - filament left - twisted fibrils with observable left - twisted morphology , while right - twisted fibrils do not braid and instead adopt a flat tape - like morphology consisting of parallel side - by - side filaments . further in the case of insulin , on the basis of the observation that reversed fibrils spontaneously convert to normal fibrils upon raising the ph into the normal fibril ph range whereas normal fibrils do not convert to reversed fibrils when the ph is correspondingly reduced to the reversed fibril ph range , we have concluded that normal left - twisted mature fibrils are more thermodynamically stable , at least for insulin , than flat tape - like fibrils . at the same time , the pi of het - s ( 218289 ) is 9.19 , while it exhibits the opposite ph chirality dependence of lysozyme ( pi 10.610.9 ) . finally , het - s ( 218289 ) and ttr ( 105115 ) exhibit similar ph dependence of fibril chirality ( normal at low ph and reversed at high ph ) . nevertheless , their pis are substantially different : 9.19 for het - s ( 218289 ) and 6.09 for ttr ( 105115 ) . although the focus here is to relate the sign pattern of the enhanced vcd to the major classes of fibril morphology , inspection of the ir absorption spectra for fibrils described in figures 1 , 3 , 5 , and 7 reveals variations in a band in the 16501670 region that also shows intensity differences that correlate with vcd sign and fibril morphology . not as dramatic as the vcd sign pattern , there is likely interesting information about the differences between left - twisted and flat tape - like fibrils associated with these ir bands . finally , examination of the structural organization of the opposite chiral polymorphs using duvrr and ir spectroscopy shows that insulin , lysozyme , and ttr ( 105115 ) fibril polymorphs have the same secondary structure . at the same time , we have found that normal and reversed vcd chiral polymorphs of het - s ( 218289 ) fibrils have small differences secondary structure distributions with the same cross--core structure . a corresponding 2 intrastrand helical twist in the opposite direction , corresponding to a right - hand helical filament , reversed the sign of the enhanced vcd couplet and corresponds to our reversed vcd . most likely , the sensitivity of amino - acid side chains to the aqueous solvent is responsible for this ph sensitivity , and apparently , differing arrays of constituent side - chains , exposed or not to the solvent , results in different ph sensitivity ( high versus low ) for the sense of filament chirality observed . as far as we are aware , the chirality of individual fibril filaments lies below the sensitivity of afm or sem imaging , but can be observed with vcd at the initial and subsequent stages of fibril formation . it is known from afm and sem images that fibril filaments form higher levels of structure subsequent to the formation of individual filaments . this is particularly true for the left - twisted morphologies composed of multifilament chiral braids of left - helical filaments and to a lesser degree for the flat morphologies of side - by - side associations of right - helical filaments . an important unanswered question is why only left - helical filaments form braids corresponding to the left - twisted fibril morphology as illustrated diagrammatically in figure 9 . a model calculation of this property , the appearance of left - twisted braids and nonappearance of right - twisted braids of helical filaments , again involves consideration of the structure and influence of amino acid side chains . diagram illustrating the progression from folded protein to individual straight filament with chirality below the senstivity of afm / sem and on to mature multifilament fibrils with right helical filaments forming flat tape - like fibrils of straight filaments and left helical filaments forming , possibly more stable , left twisted braids of filaments . nevertheless , it seems reasonable that chirality along the filament axis , as emphasized in this paper ( see figure 9 ) , as opposed to the more local strand or intersheet twist chirality , is the form of filament chirality that can be extended most easily to the supramolecular level of filament structure and hence to vcd enhanced by up to 2 orders of magnitude relative to the isolated - protein vcd intensity level . it is our view that the realization of a model , even at the course grain level , that is capable of rationalizing the correlation of left - helical filaments to left - twisted fibril morphology and right - helical filaments to flat fibril morphology , is beyond the reach of near - term possibilities and must await further advances in our understanding of the unusual sensitivity of vcd to filament and mature fibril chirality as well as the many chiral degrees of freedom that may influence fibril morphology . thus , the interfilament interactions of the left - twisted braided fibril consisting of left - helical filaments are stronger than the corresponding side - by - side interactions of the flat tape - like fibrils composed of right - helical filaments . the results of the present study , and the absence of any observable chirality by afm for flat tape - like fibrils , extends , beyond the case of insulin , the correlation of the sign of vcd intensity pattern with the two major classes of amyloid fibril morphology , round left - twisted and flat tape - like . finally , there is wide variability in the ph dependence of filament chirality , being one sense ( reversed , right - handed at lower ph and normal , left - handed at higher ph ) for the proteins insulin , lysozyme , and apo--lactalbumin , and the opposite sense ( normal , left - handed at lower ph and reversed right - handed at higher ph ) for the peptide fragments het - s ( 218289 ) from prion protein , and ttr ( 105115 ) from transthyretin . namely , yes , the chirality of individual amyloid fibril filaments does indeed appear to be the underlying cause of the major morphological differences ( round twisted versus flat tape - like ) in all amyloid fibrils explored to date by vcd .

hypertension ( htn ) and type 2 diabetes mellitus ( t2 dm ) are common diseases associated with obesity and the metabolic syndrome . approximately 26 million people in the united states have diabetes and it is estimated that 67% of those individuals also have htn . independently of t2 dm , htn affects approximately 30% of the us adult population . htn and t2 dm share several common vascular complications such as coronary artery disease , renal disease , and stroke . htn and t2 dm are strong predictors of cardiovascular disease and can also have a significant impact on brain health . white matter ( wm ) health is inversely associated with age . whereas t2 dm is frequently associated with volume changes , particularly in the hippocampus [ 6 , 7 ] , both htn and t2 dm are known to result in similarly prominent effects on the integrity of wm [ 8 , 9 ] . in experimental rodent models , diabetes and htn have been individually shown to have a differential impact on both cerebral gray and white matter . the impact of htn on the brain might be further complicated by co - morbid t2 dm [ 6 , 11 ] . given that retinal and cerebral microvessels share developmental and physiological controls , it is no surprise that previous studies link retinal vessel integrity to certain aspects of brain health . in particular , narrowed retinal arteriolar diameters are robust indicator of cerebrovascular health and have been found to be a risk factor for htn . moreover , there is evidence that retinal vessel diameter is affected by insulin resistance and is related to brain atrophy [ 16 , 17 ] . however , the role of arterial vessel health in explaining wm integrity in the presence of htn and/or t2 dm remains to be further developed . first , by using a novel and statistically conservative voxelwise analysis method , and after accounting for wm hyperintensities , we wanted to ascertain by using diffusion tensor imaging ( dti ) which areas of wm had reductions in microstructural integrity associated with htn . secondly , we wanted to evaluate whether t2 dm has additive deleterious effects on wm health in the presence of htn . finally , given that retinal vessel integrity can be used as a noninvasive marker of cerebral vascular health [ 17 , 18 ] , we also wanted to ascertain whether there are relationships between retinal arteriolar integrity and the anticipated reductions in dti - based cerebral wm integrity among hypertensive individuals . sixty two middle - aged and elderly community - residing individuals , 33 fulfilling criteria for htn ( see below for definition ) and 29 not , were consecutively recruited via web advertisements and referrals from collaborating endocrinologist as part of an ongoing nih - sponsored study of the effects of diabetes and aging on the brain . all participants had a minimum of a high school education ( 1221 years for both groups ) and no impairments in their day - to - day functioning and were medically stable ( other than some of them having diabetes , hypertension , or dyslipidemia ) and free of psychiatric illness or significant vascular disease . individuals were excluded if they had a history of current insulin treatment , so as to avoid the possible confounding effects of severe hypoglycemic episodes , or if they had uncontrolled hypertension ( blood pressure ( bp ) > 150/90 mm / hg ) , significant coronary ischemic disease detected on the electrocardiogram or a modified hachinski ischemia scale score > 4 , any focal neurological signs , current diagnosis or past history of stroke or significant head trauma , or evidence of tumor on the structural mr scan . in an extensive 8-hour evaluation , performed over 3 separate days , we collected medical , psychiatric , and endocrine data and performed an mri of the brain . the psychiatric evaluation ensured that our population did not include significant psychiatric illness such as depression or evidence of a degenerative process . the patients were also free of significant vascular disease ( hachinski score below 4 ; ) . the new york university school of medicine internal review board approved this study and each patient gave informed written consent . individuals with t2 dm met one or more of the following criteria : ( 1 ) fasting blood glucose 6.99 mmol / l on two separate occasions or ( 2 ) 2-hour blood glucose level > 11.10 mmol / l during a 75 g oral glucose tolerance test or ( 3 ) a prior diagnosis of t2 dm . nondiabetic participants had normal fasting glucose levels and showed no evidence of obvious insulin resistance ( ir ) as demonstrated by their fasting glucose and insulin levels and on the intravenous glucose tolerance test . participants were classified as having htn if they were receiving anti - htn pharmacological treatment or had a sitting bp above the ncep cut - off ( systolic bp 130 mmhg or a diastolic bp 85 mmhg ) . bp was determined using a manual sphygmomanometer by a physician during the second visit after an oral or intravenous glucose tolerance test . briefly , the participant 's eyes are allowed to acclimate to a dimly lit room for 10 minutes in order to nonpharmacologically dilate the pupil sufficiently to view the optic disk and the retinal vessels . the images were then loaded on to a computer , cropped , red - free filtered , and contrast - enhanced by grayscale conversion . using in - house image analysis software an outline of the optic disk the operator then creates two more concentric circles at 0.5 and 1.0 disc diameters out from the original optic disk outline . this creates two zones , one between the original disc outline and the 0.5 outline and one between the 0.5 outline and the 1.0 outline . the larger of the two zones ( between the 0.5 and 1.0 disc outlines ) is where the arterioles and venules are identified for measurement . in this zone straight segments of vessel are selected and five equidistant lines orthogonal to the vessel are applied . in an automated fashion , as the vessel is traversed orthogonal to its long axis , the pixel intensity is used to calculate the average width of the vessel . this process is repeated on the 6 largest arterioles and 6 largest venules in the selected zone . to account for branching coefficient , these measurements are combined using the revised parr - hubbard formula to produce the central vessel equivalent . in our analyses of these data , we accounted for individual variations in vessel size by residualizing the arteriolar diameters to venular diameters . t1-weighted magnetization - prepared rapid acquisition gradient echo ( mprage ) sequence ( tr 1300 ms ; te 4.38 ms ; ti 800 ms ; fov 250 250 ; 192 coronal slices ; slice thickness 1.2 mm ; nex 1 ; flip angle 15 ) was used for structural imaging . t2-weighted sequence ( tr 9000 ms ; te 94 ms ; ti 2000 ms ; fov 210 210 ; 50 axial slices ; slice thickness 3 mm ) was used to spatially correct dti images ( please see section 2.6 ) . in order to account for primary neurological and wm disease , fluid attenuated inversion recovery ( flair ; tr 9000 ms ; te 97 ms ; fov 210 210 ; 1 average and 2 concatenations ; 50 axial slices ; slice thickness 3 mm ; flip angle 145 ) sequence was combined with the mprage and assessed using the modified fazekas scale . a dti echo planar sequence ( tr 6100 ms ; te 75 ms ; delay in tr = 0 ; b values 0 , 1000 ; 6 directions ; fov 210 210 ; 4 averages and 1 concatenation ; 50 axial slices ; voxel size 1.64 1.64 3 mm ) was acquired in order to analyze fractional anisotropy ( fa ) . dti images were utilized to generate fa maps that were then registered to a structurally relevant mprage image using automated registration toolkit 2 ( art2 ) as previously described [ 9 , 22 ] . in short , these images were then normalized to the talairach space to obtain a 3d warp field that includes transformation parameters to change the mprage to target image . an mprage transformation matrix was obtained by applying a rigid body registration of the t2-weighted volume to the mprage volume . another transformation matrix was created to correct for registration errors caused by the participant 's head movements . next , using the skull - stripped t2 image as a guide , the b0 image was iteratively warped to correct for spatial distortions and that produced a 2d warp field with spatial transformation and distortion correction parameters . the fa values and overlay maps were generated from the native dti images and then these maps were spatially corrected and normalized to standard space using the combination of the four transformations obtained in the previous steps . furthermore , the flair image , which is very sensitive to wm hyperintensities , was also used as a covariate to control for nonclinically relevant very subtle wm hyperintensities after the image had been normalized to the target template using the same parameters applied to the fa maps . to examine group differences , the chi - square test of independence was utilized for data in percentage and nominal variables . in order to adjust for overall vessel diameter , retinal arteriolar diameter was subjected to a linear regression analysis with retinal venular diameter to produce a residual mean of arteriolar diameter that accounts for individual differences in vessel diameter . a two - way factorial anova evaluated the contributions of htn ( htn versus normotensive ) and t2 dm ( t2 dm versus no t2 dm ) and their interaction effect on retinal arteriolar diameter , after accounting for age and bmi . the group fa maps were created by averaging the corrected and normalized fa maps and two - tailed voxelwise analysis of covariance ( vancova ) analyses were performed with age and wm hyperintensities on the flair image as covariates . given that htn is highly associated with obesity , we also controlled for bmi in those analyses . the association between wm fa and retinal arteriolar diameter residualized to venular diameter was evaluated among individuals with htn using a voxelwise correlation analysis with age and bmi as covariates . a wm mask was used to restrict all voxelwise fa analyses to wm regions . in an effort to reduce the likelihood of making type i errors and to adjust for multiple comparisons , we limited the accepted voxels showing statistical significance to those having at least 100 contiguous significant voxels in the same direction ( equivalent to at least 0.1 cc in volume ) . we also chose a conservative false discovery rate of less than one percent utilizing procedures previously described . the hypertensive and normotensive groups were well matched on age and did not differ on sex or ethnicity distribution . descriptors and endocrine data can be found on table 1 . as expected the hypertensive group had significantly higher rates of obesity and t2 dm . despite the fact that 29/33 of our hypertensive participants were on antihypertensive medication , they still had significantly higher systolic but not diastolic bp than the group without htn . consistent with the existing literature linking obesity and htn , we found that our hypertensive participants had significantly higher bmi than our normotensive participants . of those participants who also had a diagnosis of t2 dm ( 22 in the htn and 8 in the normotensive group , resp . ) , all but one in the normotensive group were on diabetes medications . the groups did not differ in lipid concentrations , likely due to the fact that significantly more hypertensive participants ( 22/33 ) as opposed to only 2/29 in the normotensive group were being treated with cholesterol - lowering medications . relative to normotensive participants , those with htn had significantly higher ratings of periventricular wm hyperintensities ( pwmh ) and higher , though not statistically significant , ratings of deep wm hyperintensities ( dwmh ; see table 1 ) . additionally , we wanted to explore whether there were subtle changes in wm microstructure even after accounting for those wm hyperintensities seen on the flair image by using a voxelwise dti fa approach . one control case was excluded from the dti fa analysis due to uncorrectable spatial distortions on the fa map , leaving 33 hypertensive and 28 normotensive participants for those group comparisons . since both bp groups had individuals with t2 dm , which is associated with an increased incidence of wm lesions , in addition to age , bmi , and the flair image as covariates we also controlled for a diagnosis of t2 dm in the dti fa analyses . the result revealed a total of 10 clusters of significant group differences ( overall 2,747 voxels or 2.75 cc in volume ; p < 0.01 ; table 2 ) , seven of which showed fa reduction among participants with htn relative to the normotensive ones ( 2,344 voxels or 2.34 cc in volume ) . the largest five clusters showing fa reductions among hypertensive participants were located bilaterally ( 2,121 voxels ) in the internal and external capsules ( ic and ec ; see figure 1 ) and the remaining two were found in the right prefrontal region ( 114 voxels ) and the left cerebellar peduncle ( 109 voxels ) . the three clusters showing higher fa values among hypertensive participants were relatively small and were located in regions where there were substantial gray matter contaminations . to ascertain whether t2 dm contributed to further wm damage among hypertensive participants , the hypertensive group was further divided based on whether or not they also had t2 dm ( 22 with htn and t2 dm versus 11 with just htn ; see table 3 for group descriptors ) . in addition to having significantly higher fasting glucose and hemoglobin a1c ( hba1c ) , the hypertensive group with t2 dm also had significantly higher bmi . the groups were comparable in bp , which is not surprising , as individuals with both htn and t2 dm are more likely to receive rigorous pharmacological treatment . all participants with htn and co - morbid t2 dm were receiving antihypertensive medications whereas fewer , 7/11 , of those without t2 dm were being treated pharmacologically . despite the fact that more participants with htn and t2 dm were on cholesterol - lowering pharmacological treatment ( 16/22 versus 6/11 ) , their high - density lipoprotein ( hdl ) levels were significantly lower ( p < 0.05 ) and their triglyceride levels were higher , although not statistically significant , than those of individuals without t2 dm ( p > 0.39 ) . there were no differences in the fazekas scores between the groups ( see table 3 ) . nevertheless , the voxelwise fa comparisons uncovered a total of sixteen clusters all showing wm fa reduction ( overall 4,021 voxels or 4.02 cc in volume ; p < 0.01 ) among participants with htn and t2 dm relative to those with only htn , independent of age , bmi , and wm hyperintensities on the flair image , thus suggesting a widespread distribution of wm abnormalities ( see table 4 and figure 2 for those clusters of 200 voxels in size ) . similar to the results reported above for the htn yes / no group comparison , significant substantive number of clusters were identified in the ic and ec ( total 988 voxels ) . also , two clusters were found bilaterally in the arcuate fasciculi ( total 645 voxels ) , which connect the frontal and temporal lobes . two clusters were identified bilaterally in the parietal lobe ( total 686 voxels ) , two in the left occipital lobe ( total 414 voxels ) , two in the temporal lobe ( total 294 voxels ) , one in the left the corpus callosum ( 228 voxels ) , and one in the left frontal lobe ( 151 voxels ) . the remaining two clusters were located in the subcortical regions ( total 615 voxels ) . retinal vessel diameter ( arterioles and venules ) was measured for all but three participants ( two of those excluded had poor retinal image quality and one had cataracts ) . the factorial anova analysis revealed a significant htn effect ( htn , 170.98 16.51 m versus normotensive,184.63 20.43 m , f = 7.29 , p < 0.01 , p = 0.121 ) , with reduced retinal arteriolar diameter in htn , showing a medium - large effect size . the t2 dm effect was also significant ( t2 dm , 173.71 18.42 m versus no t2 dm , 180.84 20.23 m , f = 5.10 , p = 0.03 , p = 0.088 ) , with the effect size being medium - large . please note that the means and standard deviations in the previous sentence are the raw arteriolar diameters prior to residualization , but the statistics presented were derived from the comparisons of the residualized values . the htn t2 dm interaction effect was significant as well with a medium - large effect size ( f = 4.13 , p = 0.05 , p = 0.072 ; see figure 3 ) . as seen on figure 3 , participants with htn and/or t2 dm had distinctively smaller retinal arteriolar diameter than those without these conditions . in light of these findings , we further explored whether the observed reduction in retinal arteriolar size ( residualized to venular diameter ) could explain the lower wm microstructural integrity among hypertensive participants . using the voxelwise correlational approach outlined in the methods section , we identified a total of 15 clusters showing significant association between the residualized arteriolar diameter and wm fa values ( overall 3,748 voxels or 3.75 cc in volume , p < 0.01 ; see table 5 ) , twelve of which demonstrated significant positive associations ( overall 2,846 voxels or 2.85 cc in volume ) . these correlations were independent of both age and bmi . nine of the clusters were located in the frontal regions ( total 1892 voxels ) , two in the right optic radiation ( total 800 voxels ) , and one in the left parietal lobe ( 154 voxels ) . the four largest clusters demonstrating significant correlation of at least 200 voxels in size are illustrated in figure 4 . the three clusters in the opposite direction , namely , demonstrating significant inverse correlation between fa and residualized arteriolar diameter , were located in right superior temporal wm ( 387 voxels ) , left temporal wm ( 327 voxels ) , and right prefrontal wm ( 188 voxels ) . we found that among middle - aged and elderly adults , htn , in addition to being associated with increased incidence of periventricular wm lesions , also affects the microstructural integrity of major wm fiber tracts . these more diffuse effects are still present despite the fact that 29/33 of the htn participants were receiving pharmacological antihypertensive treatment and had bp under reasonable control at the time of the study . moreover , individuals with htn and co - morbid t2 dm manifest larger wm microstructural abnormalities than those with htn alone . we also demonstrate , for the first time , that the retinal arterial narrowing in htn is related to extensive reductions in wm microstructural integrity , with the frontal lobe areas being predominantly affected . this is shown in figure 3 , which represents the htn t2 dm interaction effect on retinal arteriolar width ( a measure of arteriolar health ) . we demonstrated the impact of htn on wm integrity using different measures of wm health . wm hyperintensity assessments using the fazekas scale showed significantly higher ratings among individuals with htn , and this is consistent with previous work . we also found significant alterations in cerebral wm microstructural integrity as measured by fa in participants with htn , and these findings were independent of age , obesity , and the wm hyperintensities seen on the flair image . some studies show no significant differences in whole brain mean fa , others found differences in fa only in the optic radiation , while some found more dramatic differences in fa in the frontal periventricular and parieto - occipital areas . here , we found that among participants with htn there were distinct areas of reduced fa primarily in the ic and ec bilaterally . our findings may be somewhat different from those previously reported in that both our hypertensive and normotensive groups contained individuals with t2 dm ; t2 dm and htn likely have different effects on the brain . nevertheless , in order to adjust for these potential additive effects , we also accounted for a diagnosis of t2 dm in our dti voxelwise htn group fa comparisons . we provide preliminary evidence that participants with both htn and t2 dm have more widespread , albeit subtle , deficits in wm microstructural integrity than those with only htn , despite being in overall good bp control . furthermore , these effects were also independent of age , overt wm pathology ( hyperintensities present on the flair image ) , and bmi . to our knowledge , this is the first report on the co - morbid impact of htn and t2 dm on wm microstructure . although this data set is relatively small and our findings and conclusions should be considered preliminary , determining the combined effects of htn and t2 dm may be valuable towards understanding the full influence of the metabolic syndrome on the brain . the preliminary results presented here are supported by rodent models , where both diabetes and htn were found to contribute to microstructural wm abnormalities , but the contribution of diabetes may have been larger . we found that htn and t2 dm are independently related to decreased retinal arteriolar diameter , after adjusting for vessel ( venular ) diameter . our result showed that individuals with htn and/or t2 dm have distinctly smaller retinal arteriolar diameter than those with neither conditions , and the group with both conditions was most affected . importantly , we demonstrated that the observed retinal arteriolar narrowing was related to widespread subclinical wm pathology as reflected by reductions in the mri dti - based fa values , and these abnormalities were found predominately in the frontal lobe . although we also uncovered small regions of negative associations between retinal arteriolar diameter and wm fa , primarily in the temporal lobe , over 75% of the significant voxels showed positive associations . given that the subgroups were relatively small , future studies should clarify these relationships with larger samples . given the developmental and functional links between retinal arterioles and brain microvasculature , the decrease in retinal arteriolar diameter may suggest that parallel pathology exists in cerebral arterioles , which by disrupting blood flow to the cerebral wm may be partially accounting for the reductions in fa reported here . the added wm pathology from co - morbid t2 dm was not surprising given the likely vascular origin for this wm pathology and the emerging evidence of cerebrovascular reactivity deficits , carotid stiffening , and intima - media thickening associated with t2 dm and ir [ 30 , 31 ] . we propose that in t2 dm , ir , and associated inflammation contribute to impairments in cerebral vascular reactivity which , when coupled with other adverse mechanisms such as increased oxidative stress , in turn may lead to neuronal and wm damage . a significant strength of this report is derived from the use of a voxelwise dti analysis procedure , which allows for precise determination of the location of wm changes utilizing conservative statistics . previous reports analyzed the dti based on average fa values of larger structures , which may have overestimated the actual brain abnormalities . a second strength of the study is our very conservative statistical approach ; by covarying out age and bmi effects on wm , as well as the overt wm damage seen on the flair image , we were able to capture additional more subtle wm microstructural damage . a limitation of this study is the relatively small number of participants , especially in the htn / t2 dm versus htn only analysis . nevertheless , despite the relatively small numbers of subjects , the dti results , which used very conservative statistical thresholds , were consistently in the predicted direction ; those subjects with htn and t2 dm had significant reductions in their wm microstructural integrity relative to those with only htn . although the relationship between hypertension and cerebral white matter damage may be influenced by ethnicity as previously reported , given our relatively small sample size , it was not feasible to split the analysis by ethnicity . another limitation is that we do not have information regarding smoking in our participants , and details regarding hypertension and diabetes medications or antiplatelet use were only available for 35/62 of the participants . antihypertensive medications , such as diuretics , have effects on cerebral autoregulation , but only two of our subjects reported diuretic use , so we do not think that this significantly contributed to our findings . future studies should utilize autoregulation or cerebral blood flow assessments to ascertain their possible effects on wm abnormalities . in summary , this study suggests that t2 dm adds to the damaging effects of htn on wm microstructural integrity , with the frontal lobes being particularly vulnerable . ascertaining dti - based fa values , we demonstrated subclinical diffuse alterations in wm microstructural integrity above and beyond the wm areas showing ischemic changes ( hyperintensities ) . although we accounted for age in our statistics , this statistical adjustment may not account for differences in subtle age - associated rates of atherosclerosis . studying these much younger individuals would allow us to differentiate more acute effects from more chronic ones associated with atherosclerosis and/or more advanced forms of vascular disease .

we examined 33 hypertensive ( 22 with comorbid type 2 diabetes mellitus ( t2 dm ) ) and 29 normotensive ( 8 with t2 dm ) middle - aged and elderly adults , comparable in age and education . relative to normotensive participants , those with hypertension , in addition to a higher prevalence of periventricular white matter ( wm ) lesions , had significantly lower wm microstructural integrity of major fiber tracts as seen with mri - based diffusion tensor imaging . among participants with hypertension , those with co - morbid t2 dm ( n = 22 ) had more widespread wm pathology than those without t2 dm ( n = 11 ) . furthermore and consistent with previous research , both hypertension and t2 dm were related to decreased retinal arterial diameter . further exploratory analysis demonstrated that the observed retinal arteriolar narrowing among individual with hypertension was associated with widespread subclinical losses in wm microstructural integrity and these associations were present predominantly in the frontal lobe . we found that t2 dm adds to the damaging effects of hypertension on cerebral wm , and notably these effects were independent of age and body mass index . given that the decrease in retinal arteriolar diameter may be a biomarker for parallel pathology in cerebral arterioles , our data suggest that the frontal lobe may be particularly vulnerable to microvascular damage in the presence of hypertension and t2 dm .

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

hypertension ( htn ) and type 2 diabetes mellitus ( t2 dm ) are common diseases associated with obesity and the metabolic syndrome . approximately 26 million people in the united states have diabetes and it is estimated that 67% of those individuals also have htn . htn and t2 dm share several common vascular complications such as coronary artery disease , renal disease , and stroke . htn and t2 dm are strong predictors of cardiovascular disease and can also have a significant impact on brain health . white matter ( wm ) health is inversely associated with age . whereas t2 dm is frequently associated with volume changes , particularly in the hippocampus [ 6 , 7 ] , both htn and t2 dm are known to result in similarly prominent effects on the integrity of wm [ 8 , 9 ] . the impact of htn on the brain might be further complicated by co - morbid t2 dm [ 6 , 11 ] . given that retinal and cerebral microvessels share developmental and physiological controls , it is no surprise that previous studies link retinal vessel integrity to certain aspects of brain health . in particular , narrowed retinal arteriolar diameters are robust indicator of cerebrovascular health and have been found to be a risk factor for htn . moreover , there is evidence that retinal vessel diameter is affected by insulin resistance and is related to brain atrophy [ 16 , 17 ] . however , the role of arterial vessel health in explaining wm integrity in the presence of htn and/or t2 dm remains to be further developed . first , by using a novel and statistically conservative voxelwise analysis method , and after accounting for wm hyperintensities , we wanted to ascertain by using diffusion tensor imaging ( dti ) which areas of wm had reductions in microstructural integrity associated with htn . secondly , we wanted to evaluate whether t2 dm has additive deleterious effects on wm health in the presence of htn . finally , given that retinal vessel integrity can be used as a noninvasive marker of cerebral vascular health [ 17 , 18 ] , we also wanted to ascertain whether there are relationships between retinal arteriolar integrity and the anticipated reductions in dti - based cerebral wm integrity among hypertensive individuals . sixty two middle - aged and elderly community - residing individuals , 33 fulfilling criteria for htn ( see below for definition ) and 29 not , were consecutively recruited via web advertisements and referrals from collaborating endocrinologist as part of an ongoing nih - sponsored study of the effects of diabetes and aging on the brain . all participants had a minimum of a high school education ( 1221 years for both groups ) and no impairments in their day - to - day functioning and were medically stable ( other than some of them having diabetes , hypertension , or dyslipidemia ) and free of psychiatric illness or significant vascular disease . individuals were excluded if they had a history of current insulin treatment , so as to avoid the possible confounding effects of severe hypoglycemic episodes , or if they had uncontrolled hypertension ( blood pressure ( bp ) > 150/90 mm / hg ) , significant coronary ischemic disease detected on the electrocardiogram or a modified hachinski ischemia scale score > 4 , any focal neurological signs , current diagnosis or past history of stroke or significant head trauma , or evidence of tumor on the structural mr scan . in an extensive 8-hour evaluation , performed over 3 separate days , we collected medical , psychiatric , and endocrine data and performed an mri of the brain . individuals with t2 dm met one or more of the following criteria : ( 1 ) fasting blood glucose 6.99 mmol / l on two separate occasions or ( 2 ) 2-hour blood glucose level > 11.10 mmol / l during a 75 g oral glucose tolerance test or ( 3 ) a prior diagnosis of t2 dm . briefly , the participant 's eyes are allowed to acclimate to a dimly lit room for 10 minutes in order to nonpharmacologically dilate the pupil sufficiently to view the optic disk and the retinal vessels . the images were then loaded on to a computer , cropped , red - free filtered , and contrast - enhanced by grayscale conversion . in this zone straight segments of vessel are selected and five equidistant lines orthogonal to the vessel are applied . dti images were utilized to generate fa maps that were then registered to a structurally relevant mprage image using automated registration toolkit 2 ( art2 ) as previously described [ 9 , 22 ] . in short , these images were then normalized to the talairach space to obtain a 3d warp field that includes transformation parameters to change the mprage to target image . the fa values and overlay maps were generated from the native dti images and then these maps were spatially corrected and normalized to standard space using the combination of the four transformations obtained in the previous steps . furthermore , the flair image , which is very sensitive to wm hyperintensities , was also used as a covariate to control for nonclinically relevant very subtle wm hyperintensities after the image had been normalized to the target template using the same parameters applied to the fa maps . in order to adjust for overall vessel diameter , retinal arteriolar diameter was subjected to a linear regression analysis with retinal venular diameter to produce a residual mean of arteriolar diameter that accounts for individual differences in vessel diameter . a two - way factorial anova evaluated the contributions of htn ( htn versus normotensive ) and t2 dm ( t2 dm versus no t2 dm ) and their interaction effect on retinal arteriolar diameter , after accounting for age and bmi . the group fa maps were created by averaging the corrected and normalized fa maps and two - tailed voxelwise analysis of covariance ( vancova ) analyses were performed with age and wm hyperintensities on the flair image as covariates . given that htn is highly associated with obesity , we also controlled for bmi in those analyses . the association between wm fa and retinal arteriolar diameter residualized to venular diameter was evaluated among individuals with htn using a voxelwise correlation analysis with age and bmi as covariates . the hypertensive and normotensive groups were well matched on age and did not differ on sex or ethnicity distribution . as expected the hypertensive group had significantly higher rates of obesity and t2 dm . despite the fact that 29/33 of our hypertensive participants were on antihypertensive medication , they still had significantly higher systolic but not diastolic bp than the group without htn . consistent with the existing literature linking obesity and htn , we found that our hypertensive participants had significantly higher bmi than our normotensive participants . of those participants who also had a diagnosis of t2 dm ( 22 in the htn and 8 in the normotensive group , resp . ) the groups did not differ in lipid concentrations , likely due to the fact that significantly more hypertensive participants ( 22/33 ) as opposed to only 2/29 in the normotensive group were being treated with cholesterol - lowering medications . relative to normotensive participants , those with htn had significantly higher ratings of periventricular wm hyperintensities ( pwmh ) and higher , though not statistically significant , ratings of deep wm hyperintensities ( dwmh ; see table 1 ) . additionally , we wanted to explore whether there were subtle changes in wm microstructure even after accounting for those wm hyperintensities seen on the flair image by using a voxelwise dti fa approach . one control case was excluded from the dti fa analysis due to uncorrectable spatial distortions on the fa map , leaving 33 hypertensive and 28 normotensive participants for those group comparisons . since both bp groups had individuals with t2 dm , which is associated with an increased incidence of wm lesions , in addition to age , bmi , and the flair image as covariates we also controlled for a diagnosis of t2 dm in the dti fa analyses . the result revealed a total of 10 clusters of significant group differences ( overall 2,747 voxels or 2.75 cc in volume ; p < 0.01 ; table 2 ) , seven of which showed fa reduction among participants with htn relative to the normotensive ones ( 2,344 voxels or 2.34 cc in volume ) . the largest five clusters showing fa reductions among hypertensive participants were located bilaterally ( 2,121 voxels ) in the internal and external capsules ( ic and ec ; see figure 1 ) and the remaining two were found in the right prefrontal region ( 114 voxels ) and the left cerebellar peduncle ( 109 voxels ) . to ascertain whether t2 dm contributed to further wm damage among hypertensive participants , the hypertensive group was further divided based on whether or not they also had t2 dm ( 22 with htn and t2 dm versus 11 with just htn ; see table 3 for group descriptors ) . in addition to having significantly higher fasting glucose and hemoglobin a1c ( hba1c ) , the hypertensive group with t2 dm also had significantly higher bmi . the groups were comparable in bp , which is not surprising , as individuals with both htn and t2 dm are more likely to receive rigorous pharmacological treatment . all participants with htn and co - morbid t2 dm were receiving antihypertensive medications whereas fewer , 7/11 , of those without t2 dm were being treated pharmacologically . despite the fact that more participants with htn and t2 dm were on cholesterol - lowering pharmacological treatment ( 16/22 versus 6/11 ) , their high - density lipoprotein ( hdl ) levels were significantly lower ( p < 0.05 ) and their triglyceride levels were higher , although not statistically significant , than those of individuals without t2 dm ( p > 0.39 ) . there were no differences in the fazekas scores between the groups ( see table 3 ) . nevertheless , the voxelwise fa comparisons uncovered a total of sixteen clusters all showing wm fa reduction ( overall 4,021 voxels or 4.02 cc in volume ; p < 0.01 ) among participants with htn and t2 dm relative to those with only htn , independent of age , bmi , and wm hyperintensities on the flair image , thus suggesting a widespread distribution of wm abnormalities ( see table 4 and figure 2 for those clusters of 200 voxels in size ) . similar to the results reported above for the htn yes / no group comparison , significant substantive number of clusters were identified in the ic and ec ( total 988 voxels ) . also , two clusters were found bilaterally in the arcuate fasciculi ( total 645 voxels ) , which connect the frontal and temporal lobes . two clusters were identified bilaterally in the parietal lobe ( total 686 voxels ) , two in the left occipital lobe ( total 414 voxels ) , two in the temporal lobe ( total 294 voxels ) , one in the left the corpus callosum ( 228 voxels ) , and one in the left frontal lobe ( 151 voxels ) . the remaining two clusters were located in the subcortical regions ( total 615 voxels ) . the factorial anova analysis revealed a significant htn effect ( htn , 170.98 16.51 m versus normotensive,184.63 20.43 m , f = 7.29 , p < 0.01 , p = 0.121 ) , with reduced retinal arteriolar diameter in htn , showing a medium - large effect size . the t2 dm effect was also significant ( t2 dm , 173.71 18.42 m versus no t2 dm , 180.84 20.23 m , f = 5.10 , p = 0.03 , p = 0.088 ) , with the effect size being medium - large . please note that the means and standard deviations in the previous sentence are the raw arteriolar diameters prior to residualization , but the statistics presented were derived from the comparisons of the residualized values . the htn t2 dm interaction effect was significant as well with a medium - large effect size ( f = 4.13 , p = 0.05 , p = 0.072 ; see figure 3 ) . as seen on figure 3 , participants with htn and/or t2 dm had distinctively smaller retinal arteriolar diameter than those without these conditions . in light of these findings , we further explored whether the observed reduction in retinal arteriolar size ( residualized to venular diameter ) could explain the lower wm microstructural integrity among hypertensive participants . using the voxelwise correlational approach outlined in the methods section , we identified a total of 15 clusters showing significant association between the residualized arteriolar diameter and wm fa values ( overall 3,748 voxels or 3.75 cc in volume , p < 0.01 ; see table 5 ) , twelve of which demonstrated significant positive associations ( overall 2,846 voxels or 2.85 cc in volume ) . these correlations were independent of both age and bmi . nine of the clusters were located in the frontal regions ( total 1892 voxels ) , two in the right optic radiation ( total 800 voxels ) , and one in the left parietal lobe ( 154 voxels ) . the three clusters in the opposite direction , namely , demonstrating significant inverse correlation between fa and residualized arteriolar diameter , were located in right superior temporal wm ( 387 voxels ) , left temporal wm ( 327 voxels ) , and right prefrontal wm ( 188 voxels ) . we found that among middle - aged and elderly adults , htn , in addition to being associated with increased incidence of periventricular wm lesions , also affects the microstructural integrity of major wm fiber tracts . moreover , individuals with htn and co - morbid t2 dm manifest larger wm microstructural abnormalities than those with htn alone . we also demonstrate , for the first time , that the retinal arterial narrowing in htn is related to extensive reductions in wm microstructural integrity , with the frontal lobe areas being predominantly affected . this is shown in figure 3 , which represents the htn t2 dm interaction effect on retinal arteriolar width ( a measure of arteriolar health ) . wm hyperintensity assessments using the fazekas scale showed significantly higher ratings among individuals with htn , and this is consistent with previous work . we also found significant alterations in cerebral wm microstructural integrity as measured by fa in participants with htn , and these findings were independent of age , obesity , and the wm hyperintensities seen on the flair image . some studies show no significant differences in whole brain mean fa , others found differences in fa only in the optic radiation , while some found more dramatic differences in fa in the frontal periventricular and parieto - occipital areas . here , we found that among participants with htn there were distinct areas of reduced fa primarily in the ic and ec bilaterally . our findings may be somewhat different from those previously reported in that both our hypertensive and normotensive groups contained individuals with t2 dm ; t2 dm and htn likely have different effects on the brain . nevertheless , in order to adjust for these potential additive effects , we also accounted for a diagnosis of t2 dm in our dti voxelwise htn group fa comparisons . we provide preliminary evidence that participants with both htn and t2 dm have more widespread , albeit subtle , deficits in wm microstructural integrity than those with only htn , despite being in overall good bp control . furthermore , these effects were also independent of age , overt wm pathology ( hyperintensities present on the flair image ) , and bmi . to our knowledge , this is the first report on the co - morbid impact of htn and t2 dm on wm microstructure . although this data set is relatively small and our findings and conclusions should be considered preliminary , determining the combined effects of htn and t2 dm may be valuable towards understanding the full influence of the metabolic syndrome on the brain . we found that htn and t2 dm are independently related to decreased retinal arteriolar diameter , after adjusting for vessel ( venular ) diameter . our result showed that individuals with htn and/or t2 dm have distinctly smaller retinal arteriolar diameter than those with neither conditions , and the group with both conditions was most affected . importantly , we demonstrated that the observed retinal arteriolar narrowing was related to widespread subclinical wm pathology as reflected by reductions in the mri dti - based fa values , and these abnormalities were found predominately in the frontal lobe . although we also uncovered small regions of negative associations between retinal arteriolar diameter and wm fa , primarily in the temporal lobe , over 75% of the significant voxels showed positive associations . given that the subgroups were relatively small , future studies should clarify these relationships with larger samples . given the developmental and functional links between retinal arterioles and brain microvasculature , the decrease in retinal arteriolar diameter may suggest that parallel pathology exists in cerebral arterioles , which by disrupting blood flow to the cerebral wm may be partially accounting for the reductions in fa reported here . the added wm pathology from co - morbid t2 dm was not surprising given the likely vascular origin for this wm pathology and the emerging evidence of cerebrovascular reactivity deficits , carotid stiffening , and intima - media thickening associated with t2 dm and ir [ 30 , 31 ] . we propose that in t2 dm , ir , and associated inflammation contribute to impairments in cerebral vascular reactivity which , when coupled with other adverse mechanisms such as increased oxidative stress , in turn may lead to neuronal and wm damage . a second strength of the study is our very conservative statistical approach ; by covarying out age and bmi effects on wm , as well as the overt wm damage seen on the flair image , we were able to capture additional more subtle wm microstructural damage . a limitation of this study is the relatively small number of participants , especially in the htn / t2 dm versus htn only analysis . nevertheless , despite the relatively small numbers of subjects , the dti results , which used very conservative statistical thresholds , were consistently in the predicted direction ; those subjects with htn and t2 dm had significant reductions in their wm microstructural integrity relative to those with only htn . although the relationship between hypertension and cerebral white matter damage may be influenced by ethnicity as previously reported , given our relatively small sample size , it was not feasible to split the analysis by ethnicity . another limitation is that we do not have information regarding smoking in our participants , and details regarding hypertension and diabetes medications or antiplatelet use were only available for 35/62 of the participants . antihypertensive medications , such as diuretics , have effects on cerebral autoregulation , but only two of our subjects reported diuretic use , so we do not think that this significantly contributed to our findings . in summary , this study suggests that t2 dm adds to the damaging effects of htn on wm microstructural integrity , with the frontal lobes being particularly vulnerable . ascertaining dti - based fa values , we demonstrated subclinical diffuse alterations in wm microstructural integrity above and beyond the wm areas showing ischemic changes ( hyperintensities ) .

from an evolutionary point of view , the integration of the circuits controlling metabolism and reproduction is essential for the survival and perpetuation of the species . wasting of energy on reproduction , in situations of deficit in food availability , may threaten the survival of the individuals and their progeny . thus , in times of famine or under certain pathological conditions , which can not ensure the correct utilization of the metabolic resources ( anorexia nervosa , obesity , diabetes , lipodystrophies , etc . ) , the body has to define priorities to preserve physiological processes essential for survival . in this sense , key physiological activities as blood circulation or neural activity can not be compromised , whereas others , such as locomotion , thermoregulation , or growth can be reduced in conditions of metabolic stress , as occurs in hibernating animals . in the above situations , reproduction is totally dispensable or even incompatible with survival since , besides the high energetic cost required to maintain the fertility , pregnancy and nursing limit considerably the mobility for food seeking . in wildlife , animals are more dependent on seasonal fluctuations in food availability than their counterparts living under domestic conditions or humans . however , the increased prevalence in developed countries of metabolic pathologies ( ranging from anorexia to obesity and metabolic syndrome ) makes the study of the metabolic control of reproduction of special interest , in part due to economic reasons . in this sense , it is interesting that the increase in the incidence of metabolic disorders during the last decades coincides also with increased rates of infertility , although direct association between these two phenomena has not been demonstrated so far [ 2 , 3 ] . of note , besides the elevated cost derived from the treatment of illness associated with metabolic disorders such as diabetes , hypertension , and other cardiovascular problems , the cost of a successful treatment by means of in vitro fertilization ranges from 19,588 to 134,190 . changes in energy stores produce long- and short - term fluctuations in hormonal ( leptin , insulin , and ghrelin ) as well as nutritional ( glucose , lipids ) signals that feedback mainly to the cns to regulate metabolism and fertility . however , although many of the neural circuits controlling energy homeostasis are well characterized , the elements conveying nutritional information to the reproductive axis are yet to be conclusively defined . the reproductive axis comprises three major elements , the hypothalamus , the pituitary , and the gonads , which form the so - called hpg axis . gonadotropin - releasing hormone ( gnrh ) neurons , located in the preoptic area ( poa ) of the hypothalamus , induce gonadotropin stimulation at the pituitary , which subsequently increases gonadal hormone secretion . the coordinated actions of these elements , together with other peripheral factors , allow the integration of endogenous and environmental information to ultimately produce gametes and modulate sex behaviour . gnrh neurons are located in an excellent position to be considered as the best candidate to relay metabolic information to the downstream elements of the hpg axis since ( i ) gnrh neurons are the final output of the brain controlling the reproduction , ( ii ) gnrh neurons can sense peripheral signals because they send multiple projections to the organum vasculosum of the lamina terminalis ( ovlt ) and median eminence where the permeability of the blood - brain barrier is very high , and ( iii ) gnrh mrna and the patterns of gnrh secretion change in situations of metabolic stress , suggesting that gnrh neurons are influenced by nutritional reserves . however , there is a growing body of evidence demonstrating that gnrh neurons may not be targeted directly by the above metabolic factors . in support of this idea , recent analyses demonstrated that the signal transducer and activator of transcription 3 ( stat3 ) is not presented in gnrh neurons after leptin administration , and that gnrh neurons lack leptin receptor ( lepr ) , assessed by single - cell pcr . further proof for the absence of direct actions of leptin on gnrh neurons came from functional genomic studies involving selective ablation of lepr in gnrh neurons or in all forebrain neurons . these experiments demonstrated that elimination of leptin signalling in gnrh neurons does not have any impact on reproduction , whereas lepr ablation in all forebrain neurons impaired fertility . along with leptin , insulin is another potential candidate to relay metabolic information to the reproductive system . in fact , neuron - specific insulin receptor - knockout ( nirko ) mice exhibit hypogonadism of central origin . however , the direct influence of insulin on gnrh neurons or even the presence of insulin receptors ( ir ) in this neuronal population has been extensively questioned . recent analyses have confirmed that , although ir is expressed in gnrh neurons , the selective ablation of ir on this neuronal population , by breeding ir mice onto a gnrh - cre background , does not alter the normal timing of puberty or fertility . these data suggest that the positive effect of insulin on reproduction is not mediated directly via gnrh neurons . compelling evidences have highlighted the negative effect of ghrelin at different levels of the reproductive axis . in this sense , chronic administration of ghrelin to peripuberal rats inhibits gonadotropin release and mimics the delay on puberty onset produced by situations of energy deficit , such as chronic undernutrition , conditions in which endogenous ghrelin levels are expected to be elevated . besides its central effects , it has been demonstrated that ghrelin can also modulate the reproductive function through its actions on the pituitary and gonads [ 8 , 10 ] . however , conclusive demonstration of the expression of this receptor in gnrh neurons remains elusive . nonetheless , studies in rats of the inhibitory effects of ghrelin on gnrh pulsatility ( as evidenced by an increase of gnrh interpulse intervals ) demonstrated that this can be blockaded by an npy y5r antagonist . this suggests that ghrelin effects on gnrh neuron are mediated , at least in part , by afferent neurons ( probably npy neurons ) . the above lines of evidence indicate that , although gnrh neurons are ultimately affected by metabolic signals , some key peripheral indicators of the energy status do not target this information directly on gnrh neurons . some of the potential candidate afferents responsible for transmitting metabolic information to gnrh neurons will be reviewed in the following sections . kisspeptins , encoded by the kiss1 gene , are peptides derived from a common precursor named kisspeptin-54 , which was originally termed metastin by its ability to suppress melanoma metastasis . these peptides share a common rf - amide carboxyl - terminal region , which is essential for the activation of its g - protein - coupled receptor , gpr54 . both kiss1 and gpr54 genes have been phylogenetically well conserved , certifying the importance of this system . in 2003 , two independent studies uncovered the indispensable role of kisspeptins and its receptor for the normal timing of the maturational events controlling the reproductive function . these two reports identified that inactivating mutations of gpr54 causes hypogonadotropic hypogonadism , suggesting that kisspeptin signaling plays a major role in the control of the reproductive axis [ 14 , 15 ] . since then , a large number of papers have confirmed and extended this initial observation , thus deepening into the molecular and physiological mechanisms involved in the control of the reproductive function by the kiss1/gpr54 system . in rodents , one of these populations is located in the so - called rostral periventricular area of the third ventricle ( rp3v ) , which comprises the anteroventral periventricular nucleus ( avpv ) , the rostral preoptic periventricular nucleus ( rpvpo ) , and the caudal preoptic periventricular nucleus ( cpvpo ) . several studies have demonstrated that this population is activated by estradiol and sends projections to gnrh neurons in the poa suggesting that rp3v kiss1 neurons are potentially involved in conveying the estrogen - positive feedback [ 17 , 18 ] . the other population , located in the arcuate nucleus ( arc ) , shows diametrically opposite responses to estradiol , as arc kiss1 expression is inhibited by estrogen . this circumstance , together with the fact that arc kiss1 neurons also project ( albeit to a lesser extent ) to gnrh neurons in the poa , suggests that this neuronal population could be involved in mediating estrogen - negative feedback [ 19 , 20 ] . of note , however , although this assumption has been accepted for years , recent data from electrophysiological recordings have failed to document electrical activation of arc kiss1 neurons after withdrawal of the inhibitory effect of gonadal steroids in mice . kiss1 neurons have been also proposed as mediators for relaying metabolic information to gnrh neurons . fasting , which reduces significantly lh secretion , is correlated with diminished kiss1 expression [ 2325 ] . chronic administration of kisspeptin partially rescues the delay on puberty onset caused by chronic undernutrition . anovulation and reduced levels of gnrh / lh during lactation , caused by the negative energy balance due to milk production , are associated with reduced levels of kiss1 mrna in the arc . infertile leptin - deficient mice ( ob / ob ) show low levels of kiss1 mrna , and leptin injection recovers , in some extent , kiss1 expression and fertility . likewise , streptozotocin - induced diabetic rats , which are leaner and hypoleptinemic , display low levels of kiss1 mrna in the hypothalamus . however , chronic injections of kisspeptin-10 partially rescued the negative effects of insulin deficiency on reproductive parameters ( lh and testosterone secretion and prostate and testis weights ) . all in all , the above data point out that conditions of negative energy balance and metabolic stress that suppress the function of the hpg axis are associated with a detectable inhibition of the hypothalamic kiss1 system . despite this solid evidence , although previous studies assumed that almost half of the arcuate kiss1 neurons express lepr , recent data demonstrated that the ablation of this receptor on kiss1 neurons or its restoration on lepr - deficient mice does not have a detectable impact on reproductive function [ 30 , 31 ] . in fact , a recent paper suggested that only a low percentage of kiss1 neurons expresses lepr , only after completion of puberty onset . these results suggest that leptin may not act directly on kiss1 neurons to control the reproductive function . however , it is highly plausible that leptin could modulate the activity of kiss1 neurons through afferent inputs . in this context , it has been demonstrated that leptin signalling in the ventral premmamillary nucleus ( pmv ) is essential for puberty and fertility in mice , and lepr - expressing cells in this nucleus send projections to kiss1 neurons . this arises the possibility that kiss1 neurons could act as downstream mediators for leptin actions on the pmv . in this sense , recent data demonstrated that targeted lesion of the pmv disrupts kiss1 and gnrh expression during the proestrus - to - estrus transition in rats . on the other hand , recent analysis revealed that kiss1 neurons express ir , suggesting that kiss1 neurons could sense metabolic status by receiving information about insulin fluctuations in bloodstream , to regulate reproduction . in fact , ir ablation from kiss1 neurons , by cre - loxp strategy , caused a moderate delay of puberty onset and a reduction of lh secretion in both sexes . these data suggest that insulin signalling in kiss1 neurons exerts , to some extent , a positive influence for the initiation of the puberty , although in absence of this stimulus , compensatory mechanisms are activated to complete the maturation of the reproductive axis . in addition , the orexigenic hormone , ghrelin , which may play a major role disrupting fertility in situations of energy insufficiency , has been demonstrated to modulate kiss1 expression [ 35 , 36 ] . of note , intravenous injection of ghrelin has been shown to produce a significant decrease in lh pulsatility and kiss1 expression in the median poa ( an area that includes the rp3v ) of adult rats . these data suggest that kiss1 neurons could contribute to mediate the negative effects of ghrelin on reproduction . the above lines of evidence situate kiss1 neurons as an excellent candidate responsible for the integration of metabolism and reproduction . however , many of the responses to metabolic hormones such as leptin , insulin , and ghrelin could be mediated by afferent inputs to kiss1 neurons rather than by direct actions on this neuronal population . proopiomelanocortin ( pomc ) is a precursor protein that can be cleaved at different sites to generate several peptides with different biological activity . among others , -msh , -endorphin , and acth are neuropeptides derived from the pomc precursor . in the brain , one of them is located in the nucleus of the solitary tract ( nts ) . this is a small population comprised by nearly 190 neurons scattered throughout the dorsomedial and medial parts of the nts . a recent study suggested that this population could be involved in short - term feeding suppression . however , selective ablation of nts pomc neurons does not reproduce the obese phenotype showed by the global pomc - null mice [ 38 , 39 ] . in addition , it has been demonstrated that these neurons do not respond to leptin . such evidences suggest that pomc neurons located in the nts may have a marginal contribution to the global control of the energy homeostasis exerted by the pomc system . further analysis will be necessary to unveil the function of the nts pomc neurons . the other population is located in the arc . unlike nts pomc neurons , the function and phenotype of arc pomc neurons have been extensively studied . this neuronal population coexpresses multiple neuropeptides ( cart , dynorphin , and vgf ) and neurotransmitters ( gaba , glutamate , acetylcholine ) [ 4043 ] . in addition , arc pomc neurons express a wide range of receptors , such as lepr , ir , and npy y1-r , that confer the ability to sense peripheral and central signals involved in the control of metabolic homeostasis . in fact , selective ablation of arc pomc neurons mimics the phenotype of the global pomc - null mice , producing increased food intake and reduced energy expenditure , resulting ultimately in obesity . these lines of evidences situate pomc neurons as a central node for sensing body energy reserves and thus as key elements to finely tune the mechanisms involved in the control of food intake and energy expenditure to keep energy homeostasis . accordingly , pomc mrna expression in the arc is reduced in leptin - deficient ob / ob mice , and leptin administration rescues this expression to that found in control mice . also , lepr deletion from pomc - expressing neurons disrupts body weight homeostasis , synapsis plasticity and produces hyperleptinemia [ 45 , 46 ] . in the same vein , although ir ablation in pomc neurons does not cause any impact on body weight or glucose regulation , probably due to compensatory mechanisms , double lepr and ir deletion on pomc neurons produce higher negative impact on metabolic parameters than that found in the lepr ko mice . the above lines of evidence demonstrate that arc pomc neurons are key elements in the control of body weight and metabolism . this observation makes also pomc neurons a good candidate to relay metabolic information to gnrh neurons . admittedly , information about the role of pomc products in the control of the reproductive axis is scarce and , in some cases , controversial . yet , there is growing evidence suggesting that pomc neurons do participate in conveying metabolic information to gnrh neurons . in this context , whereas early studies demonstrated that independent ablation of lepr or ir on pomc neurons does not disrupt fertility [ 45 , 47 ] , mutant mice lacking both receptors in pomc neurons show severe reproductive deficiencies . moreover , immunohistochemical analyses demonstrated that pomc neurons project and make synaptic contacts with gnrh perikarya and nerve terminals , suggesting direct actions of pomc - derived peptides on gnrh excitability [ 49 , 50 ] . overall , the above data suggest that arcuate pomc neurons could act as a mediator for leptin and insulin actions upon gnrh neurons . in this sense , watanobe showed , using push - pull perfusion techniques , that leptin infusion in the poa and median eminence stimulates gnrh / lh release , and this effect is preceded by an increase in -msh secretion . early pharmacological studies revealed that -msh is able to elicit a robust increase in lh in different mammalian species [ 5254 ] , as well as to stimulate sexual receptivity and lordosis behaviour in female rats . however , discrepancies about the effect of -msh on lh can be found in the literature . in this context , there are data showing diminished or unchanged lh levels after -msh administration in rats [ 56 , 57 ] . from these studies , it is apparent that steroid environment and administration site could influence lh response to -msh . in this sense , recent data demonstrated that melanocortin receptors 3 and 4 ( mc3/4r ) agonist , melanotan ii , increases gnrh pulse generator activity in goats and this effect can be attenuated by estradiol . this finding also suggests that the central actions of -msh on reproduction are mediated probably via mc3r and mc4r . unlike the clear role of mc3r and mc4r on metabolism , evidenced by the fact that mc4r ko and to a lesser extent mc3r ko mice develop obvious metabolic disorders [ 60 , 61 ] data derived from mc4r mutants demonstrated that although mc4r - deficient mice are fertile , females are poor breeders exhibiting reduced ovulation rates . moreover , males display erectile dysfunction and disturbed copulatory behaviour . in the same way , mc3r ko males are fertile though females display a certain degree of subfertility . overall , it seems that although both mc3r and mc4r are involved in the control of reproduction , the lack of one type of receptor could be partially compensated by the other , resulting in a milder phenotype . accordingly , mice with functional blockade of both mc3r and mc4r signalling pathways , by overexpression of its endogenous antagonist , agouti - related peptide ( agrp ) , are infertile . however , whether gnrh neurons are direct targets of melanocortin actions remained unknown until very recently , when electrophysiological recordings of gnrh neurons demonstrated that -msh increases cell firing in most of gnrh neurons through postsynaptic activation of both mc3r and mc4r . almost simultaneously , israel et al . confirmed by single - cell rt - pcr that gnrh neurons express mc4r and showed that restoration of melanocortin signalling in leptin - deficient db / db mice recovers the normal timing of puberty onset and fertility , suggesting that melanocortin signalling is essential for leptin actions on gnrh neurons . in good agreement , our preliminary studies have documented that the positive effects of leptin on puberty onset , in rats subjected to 20% daily caloric restriction , can be blocked to a large extent by the mc3/4r antagonist shu9119 ( manuscript in preparation ) . overall , it seems that pomc neurons convey leptin actions on gnrh neurons directly through mc3/4r pathways . in any case , the existence also of indirect intermediaries between pomc and gnrh neurons can not be discarded . although -endorphin is also derived from the pomc precursor , lines of evidence in the literature attribute to these neuropeptide effects diametrically opposite , in terms of metabolic and reproductive control , to those found for other pomc - derived peptides ( such as -msh , -msh , and acth ) . -endorphin mediates its actions mostly via -opioid receptor , although it displays also relatively high affinity by - , and -subtypes of opioid receptors . pharmacological experiments indicated that -endorphin increases food intake and body weight gain , whereas the opioid receptor antagonist , naloxone , inhibits feeding behaviour [ 68 , 69 ] . albeit there is consensus in the pharmacological studies about the stimulatory actions of -endorphin on food intake , paradoxically , mutant male mice retaining all the pomc - derived peptides except -endorphin show increased food consumption and are obese . several analyses revealed that -endorphin inhibits basal gnrh / gonadotropin secretion in different species and physiological conditions [ 7175 ] , as well as the electrical activity of a subpopulation of murine gnrh neurons . also , central injection of -endorphin is able to block the preovulatory surge of lh and to inhibit sexual behaviour . interestingly , naloxone administration to amenorrhoeic women is able to elicit a potent lh response , suggesting that excessive opioid activity could be involved in some pathophysiological conditions resulting in amenorrhea . intriguingly , mutant mice lacking -endorphin display a normal reproductive phenotype , showing normal puberty onset and fertility , which could be attributed to compensatory mechanisms mediated by other opioids . the fact that -endorphin is able to negatively modulate gnrh / gonadotropin release opens up the possibility that pomc neurons could integrate also negative inputs to suppress gnrh activity via this opioid . in this sense , recent data suggested that the signal of energy insufficiency , ghrelin , suppresses the reproductive axis via -endorphin . thus , ogata et al . showed recently that central administration of ghrelin reduces significantly lh concentration and pulse frequency , whereas naloxone is able to block this effect . however , whether -endorphin acts directly on gnrh neurons or via other intermediate neurons is still under discussion . in this context , while electrophysiological recordings in guinea pig demonstrated that the -opioid receptor agonist , damgo , inhibits gnrh neurons postsynaptically , data from the teleost fish , medaka , indicated that -endorphin reduces action potential firing in gnrh neurons via indirect mechanisms . on the other hand , there is a large number of data demonstrating that rat gnrh neurons do not express opioid receptors [ 86 , 87 ] . admittedly , part of the above discrepancies could be attributed to interspecies differences . regarding the possible indirect effects of -endorphin on gnrh neurons , pharmacological analyses suggested that -endorphin modulates gnrh release via glutamate - nitric oxide pathway . cart - immunoreactive neurons have been identified in many different hypothalamic ( paraventricular , arcuate , dorsomedial , and ventral premmamillary nuclei , as well as lateral hypothalamic area ) and extrahypothalamic nuclei ( central amygdala ) [ 8991 ] . however , the arc pomc / cart population has received special attention due to its potential role in regulating food intake and reproduction , while little is known about the intervention of other cart populations in the integration of these two systems . however , the receptor responsible for cart actions has not been identified yet . unlike other arc pomc neuropeptides , nonetheless , data from in vitro incubation of hypothalamic explants showed that both cart and leptin are able to stimulate gnrh secretion by reducing interpulse intervals . interestingly , coadministration of an anti - cart antiserum completely blocked cart effects on gnrh pulsatility whereas partially abrogated leptin actions [ 94 , 95 ] . this means that arc pomc neurons can mediate leptin actions on gnrh neurons , at least partially , through cart secretion . intriguingly , very recent analysis demonstrated that cart could stimulate gnrh excitability both postsynaptically and presynaptically . altogether , the above lines of evidence suggest that pomc neurons are key elements for conveying a large variety of metabolic inputs , ranging from signals of nutrient deficiency to energy sufficiency cues , to control the reproductive axis by secreting a wide diversity of neuropeptides with different , in many cases opposite , actions . npy is member of a family of peptides that include also peptide yy ( pyy ) and the pancreatic polypeptide ( pp ) . in rats , npy - expressing neurons are widespread through different areas across the brain including , among others , the olfactory bulb , striatum , hypothalamus , spinal cord , and pineal gland , being the arc and the paraventricular nucleus , within the hypothalamus , two of the nuclei containing higher concentrations of npy neurons and fibers . the arc population coexpresses also agrp and has been postulated as key element in the control of feeding behaviour . in fact , in 2011 aponte and co - workers demonstrated in an extremely elegant study that activation of arcuate npy / agrp neurons , using optogenetic tools , produces rapid stimulation of seeking behaviour and compulsive feeding in mice . interestingly , although agrp is the endogenous antagonist of mc3/4r , this effect was demonstrated to be independent of melanocortin signalling suppression . these evidences suggest that arc npy neurons are essential to stimulate food consumption in situations of negative energy balance . so far , five different npy receptors ( y1 , y2 , y4 , y5 , and y6 ) have been identified , which show different affinity for the various peptides of the npy family [ 100103 ] . these receptors present different distribution patterns within the brain and participate in the regulation of multiple functions [ 104 , 105 ] . regarding the potential role of npy in reproduction , arc npy neurons send projections to gnrh perikarya and nerve terminals and have been suggested to participate in conveying information about energy insufficiency to the reproductive axis . in fact , food restriction markedly increases npy mrna , and this is well correlated with reduced lh release . in addition , the infertile phenotype displayed by ob / ob mice has been associated with high levels of npy mrna . indeed , deficiency of either npy or its y1 or y4 receptors in these animals rescues fertility . despite these compelling lines of evidence , in fact , pharmacological studies showed opposite effects of npy on lh release depending on the steroid milieu and receptor agonist used . thus , whereas npy inhibited lh release in intact and castrated animals [ 108 , 109 ] , this neuropeptide induced opposite stimulatory effects on steroid - primed ovariectomized rats . interestingly , npy ko mice do not display any reproductive alterations under normal conditions . however , fasting does not induce the expected decay in lh levels in these animals , suggesting that npy signalling is needed for transmitting metabolic information when the conditions for reproduction are unfavourable due to reduced energy availability . a recent electrophysiological study evaluated the role of npy receptors on gnrh neuronal activity by subtractive analysis . to address such a wide spectrum of potential effects , a selection of npy receptor agonists was used to determine the possible influence of each individual receptor on gnrh activity . this procedure allowed to identify that y1r activation inhibits murine gnrh neurons . in good agreement , previous data showed that y1r activation significantly decreases the number of calcium transients in gnrh neurons from nasal explants . so , differences between species may exist for the type of receptor that mediates the inhibitory actions of npy on gnrh neurons . on the other hand , y4 receptor activation by different agonists resulted in a potent postsynaptic stimulation of gnrh neurons . overall , the former discrepancies about the dual inhibitory / stimulatory effect of npy on lh secretion could be due to differences in the ratio y1r / y4r in the model evaluated . further analysis will be necessary to clarify this phenomenon . as it was mentioned before , arc npy neurons also co - express agrp . . however , the mechanisms through which npy and agrp influence feeding behaviour are different . accordingly , agrp increases food intake , at least partially , by blocking the anorexigenic effect of endogenous mc3/4r activation by melanocortins . taking in account the clear stimulatory effect exerted by melanocortins on reproduction , opposite results might be expected for agrp . moreover , electrical recordings in gnrh neurons demonstrated that agrp administration prevents the excitatory effect of the mc3/4r agonist , melanotan ii . however , later results revealed that agrp exhibits also stimulatory effects in a small subpopulation of gnrh neurons . this paradoxical effect could be mediated via a mechanism independent of mcr3/4 receptors , a possibility that had been already considered in the context of other studies , which showed agrp actions unexplained via mcr3/4 blockade [ 118 , 119 ] . overall , the above data suggest that arc npy neurons are able to module reproductive function through different mechanism involving npy secretion and/or inhibition of melanocortin signalling by agrp in situations of energy deficiency . reproductive function is highly dependent on nutrient availability . to ensure an efficient utilization of the energy stores , redundant pathways are necessary . these circuits must detect situations of metabolic stress as to be able to derive energy resources to maintain essential physiological functions , while partially or totally suppressing reproduction until more favourable conditions are achieved . of note , data in the literature suggest that many of metabolic signals informing the reproductive brain do no act directly on gnrh neurons , the final output in the brain controlling reproduction . in fact , in recent years , the existence of different neuronal populations that are able to sense peripheral and central indicators of the energy status to convey this information to gnrh neurons has been exposed by a large number of experimental studies . as reviewed herein , arc pomc , npy , and kiss1 neurons have been proposed as key intermediary elements to carry out this function . interestingly , there are solid lines of evidence suggesting that these neurons make direct contacts and are able to modulate the activity of each other [ 96 , 120 , 121 ] . this raises the possibility of the existence of a complex network of interconnected neurons that is involved in the precise sensing of the metabolic status and in the transmission of this information to gnrh neurons to consequently modulate the reproductive function . on the basis of the evidence summarized here , it is tenable to postulate that kiss1 , npy , and pomc neurons are prominent elements of such a complex neuronal network admittedly , however , although for sake of concision this review has focussed only in this selected group of neuronal populations , it is likely that other partners exist on such circuitry responsible for central metabolic - reproductive interactions , such as neurons expressing galanin - like peptide ( galp ) , melanin - concentrating hormone ( mch ) , orexins , or corticotropin - releasing hormone ( crf ) [ 122 , 123 ] . while the evidence so far available suggests that the roles of the latter neuropeptides in the control of the hpg axis are less prominent , it remains a challenge for the future to decipher how major and subordinate metabolic regulators interplay with and impinge on the central elements of the reproductive axis .

reproductive function is regulated by a plethora of signals that integrate physiological and environmental information . among others , metabolic factors are key components of this circuit since they inform about the propitious timing for reproduction depending on energy availability . this information is processed mainly at the hypothalamus that , in turn , modulates gonadotropin release from the pituitary and , thereby , gonadal activity . metabolic hormones , such as leptin , insulin , and ghrelin , act as indicators of the energy status and convey this information to the reproductive axis regulating its activity . in this review , we will analyse the central mechanisms involved in the integration of this metabolic information and their contribution to the control of the reproductive function . particular attention will be paid to summarize the participation of gnrh , kiss1 , npy , and pomc neurons in this process and their possible interactions to contribute to the metabolic control of reproduction .

1. Introduction 2. GnRH Neurons and the Metabolic Control of Reproduction 3. Kiss1 Neurons and the Metabolic Control of Reproduction 4. Proopiomelanocortin Neurons and the Metabolic Control of Reproduction 5. NPY Neurons and the Metabolic Control of Reproduction 6. Conclusions

from an evolutionary point of view , the integration of the circuits controlling metabolism and reproduction is essential for the survival and perpetuation of the species . wasting of energy on reproduction , in situations of deficit in food availability , may threaten the survival of the individuals and their progeny . thus , in times of famine or under certain pathological conditions , which can not ensure the correct utilization of the metabolic resources ( anorexia nervosa , obesity , diabetes , lipodystrophies , etc . ) in this sense , key physiological activities as blood circulation or neural activity can not be compromised , whereas others , such as locomotion , thermoregulation , or growth can be reduced in conditions of metabolic stress , as occurs in hibernating animals . in the above situations , reproduction is totally dispensable or even incompatible with survival since , besides the high energetic cost required to maintain the fertility , pregnancy and nursing limit considerably the mobility for food seeking . however , the increased prevalence in developed countries of metabolic pathologies ( ranging from anorexia to obesity and metabolic syndrome ) makes the study of the metabolic control of reproduction of special interest , in part due to economic reasons . in this sense , it is interesting that the increase in the incidence of metabolic disorders during the last decades coincides also with increased rates of infertility , although direct association between these two phenomena has not been demonstrated so far [ 2 , 3 ] . of note , besides the elevated cost derived from the treatment of illness associated with metabolic disorders such as diabetes , hypertension , and other cardiovascular problems , the cost of a successful treatment by means of in vitro fertilization ranges from 19,588 to 134,190 . changes in energy stores produce long- and short - term fluctuations in hormonal ( leptin , insulin , and ghrelin ) as well as nutritional ( glucose , lipids ) signals that feedback mainly to the cns to regulate metabolism and fertility . however , although many of the neural circuits controlling energy homeostasis are well characterized , the elements conveying nutritional information to the reproductive axis are yet to be conclusively defined . the reproductive axis comprises three major elements , the hypothalamus , the pituitary , and the gonads , which form the so - called hpg axis . gonadotropin - releasing hormone ( gnrh ) neurons , located in the preoptic area ( poa ) of the hypothalamus , induce gonadotropin stimulation at the pituitary , which subsequently increases gonadal hormone secretion . the coordinated actions of these elements , together with other peripheral factors , allow the integration of endogenous and environmental information to ultimately produce gametes and modulate sex behaviour . gnrh neurons are located in an excellent position to be considered as the best candidate to relay metabolic information to the downstream elements of the hpg axis since ( i ) gnrh neurons are the final output of the brain controlling the reproduction , ( ii ) gnrh neurons can sense peripheral signals because they send multiple projections to the organum vasculosum of the lamina terminalis ( ovlt ) and median eminence where the permeability of the blood - brain barrier is very high , and ( iii ) gnrh mrna and the patterns of gnrh secretion change in situations of metabolic stress , suggesting that gnrh neurons are influenced by nutritional reserves . however , there is a growing body of evidence demonstrating that gnrh neurons may not be targeted directly by the above metabolic factors . in support of this idea , recent analyses demonstrated that the signal transducer and activator of transcription 3 ( stat3 ) is not presented in gnrh neurons after leptin administration , and that gnrh neurons lack leptin receptor ( lepr ) , assessed by single - cell pcr . along with leptin , insulin is another potential candidate to relay metabolic information to the reproductive system . however , the direct influence of insulin on gnrh neurons or even the presence of insulin receptors ( ir ) in this neuronal population has been extensively questioned . compelling evidences have highlighted the negative effect of ghrelin at different levels of the reproductive axis . in this sense , chronic administration of ghrelin to peripuberal rats inhibits gonadotropin release and mimics the delay on puberty onset produced by situations of energy deficit , such as chronic undernutrition , conditions in which endogenous ghrelin levels are expected to be elevated . besides its central effects , it has been demonstrated that ghrelin can also modulate the reproductive function through its actions on the pituitary and gonads [ 8 , 10 ] . however , conclusive demonstration of the expression of this receptor in gnrh neurons remains elusive . nonetheless , studies in rats of the inhibitory effects of ghrelin on gnrh pulsatility ( as evidenced by an increase of gnrh interpulse intervals ) demonstrated that this can be blockaded by an npy y5r antagonist . the above lines of evidence indicate that , although gnrh neurons are ultimately affected by metabolic signals , some key peripheral indicators of the energy status do not target this information directly on gnrh neurons . some of the potential candidate afferents responsible for transmitting metabolic information to gnrh neurons will be reviewed in the following sections . in 2003 , two independent studies uncovered the indispensable role of kisspeptins and its receptor for the normal timing of the maturational events controlling the reproductive function . these two reports identified that inactivating mutations of gpr54 causes hypogonadotropic hypogonadism , suggesting that kisspeptin signaling plays a major role in the control of the reproductive axis [ 14 , 15 ] . since then , a large number of papers have confirmed and extended this initial observation , thus deepening into the molecular and physiological mechanisms involved in the control of the reproductive function by the kiss1/gpr54 system . in rodents , one of these populations is located in the so - called rostral periventricular area of the third ventricle ( rp3v ) , which comprises the anteroventral periventricular nucleus ( avpv ) , the rostral preoptic periventricular nucleus ( rpvpo ) , and the caudal preoptic periventricular nucleus ( cpvpo ) . several studies have demonstrated that this population is activated by estradiol and sends projections to gnrh neurons in the poa suggesting that rp3v kiss1 neurons are potentially involved in conveying the estrogen - positive feedback [ 17 , 18 ] . the other population , located in the arcuate nucleus ( arc ) , shows diametrically opposite responses to estradiol , as arc kiss1 expression is inhibited by estrogen . this circumstance , together with the fact that arc kiss1 neurons also project ( albeit to a lesser extent ) to gnrh neurons in the poa , suggests that this neuronal population could be involved in mediating estrogen - negative feedback [ 19 , 20 ] . kiss1 neurons have been also proposed as mediators for relaying metabolic information to gnrh neurons . anovulation and reduced levels of gnrh / lh during lactation , caused by the negative energy balance due to milk production , are associated with reduced levels of kiss1 mrna in the arc . infertile leptin - deficient mice ( ob / ob ) show low levels of kiss1 mrna , and leptin injection recovers , in some extent , kiss1 expression and fertility . likewise , streptozotocin - induced diabetic rats , which are leaner and hypoleptinemic , display low levels of kiss1 mrna in the hypothalamus . despite this solid evidence , although previous studies assumed that almost half of the arcuate kiss1 neurons express lepr , recent data demonstrated that the ablation of this receptor on kiss1 neurons or its restoration on lepr - deficient mice does not have a detectable impact on reproductive function [ 30 , 31 ] . these results suggest that leptin may not act directly on kiss1 neurons to control the reproductive function . in this context , it has been demonstrated that leptin signalling in the ventral premmamillary nucleus ( pmv ) is essential for puberty and fertility in mice , and lepr - expressing cells in this nucleus send projections to kiss1 neurons . this arises the possibility that kiss1 neurons could act as downstream mediators for leptin actions on the pmv . in this sense , recent data demonstrated that targeted lesion of the pmv disrupts kiss1 and gnrh expression during the proestrus - to - estrus transition in rats . these data suggest that insulin signalling in kiss1 neurons exerts , to some extent , a positive influence for the initiation of the puberty , although in absence of this stimulus , compensatory mechanisms are activated to complete the maturation of the reproductive axis . of note , intravenous injection of ghrelin has been shown to produce a significant decrease in lh pulsatility and kiss1 expression in the median poa ( an area that includes the rp3v ) of adult rats . the above lines of evidence situate kiss1 neurons as an excellent candidate responsible for the integration of metabolism and reproduction . however , many of the responses to metabolic hormones such as leptin , insulin , and ghrelin could be mediated by afferent inputs to kiss1 neurons rather than by direct actions on this neuronal population . among others , -msh , -endorphin , and acth are neuropeptides derived from the pomc precursor . in the brain , one of them is located in the nucleus of the solitary tract ( nts ) . this is a small population comprised by nearly 190 neurons scattered throughout the dorsomedial and medial parts of the nts . a recent study suggested that this population could be involved in short - term feeding suppression . such evidences suggest that pomc neurons located in the nts may have a marginal contribution to the global control of the energy homeostasis exerted by the pomc system . further analysis will be necessary to unveil the function of the nts pomc neurons . the other population is located in the arc . unlike nts pomc neurons , the function and phenotype of arc pomc neurons have been extensively studied . in addition , arc pomc neurons express a wide range of receptors , such as lepr , ir , and npy y1-r , that confer the ability to sense peripheral and central signals involved in the control of metabolic homeostasis . in fact , selective ablation of arc pomc neurons mimics the phenotype of the global pomc - null mice , producing increased food intake and reduced energy expenditure , resulting ultimately in obesity . these lines of evidences situate pomc neurons as a central node for sensing body energy reserves and thus as key elements to finely tune the mechanisms involved in the control of food intake and energy expenditure to keep energy homeostasis . accordingly , pomc mrna expression in the arc is reduced in leptin - deficient ob / ob mice , and leptin administration rescues this expression to that found in control mice . in the same vein , although ir ablation in pomc neurons does not cause any impact on body weight or glucose regulation , probably due to compensatory mechanisms , double lepr and ir deletion on pomc neurons produce higher negative impact on metabolic parameters than that found in the lepr ko mice . the above lines of evidence demonstrate that arc pomc neurons are key elements in the control of body weight and metabolism . this observation makes also pomc neurons a good candidate to relay metabolic information to gnrh neurons . admittedly , information about the role of pomc products in the control of the reproductive axis is scarce and , in some cases , controversial . yet , there is growing evidence suggesting that pomc neurons do participate in conveying metabolic information to gnrh neurons . in this context , whereas early studies demonstrated that independent ablation of lepr or ir on pomc neurons does not disrupt fertility [ 45 , 47 ] , mutant mice lacking both receptors in pomc neurons show severe reproductive deficiencies . overall , the above data suggest that arcuate pomc neurons could act as a mediator for leptin and insulin actions upon gnrh neurons . in this sense , watanobe showed , using push - pull perfusion techniques , that leptin infusion in the poa and median eminence stimulates gnrh / lh release , and this effect is preceded by an increase in -msh secretion . however , discrepancies about the effect of -msh on lh can be found in the literature . in this sense , recent data demonstrated that melanocortin receptors 3 and 4 ( mc3/4r ) agonist , melanotan ii , increases gnrh pulse generator activity in goats and this effect can be attenuated by estradiol . this finding also suggests that the central actions of -msh on reproduction are mediated probably via mc3r and mc4r . overall , it seems that although both mc3r and mc4r are involved in the control of reproduction , the lack of one type of receptor could be partially compensated by the other , resulting in a milder phenotype . however , whether gnrh neurons are direct targets of melanocortin actions remained unknown until very recently , when electrophysiological recordings of gnrh neurons demonstrated that -msh increases cell firing in most of gnrh neurons through postsynaptic activation of both mc3r and mc4r . overall , it seems that pomc neurons convey leptin actions on gnrh neurons directly through mc3/4r pathways . although -endorphin is also derived from the pomc precursor , lines of evidence in the literature attribute to these neuropeptide effects diametrically opposite , in terms of metabolic and reproductive control , to those found for other pomc - derived peptides ( such as -msh , -msh , and acth ) . -endorphin mediates its actions mostly via -opioid receptor , although it displays also relatively high affinity by - , and -subtypes of opioid receptors . albeit there is consensus in the pharmacological studies about the stimulatory actions of -endorphin on food intake , paradoxically , mutant male mice retaining all the pomc - derived peptides except -endorphin show increased food consumption and are obese . the fact that -endorphin is able to negatively modulate gnrh / gonadotropin release opens up the possibility that pomc neurons could integrate also negative inputs to suppress gnrh activity via this opioid . in this sense , recent data suggested that the signal of energy insufficiency , ghrelin , suppresses the reproductive axis via -endorphin . in this context , while electrophysiological recordings in guinea pig demonstrated that the -opioid receptor agonist , damgo , inhibits gnrh neurons postsynaptically , data from the teleost fish , medaka , indicated that -endorphin reduces action potential firing in gnrh neurons via indirect mechanisms . admittedly , part of the above discrepancies could be attributed to interspecies differences . cart - immunoreactive neurons have been identified in many different hypothalamic ( paraventricular , arcuate , dorsomedial , and ventral premmamillary nuclei , as well as lateral hypothalamic area ) and extrahypothalamic nuclei ( central amygdala ) [ 8991 ] . however , the arc pomc / cart population has received special attention due to its potential role in regulating food intake and reproduction , while little is known about the intervention of other cart populations in the integration of these two systems . altogether , the above lines of evidence suggest that pomc neurons are key elements for conveying a large variety of metabolic inputs , ranging from signals of nutrient deficiency to energy sufficiency cues , to control the reproductive axis by secreting a wide diversity of neuropeptides with different , in many cases opposite , actions . in rats , npy - expressing neurons are widespread through different areas across the brain including , among others , the olfactory bulb , striatum , hypothalamus , spinal cord , and pineal gland , being the arc and the paraventricular nucleus , within the hypothalamus , two of the nuclei containing higher concentrations of npy neurons and fibers . the arc population coexpresses also agrp and has been postulated as key element in the control of feeding behaviour . in fact , in 2011 aponte and co - workers demonstrated in an extremely elegant study that activation of arcuate npy / agrp neurons , using optogenetic tools , produces rapid stimulation of seeking behaviour and compulsive feeding in mice . so far , five different npy receptors ( y1 , y2 , y4 , y5 , and y6 ) have been identified , which show different affinity for the various peptides of the npy family [ 100103 ] . regarding the potential role of npy in reproduction , arc npy neurons send projections to gnrh perikarya and nerve terminals and have been suggested to participate in conveying information about energy insufficiency to the reproductive axis . despite these compelling lines of evidence , in fact , pharmacological studies showed opposite effects of npy on lh release depending on the steroid milieu and receptor agonist used . however , fasting does not induce the expected decay in lh levels in these animals , suggesting that npy signalling is needed for transmitting metabolic information when the conditions for reproduction are unfavourable due to reduced energy availability . overall , the former discrepancies about the dual inhibitory / stimulatory effect of npy on lh secretion could be due to differences in the ratio y1r / y4r in the model evaluated . moreover , electrical recordings in gnrh neurons demonstrated that agrp administration prevents the excitatory effect of the mc3/4r agonist , melanotan ii . however , later results revealed that agrp exhibits also stimulatory effects in a small subpopulation of gnrh neurons . this paradoxical effect could be mediated via a mechanism independent of mcr3/4 receptors , a possibility that had been already considered in the context of other studies , which showed agrp actions unexplained via mcr3/4 blockade [ 118 , 119 ] . overall , the above data suggest that arc npy neurons are able to module reproductive function through different mechanism involving npy secretion and/or inhibition of melanocortin signalling by agrp in situations of energy deficiency . reproductive function is highly dependent on nutrient availability . to ensure an efficient utilization of the energy stores , redundant pathways are necessary . of note , data in the literature suggest that many of metabolic signals informing the reproductive brain do no act directly on gnrh neurons , the final output in the brain controlling reproduction . in fact , in recent years , the existence of different neuronal populations that are able to sense peripheral and central indicators of the energy status to convey this information to gnrh neurons has been exposed by a large number of experimental studies . as reviewed herein , arc pomc , npy , and kiss1 neurons have been proposed as key intermediary elements to carry out this function . this raises the possibility of the existence of a complex network of interconnected neurons that is involved in the precise sensing of the metabolic status and in the transmission of this information to gnrh neurons to consequently modulate the reproductive function . on the basis of the evidence summarized here , it is tenable to postulate that kiss1 , npy , and pomc neurons are prominent elements of such a complex neuronal network admittedly , however , although for sake of concision this review has focussed only in this selected group of neuronal populations , it is likely that other partners exist on such circuitry responsible for central metabolic - reproductive interactions , such as neurons expressing galanin - like peptide ( galp ) , melanin - concentrating hormone ( mch ) , orexins , or corticotropin - releasing hormone ( crf ) [ 122 , 123 ] . while the evidence so far available suggests that the roles of the latter neuropeptides in the control of the hpg axis are less prominent , it remains a challenge for the future to decipher how major and subordinate metabolic regulators interplay with and impinge on the central elements of the reproductive axis .

chemical senses during a meal are a cue to recognize food ingestion and to determine its composition of nutrients when chemical substances stimulate sensory organs . olfaction and taste are typically known as chemical senses , and subsequent visceral senses by digested products in the alimentary tract is as an intake signal of nutrients which were absorbed into the inner body sensory organs such as blood vessels , lymph node , liver , kidney , and brain . major physiological roles of chemical senses include recognition of food intake , smooth digestion and absorption , and maintenance for homeostasis of each nutrient in the blood as well as the brain after a meal . additionally , regulation of glucose and free fatty acid concentration in the blood and related hormones released from specific tissues can also primarily be occurring by chemical senses for control of appetite and satiety for foods intake as a major marker for energy balance in the living body . the concentration of each amino acid in the blood as well as the brain are metabolically controlled within certain limits all day long , regardless of the amount and the quality of meals containing any dietary protein . as such , attention is focused on visual sense , texture , olfaction and taste during the meals , to judge whether it is food or not , which nutrients are included in what amount , and whether there has been some changes from past experience , retrieving previous memory . once the brain judges there would be no problem based on the past experience of similar food , the esophagus opens and food is swallowed with saliva , which includes electrolytes , and other nutrients such as amino acids and digestive enzyme such as -amylase that digests starch to amylose . a aense of taste profile adapts to intake these nutrients and forms boundary concentrations to feel the particular taste such as sweetness for energy , saltiness for electrolytes , and umami taste for protein . in other words , in order to feel the taste of food , in parallel with the composition of nutrients , they must be more concentrated rather than those included in the saliva , which is the reason that foods rich in nutrients but have no smell or taste are hard either to stimulate appetite or to reach satiety . for example , we can eat cooked rice which has little taste , because we used to get it . but actually we were able to eat the rice with other palatable food although it is not easy to eat the rice alone . recent research has exposed that l - glutamate ( glu ) , an amino acid , has multiple physiological functions in our body during and after a meal . as an essential substrate in the intermediary metabolism , therefore , free glu is present in most organs and tissues ( skeletal muscles , brain , kidneys , and liver ) in substantial concentrations [ 1 , 2 ] . glu plays an important role for energy metabolism and the substance for metabolism of other amino acids , glutathione , and body proteins . in the brain , glu , which is locally produced completely de novo from glucose , acts as a major excitatory neurotransmitter , and its activity regulates synaptic plasticity , learning , memory , motor activity , and neural development . but dietary glu is almost impermeable into the circulating blood . also there is the blood brain barrier against glu in between blood and brain so as to not to incorporate into it the brain . this glu plays independently of dietary glu in the alimentary tracts as a glu signaling mainly , a major portion of glu is metabolized for energy sources and partially for substrate for protein synthesis . in the oral cavity , glu in foods elicits a unique taste termed umami taste , which is generally thought to be a signal for dietary protein ingestion in the brain . in addition to the gustatory roles of glu as a typical umami taste material , recent studies have unveiled the postingestive significance of glu on various physiological functions such as the process of digestion , nutrient absorption and metabolism , and energy homeostasis via brain activation . these effects might be mediated via luminal gut glu sensors functionally linked to the afferent branches of the vagus nerve as visceral sensations , or particularly via the afferent sensory nerves in the oral cavity as a taste sensation . moreover , glu acts as a reinforcer after ingestion via vagal afferent activation in the gut . for example , we recently observed that an intragastric infusion of 60 mm glu aqueous solution induced a conditioned flavor preference ( cfp ) in rats [ 3 , 4 ] . it is strongly suggested that the sense of glu mortified food intake into the brain to prepare to operate efficient digestion in parallel with memory functions due to expectation for normal appetite for foods and maintenance for homeostasis . in recent years , umami taste was categorized as a basic taste like others , i.e. , sweetness , saltiness , bitterness and sourness . typical umami taste material is monosodium l - glutamate ( msg ) that is generally used as seasoning of umami taste material beyond 2.5 million tons per year , and the consumption is still increasing by several percent per year . this fact strongly suggests that msg plays physiological roles in the living body including humans during and after meals particularly in the alimentary organs as visceral information via the gut brain axis except the umami sensation orally . thus , this review was conducted to summarize recent progress in efficacies of glu signaling in gut including gut brain communication for a healthier life . the relative strengths of different stimuli inducing the parotid salivary flow are as follows : citric acid ( sour ) msg ( umami ) > nacl ( salty ) > sucrose ( sweet ) magnesium sulfate ( bitter ) . the role of saliva is not only to lubricate food for mastication and swallowing but also to initiate the digestion of nutrients , i.e. , carbohydrates and fats , because it contains the digestive enzymes such as -amylase and lipase . sweet taste stimulation with sucrose and glucose in iso - tonic solution increases the efferent activity of the pancreatic and the hepatic vagus nerves in rats , whereas a very salty taste solution containing a high concentration of nacl suppresses such activity because of aversive foods stimuli for the expected disorder of homeostasis [ 710 ] . in addition , sweet taste stimulation elicits insulin release prior to increasing plasma glucose levels , a process called cephalic - phase insulin release [ 1113 ] . by contrast , sweet taste stimulation was observed to suppress vagal gastric efferent ( vge ) activity and the efferent activity of the adrenal , pancreatic and hepatic excitation of sympathetic nerves , whereas salty taste stimulation was shown to increase these activities [ 9 , 14 ] . moreover , sweet taste signals stimulate gastric acid secretion ( ph 0.56 ) via the vagus nerve excitation . umami taste stimulation produced by a msg isotonic solution ( 150 mm ) , was able to activate vge activity in overnight fasted rats and the efferent activity of the pancreatic and hepatic vagus nerves ( fig . 1 ) [ 10 , 16 , 17 ] , in association with an increase in insulin secretion . however , it has been reported that msg aqueous solution without food ingredients does not elicit cephalic - phase insulin release , so further study to evaluate certain permissive effect will be necessary in the case of food intake with or without msg . in addition , glu stimulation orally enhanced the sympathetic efferent activity into the adipose tissue in overnight fasted rats , markedly suggesting that recognition of food intake by glu signaling via vagal afferent excitation reduced lipolysis during food deprivation or overnight fasting.fig . 1reflex activation of vagal gastric and pancreatic nerve activity stimulated through oral , gastric , intestinal and hepatoportal glutamate sensors . reproduced from niijima reflex activation of vagal gastric and pancreatic nerve activity stimulated through oral , gastric , intestinal and hepatoportal glutamate sensors . in the gastrointestinal ( gi ) tract , various nutrients are detected and absorbed through the luminal layer . nutrients also regulate the activity of vagal afferent nerves and the release of gi peptides , including cholecystokinin ( cck ) , peptide yy , glucagon - like peptide-1 ( glp-1 ) and -2 , leptin , ghrelin , and others [ 2023 ] due to efficient usage of adsorbed nutrients and their homeostasis in the living body after a meal . it was thought for a long time that the vagal gastric afferents ( vga ) in the stomach could detect only gastric distension and not individual nutrients as chemical senses . this is important in the field of gastric nutrient perception because these data strongly suggest that chemical perception , in particular an amino acid - sensing system , exists in the gastric mucosa . interestingly , among the 20 kinds of amino acids , glu alone stimulates the nerve end of rat vga ( fig . 2 ) . there are no responses to any sugars including glucose and sodium chloride , luminal perfusion in rats under the peripheral anesthetic treatment with lidocaine , abolished the glu - evoked vga activation , indicating that this response is a chemical event within the tissue of gastric mucosa . furthermore , the glu response was blocked by either depletion of serotonin ( 5-ht ) using parachlorophenylalanine ( pcpa ) treatment for 5ht type 3 ( 5-ht3 ) receptors in the nerve end of vagal afferent , or inhibition of the nitric oxide ( no ) synthetase using l - name treatment . the vagal afferent response was also mimicked by luminal perfusion with an no donor such as sodium nitroprusside . in addition , no donor - induced afferent activation was abolished by 5-ht3 receptor blockage . this finding strongly supports the possibility of intercellular communication in the rat gastric mucosa between mucosal cells and the vagus nerve - end using no and 5-ht as potent stimuli during digestion processes . more than 90 % of 5-ht throughout the body is concentrated and localized in the enterochromaffin ( ec ) cells of the gi mucosa . the physiological role of mucosal 5-ht from ec cells serves a paracrine function by specifically recognizing glu in the lumen of the stomach , which is similar to the physiological role , as reported in the duodenal glucose sensing system.fig . 2vagal gastric afferent ( vga ) responses to intragastric infusion of various amino acid solutions . each aqueous solution ( 150 mmol / l , 2 ml / rat ) was intubated to rat stomach , and the mean value of discharge rate above baseline at 20 min was plotted . each column and horizontal bar represents mean sem from 5 rats . * * p < 0.05 versus saline ( kruskal wallis test ) . vagal gastric afferent ( vga ) responses to intragastric infusion of various amino acid solutions . each aqueous solution ( 150 mmol / l , 2 ml / rat ) was intubated to rat stomach , and the mean value of discharge rate above baseline at 20 min was plotted . each column and horizontal bar represents mean sem from 5 rats . * * p < 0.05 versus saline ( kruskal wallis test ) . reproduced from uneyama et al . our understanding of the sensing of nutrients in the gut , luminal layer may contains particular cells to be referred to as an intestinal sensor cell as originally proposed in the 1970s by fujita et al . this hypothesis suggests that nutrient - sensing cells are distributed in the gastric antrum or duodenal mucosa and that when these cells interact with luminal nutrients in the digested foods , they enhance to release hormones in an endocrine or paracrine manner to transfer information about luminal nutrient content to other organs , including the brain either via endocrine or vagal pathways . however , these cells involved in the gut nutrient perception system remained to be identified for a long time . in 1996 , hfer et al . reported that taste - like cells similar to the taste cells in the oral cavity are distributed in the gastric and intestinal mucosa and they proposed that these taste - like cells represent the unknown sensor cells . subsequently , with the development of molecular biology techniques in the field of taste research , several taste receptors responding to different amino acids have been identified . we now know that metabotropic glu receptors ( mglurs ) , a calcium sensing receptor ( casr ) , and a taste receptor ( a heterodimer of t1r1 and t1r3 : t1r1/t1r3 ) are all linked to amino acid sensation in the tongue . although the molecule(s ) that sense glu in the gastric mucosa is / are still unclear , intragastric infusion of msg causes a vago - vagal reflex , which increases vge , as well as vagal pancreatic and celiac efferent activities ( fig . 1 ) [ 17 , 27 ] . interestingly , inosine 5-monophosphate , another umami substance that enhances msg binding to taste receptors and thus enhances umami taste intensity synergistically , also activates vga and increases vagal celiac efferent activity in an additional manner . assuming the coexistence of free glu with dietary protein is very common in foods , these findings suggest that a specific glu - sensing system in the stomach could contribute to the gastric phase of protein digestion and could integrate nutrient information in the brain by vagal afferent excitation . we defined recently that the possible glu - sensing system in the stomach is a metabotropic glutamate receptor type 1 ( mglur1 ) in mucosal cells that is to express no synthetase to stimulate 5-ht release through the vagal afferents excitation . there is little contribution by ec cells expressing tir1/tir3 to induce 5-ht release < 5 % in total . recently , we defined that free glu concentration in saliva and pancreatic juice is several times higher ti than in its blood level . this digestive juice derived glu possibly collaborates with dietary glu to act as glu signaling in alimentary tracts . in contrast to what occurs in the small intestine , there are many reports that intraduodenal infusion of amino acids or oligo - peptides alters the vagal celiac afferent ( vca ) activity . sharma and nasset observed an apparent increase in the mesenteric afferent activity in either whole - nerve or multifiber preparations from the gi tract following amino acid infusions in cats . using a unitary recording technique in the nodose ganglion , jeanningros and colleagues subsequently revealed in detail the response of vca to various kinds of amino acid infusions in the cat small intestine . their report described many sensors responsive to arginine , leucine , and other amino acids [ 29 , 30 ] . recently , we re - examined the luminal amino acid sensitivity of vca in rats . intraintestinal infusion of msg , lysine , leucine , and other amino acids evoked excitatory responses in vca . in contrast to the responses in these amino acids , the same treatment with glycine , methionine , and certain other amino acids led to the depression of afferent nerve activity . in rats , duodenal infusions of protein hydrolysates also increased mesenteric afferent activity [ 32 , 33 ] . schwartz and moran revealed that duodenal intubation of protein hydrolysates ( e.g. , peptone ) stimulated vagal celiac afferents , indicating that an amino acid sensor or oligopeptide sensor might exist in the rat luminal duodenum . however , the mechanisms underlying such sensations are not fully understood , and further research is needed . changes in the vca activity induce autonomic reflexes and regulate various visceral functions , oral and intra - gastric and intestinal infusions of msg isotonic solution in rats resulted in an increase in vge , vagal pancreatic efferent activity [ 16 , 17 ] , and lysine evoked long - lasting enhancement of vge activity in lysine - deficient rats . on the other hand , the intraintestinal infusion of glycine inhibited vge activity . in addition , introduction of a glucose solution into the intestine increased vca activity ; the sensing mechanism underlying glucose effects has been described in another review . a glucose solution also suppressed sympathetic adrenal efferent activity and enhanced vagal pancreatic efferent activity . these observations support our hypothesis that the vagal gi afferent signals , particularly gastric branch by glu signaling , are to recognize food intake , regulate gastric emptying , gi motility , metabolic control for homeostasis and both appetite and satiety for food [ 35 , 36 ] . from our recent studies , in addition to autonomic reflexes , the effects of ingested nutrients in the alimentary tracts are processed in the forebrain , which determines whether food is good or not for content of required nutrients and maintenance of homeostasis and subsequently regulates feeding behavior in a palatable manner . to investigate which regions of the rat brain respond to ingested nutrients , we used an functional mri ( fmri , 4.7 t ) technique . the merit of fmr is that activated areas in the well - trained rat whole brain can be investigated simultaneously by non - invasive awake conditions without unpleasant stresses , like any painful treatment . an intragastric intubation of 60 mm msg , or isocaloric ( 60 mm ) glucose and an equimolar nacl solution have been shown to activate distinct forebrain regions ( fig . 3 ) [ 37 , 38 ] . an intragastric load of msg significantly activated several brain regions including the hippocampus ( memory function ) , the amygdala central body ( preference center for nutrients ) , the lateral hypothalamus ( feeding center ) , the dorsomedial hypothalamus ( basic metabolism regulator ) , and the medial preoptic ( body temperature regulator ) . on the other hand , an intragastric infusion of glucose activated the insular cortex , amygdala , nucleus accumbens ( which is the terminal dopaminergic projection for indulgence or craving ) , and the lateral and ventromedial ( satiety center ) hypothalamus . we also investigated brain responses to an intragastric load of corn oil emulsion , which activated the amygdala , the lateral hypothalamus , the hippocampus , and the ventral tegmental area . the changes of brain function after 60 mm msg intubation into the stomach to cause thermogenesis for energy expenditure as diet induced thermogenesis during and after a meal . there was a concern about prevention of obesity development , in the model of rats , fed a high sugar and fat diet for 1 year from the wearing period . control animals offered to drinking water alone , became typically obesity but similar ones were freely available to drink 1 % ( w / v ) msg solution and water in a choice paradigm , and dominantly preferred the msg solution . glu signaling during digestion processes may enhance thermogenesis and force excessive intake of every source from an experimental diet to be cured as energy expenditure suggesting that glu is effective in not developing obesity when overeating occurs .fig . 3activated area of rat forebrain specific to the stimulation of treatment with post - oral nutrients ( glucose , msg , nacl 60 mm and nacl 150 mm ) compared with common control images which are averaged during 10 min before administration . t - map images ( bregma + 2.0 ) depict activated brain regions at pre - administration ( 5 min ) , 10 , 20 , and 40 min after the onset of intragastric infusion . acc anterior cingulate cortex , cpu caudate putamen , icx insular cortex , nac nucleus accumbens . color bar , t value . reproduced from tsurugizawa et al . activated area of rat forebrain specific to the stimulation of treatment with post - oral nutrients ( glucose , msg , nacl 60 mm and nacl 150 mm ) compared with common control images which are averaged during 10 min before administration . t - map images ( bregma + 2.0 ) depict activated brain regions at pre - administration ( 5 min ) , 10 , 20 , and 40 min after the onset of intragastric infusion . acc anterior cingulate cortex , cpu caudate putamen , icx insular cortex , nac nucleus accumbens . behavioral studies also showed that ingested glutamate or glucose ( 60 mm in each ) in water or corn oil emulsion have positive postingestive effects with regard to flavor preference learning in rats [ 3 , 39 , 41 ] . in rodents and humans , the preference for the flavor of an ingested solution can be increased by repeatedly pairing it with ingestion or intragastric infusion of certain solutions containing particular nutrients to be required physiologically . behavioral studies revealed that the intragastric infusion of liquid containing carbohydrates , lipids , and alcohols induces cfp in rodents [ 41 , 42 ] . in addition , we previously showed that an intragastric load of 60 mm msg evoked cfp in rats . isocaloric ( 60 mm ) glucose and equimolar ( 60 mm ) nacl solution did not evoke cfp ( fig . 4 ) [ 3 , 4 ] , although 480 mm hypertonic glucose aqueous solution did evoke cfp because of an unpleasant symptom occurring . much higher concentrations of glucose lead to an increase in blood glucose and insulin release . these results indicate that the preference for the flavored solution paired with a gut infusion of msg is coupled with memory function operation to establish cfp due to neither a caloric effect nor hyperosmotic pressure effect . based on the results of functional brain imaging and cfp behavioral studies , the brain regions commonly activated in response to the intragastric infusion of either msg or glucose in water ( 60 mm ) or corn oil emulsion include the anterior cingulate cortex , insular cortex , amygdala , caudate - putamen , hippocampus , and the lateral hypothalamus [ 38 , 39 ] . thus , these regions should be related to cfp . in particular , the lateral hypothalamus is an important area for regulating intake of food or liquid containing nutrients.fig . 4mean intake of conditioned stimulus ( filled bar cs+ , flavored water with intragastric infusion of msg , nacl , or glucose ; and open bar cs , flavored water with intragastric infusion of water ) solutions in the pre - test and test periods . mean percent intakes of the cs+ solution are shown above the bars . reproduced from uematsu et al . mean intake of conditioned stimulus ( filled bar cs+ , flavored water with intragastric infusion of msg , nacl , or glucose ; and open bar cs , flavored water with intragastric infusion of water ) solutions in the pre - test and test periods . mean percent intakes of the cs+ solution are shown above the bars . reproduced from uematsu et al . a previous report revealed that lesions of the lateral hypothalamus diminished cfp induced by intragastric infusion of a glucose solution . the glucose - sensitive neurons that exist in the ventromedial hypothalamus are activated as the intracellular glucose levels increase as a satiety signal . dopaminergic projections from the ventrotegmental area to the nucleus accumbens , the amygdala , and the lateral hypothalamus are related to the preference for , or addiction to , ingested energy sources , such as glucose - like sugars and corn oil . some studies showed that sugar intake increased dopamine release in the nucleus accumbens shell region in rats , reportedly causing them to become an addicted behavior to all sugars . on the other hand , intragastric infusion of msg and nacl 3 ) , and lesions of neurons in the ventrotegmental area bilaterally using 6 hydroxy dopamine do not affect the preference for msg and nacl in rats . these results show that the postingestive effects of glu and nacl differ from those of sugars and lipids . similar rats under lysine deficiency strongly preferred lysine solution with bitterness quantitatively in a choice paradigm among whole amino acids . it is manifestthat any amino acid and nacl are independent to indulgence and/or addiction like sugars and lipids . another advantage of fmri is that it has better temporal resolution than an alternative monitoring technique , c - fos labeling . the time course of particular brain area activation is different for msg and for glucose , as revealed by fmri ( fig . 5 ) . an intragastric infusion of 60 mm msg induced the vagus afferent activation in most of the brain during the infusion period . in contrast , intragastric infusion of 60 mm glucose induces long - lasting activation for more than 1 h. these different temporal and regional activation patterns in the brain are due to distinctive signaling pathways between the gut and brain and they result in distinctive effects on postingestive behavior.fig . ingested food is digested and absorbed in free form as small molecular nutrients in the gi tract . the afferent vagus nerve , which innervates the entire gi tract and projects to the nucleus of the solitary tract ( nts ) , is activated by each of these free form nutrients during digestion processes . in parallel , in addition to the process of absorption and metabolism in the gut , recent studies have indicated that the stomach , duodenum , and other small intestine express to localize chemosensing taste receptors in the luminal layer , and other g - protein - coupled receptors ( gpcrs ) . the t1r receptors , which are responsible for the chemoreception of the sweet and umami tastes , and the family of t2r receptors , which mediate the chemoreception of the bitter taste , are both expressed in the gut [ 46 , 47 ] . a fatty acid receptor , gpr120 , exists in both the oral cavity and the gi tract , and these acids interact with it to induce the release of circulating glp-1 . furthermore , free fatty acids also interact with another receptor , gpr40 , in the gi tract to promote secretion of glp-1 and cholecystokinin ( cck ) . the glp-1 and cck evoke c - fos positive immunoreactivity in several brain regions , including the amygdala [ 5153 ] . recently , we demonstrated that fluctuations in insulin following the intragastric administration of glucose correlate with fmri responses in the amygdala , the ventromedial hypothalamus , and the nucleus accumbens . electrophysiological studies have shown that intragastric and enteric delivery of amino acids and lipids both activate the afferent vagus nerve as described above [ 3032 , 54 , 55 ] . these reports indicate that the vagus afferent response is important for the transmission of each dietary nutrient information in the gut to the brain . interestingly , behavioral studies have shown that abdominal vagotomy eliminates cfp in response to intragastric infusion of msg but does not affect cfp response in the case of carbohydrates in rats . an fmri study showed that total and partial abdominal vagotomy diminished glu - induced activation in the nts and the hypothalamus , whereas total vagotomy did not affect glucose - induced brain activation . instead these results from fmri studies in vagotomized rats are consistent with postingestive behavior studies , indicating that internal signals in response to glu mainly involve the vagus nerve , whereas those in response to glucose at least partially involve insulin release . finally , there are distinct postingestive effects in response to different nutrients , resulting in the activation of forebrain regions . the spatial and temporal patterns of brain activation could link with postingestive behavioral and physiological effects underlying the maintenance of homeostasis following a meal . weaning is a critical period for infants , because of changes of breast milk to foods exposing the infant to a risky environment including pathogenic infections . recently , we analyzed free glutamate content in the pancreatic juice several fold higher than the plasma level . the localization of free glutamate in the pancreas using a specific antibody was seen so that zymogen granule in the acinar cells was positive . during exocytosis of the zymogen granule as digestive juices , also free glutamate is to be released . other acinar cell membranes expresses mglur1 to receive free glutamate stimuli due to exocytosis of zymogen granules . the cascade type of pancreatic juice secretion should be triggered by released free glu in the pancreas . these endocrine cells also express metabotropic glutamate receptors type 4 and 8 receiving free glu stimuli released from the vagus nerve - end like cephalic phase stimuli . so , exocrine and endocrine systems to release digestive enzymes as well as pancreatic hormones have a common fashion in the digestive organs to be controlled by free glutamate stimuli . intracellular glutamine is enzymatically metabolized to glutamate and yielded free glutamate to be incorporated into the zymogen granules . the free form of glutamate in the digestive juice directly stimulate the alimentary tract to regulate digestion and absorption normally , through ph control and mucin release for the barrier system . the capacity of biosynthesis of free glutamate is immature in infants and retarded in the elderly . so , the propriate level of dietary glutamine as a precursor of glutamate and free glutamate itself are beneficial for normal digestive function . we tried to use piglets similar to a human infants model so that just weaned piglets are anorexigenic is concomitant with negative energy balance . they used to display diarrhea and subsequent dehydration , and subsequently malnutritional piglet growth is to be retarded . from the point of digestive physiology , switching from a milk formula to a solid diet as described above , glu has a potential for the intestine to release mucin due to establishment of a barriers system against microbiota . therefore , supplementation liquid for weaning piglets containing glu and l - glutamine ( gln ) as a precursor for glu adding to the weaning diet results in a significant improvement of the diarrhea index and their state of nutrition . habituation studies employed elderly subjects to be offered rice glue containing 0.5 ( w / v ) msg for 3 months . double blind and cross - over studies yield improvement in the quality of life concerning emotion and appetite for foods , reduced incidence of diarrhea and vomiting , peripheral circulation to cure bed thaw lesion , etc . reflecting the basic metabolic rate enhanced by glu signaling in gut . free glu signaling during a meal and subsequent processes in the alimentary organs plays important physiological roles in the perception of umami taste , visceral information and regulation of endo - and exocrine systems , and excitatory neurotransmission . but there are no way to entry dietary free glu into the circulating blood as well as brain cerebrospinal fluid because of both the intestinal hepatic barrier and the blood brain barrier are not permeable for free glu . glu signaling by dietary glu is positively effective in the oral cavity through alimentary tracts via several receptors . in our series of studies reviewed here , we showed that dietary glu also stimulates glu sensors in the stomach and intestines , producing local effects on gut function for ingested food digestion . moreover , via the release of signaling molecules no and 5-ht , the presence of glu in the gut leads to the activation of the vagal afferent nerve which in turn modulates a number of target areas in the brain , particularly the gastric vagal afferent responds to glu alone rather than electrolytes , sugars and other amino acids , suggesting that glu signaling in the stomach is to be a signal for food intake controlling whole processes of digestion and absorption of yielded nutrients . in addition , we described the postingestive effects of glu and showed that they were different from those elicited by glucose and lipids . previous fmri observation in the brain and behavioral studies in rodents have indicated that glu has positive postingestive effects through the vagal afferent nerve that controls appetite and satiety for food . however , glu does not have the same reinforcing properties of craving and/or addictive substances such as sugars and alcohol . altogether , these findings indicate that dietary glu influences numerous physiological functions , suggesting a broad , integrative role for dietary glu in body homeostasis . this newly developed method for brain functional change observation in well - trained awake rats using a fmri after nutrient stimulation in the gut by gastric intubation into the stomach was recently confirmed by c - fos expression in similar rats , suggesting that gut brain communication through chemical senses of nutrient , particularly glu , becomes available to research using awake animals in connection with non - invasive treatment [ 39 , 58 ] due to an understanding why rats , fed a high - fat and high - sugar diet , do not become obese when glu solution is available to drink with this food intake through higher thermogenesis due to energy expenditure . dietary glu and glutamine as a precursor for glu signaling are positively promoted as a function of the alimentary tracts in infants as well as the elderly because of the biosynthesis capacity of free glu is not sufficient in the exo- and endocrine organs .

dietary glutamate ( glu ) stimulates to evoke the umami taste , one of the five basic tastes , enhancing food palatability . but it is also the main gut energy source for the absorption and metabolism for each nutrient , thus , only a trace amount of glu reaches the general circulation . recently , we demonstrated a unique gut sensing system for free glu ( glutamate signaling ) . glu is the only nutrient among amino acids , sugars and electrolytes that activates rat gastric vagal afferents from the luminal side specifically via metabotropic glu receptors type 1 on mucosal cells releasing mucin and nitrite mono - oxide ( no ) , then no stimulates serotonin ( 5ht ) release at the enterochromaffin cell . finally released 5ht stimulates 5ht3 receptor at the nerve end of the vagal afferent fiber . functional magnetic resonance imaging ( f - mri , 4.7 t ) analysis revealed that luminal sensing with 1 % ( w / v ) monosodium l - glutamate ( msg ) in rat stomach activates both the medial preoptic area ( body temperature controller ) and the dorsomedial hypothalamus ( basic metabolic regulator ) , resulting in diet - induced thermogenesis during mealing without changes of appetite for food . interestingly , rats were forced to eat a high fat and high sugar diet with free access to 1 % ( w / w ) msg and water in a choice paradigm and showed the strong preference for the msg solution and subsequently , they displayed lower fat deposition , weight gain and blood leptin . on the other hand , these brain functional changes by the f - mri signal after 60 mm msg intubation into the stomach was abolished in the case of total vagotomized rats , suggesting that luminal glutamate signaling contributes to control digestion and thermogenesis without obesity .

Introduction Gustatory glutamate stimuli regulate autonomic nerve activity Gut nutrient stimuli control vagus nervous activity in the stomach during a meal Chemical senses in alimentary tracts to regulate metabolism of absorbed nutrients Brain activation after gut nutrient stimulation Signaling mechanisms of the gutbrain axis promotes control of metabolism for homeostasis Glutamate plays a role to promote digestive juice secretion and bowel movement Conclusions

olfaction and taste are typically known as chemical senses , and subsequent visceral senses by digested products in the alimentary tract is as an intake signal of nutrients which were absorbed into the inner body sensory organs such as blood vessels , lymph node , liver , kidney , and brain . major physiological roles of chemical senses include recognition of food intake , smooth digestion and absorption , and maintenance for homeostasis of each nutrient in the blood as well as the brain after a meal . additionally , regulation of glucose and free fatty acid concentration in the blood and related hormones released from specific tissues can also primarily be occurring by chemical senses for control of appetite and satiety for foods intake as a major marker for energy balance in the living body . the concentration of each amino acid in the blood as well as the brain are metabolically controlled within certain limits all day long , regardless of the amount and the quality of meals containing any dietary protein . in other words , in order to feel the taste of food , in parallel with the composition of nutrients , they must be more concentrated rather than those included in the saliva , which is the reason that foods rich in nutrients but have no smell or taste are hard either to stimulate appetite or to reach satiety . for example , we can eat cooked rice which has little taste , because we used to get it . recent research has exposed that l - glutamate ( glu ) , an amino acid , has multiple physiological functions in our body during and after a meal . as an essential substrate in the intermediary metabolism , therefore , free glu is present in most organs and tissues ( skeletal muscles , brain , kidneys , and liver ) in substantial concentrations [ 1 , 2 ] . glu plays an important role for energy metabolism and the substance for metabolism of other amino acids , glutathione , and body proteins . but dietary glu is almost impermeable into the circulating blood . this glu plays independently of dietary glu in the alimentary tracts as a glu signaling mainly , a major portion of glu is metabolized for energy sources and partially for substrate for protein synthesis . in the oral cavity , glu in foods elicits a unique taste termed umami taste , which is generally thought to be a signal for dietary protein ingestion in the brain . in addition to the gustatory roles of glu as a typical umami taste material , recent studies have unveiled the postingestive significance of glu on various physiological functions such as the process of digestion , nutrient absorption and metabolism , and energy homeostasis via brain activation . these effects might be mediated via luminal gut glu sensors functionally linked to the afferent branches of the vagus nerve as visceral sensations , or particularly via the afferent sensory nerves in the oral cavity as a taste sensation . for example , we recently observed that an intragastric infusion of 60 mm glu aqueous solution induced a conditioned flavor preference ( cfp ) in rats [ 3 , 4 ] . it is strongly suggested that the sense of glu mortified food intake into the brain to prepare to operate efficient digestion in parallel with memory functions due to expectation for normal appetite for foods and maintenance for homeostasis . typical umami taste material is monosodium l - glutamate ( msg ) that is generally used as seasoning of umami taste material beyond 2.5 million tons per year , and the consumption is still increasing by several percent per year . this fact strongly suggests that msg plays physiological roles in the living body including humans during and after meals particularly in the alimentary organs as visceral information via the gut brain axis except the umami sensation orally . sweet taste stimulation with sucrose and glucose in iso - tonic solution increases the efferent activity of the pancreatic and the hepatic vagus nerves in rats , whereas a very salty taste solution containing a high concentration of nacl suppresses such activity because of aversive foods stimuli for the expected disorder of homeostasis [ 710 ] . by contrast , sweet taste stimulation was observed to suppress vagal gastric efferent ( vge ) activity and the efferent activity of the adrenal , pancreatic and hepatic excitation of sympathetic nerves , whereas salty taste stimulation was shown to increase these activities [ 9 , 14 ] . umami taste stimulation produced by a msg isotonic solution ( 150 mm ) , was able to activate vge activity in overnight fasted rats and the efferent activity of the pancreatic and hepatic vagus nerves ( fig . however , it has been reported that msg aqueous solution without food ingredients does not elicit cephalic - phase insulin release , so further study to evaluate certain permissive effect will be necessary in the case of food intake with or without msg . in addition , glu stimulation orally enhanced the sympathetic efferent activity into the adipose tissue in overnight fasted rats , markedly suggesting that recognition of food intake by glu signaling via vagal afferent excitation reduced lipolysis during food deprivation or overnight fasting.fig . in the gastrointestinal ( gi ) tract , various nutrients are detected and absorbed through the luminal layer . nutrients also regulate the activity of vagal afferent nerves and the release of gi peptides , including cholecystokinin ( cck ) , peptide yy , glucagon - like peptide-1 ( glp-1 ) and -2 , leptin , ghrelin , and others [ 2023 ] due to efficient usage of adsorbed nutrients and their homeostasis in the living body after a meal . it was thought for a long time that the vagal gastric afferents ( vga ) in the stomach could detect only gastric distension and not individual nutrients as chemical senses . this is important in the field of gastric nutrient perception because these data strongly suggest that chemical perception , in particular an amino acid - sensing system , exists in the gastric mucosa . interestingly , among the 20 kinds of amino acids , glu alone stimulates the nerve end of rat vga ( fig . furthermore , the glu response was blocked by either depletion of serotonin ( 5-ht ) using parachlorophenylalanine ( pcpa ) treatment for 5ht type 3 ( 5-ht3 ) receptors in the nerve end of vagal afferent , or inhibition of the nitric oxide ( no ) synthetase using l - name treatment . in addition , no donor - induced afferent activation was abolished by 5-ht3 receptor blockage . this finding strongly supports the possibility of intercellular communication in the rat gastric mucosa between mucosal cells and the vagus nerve - end using no and 5-ht as potent stimuli during digestion processes . more than 90 % of 5-ht throughout the body is concentrated and localized in the enterochromaffin ( ec ) cells of the gi mucosa . the physiological role of mucosal 5-ht from ec cells serves a paracrine function by specifically recognizing glu in the lumen of the stomach , which is similar to the physiological role , as reported in the duodenal glucose sensing system.fig . each aqueous solution ( 150 mmol / l , 2 ml / rat ) was intubated to rat stomach , and the mean value of discharge rate above baseline at 20 min was plotted . each aqueous solution ( 150 mmol / l , 2 ml / rat ) was intubated to rat stomach , and the mean value of discharge rate above baseline at 20 min was plotted . our understanding of the sensing of nutrients in the gut , luminal layer may contains particular cells to be referred to as an intestinal sensor cell as originally proposed in the 1970s by fujita et al . this hypothesis suggests that nutrient - sensing cells are distributed in the gastric antrum or duodenal mucosa and that when these cells interact with luminal nutrients in the digested foods , they enhance to release hormones in an endocrine or paracrine manner to transfer information about luminal nutrient content to other organs , including the brain either via endocrine or vagal pathways . however , these cells involved in the gut nutrient perception system remained to be identified for a long time . subsequently , with the development of molecular biology techniques in the field of taste research , several taste receptors responding to different amino acids have been identified . we now know that metabotropic glu receptors ( mglurs ) , a calcium sensing receptor ( casr ) , and a taste receptor ( a heterodimer of t1r1 and t1r3 : t1r1/t1r3 ) are all linked to amino acid sensation in the tongue . interestingly , inosine 5-monophosphate , another umami substance that enhances msg binding to taste receptors and thus enhances umami taste intensity synergistically , also activates vga and increases vagal celiac efferent activity in an additional manner . assuming the coexistence of free glu with dietary protein is very common in foods , these findings suggest that a specific glu - sensing system in the stomach could contribute to the gastric phase of protein digestion and could integrate nutrient information in the brain by vagal afferent excitation . we defined recently that the possible glu - sensing system in the stomach is a metabotropic glutamate receptor type 1 ( mglur1 ) in mucosal cells that is to express no synthetase to stimulate 5-ht release through the vagal afferents excitation . recently , we defined that free glu concentration in saliva and pancreatic juice is several times higher ti than in its blood level . in contrast to what occurs in the small intestine , there are many reports that intraduodenal infusion of amino acids or oligo - peptides alters the vagal celiac afferent ( vca ) activity . sharma and nasset observed an apparent increase in the mesenteric afferent activity in either whole - nerve or multifiber preparations from the gi tract following amino acid infusions in cats . recently , we re - examined the luminal amino acid sensitivity of vca in rats . on the other hand , the intraintestinal infusion of glycine inhibited vge activity . these observations support our hypothesis that the vagal gi afferent signals , particularly gastric branch by glu signaling , are to recognize food intake , regulate gastric emptying , gi motility , metabolic control for homeostasis and both appetite and satiety for food [ 35 , 36 ] . from our recent studies , in addition to autonomic reflexes , the effects of ingested nutrients in the alimentary tracts are processed in the forebrain , which determines whether food is good or not for content of required nutrients and maintenance of homeostasis and subsequently regulates feeding behavior in a palatable manner . to investigate which regions of the rat brain respond to ingested nutrients , we used an functional mri ( fmri , 4.7 t ) technique . an intragastric load of msg significantly activated several brain regions including the hippocampus ( memory function ) , the amygdala central body ( preference center for nutrients ) , the lateral hypothalamus ( feeding center ) , the dorsomedial hypothalamus ( basic metabolism regulator ) , and the medial preoptic ( body temperature regulator ) . on the other hand , an intragastric infusion of glucose activated the insular cortex , amygdala , nucleus accumbens ( which is the terminal dopaminergic projection for indulgence or craving ) , and the lateral and ventromedial ( satiety center ) hypothalamus . the changes of brain function after 60 mm msg intubation into the stomach to cause thermogenesis for energy expenditure as diet induced thermogenesis during and after a meal . there was a concern about prevention of obesity development , in the model of rats , fed a high sugar and fat diet for 1 year from the wearing period . control animals offered to drinking water alone , became typically obesity but similar ones were freely available to drink 1 % ( w / v ) msg solution and water in a choice paradigm , and dominantly preferred the msg solution . glu signaling during digestion processes may enhance thermogenesis and force excessive intake of every source from an experimental diet to be cured as energy expenditure suggesting that glu is effective in not developing obesity when overeating occurs .fig . behavioral studies also showed that ingested glutamate or glucose ( 60 mm in each ) in water or corn oil emulsion have positive postingestive effects with regard to flavor preference learning in rats [ 3 , 39 , 41 ] . in addition , we previously showed that an intragastric load of 60 mm msg evoked cfp in rats . these results indicate that the preference for the flavored solution paired with a gut infusion of msg is coupled with memory function operation to establish cfp due to neither a caloric effect nor hyperosmotic pressure effect . based on the results of functional brain imaging and cfp behavioral studies , the brain regions commonly activated in response to the intragastric infusion of either msg or glucose in water ( 60 mm ) or corn oil emulsion include the anterior cingulate cortex , insular cortex , amygdala , caudate - putamen , hippocampus , and the lateral hypothalamus [ 38 , 39 ] . a previous report revealed that lesions of the lateral hypothalamus diminished cfp induced by intragastric infusion of a glucose solution . dopaminergic projections from the ventrotegmental area to the nucleus accumbens , the amygdala , and the lateral hypothalamus are related to the preference for , or addiction to , ingested energy sources , such as glucose - like sugars and corn oil . some studies showed that sugar intake increased dopamine release in the nucleus accumbens shell region in rats , reportedly causing them to become an addicted behavior to all sugars . on the other hand , intragastric infusion of msg and nacl 3 ) , and lesions of neurons in the ventrotegmental area bilaterally using 6 hydroxy dopamine do not affect the preference for msg and nacl in rats . these results show that the postingestive effects of glu and nacl differ from those of sugars and lipids . similar rats under lysine deficiency strongly preferred lysine solution with bitterness quantitatively in a choice paradigm among whole amino acids . it is manifestthat any amino acid and nacl are independent to indulgence and/or addiction like sugars and lipids . an intragastric infusion of 60 mm msg induced the vagus afferent activation in most of the brain during the infusion period . the afferent vagus nerve , which innervates the entire gi tract and projects to the nucleus of the solitary tract ( nts ) , is activated by each of these free form nutrients during digestion processes . in parallel , in addition to the process of absorption and metabolism in the gut , recent studies have indicated that the stomach , duodenum , and other small intestine express to localize chemosensing taste receptors in the luminal layer , and other g - protein - coupled receptors ( gpcrs ) . the t1r receptors , which are responsible for the chemoreception of the sweet and umami tastes , and the family of t2r receptors , which mediate the chemoreception of the bitter taste , are both expressed in the gut [ 46 , 47 ] . a fatty acid receptor , gpr120 , exists in both the oral cavity and the gi tract , and these acids interact with it to induce the release of circulating glp-1 . recently , we demonstrated that fluctuations in insulin following the intragastric administration of glucose correlate with fmri responses in the amygdala , the ventromedial hypothalamus , and the nucleus accumbens . these reports indicate that the vagus afferent response is important for the transmission of each dietary nutrient information in the gut to the brain . interestingly , behavioral studies have shown that abdominal vagotomy eliminates cfp in response to intragastric infusion of msg but does not affect cfp response in the case of carbohydrates in rats . an fmri study showed that total and partial abdominal vagotomy diminished glu - induced activation in the nts and the hypothalamus , whereas total vagotomy did not affect glucose - induced brain activation . finally , there are distinct postingestive effects in response to different nutrients , resulting in the activation of forebrain regions . recently , we analyzed free glutamate content in the pancreatic juice several fold higher than the plasma level . these endocrine cells also express metabotropic glutamate receptors type 4 and 8 receiving free glu stimuli released from the vagus nerve - end like cephalic phase stimuli . the free form of glutamate in the digestive juice directly stimulate the alimentary tract to regulate digestion and absorption normally , through ph control and mucin release for the barrier system . from the point of digestive physiology , switching from a milk formula to a solid diet as described above , glu has a potential for the intestine to release mucin due to establishment of a barriers system against microbiota . therefore , supplementation liquid for weaning piglets containing glu and l - glutamine ( gln ) as a precursor for glu adding to the weaning diet results in a significant improvement of the diarrhea index and their state of nutrition . habituation studies employed elderly subjects to be offered rice glue containing 0.5 ( w / v ) msg for 3 months . double blind and cross - over studies yield improvement in the quality of life concerning emotion and appetite for foods , reduced incidence of diarrhea and vomiting , peripheral circulation to cure bed thaw lesion , etc . free glu signaling during a meal and subsequent processes in the alimentary organs plays important physiological roles in the perception of umami taste , visceral information and regulation of endo - and exocrine systems , and excitatory neurotransmission . but there are no way to entry dietary free glu into the circulating blood as well as brain cerebrospinal fluid because of both the intestinal hepatic barrier and the blood brain barrier are not permeable for free glu . in our series of studies reviewed here , we showed that dietary glu also stimulates glu sensors in the stomach and intestines , producing local effects on gut function for ingested food digestion . moreover , via the release of signaling molecules no and 5-ht , the presence of glu in the gut leads to the activation of the vagal afferent nerve which in turn modulates a number of target areas in the brain , particularly the gastric vagal afferent responds to glu alone rather than electrolytes , sugars and other amino acids , suggesting that glu signaling in the stomach is to be a signal for food intake controlling whole processes of digestion and absorption of yielded nutrients . in addition , we described the postingestive effects of glu and showed that they were different from those elicited by glucose and lipids . previous fmri observation in the brain and behavioral studies in rodents have indicated that glu has positive postingestive effects through the vagal afferent nerve that controls appetite and satiety for food . altogether , these findings indicate that dietary glu influences numerous physiological functions , suggesting a broad , integrative role for dietary glu in body homeostasis . this newly developed method for brain functional change observation in well - trained awake rats using a fmri after nutrient stimulation in the gut by gastric intubation into the stomach was recently confirmed by c - fos expression in similar rats , suggesting that gut brain communication through chemical senses of nutrient , particularly glu , becomes available to research using awake animals in connection with non - invasive treatment [ 39 , 58 ] due to an understanding why rats , fed a high - fat and high - sugar diet , do not become obese when glu solution is available to drink with this food intake through higher thermogenesis due to energy expenditure . dietary glu and glutamine as a precursor for glu signaling are positively promoted as a function of the alimentary tracts in infants as well as the elderly because of the biosynthesis capacity of free glu is not sufficient in the exo- and endocrine organs .

there are many different definitions for spirituality , but the following is provided by cook as a working definition : " spirituality is a distinctive , potentially creative and universal dimension of human experience arising both within the inner subjective awareness of individuals and within communities , social groups and traditions . it may be experienced as a relationship which is intimately inner , immanent and personal , within the self and others , and/or as relationship with that which is wholly other , transcendent and beyond the self . it is experienced as being of fundamental or ultimate importance and is thus concerned with matters of meaning and purpose in life , truth and values " ( 1 ) . human beings are created of two physical and spiritual dimensions , and in the holistic care mechanism , both of the dimensions should be addressed ( 2 ) . the world health organization ( who ) definition of supportive care of patients with serious diseases answers the spiritual needs of these patients alongside physical and psychosocial needs ( 3 ) . studies also have demonstrated that at least half of patients with chronic illnesses and their families need spiritual services . on the one hand , there is a strong association between emotional and spiritual needs of patients , their general satisfaction of treatment , and the reduction of symptoms such as anxiety , pain , and depression ; on the other hand , it was seen that most health care providers were not educated enough in offering spiritual services ( 4 - 7 ) . several questionnaires are available to assess spiritual needs , though they might have some shortcomings ( 8) . first , the applied questionnaires mostly contain different religious purports and are not applicable to all communities . second , they are considered a detailed part of a more general questionnaire about spiritual assessment . finally , the main interest of the questionnaire designers is in nursing rather than the medicine domain ( 9 ) . therefore , to improve the precision of assessment , new questionnaires are being developed by different research groups . knowing how these questionnaires are made , their strengths and weaknesses , and their theoretical bases can provide the groundwork for creating future questionnaires . the purpose of this study was to collect comprehensive information about all of the scales that currently exist in this field . therefore , this study may open a path for future research based on the present weaknesses . an electronic search was performed with no limitations on time span ( until june 2015 ) or language in databases including pubmed , scopus , ovid , proquest and google scholar . in order to design the search order , first , the word need and all its synonyms were selected from www.thesaurus.com as well as oxford and longman dictionaries . each of them was then searched in combination with derivations of the words spirit and religion in the title and keywords fields of articles . for example , the search query in pubmed was as follows : ( spirit*[title ] or religi*[ttitle ] ) and ( need*[title ] or wish*[title ] or obligat*[title ] or essenti*[title ] or requir*[title ] or demand*[title ] or want*[title ] or necess*[title ] or requisi*[title ] or intere*[title ] ) the titles and abstracts of the papers were studied by the authors of the present paper . the criteria for selecting papers for the final report included : the existence of one search word in the title or keyword , needs recognition being mentioned as the main research objective in the abstract , and research leading to the generation of a new questionnaire . the information was selected , and finally all the references of the selected papers were reviewed . if they met the criteria , they were added to the selected papers in the following way : the keywords were reviewed , and new synonyms were added and searched again ( the final form is mentioned as above ) . tables 1 and 2 lists the instruments and their psychometric properties in detail and table 3 displays their strong and weak points . an electronic search was performed with no limitations on time span ( until june 2015 ) or language in databases including pubmed , scopus , ovid , proquest and google scholar . in order to design the search order , first , the word need and all its synonyms were selected from www.thesaurus.com as well as oxford and longman dictionaries . each of them was then searched in combination with derivations of the words spirit and religion in the title and keywords fields of articles . for example , the search query in pubmed was as follows : ( spirit*[title ] or religi*[ttitle ] ) and ( need*[title ] or wish*[title ] or obligat*[title ] or essenti*[title ] or requir*[title ] or demand*[title ] or want*[title ] or necess*[title ] or requisi*[title ] or intere*[title ] ) the titles and abstracts of the papers were studied by the authors of the present paper . the criteria for selecting papers for the final report included : the existence of one search word in the title or keyword , needs recognition being mentioned as the main research objective in the abstract , and research leading to the generation of a new questionnaire . full texts of the selected papers were studied . the information was selected , and finally all the references of the selected papers were reviewed . if they met the criteria , they were added to the selected papers in the following way : the keywords were reviewed , and new synonyms were added and searched again ( the final form is mentioned as above ) . tables 1 and 2 lists the instruments and their psychometric properties in detail and table 3 displays their strong and weak points . this scale was developed by galek et al . in 2005 in the usa and the three names spiritual need survey , spiritual need scale and patient spiritual needs assessment scale were applied for its introduction in different studies ( 9 - 11 ) . this questionnaire had no special religious procedure and was based on the results of 12 qualitative , 7 quantitative , and 3 theoretical studies , using a systematic review method and had 29 yes / no questions originally ( 9 ) . in other studies , the developers modified the original tool and introduced the 28-item ( 10 ) and 24-item ( 11 ) versions of the questionnaire . this questionnaire was designed by hermann in 2006 in the usa in a qualitative study on patients diagnosed with cancer in the final stage of life . this questionnaire , which did not have any special religious procedure , was originally a special needs questionnaire design for patient with special problems . the aim was to investigate only spiritual needs and no other topic related to spirituality . maslow s theory of motivation is the theoretical basis of this questionnaire , but with the assumption that spiritual needs exist at all of maslow s levels , with the move toward deeper levels of fulfillment of more complex and abstract needs . in sni , for each question there was also the survey of reply to the needs along with the survey of needs existence ( according to a yes / no to answer the question : " is this need being met in your life now ? " ) ( 13 ) . sni was normalized in a group of caregivers of patients with cancer and demonstrated the reliability and validity in the group without illness ( 21 ) . this tool was created by taylor in 2006 in the usa to investigate the spiritual needs prevalent in patients with cancer and their caregivers based on a qualitative and then quantitative study ( 14 , 22 , 23 ) . the stem of all the questions in this self - report tool started with " how important is it now to " , and at the end of each group of questions , an item asked : " how important it is to have my ( or my loved one ) nurse help me satisfy these spiritual interests ? " ( 14 ) . because need may transmit a negative concept , authors used the word interest to avoid misunderstanding and to decrease patient denial in reporting their needs . another important difference between this questionnaire and others was the need to review beliefs , which involved a cognitive involvement with concepts such as unfairness of what has been happening , correctness of beliefs about god , why did i / we deserve this ? and finally , receiving a response or gaining acceptance ( 14 ) . this scale was designed by yong et al . in 2008 in korea with the assumption that spiritual needs vary across cultures . to extract key concepts of spiritual needs , both an extensive literature review on spiritual needs and semistructured interviews with 10 hospitalized participants were conducted . sns had five sub - constructs which were similar to sub - constructs of psnas , but which had one more construct , appreciation of beauty . on the other hand , the constructs of spirit yielded two other different constructs , positive perspective and reviewing beliefs , which were not included in the constructs of sns . this questionnaire , which may be the most important assigned questionnaire for the evaluation of spiritual needs of particular patients , was designed in 2010 by bssing et al . in germany . it was , in fact , a continuation of the dedication of assessing the spiritual needs of special patient populations . the reason for selecting patients with chronic illnesses such as chronic pain and cancer was that for these patients , one important method of coping with illness was the use of religion and spirituality . most previous questionnaires focused on the spiritual needs of patients close to death as opposed to those with chronic illnesses . a novel aspect of spnq was the need of actively giving , which is the need of having an active role in life instead of the role of an afflicted patient ( 16 ) . spnq has been used in different settings ( 25 ) and also normalized in malaysia , nigeria , poland , france , china , and england and is now normalized in iran . in each country , the questionnaire has been normalized with minor changes and the priority of needs has been reported differently ( 26 ) . this questionnaire was developed in 2012 by sharma et al . in the usa and was introduced as a complete tool for spiritual needs assessment in the published paper . the developers tried to merge all the existing data in literature with experts opinions and also their clinical experiences . this short questionnaire consisted of 23 questions with scoring ranged between 23 and 89 , such that the closer the score to 89 , the greater the need . this questionnaire placed needs into three main groups and appeared as an integration of the previous needs questionnaires ( 17 ) . a noteworthy point is that in the chinese normalization , researchers found that some items that were considered as spiritual needs by western patients were not considered as such for chinese patients ( 27 ) . this questionnaire was developed by vilalta et al . in 2014 in spain . with the aim of designing a simple , effective and easily tool , they performed a comprehensive literature review and selected the most common repeated typologies of spiritual needs . after incorporating them , in collaboration with experts who were specialist in theology , ethics , bioethics , psychology and oncology we did not find any published data about the questionnaire s psychometric evaluation in databases we searched until the time the present article was being written , with the exception of its face validity and a pilot study involving 10 patients ( 18 ) . this questionnaire was developed by wu et al . in 2015 in taiwan . with the aim of designing a specific tool to assess spiritual needs in acute care hospital patients with different religious beliefs , they merged items obtained from 149 published articles and three patients spiritual needs instruments . after incorporating them the meaning and hope component referred to the domains of self , nature and environmental aspects of spiritual well - being , and the caring and respect component referred to the domain of communal relationship with others ( 19 ) . this scale was developed by galek et al . in 2005 in the usa and the three names spiritual need survey , spiritual need scale and patient spiritual needs assessment scale were applied for its introduction in different studies ( 9 - 11 ) . this questionnaire had no special religious procedure and was based on the results of 12 qualitative , 7 quantitative , and 3 theoretical studies , using a systematic review method and had 29 yes / no questions originally ( 9 ) . in other studies , the developers modified the original tool and introduced the 28-item ( 10 ) and 24-item ( 11 ) versions of the questionnaire . this questionnaire was designed by hermann in 2006 in the usa in a qualitative study on patients diagnosed with cancer in the final stage of life . this questionnaire , which did not have any special religious procedure , was originally a special needs questionnaire design for patient with special problems . the aim was to investigate only spiritual needs and no other topic related to spirituality . maslow s theory of motivation is the theoretical basis of this questionnaire , but with the assumption that spiritual needs exist at all of maslow s levels , with the move toward deeper levels of fulfillment of more complex and abstract needs . in sni , for each question there was also the survey of reply to the needs along with the survey of needs existence ( according to a yes / no to answer the question : " is this need being met in your life now ? " ) ( 13 ) . sni was normalized in a group of caregivers of patients with cancer and demonstrated the reliability and validity in the group without illness ( 21 ) . this tool was created by taylor in 2006 in the usa to investigate the spiritual needs prevalent in patients with cancer and their caregivers based on a qualitative and then quantitative study ( 14 , 22 , 23 ) . the stem of all the questions in this self - report tool started with " how important is it now to " , and at the end of each group of questions , an item asked : " how important it is to have my ( or my loved one ) nurse help me satisfy these spiritual interests ? " ( 14 ) . because need may transmit a negative concept , authors used the word interest to avoid misunderstanding and to decrease patient denial in reporting their needs . another important difference between this questionnaire and others was the need to review beliefs , which involved a cognitive involvement with concepts such as unfairness of what has been happening , correctness of beliefs about god , why did i / we deserve this ? and finally , receiving a response or gaining acceptance ( 14 ) . this scale was designed by yong et al . in 2008 in korea with the assumption that spiritual needs vary across cultures . to extract key concepts of spiritual needs , both an extensive literature review on spiritual needs and semistructured interviews with 10 hospitalized participants sns had five sub - constructs which were similar to sub - constructs of psnas , but which had one more construct , appreciation of beauty . on the other hand , the constructs of spirit yielded two other different constructs , positive perspective and reviewing beliefs , which were not included in the constructs of sns . this questionnaire , which may be the most important assigned questionnaire for the evaluation of spiritual needs of particular patients , was designed in 2010 by bssing et al . in germany . it was , in fact , a continuation of the dedication of assessing the spiritual needs of special patient populations . the reason for selecting patients with chronic illnesses such as chronic pain and cancer was that for these patients , one important method of coping with illness was the use of religion and spirituality . most previous questionnaires focused on the spiritual needs of patients close to death as opposed to those with chronic illnesses . a novel aspect of spnq was the need of actively giving , which is the need of having an active role in life instead of the role of an afflicted patient ( 16 ) . spnq has been used in different settings ( 25 ) and also normalized in malaysia , nigeria , poland , france , china , and england and is now normalized in iran . in each country , the questionnaire has been normalized with minor changes and the priority of needs has been reported differently ( 26 ) . this questionnaire was developed in 2012 by sharma et al . in the usa and was introduced as a complete tool for spiritual needs assessment in the published paper . the developers tried to merge all the existing data in literature with experts opinions and also their clinical experiences . this short questionnaire consisted of 23 questions with scoring ranged between 23 and 89 , such that the closer the score to 89 , the greater the need . this questionnaire placed needs into three main groups and appeared as an integration of the previous needs questionnaires ( 17 ) . a noteworthy point is that in the chinese normalization , researchers found that some items that were considered as spiritual needs by western patients were not considered as such for chinese patients ( 27 ) . this questionnaire was developed by vilalta et al . in 2014 in spain . with the aim of designing a simple , effective and easily tool , they performed a comprehensive literature review and selected the most common repeated typologies of spiritual needs . after incorporating them , in collaboration with experts who were specialist in theology , ethics , bioethics , psychology and oncology we did not find any published data about the questionnaire s psychometric evaluation in databases we searched until the time the present article was being written , with the exception of its face validity and a pilot study involving 10 patients ( 18 ) . this questionnaire was developed by wu et al . in 2015 in taiwan . with the aim of designing a specific tool to assess spiritual needs in acute care hospital patients with different religious beliefs , they merged items obtained from 149 published articles and three patients spiritual needs instruments . after incorporating them the meaning and hope component referred to the domains of self , nature and environmental aspects of spiritual well - being , and the caring and respect component referred to the domain of communal relationship with others ( 19 ) . a distinctive feature of this study was collecting the detailed characteristics of the instruments with their strong and weak points . therefore , those interested in spiritual needs can choose the appropriate instrument regarding the constructs assessed by these instruments . however , the followings are some pitfalls seem to exist in this field of research : a , the similarity of all the aforementioned questionnaires assessed spiritual needs of patients in critical conditions , mostly patients with cancer , and designed questionnaires to answer these specific patients needs . on the other hand , cancer can significantly increase and change patients spiritual needs , because patients rely on spirituality as a strong method to deal with cancer ( 10 , 28 , 29 ) . further studies are recommended to design appropriate instruments for other patient groups and the healthy population ; b , for assessing spiritual needs , one should understand the voice that lies deep within the respondent that may reveal something of the respondent s inner self . therefore , despite the advantages of questionnaires in research , clinicians should be cautious of this point and avoid applying instruments stereotypically ( 30 ) ; c , with the exception of the questionnaires mentioned in this paper , there were many qualitative studies to evaluate spiritual needs that considered other variables . some examples include transcendental needs , mystical needs ( including desire and mystical consciousness ) , which involve abstract aspects of spiritual needs ( 30 ) , and needs such as life beyond the individual , need for continuity and after life , need for truth , and need for freedom and to be free ( 18 ) . these points highlight the need to study and develop new questionnaires that cover more dimensions of spiritual needs ; d , in most of these questionnaires , normalization information was incomplete . in some , the sample volume was not only low , but often used in limited studies , and in most of them , the theoretical bases of the questionnaire design were not mentioned . therefore , using these questionnaires in clinical daily work should be performed with care until further research is conducted ; e , some concepts of these questionnaires are culture - dependent . for example , in western cultures , listening to music is a spiritual need , while in iran listening to the quran is more significant . furthermore , depending on the culture in which the questionnaire is used , the priority of needs will change . in the chinese version of snap , for example , the first priority is the need of actively giving ( not inner peace that is seen in the german normalization ) ( 26 ) . in iran , on the other hand , the significant need is going to the prayer hall and participating in mass prayer ( 2 , 30 - 32 ) . in korea , in contrast with western cultures , some patients are not comfortable in talking about death and unfinished works at the end of life ( 15 ) . this is important from two perspectives : first , one construct can be measured by different factors in different cultures . second , because of cultural differences and the relationship between spiritual needs and culture , it can be recommended to design culture - specific questionnaires ; f , historically , a criticism in questionnaire design is the reductionist approach . it seems that the concept of spirituality varies from religious to secular , and this topic is seen in spiritual needs surveys . for example , in the aforementioned questionnaires , general concepts such as the need of meaning discovery in illness are weak , and strong concepts such as life beyond the individual were measured by variables such as talking with others . for example , in top sense , the beauty can achieve ( that is kind of intuitive beauty ) ( 33 ) , while the concept of life beyond the individual was simply about talking with others . this point should be considered important in culture - specific questionnaire design , especially in eastern cultures where religious concepts have a strong link to spiritual concepts ( 18 , 34 , 35 ) . first , there were not enough references such as congress papers , abstracts , theses , etc . the reason was the unavailability of details because of lack of publication in research sites . second , the concept of spiritual needs is an extensive one for the introduction of which several words can be used and may be overlooked in the keywords . furthermore , there were many questionnaires that assess different dimensions of spirituality including spiritual needs . we deleted them from our study because our focus was on the questionnaires that assessed spiritual needs specifically . in conclusion , although most of the studies showed that the need of relationship was the center of spiritual needs , the complexity of these needs can not be summarized in this concept ( 25 , 36 ) . therefore , the assessment of questionnaires should be conducted with a great deal of care and consideration . this is an aspect that has been ignored in most questionnaires and has led to the lack of harmony in terms of content and assessment .

contextthe objective of the present review was to collect published spiritual needs questionnaires and to present a clear image of the research condition of this domain.evidence acquisitionfirst , an electronic search was conducted with no limits on time span ( until june 2015 ) or language in the following databases : pubmed , scopus , ovid , proquest and google scholar . all derivations of the keywords religion and spiritual alongside need and its synonyms were included in the search . researches that introduced new tools was then selected and included in the study . due to the limited quantity of questionnaires in this domain and with no consideration given to the existence or lack of exact standardization information , all of the questionnaires were included in the final report.resultseight questionnaires were found : patients spiritual needs assessment scale ( psnas ) , spiritual needs inventory ( sni ) , spiritual interests related to illness tool ( spirit ) , spiritual needs questionnaire ( spnq ) , spiritual needs assessment for patients ( snap ) , spiritual needs scale ( sns ) , spiritual care needs inventory ( scni ) , and spiritual needs questionnaire for palliative care.conclusionsthese questionnaires have been designed from a limited medical perspective and often involve cultural concepts which complicate their cross - cultural applicability .

1. Context 2. Evidence Acquisition 2.1. Step One 2.2. Step Two 2.3. Step Three 3. Results 3.1. Patients Spiritual Needs Assessment Scale (PSNAS) 3.2. Spiritual Needs Inventory (SNI) 3.3. Spiritual Interests Related to Illness Tool (SpIRIT) 3.4. Spiritual Needs Scale (SNS) 3.5. Spiritual Needs Questionnaire (SpNQ) 3.6. Spiritual Needs Assessment for Patients (SNAP) 3.7. Spiritual Needs Questionnaire for Palliative Care 3.8. Spiritual Care Needs Inventory (SCNI) 4. Discussion

there are many different definitions for spirituality , but the following is provided by cook as a working definition : " spirituality is a distinctive , potentially creative and universal dimension of human experience arising both within the inner subjective awareness of individuals and within communities , social groups and traditions . it may be experienced as a relationship which is intimately inner , immanent and personal , within the self and others , and/or as relationship with that which is wholly other , transcendent and beyond the self . human beings are created of two physical and spiritual dimensions , and in the holistic care mechanism , both of the dimensions should be addressed ( 2 ) . the world health organization ( who ) definition of supportive care of patients with serious diseases answers the spiritual needs of these patients alongside physical and psychosocial needs ( 3 ) . studies also have demonstrated that at least half of patients with chronic illnesses and their families need spiritual services . on the one hand , there is a strong association between emotional and spiritual needs of patients , their general satisfaction of treatment , and the reduction of symptoms such as anxiety , pain , and depression ; on the other hand , it was seen that most health care providers were not educated enough in offering spiritual services ( 4 - 7 ) . several questionnaires are available to assess spiritual needs , though they might have some shortcomings ( 8) . first , the applied questionnaires mostly contain different religious purports and are not applicable to all communities . second , they are considered a detailed part of a more general questionnaire about spiritual assessment . finally , the main interest of the questionnaire designers is in nursing rather than the medicine domain ( 9 ) . knowing how these questionnaires are made , their strengths and weaknesses , and their theoretical bases can provide the groundwork for creating future questionnaires . the purpose of this study was to collect comprehensive information about all of the scales that currently exist in this field . therefore , this study may open a path for future research based on the present weaknesses . an electronic search was performed with no limitations on time span ( until june 2015 ) or language in databases including pubmed , scopus , ovid , proquest and google scholar . in order to design the search order , first , the word need and all its synonyms were selected from www.thesaurus.com as well as oxford and longman dictionaries . each of them was then searched in combination with derivations of the words spirit and religion in the title and keywords fields of articles . for example , the search query in pubmed was as follows : ( spirit*[title ] or religi*[ttitle ] ) and ( need*[title ] or wish*[title ] or obligat*[title ] or essenti*[title ] or requir*[title ] or demand*[title ] or want*[title ] or necess*[title ] or requisi*[title ] or intere*[title ] ) the titles and abstracts of the papers were studied by the authors of the present paper . the criteria for selecting papers for the final report included : the existence of one search word in the title or keyword , needs recognition being mentioned as the main research objective in the abstract , and research leading to the generation of a new questionnaire . the information was selected , and finally all the references of the selected papers were reviewed . if they met the criteria , they were added to the selected papers in the following way : the keywords were reviewed , and new synonyms were added and searched again ( the final form is mentioned as above ) . tables 1 and 2 lists the instruments and their psychometric properties in detail and table 3 displays their strong and weak points . an electronic search was performed with no limitations on time span ( until june 2015 ) or language in databases including pubmed , scopus , ovid , proquest and google scholar . in order to design the search order , first , the word need and all its synonyms were selected from www.thesaurus.com as well as oxford and longman dictionaries . each of them was then searched in combination with derivations of the words spirit and religion in the title and keywords fields of articles . for example , the search query in pubmed was as follows : ( spirit*[title ] or religi*[ttitle ] ) and ( need*[title ] or wish*[title ] or obligat*[title ] or essenti*[title ] or requir*[title ] or demand*[title ] or want*[title ] or necess*[title ] or requisi*[title ] or intere*[title ] ) the titles and abstracts of the papers were studied by the authors of the present paper . the criteria for selecting papers for the final report included : the existence of one search word in the title or keyword , needs recognition being mentioned as the main research objective in the abstract , and research leading to the generation of a new questionnaire . full texts of the selected papers were studied . the information was selected , and finally all the references of the selected papers were reviewed . if they met the criteria , they were added to the selected papers in the following way : the keywords were reviewed , and new synonyms were added and searched again ( the final form is mentioned as above ) . in 2005 in the usa and the three names spiritual need survey , spiritual need scale and patient spiritual needs assessment scale were applied for its introduction in different studies ( 9 - 11 ) . this questionnaire had no special religious procedure and was based on the results of 12 qualitative , 7 quantitative , and 3 theoretical studies , using a systematic review method and had 29 yes / no questions originally ( 9 ) . in other studies , the developers modified the original tool and introduced the 28-item ( 10 ) and 24-item ( 11 ) versions of the questionnaire . this questionnaire was designed by hermann in 2006 in the usa in a qualitative study on patients diagnosed with cancer in the final stage of life . this questionnaire , which did not have any special religious procedure , was originally a special needs questionnaire design for patient with special problems . the aim was to investigate only spiritual needs and no other topic related to spirituality . maslow s theory of motivation is the theoretical basis of this questionnaire , but with the assumption that spiritual needs exist at all of maslow s levels , with the move toward deeper levels of fulfillment of more complex and abstract needs . in sni , for each question there was also the survey of reply to the needs along with the survey of needs existence ( according to a yes / no to answer the question : " is this need being met in your life now ? " ) ( 13 ) . sni was normalized in a group of caregivers of patients with cancer and demonstrated the reliability and validity in the group without illness ( 21 ) . this tool was created by taylor in 2006 in the usa to investigate the spiritual needs prevalent in patients with cancer and their caregivers based on a qualitative and then quantitative study ( 14 , 22 , 23 ) . the stem of all the questions in this self - report tool started with " how important is it now to " , and at the end of each group of questions , an item asked : " how important it is to have my ( or my loved one ) nurse help me satisfy these spiritual interests ? " ( 14 ) . because need may transmit a negative concept , authors used the word interest to avoid misunderstanding and to decrease patient denial in reporting their needs . in 2008 in korea with the assumption that spiritual needs vary across cultures . to extract key concepts of spiritual needs , both an extensive literature review on spiritual needs and semistructured interviews with 10 hospitalized participants were conducted . on the other hand , the constructs of spirit yielded two other different constructs , positive perspective and reviewing beliefs , which were not included in the constructs of sns . this questionnaire , which may be the most important assigned questionnaire for the evaluation of spiritual needs of particular patients , was designed in 2010 by bssing et al . in germany . it was , in fact , a continuation of the dedication of assessing the spiritual needs of special patient populations . the reason for selecting patients with chronic illnesses such as chronic pain and cancer was that for these patients , one important method of coping with illness was the use of religion and spirituality . most previous questionnaires focused on the spiritual needs of patients close to death as opposed to those with chronic illnesses . a novel aspect of spnq was the need of actively giving , which is the need of having an active role in life instead of the role of an afflicted patient ( 16 ) . spnq has been used in different settings ( 25 ) and also normalized in malaysia , nigeria , poland , france , china , and england and is now normalized in iran . this questionnaire was developed in 2012 by sharma et al . in the usa and was introduced as a complete tool for spiritual needs assessment in the published paper . this short questionnaire consisted of 23 questions with scoring ranged between 23 and 89 , such that the closer the score to 89 , the greater the need . this questionnaire placed needs into three main groups and appeared as an integration of the previous needs questionnaires ( 17 ) . a noteworthy point is that in the chinese normalization , researchers found that some items that were considered as spiritual needs by western patients were not considered as such for chinese patients ( 27 ) . in 2014 in spain . with the aim of designing a simple , effective and easily tool , they performed a comprehensive literature review and selected the most common repeated typologies of spiritual needs . after incorporating them , in collaboration with experts who were specialist in theology , ethics , bioethics , psychology and oncology we did not find any published data about the questionnaire s psychometric evaluation in databases we searched until the time the present article was being written , with the exception of its face validity and a pilot study involving 10 patients ( 18 ) . with the aim of designing a specific tool to assess spiritual needs in acute care hospital patients with different religious beliefs , they merged items obtained from 149 published articles and three patients spiritual needs instruments . after incorporating them the meaning and hope component referred to the domains of self , nature and environmental aspects of spiritual well - being , and the caring and respect component referred to the domain of communal relationship with others ( 19 ) . in 2005 in the usa and the three names spiritual need survey , spiritual need scale and patient spiritual needs assessment scale were applied for its introduction in different studies ( 9 - 11 ) . this questionnaire had no special religious procedure and was based on the results of 12 qualitative , 7 quantitative , and 3 theoretical studies , using a systematic review method and had 29 yes / no questions originally ( 9 ) . in other studies , the developers modified the original tool and introduced the 28-item ( 10 ) and 24-item ( 11 ) versions of the questionnaire . this questionnaire was designed by hermann in 2006 in the usa in a qualitative study on patients diagnosed with cancer in the final stage of life . this questionnaire , which did not have any special religious procedure , was originally a special needs questionnaire design for patient with special problems . the aim was to investigate only spiritual needs and no other topic related to spirituality . maslow s theory of motivation is the theoretical basis of this questionnaire , but with the assumption that spiritual needs exist at all of maslow s levels , with the move toward deeper levels of fulfillment of more complex and abstract needs . in sni , for each question there was also the survey of reply to the needs along with the survey of needs existence ( according to a yes / no to answer the question : " is this need being met in your life now ? " ) ( 13 ) . sni was normalized in a group of caregivers of patients with cancer and demonstrated the reliability and validity in the group without illness ( 21 ) . this tool was created by taylor in 2006 in the usa to investigate the spiritual needs prevalent in patients with cancer and their caregivers based on a qualitative and then quantitative study ( 14 , 22 , 23 ) . the stem of all the questions in this self - report tool started with " how important is it now to " , and at the end of each group of questions , an item asked : " how important it is to have my ( or my loved one ) nurse help me satisfy these spiritual interests ? " because need may transmit a negative concept , authors used the word interest to avoid misunderstanding and to decrease patient denial in reporting their needs . another important difference between this questionnaire and others was the need to review beliefs , which involved a cognitive involvement with concepts such as unfairness of what has been happening , correctness of beliefs about god , why did i / we deserve this ? in 2008 in korea with the assumption that spiritual needs vary across cultures . to extract key concepts of spiritual needs , both an extensive literature review on spiritual needs and semistructured interviews with 10 hospitalized participants sns had five sub - constructs which were similar to sub - constructs of psnas , but which had one more construct , appreciation of beauty . on the other hand , the constructs of spirit yielded two other different constructs , positive perspective and reviewing beliefs , which were not included in the constructs of sns . this questionnaire , which may be the most important assigned questionnaire for the evaluation of spiritual needs of particular patients , was designed in 2010 by bssing et al . it was , in fact , a continuation of the dedication of assessing the spiritual needs of special patient populations . the reason for selecting patients with chronic illnesses such as chronic pain and cancer was that for these patients , one important method of coping with illness was the use of religion and spirituality . most previous questionnaires focused on the spiritual needs of patients close to death as opposed to those with chronic illnesses . a novel aspect of spnq was the need of actively giving , which is the need of having an active role in life instead of the role of an afflicted patient ( 16 ) . spnq has been used in different settings ( 25 ) and also normalized in malaysia , nigeria , poland , france , china , and england and is now normalized in iran . this questionnaire was developed in 2012 by sharma et al . in the usa and was introduced as a complete tool for spiritual needs assessment in the published paper . this short questionnaire consisted of 23 questions with scoring ranged between 23 and 89 , such that the closer the score to 89 , the greater the need . this questionnaire placed needs into three main groups and appeared as an integration of the previous needs questionnaires ( 17 ) . a noteworthy point is that in the chinese normalization , researchers found that some items that were considered as spiritual needs by western patients were not considered as such for chinese patients ( 27 ) . in 2014 in spain . with the aim of designing a simple , effective and easily tool , they performed a comprehensive literature review and selected the most common repeated typologies of spiritual needs . after incorporating them , in collaboration with experts who were specialist in theology , ethics , bioethics , psychology and oncology we did not find any published data about the questionnaire s psychometric evaluation in databases we searched until the time the present article was being written , with the exception of its face validity and a pilot study involving 10 patients ( 18 ) . this questionnaire was developed by wu et al . in 2015 in taiwan . with the aim of designing a specific tool to assess spiritual needs in acute care hospital patients with different religious beliefs , they merged items obtained from 149 published articles and three patients spiritual needs instruments . after incorporating them the meaning and hope component referred to the domains of self , nature and environmental aspects of spiritual well - being , and the caring and respect component referred to the domain of communal relationship with others ( 19 ) . a distinctive feature of this study was collecting the detailed characteristics of the instruments with their strong and weak points . therefore , those interested in spiritual needs can choose the appropriate instrument regarding the constructs assessed by these instruments . however , the followings are some pitfalls seem to exist in this field of research : a , the similarity of all the aforementioned questionnaires assessed spiritual needs of patients in critical conditions , mostly patients with cancer , and designed questionnaires to answer these specific patients needs . on the other hand , cancer can significantly increase and change patients spiritual needs , because patients rely on spirituality as a strong method to deal with cancer ( 10 , 28 , 29 ) . further studies are recommended to design appropriate instruments for other patient groups and the healthy population ; b , for assessing spiritual needs , one should understand the voice that lies deep within the respondent that may reveal something of the respondent s inner self . therefore , despite the advantages of questionnaires in research , clinicians should be cautious of this point and avoid applying instruments stereotypically ( 30 ) ; c , with the exception of the questionnaires mentioned in this paper , there were many qualitative studies to evaluate spiritual needs that considered other variables . some examples include transcendental needs , mystical needs ( including desire and mystical consciousness ) , which involve abstract aspects of spiritual needs ( 30 ) , and needs such as life beyond the individual , need for continuity and after life , need for truth , and need for freedom and to be free ( 18 ) . these points highlight the need to study and develop new questionnaires that cover more dimensions of spiritual needs ; d , in most of these questionnaires , normalization information was incomplete . in some , the sample volume was not only low , but often used in limited studies , and in most of them , the theoretical bases of the questionnaire design were not mentioned . therefore , using these questionnaires in clinical daily work should be performed with care until further research is conducted ; e , some concepts of these questionnaires are culture - dependent . for example , in western cultures , listening to music is a spiritual need , while in iran listening to the quran is more significant . furthermore , depending on the culture in which the questionnaire is used , the priority of needs will change . in the chinese version of snap , for example , the first priority is the need of actively giving ( not inner peace that is seen in the german normalization ) ( 26 ) . in iran , on the other hand , the significant need is going to the prayer hall and participating in mass prayer ( 2 , 30 - 32 ) . second , because of cultural differences and the relationship between spiritual needs and culture , it can be recommended to design culture - specific questionnaires ; f , historically , a criticism in questionnaire design is the reductionist approach . it seems that the concept of spirituality varies from religious to secular , and this topic is seen in spiritual needs surveys . for example , in the aforementioned questionnaires , general concepts such as the need of meaning discovery in illness are weak , and strong concepts such as life beyond the individual were measured by variables such as talking with others . for example , in top sense , the beauty can achieve ( that is kind of intuitive beauty ) ( 33 ) , while the concept of life beyond the individual was simply about talking with others . this point should be considered important in culture - specific questionnaire design , especially in eastern cultures where religious concepts have a strong link to spiritual concepts ( 18 , 34 , 35 ) . first , there were not enough references such as congress papers , abstracts , theses , etc . the reason was the unavailability of details because of lack of publication in research sites . second , the concept of spiritual needs is an extensive one for the introduction of which several words can be used and may be overlooked in the keywords . furthermore , there were many questionnaires that assess different dimensions of spirituality including spiritual needs . we deleted them from our study because our focus was on the questionnaires that assessed spiritual needs specifically . in conclusion , although most of the studies showed that the need of relationship was the center of spiritual needs , the complexity of these needs can not be summarized in this concept ( 25 , 36 ) . therefore , the assessment of questionnaires should be conducted with a great deal of care and consideration . this is an aspect that has been ignored in most questionnaires and has led to the lack of harmony in terms of content and assessment .

conventional medicine and complementary and alternative medicine ( cam ) are merging into the broader field of integrative medicine . integrative medicine has been defined as healing - oriented medicine that re - emphasizes the relationship between patient and physician and that integrates the best of cam with the best of conventional medicine . some therapies traditionally considered to be cam have been validated and , having become evidence - based , are being embraced by traditional medicine ; others are being ignored by conventional medicine because science does not yet support their use . one intervention that is clearly gaining favor in the conventional medical arena is massage therapy . today , numerous rigorous scientific studies of massage have been conducted since 2000 , and many of them have shown benefit . for example , evidence in support of massage for musculoskeletal back pain in adults is so great that physicians really should at least consider it a potential therapeutic option . cerebral palsy ( cp ) describes a group of disorders of the development of movement and posture , which cause activity limitation because of non - progressive disturbances that occur in the developing fetal or infant brain . the motor disorders of cp are often accompanied by disturbances of sensation , cognition , communication , perception , and behavior . the condition is marked by musculoskeletal tone abnormality , often spasticity ( velocity - dependent tone ) . patients with cp have a wide range of symptoms , from severe motor problems with inability to stand or even sit , to a mild form of the disorder in which the motor function of just one of the patient s limbs is affected . in the general pediatric population , massage is used significantly more frequently in families in which the mother is well - educated , the father has greater disposable income , and the parents use cam themselves . however , it does include one study of 213 children with special needs surveyed in a tertiary care center in michigan . the authors reported that 25% of children with cp received massage on a regular basis . the only randomized trial of massage for patients with cp involved 20 infants with cp . in the treatment group , specifically , the authors reported that massage was associated with decreased spasticity ; improved muscle tone , range of motion , and cognition ; improved fine and gross motor skills ; and improved social functioning . however , a subsequent review of the pediatric massage literature found only modest support for improvement of muscle tone with massage , and no support for improvement of range of motion , function , or spasticity with massage . the author reported that the massaged child smiled more and appeared more relaxed . another uncontrolled qualitative evaluation of massage by parents trained to massage their children with cp was reported by powell and colleagues in england in 2006 . parents said that their children showed improved calmness , bowel habits , and verbal communication , and increased smiling , eye contact , alertness , and mobility . that study is challenged by its lack of a control group and the possible bias introduced by having parents both perform massage and report the resultant data , because the parents may be especially eager to find positive results . still , another distinct advantage is that parents are usually best able to read the child s cues . for example , a parent can distinguish between a moan of pain and a cry of happiness when the nonverbal child with cp vocalizes . cognitive deficits are not considered part of the definition of cp , but approximately 60% of people with cp also have an intellectual disability . even if cognition is not impaired , abnormal voice motor function can make speech and communication challenging or even impossible . cognitively impaired and nonverbal patients with cp may not be able to indicate the severity or the source of their pain . engel and colleagues at the university of washington surveyed verbal adults with cp about what worked for their pain . respondents reported that surgical correction of hip dislocation and massage were two of the most effective treatments for pain . although the few studies of massage in people with cp have been encouraging , more research in the form of careful randomized controlled trials ( rcts ) is needed to determine whether massage is truly beneficial for people with this disorder . one compelling reason to study massage as a treatment modality is that it has biological plausibility . it is well established that massage increases blood flow to the tissues and that enhanced blood flow encourages growth of new tissue and healing of wounds . in addition , it is possible that massage facilitates removal of lactic acid ( an anaerobic respiration byproduct that causes fatigue ) from muscle , increases endorphins , and inhibits serotonin reuptake . massage may lengthen muscle fibers and reduce the formation of fibrotic tissue , which could potentially inhibit contracture formation . the present study was designed as the first stage in a three - stage research plan . it is a feasibility study ( stage one ) , which precedes a pilot study ( stage two ) and a subsequent full - scale rct ( stage three ) , with the aim of determining whether massage can improve the health of children with cp . our feasibility study consisted of a survey with two major goals : to determine the current prevalence of massage among children with cp to determine the perceptions of families about the benefits of massage ( so as to help chose the outcomes to consider in the planned rct ) most massage studies have not used this approach ( feasibility pilot rct ) , and documentation of its use will be a valuable contribution to the field . the survey was designed in part to teach us more about the current use of massage for children with cp in the northwest united states and to help plan the next phases of the study . it was an opportunity to determine how many subjects our team can expect to recruit for subsequent phases of the project , and to determine potential recruitment barriers . to achieve those ends , we read a description of the planned rtc to patients and their families , soliciting feedback on specific aspects of the methodology and inquiring whether they would be willing to participate in future phases of the project . cerebral palsy ( cp ) describes a group of disorders of the development of movement and posture , which cause activity limitation because of non - progressive disturbances that occur in the developing fetal or infant brain . the motor disorders of cp are often accompanied by disturbances of sensation , cognition , communication , perception , and behavior . the condition is marked by musculoskeletal tone abnormality , often spasticity ( velocity - dependent tone ) . patients with cp have a wide range of symptoms , from severe motor problems with inability to stand or even sit , to a mild form of the disorder in which the motor function of just one of the patient s limbs is affected . in the general pediatric population , massage is used significantly more frequently in families in which the mother is well - educated , the father has greater disposable income , and the parents use cam themselves . however , it does include one study of 213 children with special needs surveyed in a tertiary care center in michigan . the authors reported that 25% of children with cp received massage on a regular basis . the only randomized trial of massage for patients with cp involved 20 infants with cp . in the treatment group , specifically , the authors reported that massage was associated with decreased spasticity ; improved muscle tone , range of motion , and cognition ; improved fine and gross motor skills ; and improved social functioning . however , a subsequent review of the pediatric massage literature found only modest support for improvement of muscle tone with massage , and no support for improvement of range of motion , function , or spasticity with massage . the author reported that the massaged child smiled more and appeared more relaxed . another uncontrolled qualitative evaluation of massage by parents trained to massage their children with cp was reported by powell and colleagues in england in 2006 . parents said that their children showed improved calmness , bowel habits , and verbal communication , and increased smiling , eye contact , alertness , and mobility . that study is challenged by its lack of a control group and the possible bias introduced by having parents both perform massage and report the resultant data , because the parents may be especially eager to find positive results . still , another distinct advantage is that parents are usually best able to read the child s cues . for example , a parent can distinguish between a moan of pain and a cry of happiness when the nonverbal child with cp vocalizes . cognitive deficits are not considered part of the definition of cp , but approximately 60% of people with cp also have an intellectual disability . even if cognition is not impaired , abnormal voice motor function can make speech and communication challenging or even impossible . cognitively impaired and nonverbal patients with cp may not be able to indicate the severity or the source of their pain . engel and colleagues at the university of washington surveyed verbal adults with cp about what worked for their pain . respondents reported that surgical correction of hip dislocation and massage were two of the most effective treatments for pain . although the few studies of massage in people with cp have been encouraging , more research in the form of careful randomized controlled trials ( rcts ) is needed to determine whether massage is truly beneficial for people with this disorder . it is well established that massage increases blood flow to the tissues and that enhanced blood flow encourages growth of new tissue and healing of wounds . in addition , it is possible that massage facilitates removal of lactic acid ( an anaerobic respiration byproduct that causes fatigue ) from muscle , increases endorphins , and inhibits serotonin reuptake . massage may lengthen muscle fibers and reduce the formation of fibrotic tissue , which could potentially inhibit contracture formation . the present study was designed as the first stage in a three - stage research plan . it is a feasibility study ( stage one ) , which precedes a pilot study ( stage two ) and a subsequent full - scale rct ( stage three ) , with the aim of determining whether massage can improve the health of children with cp . our feasibility study consisted of a survey with two major goals : to determine the current prevalence of massage among children with cp to determine the perceptions of families about the benefits of massage ( so as to help chose the outcomes to consider in the planned rct ) most massage studies have not used this approach ( feasibility pilot rct ) , and documentation of its use will be a valuable contribution to the field . the survey was designed in part to teach us more about the current use of massage for children with cp in the northwest united states and to help plan the next phases of the study . it was an opportunity to determine how many subjects our team can expect to recruit for subsequent phases of the project , and to determine potential recruitment barriers . to achieve those ends , we read a description of the planned rtc to patients and their families , soliciting feedback on specific aspects of the methodology and inquiring whether they would be willing to participate in future phases of the project . the institutional review board of seattle children s hospital approved the study . written informed consent and approval to review medical records were obtained from subjects willing to participate in the survey . informed assent was obtained from the child with cp whenever the child was 14 years of age or older and able to communicate understanding and an opinion . to identify eligible subjects , we reviewed the list of patients scheduled for the neurodevelopmental and neurology clinics each week from january 2007 to august 2007 . these inclusion criteria were used : the patient s medical record had to contain an entry from a developmental pediatrician or child neurologist specifying that the patient had a diagnosis of cp . children with cp who could not speak for themselves had to be accompanied by a parent or legal guardian knowledgeable enough about the child to complete the survey on the child s behalf . as the patient was being checked into an exam room , a medical assistant asked a parent or legal guardian if , while they waited to be seen by their provider , they would be interested in hearing about an opportunity to participate in a study . the medical assistant provided a written description of the study if the family desired , marked a form indicating whether the family wanted to participate , and placed that form on the chart outside the door . families desiring to hear more about the study were approached by the study s research assistant shortly thereafter to request consent and to administer the survey . surveying by the principal investigator or the research assistant took place in the exam rooms as each subject waited to be seen by the medical provider . the survey , which was written by the principal investigator , took approximately 15 minutes to complete . those unable to stay in clinic because of time constraints made arrangements to complete the survey later . most of the questions on the survey were read to the family by the research assistant , but the final page of demographic questions was completed by the families in private . this step was intentionally taken to reduce the time spent on the survey and to increase confidentiality for families , especially with regard to income . subjects were assigned to either a low - or high - income category based on combined parental income . combined parental income , reported as family income , was calculated by adding the midpoints of the income ranges reported by parents . any combined income below $ 40,000 per year was categorized as low ; any income of $ 60,000 or more was categorized as high . the data were transferred from an access spreadsheet ( microsoft corporation , redmond , wa , usa ) to an excel database ( microsoft corporation ) and then transferred to the sas software package ( version 9.1 : sas institute , cary , nc , usa ) , which was used to calculate means , medians , standard deviations , and percentages . simple cross - tabulations , chi - square tests , and analyses of variance were computed to make comparisons between groups . the customary alpha of less than 0.05 was used to determine the significance of the results . to identify eligible subjects , we reviewed the list of patients scheduled for the neurodevelopmental and neurology clinics each week from january 2007 to august 2007 . these inclusion criteria were used : the patient s medical record had to contain an entry from a developmental pediatrician or child neurologist specifying that the patient had a diagnosis of cp . children with cp who could not speak for themselves had to be accompanied by a parent or legal guardian knowledgeable enough about the child to complete the survey on the child s behalf . as the patient was being checked into an exam room , a medical assistant asked a parent or legal guardian if , while they waited to be seen by their provider , they would be interested in hearing about an opportunity to participate in a study . the medical assistant provided a written description of the study if the family desired , marked a form indicating whether the family wanted to participate , and placed that form on the chart outside the door . families desiring to hear more about the study were approached by the study s research assistant shortly thereafter to request consent and to administer the survey . surveying by the principal investigator or the research assistant took place in the exam rooms as each subject waited to be seen by the medical provider . the survey , which was written by the principal investigator , took approximately 15 minutes to complete . those unable to stay in clinic because of time constraints made arrangements to complete the survey later . most of the questions on the survey were read to the family by the research assistant , but the final page of demographic questions was completed by the families in private . this step was intentionally taken to reduce the time spent on the survey and to increase confidentiality for families , especially with regard to income . subjects were assigned to either a low - or high - income category based on combined parental income . combined parental income , reported as family income , was calculated by adding the midpoints of the income ranges reported by parents . any combined income below $ 40,000 per year was categorized as low ; any income of $ 60,000 or more was categorized as high . the data were transferred from an access spreadsheet ( microsoft corporation , redmond , wa , usa ) to an excel database ( microsoft corporation ) and then transferred to the sas software package ( version 9.1 : sas institute , cary , nc , usa ) , which was used to calculate means , medians , standard deviations , and percentages . simple cross - tabulations , chi - square tests , and analyses of variance were computed to make comparisons between groups . the customary alpha of less than 0.05 was used to determine the significance of the results . the goal of completing the entire survey with at least 100 families was achieved . of 106 families only 2 families approached about the study declined to participate , yielding a participation rate of 98% . six families ( 6% ) finished the survey at home and returned it later . among the 100 subjects eligible for analysis , ages ranged from 17 months to 21 years . severity of cp was recorded using the gross motor function classification system ( gmfcs ) . most of the children in the families approached and interviewed were at the severe end of the cp spectrum , which was in keeping with the clinic population . the more severe the child s condition , the more likely the child was to have received massage . of the analyzed subjects , 20% had never received massage , and 80% had received massage at least once . the data were analyzed so as to determine the demographic factors associated with the use of massage ( ever vs. never ) . we observed no significant differences between the two groups with regard to parental education , parental ethnicity , parental age , family income , or child s age . at the time massage was given by relatives in 68% of the families , by professional massage therapists in 23% , and by physical therapists in 49% . children whose mothers had lower incomes were more likely than children whose mothers had higher incomes to receive massage from a relative as compared with a professional . the proportion of girls who received massage from relatives was higher than the proportion of boys who received massage from relatives . parental age was not associated with whether the children received massage from a licensed massage therapist . of surveyed parents who were seeking and using massage for their children with cp , 86% believed that massage helps to relax muscles ; 71% , that it improves quality of life ; 23% , that it improves sleep ; and 30% , that it decreases their child s pain . in 29% of responding families , these are findings that have not , to our knowledge , been reported previously in the united states . for subjects that had received massage , the children s care providers were asked if massage had ever been discontinued , and if so , why . the most common reason for cessation was the time - consuming nature of massage ( 46% ) . in 11% of families , massage was stopped for various other reasons in 61% . among the other reasons , the two main ones were illness , surgery , or hospitalization ( n = 12 ) and a move by the family or the massage provider ( n = 8) . in addition to comparing children who had ever used massage with those who had never used massage , we compared current massage users with current nonusers . table 2 presents significant findings from that analysis , which revealed that the use of massage by the mother herself was significantly associated with current use of massage by the child . similarly , a more severe gmfcs level was significantly associated with current use of massage . neither the age of the child nor the age of the parents was significantly associated with current use or nonuse of massage . the type of person engaged to perform massage did appear to depend on maternal income . if maternal income was less than $ 20,000 annually , the child was significantly more likely to receive massage from a relative . the sex of the child mattered too : female children were more likely than male children to receive massage from a relative . the only unwilling person cited lack of interest and inability to commit to the time required . subjects and their caregivers were asked if they would prefer a particular geographic location for receiving massage if they were to be assigned to the treatment arm in a future rct . response options included at home , in a hospital room , at a spa , at a massage therapist s office , and other . most respondents ( 87% ) said that they preferred to receive massage therapy at home . they preferred a schedule of massages twice weekly for 5 weeks rather than once weekly for 10 weeks . we collected comments from the subjects and their families to capture their ideas about a future rct of massage for cp . some of the parents saw a future study of massage as a potential opportunity for additional insurance coverage , for the child s relief from discomfort and pain , and for their own education . almost all survey respondents expressed a clear desire to know the results of the planned trial so as to help their child . our study was designed as the beginning of a larger research plan to address the question is massage a useful modality for children with cp ? the fact that 80% of our surveyed cp patients in seattle are already using massage suggests that the modality is at least perceived as useful , that it may in fact be underutilized , and that further research is needed . pain and sleep disturbances in children with cp are especially challenging and pressing problems . of the surveyed parents who are seeking and using massage for their child with cp , 86% believed that massage helps to relax muscles , and 71% believed that it improves quality of life . this information will be valuable for the design of future studies of massage for children with cp . our results suggest that , compared with children having mild disease , those more severely affected are massaged more often . complications of cp are often a result of chronic overstimulation of muscle and lack of normal movement . more severely affected patients are more likely to suffer from constipation , sleep problems , agitation , and muscle spasms all the reasons for which parents say massage is performed . based on training time , depth of concentration , practice , and focus , experienced licensed massage therapists and physical therapists may be superior to untrained professionals in providing massage to children with cp , but this supposition has not been proven . many past pediatric massage studies have used volunteers , parents , or massage therapy students as the primary providers of treatment . more research is needed to determine whether the massage provider makes any difference with regard to specific health outcomes . the reimbursement policy in washington state is that only evidence - supported modalities of massage are covered . currently , massage is seldom covered by insurance for children with cp , and only a subset of therapists are able to perform massage when coverage is available . more research in this area is clearly needed so that , if massage is helpful , it will be accessible to children from all socioeconomic groups . second , because this study used a convenience sample of patients presenting for specialty care in an outpatient setting and because the patients surveyed generally had more severe cp , the results may not be generalizable to the entire population of children with cp . third , to keep the survey brief and to minimize the time burden on families , we obtained maternal and paternal income , but no information on insurance type ( medicaid , none , etc . ) or family size . finally , the type of massage therapy used by each family and the duration of a typical massage were not explored . an increasing number of massage modalities fall under the general heading of massage therapy . these therapies use varying pressure , pacing , and site restrictions . this variation may influence perceived benefit . family members might massage for a shorter time than do professional massage therapists , possibly influencing actual and perceived benefit . the results from this project suggest that use of massage in children with cp is prevalent among patients of the seattle children s hospital in the pacific northwest . we do not yet know whether the type of provider ( physical therapist vs. massage therapist vs. parent ) affects the quality of massage given . systematic examination of massage as a treatment modality for musculoskeletal relaxation , quality of life , pain , sleep , and other outcomes for people with cp is needed . massage looks promising as a treatment modality for all people with cp , but for families and clinicians to make scientific decisions about its usefulness , more evidence , including our planned rct , is necessary .

backgroundconventional medicine and complementary and alternative medicine ( cam ) are merging into the broader field of integrative medicine . massage is no longer considered complementary or alternative in some conventional medical circles today.purposewe aimed to determine the prevalence of massage use among children with cerebral palsy ( cp ) in the pacific northwest in the united states , the reasons that massage is being used , and the limits of recruitment for a future randomized controlled trial.methodsthis study , the first step in a three - stage research plan , was conducted at the neurodevelopmental and neurology clinics at seattle children s hospital , a tertiary pediatric hospital that provides service to patients primarily from washington , alaska , montana , and idaho . as a feasibility study ( stage one ) , it precedes a planned pilot study ( stage two ) , and subsequently , a full - scale randomized controlled trial ( stage three ) of whether massage can improve the health of children with cp . the study subjects104 families with a child with cp ranging in age from 17 months to 21 years were surveyed by the principal investigator and a research assistant in exam rooms at the hospital.resultsin the families surveyed , 80% of the children had received massage at some point . massage was currently being used in 51% , and trained professionals were providing the massage in 23% . most families use massage for musculoskeletal relaxation , to improve quality of life , and to help their children sleep . lower maternal income was associated with relatives as compared with professional massage therapists providing the massage . massage therapy use by the mother and more severe cp were significantly associated with current use of massage for the child.conclusionsmost children with cp in the pacific northwest have used massage . most parents surveyed believe that massage is helpful to their child . additional research is needed to determine whether massage should be routinely recommended for children with cp .

INTRODUCTION Cerebral Palsy Massage for Children and for Children with CP Purpose of the Current Study METHODS Prescreening and Recruitment Approach Measurement Statistical Analyses RESULTS DISCUSSION CONCLUSIONS

conventional medicine and complementary and alternative medicine ( cam ) are merging into the broader field of integrative medicine . integrative medicine has been defined as healing - oriented medicine that re - emphasizes the relationship between patient and physician and that integrates the best of cam with the best of conventional medicine . one intervention that is clearly gaining favor in the conventional medical arena is massage therapy . today , numerous rigorous scientific studies of massage have been conducted since 2000 , and many of them have shown benefit . for example , evidence in support of massage for musculoskeletal back pain in adults is so great that physicians really should at least consider it a potential therapeutic option . cerebral palsy ( cp ) describes a group of disorders of the development of movement and posture , which cause activity limitation because of non - progressive disturbances that occur in the developing fetal or infant brain . patients with cp have a wide range of symptoms , from severe motor problems with inability to stand or even sit , to a mild form of the disorder in which the motor function of just one of the patient s limbs is affected . in the general pediatric population , massage is used significantly more frequently in families in which the mother is well - educated , the father has greater disposable income , and the parents use cam themselves . however , it does include one study of 213 children with special needs surveyed in a tertiary care center in michigan . the authors reported that 25% of children with cp received massage on a regular basis . the only randomized trial of massage for patients with cp involved 20 infants with cp . in the treatment group , specifically , the authors reported that massage was associated with decreased spasticity ; improved muscle tone , range of motion , and cognition ; improved fine and gross motor skills ; and improved social functioning . however , a subsequent review of the pediatric massage literature found only modest support for improvement of muscle tone with massage , and no support for improvement of range of motion , function , or spasticity with massage . another uncontrolled qualitative evaluation of massage by parents trained to massage their children with cp was reported by powell and colleagues in england in 2006 . parents said that their children showed improved calmness , bowel habits , and verbal communication , and increased smiling , eye contact , alertness , and mobility . that study is challenged by its lack of a control group and the possible bias introduced by having parents both perform massage and report the resultant data , because the parents may be especially eager to find positive results . still , another distinct advantage is that parents are usually best able to read the child s cues . for example , a parent can distinguish between a moan of pain and a cry of happiness when the nonverbal child with cp vocalizes . cognitive deficits are not considered part of the definition of cp , but approximately 60% of people with cp also have an intellectual disability . engel and colleagues at the university of washington surveyed verbal adults with cp about what worked for their pain . respondents reported that surgical correction of hip dislocation and massage were two of the most effective treatments for pain . although the few studies of massage in people with cp have been encouraging , more research in the form of careful randomized controlled trials ( rcts ) is needed to determine whether massage is truly beneficial for people with this disorder . one compelling reason to study massage as a treatment modality is that it has biological plausibility . it is well established that massage increases blood flow to the tissues and that enhanced blood flow encourages growth of new tissue and healing of wounds . in addition , it is possible that massage facilitates removal of lactic acid ( an anaerobic respiration byproduct that causes fatigue ) from muscle , increases endorphins , and inhibits serotonin reuptake . the present study was designed as the first stage in a three - stage research plan . it is a feasibility study ( stage one ) , which precedes a pilot study ( stage two ) and a subsequent full - scale rct ( stage three ) , with the aim of determining whether massage can improve the health of children with cp . our feasibility study consisted of a survey with two major goals : to determine the current prevalence of massage among children with cp to determine the perceptions of families about the benefits of massage ( so as to help chose the outcomes to consider in the planned rct ) most massage studies have not used this approach ( feasibility pilot rct ) , and documentation of its use will be a valuable contribution to the field . the survey was designed in part to teach us more about the current use of massage for children with cp in the northwest united states and to help plan the next phases of the study . it was an opportunity to determine how many subjects our team can expect to recruit for subsequent phases of the project , and to determine potential recruitment barriers . to achieve those ends , we read a description of the planned rtc to patients and their families , soliciting feedback on specific aspects of the methodology and inquiring whether they would be willing to participate in future phases of the project . cerebral palsy ( cp ) describes a group of disorders of the development of movement and posture , which cause activity limitation because of non - progressive disturbances that occur in the developing fetal or infant brain . patients with cp have a wide range of symptoms , from severe motor problems with inability to stand or even sit , to a mild form of the disorder in which the motor function of just one of the patient s limbs is affected . in the general pediatric population , massage is used significantly more frequently in families in which the mother is well - educated , the father has greater disposable income , and the parents use cam themselves . however , it does include one study of 213 children with special needs surveyed in a tertiary care center in michigan . the authors reported that 25% of children with cp received massage on a regular basis . the only randomized trial of massage for patients with cp involved 20 infants with cp . in the treatment group , specifically , the authors reported that massage was associated with decreased spasticity ; improved muscle tone , range of motion , and cognition ; improved fine and gross motor skills ; and improved social functioning . however , a subsequent review of the pediatric massage literature found only modest support for improvement of muscle tone with massage , and no support for improvement of range of motion , function , or spasticity with massage . another uncontrolled qualitative evaluation of massage by parents trained to massage their children with cp was reported by powell and colleagues in england in 2006 . parents said that their children showed improved calmness , bowel habits , and verbal communication , and increased smiling , eye contact , alertness , and mobility . still , another distinct advantage is that parents are usually best able to read the child s cues . for example , a parent can distinguish between a moan of pain and a cry of happiness when the nonverbal child with cp vocalizes . cognitive deficits are not considered part of the definition of cp , but approximately 60% of people with cp also have an intellectual disability . engel and colleagues at the university of washington surveyed verbal adults with cp about what worked for their pain . respondents reported that surgical correction of hip dislocation and massage were two of the most effective treatments for pain . although the few studies of massage in people with cp have been encouraging , more research in the form of careful randomized controlled trials ( rcts ) is needed to determine whether massage is truly beneficial for people with this disorder . it is well established that massage increases blood flow to the tissues and that enhanced blood flow encourages growth of new tissue and healing of wounds . in addition , it is possible that massage facilitates removal of lactic acid ( an anaerobic respiration byproduct that causes fatigue ) from muscle , increases endorphins , and inhibits serotonin reuptake . the present study was designed as the first stage in a three - stage research plan . it is a feasibility study ( stage one ) , which precedes a pilot study ( stage two ) and a subsequent full - scale rct ( stage three ) , with the aim of determining whether massage can improve the health of children with cp . our feasibility study consisted of a survey with two major goals : to determine the current prevalence of massage among children with cp to determine the perceptions of families about the benefits of massage ( so as to help chose the outcomes to consider in the planned rct ) most massage studies have not used this approach ( feasibility pilot rct ) , and documentation of its use will be a valuable contribution to the field . the survey was designed in part to teach us more about the current use of massage for children with cp in the northwest united states and to help plan the next phases of the study . it was an opportunity to determine how many subjects our team can expect to recruit for subsequent phases of the project , and to determine potential recruitment barriers . to achieve those ends , we read a description of the planned rtc to patients and their families , soliciting feedback on specific aspects of the methodology and inquiring whether they would be willing to participate in future phases of the project . the institutional review board of seattle children s hospital approved the study . informed assent was obtained from the child with cp whenever the child was 14 years of age or older and able to communicate understanding and an opinion . to identify eligible subjects , we reviewed the list of patients scheduled for the neurodevelopmental and neurology clinics each week from january 2007 to august 2007 . children with cp who could not speak for themselves had to be accompanied by a parent or legal guardian knowledgeable enough about the child to complete the survey on the child s behalf . as the patient was being checked into an exam room , a medical assistant asked a parent or legal guardian if , while they waited to be seen by their provider , they would be interested in hearing about an opportunity to participate in a study . the medical assistant provided a written description of the study if the family desired , marked a form indicating whether the family wanted to participate , and placed that form on the chart outside the door . families desiring to hear more about the study were approached by the study s research assistant shortly thereafter to request consent and to administer the survey . surveying by the principal investigator or the research assistant took place in the exam rooms as each subject waited to be seen by the medical provider . the survey , which was written by the principal investigator , took approximately 15 minutes to complete . most of the questions on the survey were read to the family by the research assistant , but the final page of demographic questions was completed by the families in private . this step was intentionally taken to reduce the time spent on the survey and to increase confidentiality for families , especially with regard to income . combined parental income , reported as family income , was calculated by adding the midpoints of the income ranges reported by parents . the data were transferred from an access spreadsheet ( microsoft corporation , redmond , wa , usa ) to an excel database ( microsoft corporation ) and then transferred to the sas software package ( version 9.1 : sas institute , cary , nc , usa ) , which was used to calculate means , medians , standard deviations , and percentages . simple cross - tabulations , chi - square tests , and analyses of variance were computed to make comparisons between groups . the customary alpha of less than 0.05 was used to determine the significance of the results . to identify eligible subjects , we reviewed the list of patients scheduled for the neurodevelopmental and neurology clinics each week from january 2007 to august 2007 . children with cp who could not speak for themselves had to be accompanied by a parent or legal guardian knowledgeable enough about the child to complete the survey on the child s behalf . as the patient was being checked into an exam room , a medical assistant asked a parent or legal guardian if , while they waited to be seen by their provider , they would be interested in hearing about an opportunity to participate in a study . the medical assistant provided a written description of the study if the family desired , marked a form indicating whether the family wanted to participate , and placed that form on the chart outside the door . families desiring to hear more about the study were approached by the study s research assistant shortly thereafter to request consent and to administer the survey . surveying by the principal investigator or the research assistant took place in the exam rooms as each subject waited to be seen by the medical provider . the survey , which was written by the principal investigator , took approximately 15 minutes to complete . most of the questions on the survey were read to the family by the research assistant , but the final page of demographic questions was completed by the families in private . this step was intentionally taken to reduce the time spent on the survey and to increase confidentiality for families , especially with regard to income . combined parental income , reported as family income , was calculated by adding the midpoints of the income ranges reported by parents . the data were transferred from an access spreadsheet ( microsoft corporation , redmond , wa , usa ) to an excel database ( microsoft corporation ) and then transferred to the sas software package ( version 9.1 : sas institute , cary , nc , usa ) , which was used to calculate means , medians , standard deviations , and percentages . simple cross - tabulations , chi - square tests , and analyses of variance were computed to make comparisons between groups . the customary alpha of less than 0.05 was used to determine the significance of the results . of 106 families only 2 families approached about the study declined to participate , yielding a participation rate of 98% . among the 100 subjects eligible for analysis , ages ranged from 17 months to 21 years . most of the children in the families approached and interviewed were at the severe end of the cp spectrum , which was in keeping with the clinic population . the more severe the child s condition , the more likely the child was to have received massage . of the analyzed subjects , 20% had never received massage , and 80% had received massage at least once . the data were analyzed so as to determine the demographic factors associated with the use of massage ( ever vs. never ) . at the time massage was given by relatives in 68% of the families , by professional massage therapists in 23% , and by physical therapists in 49% . children whose mothers had lower incomes were more likely than children whose mothers had higher incomes to receive massage from a relative as compared with a professional . the proportion of girls who received massage from relatives was higher than the proportion of boys who received massage from relatives . parental age was not associated with whether the children received massage from a licensed massage therapist . of surveyed parents who were seeking and using massage for their children with cp , 86% believed that massage helps to relax muscles ; 71% , that it improves quality of life ; 23% , that it improves sleep ; and 30% , that it decreases their child s pain . in 29% of responding families , these are findings that have not , to our knowledge , been reported previously in the united states . for subjects that had received massage , the children s care providers were asked if massage had ever been discontinued , and if so , why . the most common reason for cessation was the time - consuming nature of massage ( 46% ) . in 11% of families , massage was stopped for various other reasons in 61% . among the other reasons , the two main ones were illness , surgery , or hospitalization ( n = 12 ) and a move by the family or the massage provider ( n = 8) . in addition to comparing children who had ever used massage with those who had never used massage , we compared current massage users with current nonusers . table 2 presents significant findings from that analysis , which revealed that the use of massage by the mother herself was significantly associated with current use of massage by the child . similarly , a more severe gmfcs level was significantly associated with current use of massage . neither the age of the child nor the age of the parents was significantly associated with current use or nonuse of massage . if maternal income was less than $ 20,000 annually , the child was significantly more likely to receive massage from a relative . the sex of the child mattered too : female children were more likely than male children to receive massage from a relative . subjects and their caregivers were asked if they would prefer a particular geographic location for receiving massage if they were to be assigned to the treatment arm in a future rct . response options included at home , in a hospital room , at a spa , at a massage therapist s office , and other . most respondents ( 87% ) said that they preferred to receive massage therapy at home . we collected comments from the subjects and their families to capture their ideas about a future rct of massage for cp . some of the parents saw a future study of massage as a potential opportunity for additional insurance coverage , for the child s relief from discomfort and pain , and for their own education . almost all survey respondents expressed a clear desire to know the results of the planned trial so as to help their child . our study was designed as the beginning of a larger research plan to address the question is massage a useful modality for children with cp ? the fact that 80% of our surveyed cp patients in seattle are already using massage suggests that the modality is at least perceived as useful , that it may in fact be underutilized , and that further research is needed . pain and sleep disturbances in children with cp are especially challenging and pressing problems . of the surveyed parents who are seeking and using massage for their child with cp , 86% believed that massage helps to relax muscles , and 71% believed that it improves quality of life . this information will be valuable for the design of future studies of massage for children with cp . our results suggest that , compared with children having mild disease , those more severely affected are massaged more often . more severely affected patients are more likely to suffer from constipation , sleep problems , agitation , and muscle spasms all the reasons for which parents say massage is performed . based on training time , depth of concentration , practice , and focus , experienced licensed massage therapists and physical therapists may be superior to untrained professionals in providing massage to children with cp , but this supposition has not been proven . many past pediatric massage studies have used volunteers , parents , or massage therapy students as the primary providers of treatment . more research is needed to determine whether the massage provider makes any difference with regard to specific health outcomes . the reimbursement policy in washington state is that only evidence - supported modalities of massage are covered . currently , massage is seldom covered by insurance for children with cp , and only a subset of therapists are able to perform massage when coverage is available . more research in this area is clearly needed so that , if massage is helpful , it will be accessible to children from all socioeconomic groups . second , because this study used a convenience sample of patients presenting for specialty care in an outpatient setting and because the patients surveyed generally had more severe cp , the results may not be generalizable to the entire population of children with cp . third , to keep the survey brief and to minimize the time burden on families , we obtained maternal and paternal income , but no information on insurance type ( medicaid , none , etc . ) finally , the type of massage therapy used by each family and the duration of a typical massage were not explored . an increasing number of massage modalities fall under the general heading of massage therapy . family members might massage for a shorter time than do professional massage therapists , possibly influencing actual and perceived benefit . the results from this project suggest that use of massage in children with cp is prevalent among patients of the seattle children s hospital in the pacific northwest . we do not yet know whether the type of provider ( physical therapist vs. massage therapist vs. parent ) affects the quality of massage given . systematic examination of massage as a treatment modality for musculoskeletal relaxation , quality of life , pain , sleep , and other outcomes for people with cp is needed . massage looks promising as a treatment modality for all people with cp , but for families and clinicians to make scientific decisions about its usefulness , more evidence , including our planned rct , is necessary .

reactive oxygen species ( ros ) are essential for many biological processes , including immune responses , cell signaling , neurotransmission , etc . despite the presence of enzymatic and nonenzymatic antioxidant defense mechanisms , oxidative stress may persist in cells as a result of endogenously and exogenously generated ros , which may lead to damage to macromolecules including dna , proteins and lipids . oxidative dna damage is known to be associated with the onset of many human diseases including cancer and neurodegeneration . damage to dna may compromise genomic integrity . a variety of dna lesions including modified nucleobases and 2-deoxyribose , dna strand breaks , dna dna cross - links , and dna protein cross - links can be produced . some dna lesions like phosphoglycoaldehydes and abasic sites are chemically reactive , whereas lesions like the 8,5-cyclopurine-2-deoxynucleosides are not chemically reactive and exhibit elevated stabilities compared to the corresponding unmodified nucleosides . unrepaired dna lesions are known to compromise the fidelity and/or efficiency of dna - templated processes , including dna replication and transcription . thus , identifying the dna lesions generated , evaluating their impact on dna replication and transcription , and investigating their repair are fundamental to gaining insights into their implications in human health . pseudorepair or inverted lesions , may arise from the improper chemical repair of 2-deoxyribose radicals generated from ros attack . specifically , the hydroxyl radical can abstract a hydrogen atom from the 2-deoxyribose moiety to yield carbon - centered radicals . in the presence of an h - atom donor , improper repair of these radicals formed at the c1- , c3- , and c4-positions can result in the inversion of stereochemical configuration at these carbons ( scheme 1 ) . in this review , we will summarize the research progress that has been made pertaining to the epimeric lesions of 2-deoxyribose in dna . areas to be addressed include the known formation of these lesions , their impact on stability and structure of duplex dna , and their biological consequences . additionally , we will discuss their potential therapeutic relevance , as well as future research directions about these modified nucleosides . the c1-epimers of 2-deoxyadenosine ( da ) , 2-deoxyguanosine ( dg ) , and 2-deoxyuridine ( du ) were initially reported in nucleosides . exposure of aqueous solutions of da or dg to ionizing radiation under anoxic conditions resulted in the generation of 9-(2-deoxy--d - erythro - pentofuranosyl)adenine ( -da ) and 9-(2-deoxy--d - erythro - pentofuranosyl)guanine ( -dg ) , respectively . apart from these purine nucleosides , 1-(2-deoxy--d - erythro - pentofuranosyl)uracil ( -du ) was found to form from the selectively generated c1-radical of 2-deoxyuridine . it is anticipated that both 1-(2-deoxy--d - erythro - pentofuranosyl)thymine ( -dt ) and 1-(2-deoxy--d - erythro - pentofuranosyl)cytosine ( -dc ) can also emanate from oxidative damage . the inversion from the - to -anomer at the c1-position of da in dna was initially reported when poly(da ) , poly(da - dt ) , or salmon testis dna was exposed to rays in vitro . with the exception of the loss of adenine ( which displayed a 3-fold higher level of occurrence relative to -da ) , the formation of this c1-epimer was identified as a major lesion of da in dna under anaerobic conditions . yields ( based on the total number of da residues subjected to damaging conditions ) of -da were 1.31.5% for all sequences . other lesions of da and their respective yields are ( 5r)-8,5-cyclo-2-deoxyadenosine ( 0.050.21% ) , 8-hydroxyadenine ( 0.030.10% ) , and 8-hydroxy-2-deoxyadenosine ( 0.07% ) . considering that 8,5-cyclo-2-deoxyadenosine has been observed previously in cells and tissues , it is reasonable to predict that -dns should also be formed at appreciable levels in vivo . intriguingly , lesiak and wheeler showed that -da can occur readily in the absence of thiols , which was proposed to result from either the addition of a hydrated electron followed by protonation and/or disproportionation reactions . relative to the dna damage induced by rays , exposure of da and 2-deoxyadenosine-5-monophosphate with rays resulted in significantly less amounts of -da relative to odns , indicating that the polynucleotide structure enhances the formation of -da . apart from -da , -du was identified in odns as a result of selective oxidative damage . currently , the formation of -dg , -dc , or -dt in dna remains to be determined . the c1-epimers were proposed to form from 2-deoxyribose damage occurring at the c4- and/or c1-position ( scheme 2 ) . originally , mariaggi et al . proposed that -da results from the c4-radical ( 2 ) through the hydroxyl radical - mediated hydrogen abstraction from this carbon . through this mechanism , a schiff base ( 9 ) is formed via ring opening of the sugar moiety facilitated by the protonation of the furanose oxygen . this leads to the generation of the c1-cation ( 10 ) , immediately followed by the reconstitution of the pentofuranosyl moiety in the presence of an h - atom donor , generating the two c1-epimers ( 1 and 7 ) . this mechanism for -da formation is supported by the presence of two other da damage products also proposed to originate from the c4-radical , 9-(2-deoxy--d - erythro - pentopyranosyl)adenine and 9-(2-deoxy--d - erythro - pentopyranosyl)adenine , which require a free 5-hydroxyl group for the pentopyranosyl ring formation . however , selectively generated c4-radicals of da and dt did not give rise to the formation of -da or -dt , respectively , suggesting that the c4-radical is unlikely to be a precursor for the formation of the c1-epimers . instead , the epimerization of the c1-position is more likely attributed to the formation of the c1-radical ( 4 ) ; indeed , both -du and -du were observed to form from the independently generated 2-deoxyurid-1-yl radical . the impact of the -anomer on the thermodynamic stability of duplex dna containing 2-deoxynucleosides with the normal 3-5 phosphodiester linkage has only been characterized for -da . this was accomplished by performing uv melting temperature measurements of a 9-mer duplex with a site - specifically incorporated -da being paired with the correct ( dt ) or incorrect nucleoside ( da , dg , or dc ) . in this report , the measured tm values were employed to gauge the impact of -da on g , h and s by assuming a two - state transition model . the impact of -da on duplex stability was found to vary with the pairing nucleoside of -da . the presence of a single -dadt base pair was found to increase slightly the duplex stability ( tm of 0.6 c ) relative to the canonical dadt base pair , whereas the -dadt base pair was observed to be destabilizing in other studies . discrepancies among these findings might be attributed to the different sequence contexts and/or duplex lengths used in these studies . this notion is supported by a 4.4 c range ( 49.053.4 c ) in melting temperature ( i.e. , duplex stability ) introduced by changing the nearest neighbor base - pairs . in the case of the incorrect base pairings of -da ( -dada , -dadg , and -dadc ) , significant destabilization was observed , as reflected by a drop in tm values by 7.211.0 c and a rise in g25 c values by 1.672.64 kcal / mol . the -dadg pairing resulted in the largest degree of destabilization to duplex dna ( tm = 11.0 c , g25 c = 2.64 kcal / mol ) , whereas lower extents of destabilization were found for the -dadc ( tm = 7.4 c , g25 c = 1.78 kcal / mol ) and -dada ( tm = 7.2 c , g25 c = 1.67 kcal / mol ) mispairs . aside from -da , the impact of -dg , -dt , and -dc on duplex stability was evaluated for duplexes containing two -dns with unconventional phosphodiester linkages ( 3-3 and 5-5 ) . in that study , -dc was found to be significantly more destabilizing ( tm = 10.0 c ) than -da , -dg , and -dt , which all exhibit similar effects ( average tm = 5.3 c ) . the effect of -dg , -dc , and -dt on duplex stability for the conventional 3-5 phosphodiester linkage remains to be determined . circular dichroism ( cd ) experiments demonstrated that the global conformation of duplex dna harboring an individual -da remains in b - form ( figure 1a ) . in addition , the nmr solution structure of duplex dna containing an -dadt pair revealed local changes in duplex structure ( figure 1b ) , where -da participates in both reverse watson crick base - pairing with dt and base - stacking interactions with the neighboring nucleobase ( figure 1c ) . furthermore , -da was observed to introduce a kink at the modified nucleoside site and result in an expansion of the minor groove by 18. overall , these structural perturbations were found to vary with the sequence context ( figure 1d ) . ( a ) cd spectra of double - stranded dna revealing the impact of the -da on dna secondary structure . the global b - form dna conformation observed for the unmodified duplex ( at ) is maintained for duplexes containing the -da lesion being paired with the correct ( dt ) and incorrect ( da , dc , and dg ) nucleosides ; -da is represented by . ( b ) nmr solution structure of an -da - containing 10-mer duplex dna demonstrating the b - form duplex conformation . crick base pairing interactions of a and t normally observed in b - form duplex dna and the reverse watson crick base pairing interaction observed between -da and dt in the nmr solution structure . ( d ) structural comparison highlighting the impact of the nearest - neighbor base pair on the minor - groove width and kink angle introduced by -da . duplexes ( red and orange ) containing the -da lesion were solved by nmr , while the unmodified duplex ( blue ) is a model . the minor groove is indicated by m , while the position of -da is indicated by . the nearest - neighbor base pair altered is highlighted in red , orange , or blue , respectively . so far , replication studies have been conducted only for -da . in this respect , -da was found to stall the klenow fragment in vitro , though bypass could be observed after prolonged incubation . gilbert sequencing and primer extension assays indicate that -da directs the incorporation of both correct ( dtmp ) and incorrect ( dcmp , damp and dgmp ) nucleotides , with the misincorporation of dgmp being significantly disfavored . in addition , promutagenic properties and bypass efficiency of -da were found to be sequence - dependent , particularly by the nearest neighbor base pair but independent of the exonuclease activity of the polymerase . in addition , similar results were obtained for t7 and taq dna polymerases as well as a reverse transcriptase . replication studies with the use of -da - bearing , single - stranded m13 bacteriophage showed that -da constitutes a moderate block to dna replication and introduces single nucleotide deletion in e. coli cells . although nucleotide misincorporation was not found in vivo , the frequency of single - nucleotide deletion was observed to be sequence - dependent , with mutation frequencies ranging from 1 to 26% . a misinsertion - strand - slippage mechanism was thought to contribute to the generation of single - nucleotide deletions . the impact of -dt , -dg , and -dc on dna replication , however , remains to be investigated . the c1-epimeric lesions were found to be substrates for repair enzymes of bacteria , yeast , and humans . for e. coli , a wide range of repair enzymes were evaluated , including multiple endonucleases ( i.e. , endonucleases iii , iv , viii , and deoxyinosine-3-endonuclease ) , exonuclease iii , and formamidopyrimidine n - glycosylase . among these enzymes , endonuclease iv was the only repair enzyme capable of recognizing both -da and -dt . in addition , this endonuclease activity was selective for dsdna substrates , as the corresponding ssdna constructs could not be cleaved by the enzyme . apart from the e. coli enzymes , s. cerevisiae apurinic / apyrimidinic ( ap ) endonuclease 1 ( apn1 ) and human ap endonuclease i ( ape1 ) could also recognize -da and -dt . together , these reports suggest that the repair pathway of -da and -dt is conserved among species and that -dg and -dc are likely substrates for the same repair pathway . on the grounds that the -dns are not recognized by dna glycosylases in the base excision repair ( ber ) pathway , it has been proposed that these c1-epimeric lesions are repaired by the nucleotide incision repair ( nir ) pathway , an alternative pathway to ber . the inversion of configuration at the c3-position results in the generation of the c3-epimeric lesions , i.e. , 1-(2-deoxy - r - l - threo - pentofuranosyl)-containing nucleosides , commonly referred to as xylose nucleosides . these types of lesions were first observed to form from the independently generated c3-radicals of nucleosides in the presence of an h - atom donor . the c3-epimeric lesion was observed to form in equal amounts as the properly chemically repaired c3-epimer of 2-deoxyribose . in addition , 1-(2-deoxy - r - l - threo - pentofuranosyl)thymine ( dxt ) was found to form in odns from the independently generated c3-radical under anaerobic conditions , where dxt was produced more readily in model replication - relevant architectures ( ssdna and 5-overhang ) than in dsdna . moreover , the formation of the c3-epimeric lesion competes directly with strand break formation under anaerobic conditions ( scheme 3 ) . future investigations about the in vivo formation of dxt and other dxns will provide valuable insights into the physiological relevance of the c3-epimeric lesions . formation for the c3-epimeric lesion and competing strand break pathway resulting from oxidation ( [ o ] ) in aqueous solution . alterations in both dna stability and structure have been reported for odns containing a single dxn lesion . primarily , these studies have been performed using uv melting analysis , circular dichroism ( cd ) measurements , and molecular modeling . it has been reported that the presence of a single dxt in double - stranded dna affects both dna stability and structure , though the duplex maintains b - dna geometry . in model dna replication - relevant architectures ( fork , 5-overhang , and 3-overhang ) , dxt was more destabilizing and introduced greater structural alterations when located closer to the 3-terminus of the damaged odn . the presence of an individual dxc or dxg in duplex dna also led to a significant decrease in duplex stability . initial structural characterizations of all four dxns individually , as well as the structural analysis of fully xylose duplex dna , indicate that the sugar pucker of the dxns adopts the c3-endo conformation , resembling that of ribonucleosides ( figure 2 ) . together , these reports suggest that the presence of an isolated dxn may perturb the stability and structure of duplex dna in vivo . 2-deoxyribose sugar puckers observed in duplex dna ( b and a form ) , rna , and for dxns . investigations about the impact of the c3-epimeric lesions on biological processes are limited . along this line , primer extension assays were conducted to assess the ability of dna polymerases to incorporate 2-deoxyxyloadenosine triphosphate ( dxatp ) and/or 2-deoxyxylothymidine triphosphate ( dxttp ) into dna in vitro . it was found that several different dna polymerases were capable of incorporating dxntps into the elongating strand . notably , complete primer extension was not observed , suggesting that the presence of the dxn may significantly inhibit dna replication in vivo . provided that the sugar pucker of the dxns prefers the c3-endo conformation , it is likely that the lack of complete primer extension is a result of the structural effects of the dxn lesion . future investigations seeking to obtain detailed structural data , as well as experiments measuring the bypass efficiencies of the dxn lesions , will significantly expand the current knowledge about the impact of these lesions on dna replication . endonuclease activity on dna substrates containing dxc and/or dxt was observed to vary among enzymes and positions of the dxn . ecorii was incapable of cleaving the dna backbone with the presence of dxns at the restriction recognition site , while the activity of mvai was variable and overall significantly more tolerant of the presence of the lesion than ecorii . the c4-epimeric lesions of dt and da were found to be generated in vitro in single- and double - stranded dna from independently generated radicals , while the c4-epimer of dg could be induced in an aqueous solution of dg upon exposure to -rays . similar to the c3-epimeric lesion , the formation of the c4-epimer competes with strand break generation ( scheme 4 ) . the stereoselectivity of 5 versus 1 was observed to be directly influenced by the dna structure , with significantly greater levels of 5 being generated in ssdna than dsdna . nevertheless , the levels of 5 were reported not to be affected by either the source of h - atom donor or the identity of the nucleoside ( dt or da ) from which the radical is generated , suggesting that the c4-epimeric lesions may be produced in dna at similar levels for the four canonical 2-deoxynucleosides . oxidative damage in ssdna in the presence of oxygen resulted in the epimeric mixtures of 5 and 1 , with yields ( 151% ) varying with the concentrations of h - atom donor . currently , the formation of these c4-epimeric lesions in vivo remains to be determined . the formation for the c4-epimeric lesion ( 5 ) and competing strand break pathway arising from spontaneous strand scission . investigations about the impact of the c4-epimeric lesions on dna stability are minimal , with no reports characterizing structural changes . this is likely due to the lack of facile synthetic routes to obtaining these c4-epimers . the presence of the c4-epimer of da in the duplex portion of a dangling end substrate was reported to be destabilizing , as reflected by a decrease in melting temperature of 1.2 c . crick base - pairing interactions based on the fidelity of nucleotide incorporation opposite this lesion , as discussed in section 4.3 below ; nevertheless , it remains to be assessed whether the same findings can be extended to the c4-epimers of other nucleosides . not much is known about the biological consequences of the c4-epimeric lesions with the exception of the studies assessing how this lesion affects dna replication . primer extension assay with the use of the klenow fragment revealed that the c4-epimer of da introduced a stop - site one nucleotide downstream of the lesion , though it could be bypassed after prolonged incubation . in addition , this bypass was independent of the exonuclease activity of the polymerase . intriguingly , the levels of incorporation of the incorrect dntps were found to be decreased relative to the undamaged substrate . thus , the presence of the c4-epimeric lesion of da was found not to be mutagenic in vitro but may be cytotoxic as reflected by the stalling of the klenow fragment of dna polymerase i. with respect to dna repair , the ability of the ner pathway to repair the c4-epimer of da was investigated in vitro with the use of human cell extracts . in duplex dna , the lesion was found not to be a substrate for human ner when it is paired with a dt ; however , it could be recognized by the human ner machinery when it is present in mispairs . therefore , these results suggest that the c4-epimer of da formed in at base pairs is unlikely a substrate for the ner pathway . it remains to be examined whether the c4-epimers of this and other nucleosides can be repaired by other pathways including ber and nir . currently , none of the epimeric 2-deoxyribose lesions have been detected in vivo . given the literature precedence in identifying the formation of these lesions in vitro at all three stereocenters ( c1 , c3 , and c4 ) of 2-deoxyribose , it is highly likely that these purely structural lesions can be generated in vivo from oxidative dna damage . the presence of an h - atom donor source facilitates the formation of these epimeric dna lesions . as observed at both the c1- and c4-positions , the nature of the h - atom donor does not impact lesion formation . in cells , there are a variety of potential h - atom donors that are readily available , with glutathione ( gsh ) being the major antioxidant in the nucleus , cytosol , and mitochondria . upon the formation of the 2-deoxyribose radicals , the presence of o2 readily competes with h - atom donors via generating peroxyl radicals of 2-deoxyribose , which can also be reduced by h - atom donors . thus , it is anticipated that the greatest levels of these epimeric lesions are generated under conditions and/or microenvironments where o2 levels are low and h - atom donors are prevalent . for example , the nucleus is known to contain relatively low concentrations of o2 and adequate concentrations ( 315 mm ) of gsh , which was previously demonstrated to facilitate the formation of these lesions . in the case of cancer tissues , which are often hypoxic and resistant to radiation and chemotherapeutic treatments the potential for these epimeric lesions to form under aerobic conditions in vivo is also likely ( but expected to be produced at significantly lower levels ) as demonstrated in vitro at the c4-position . several anticancer and antiviral drugs , as well as artificial metal nucleases , function by inducing strand breaks via h - atom abstraction from the 2-deoxyribose moiety . the same 2-deoxyribose radicals were previously found to yield the epimeric lesions of 2-deoxyribose . the specific 2-deoxyribose radicals generated are dictated by both dna structure and dna - oxidizer - binding interactions . in b - dna , both the c1 and c4 h - atoms reside in the minor groove , whereas the c3 h - atom is located in the major groove . therefore , drugs that bind to the minor groove and generate the c1- and/or c4-radical , as demonstrated by some enediynes and bleomycins , may foster the inversion of stereochemistry at the c1- and c4-positions ( figure 3a ) . given that these drugs bind to the minor groove of dna , accessibility of the h - atom donor to the face of the c1- and c4-radicals via the minor groove is likely inhibited , potentially facilitating the improper chemical repair of the 2-deoxyribose radicals by cellular thiols . to our knowledge , the formation of the c1- and/or c4-epimers from enediynes or bleomycins in the presence of thiols has not been reported . similarly , major - groove binders known to generate the c3-radical , such as rhodium complexes , may induce the preferential formation of the c3-epimeric lesions ( figure 3b ) . in this case , the h - atom donor may only have access to the face of the c3-radical through the minor groove , while the proper chemical repair from the face may be inhibited by the presence of the dna - binding agent targeting the major groove . potential formation of the epimeric 2-deoxyribose lesions resulting from dna binding agents . ( a ) proposed formation of the c1- and c4-epimers resulting from the h - atom abstraction by minor - groove binders . ( b ) proposed formation of the c3-epimers resulting from the c3-radical generation by major - groove binders . in considering the potential generation of these lesions by chemotherapy or radiation therapy , regard , hypoxia is known to confer resistance to chemotherapeutics and radiation therapy , where depleted o2 levels could decrease directly the toxicity of many therapeutics and serve as a signal for hypoxia - inducible transcription factors . we reason that the drug resistance manifested during hypoxia could also be attributed , in part , to the formation of these epimeric 2-deoxyribose lesions facilitated by cellular thiols , which competes directly with the formation of the intended dna strand breaks . in this regard , the formation of these lesions would prevent the generation of the dna strand breaks , which is a toxic effect of many therapeutic agents targeting cancer cells . consequently , any biological activity associated with the generation of the epimeric 2-deoxyribose lesions may be masked by the already abnormal activity of the cancer cells . as a result , the formation of these epimeric lesions may be generated as an alternative route for cancer cell survival during hypoxia , preventing lethal strand scissions at the expense of the biological consequences introduced by the epimeric lesions . thus , the formation of these epimeric 2-deoxyribose lesions in cells upon exposure to chemotherapeutic agents and ionizing radiation under normoxic and hypoxic conditions merits future investigations . the outcome of such studies may provide molecular insights into hypoxia - induced resistance to chemotherapy and radiation therapy . additionally , such studies may unveil the impact of the cellular environment on the generation of these lesions . the potential formation of the aforementioned epimeric dna lesions in vivo is well prefaced with the reported in vitro literature . demonstrating the formation of these lesions in vivo is essential for expanding the current scope of their investigations . this endeavor is challenged by the lack of alterations in chemical functional groups introduced by these lesions , necessitating a structural and/or enzymatic approach for their identification in vivo . these antibodies , however , may not recognize a single isolated c1 -anomer as generated from oxidative dna damage . thus , it would be interesting to develop antibodies that can be employed for recognizing an individual c1 -anomer or the c3 or c4 2-deoxyribose epimers in dna . another approach would be to utilize enzymatic digestions coupled with lc - ms / ms analysis . this would necessitate optimized methods to facilitate the chromatographic separation and/or mass spectrometric differentiation of these epimeric lesions from their unmodified counterparts . regardless of the approach , method development for assessing the formation of these lesions in vivo will require them to be synthetically available . the syntheses of the c1- and c3-epimers have been reported for all four dns . in the case of the c4-epimers , available synthetic routes are currently limited to da , which is generated utilizing a modified radical precursor . it should be noted that the generation of epimeric nucleosides via radical intermediates is accompanied with a high risk of byproduct formation . as a result , this approach may offer the limited yield for the desired epimer . thus , the development of facile synthetic methods for generating the full spectrum of c4-epimeric lesions will directly broaden the experimental scope of future investigations and enable the assessment about the in vivo formation of these epimeric lesions . future studies evaluating the impact of these lesions on processes such as dna replication , transcription , and repair will yield insightful results on the biological consequences for the formation of these lesions . currently , in vivo studies are limited to the c1-epimeric lesion of da in the e. coli system . if these lesions are in fact formed in vivo , understanding the repair of these lesions and the effects of these lesions on dna replication and transcription in mammalian cells should be pursued . additionally , it is possible that these lesions are formed in vivo under hypoxic conditions , in which case they may be utilized as biomarkers of hypoxia . in this review , we summarized our current knowledge of the epimeric 2-deoxyribose lesions in dna , which are the improper chemical repair products of the c1- , c3- , and c4-radicals of 2-deoxyribose . particularly , we have reviewed relevant literature highlighting the known formation of these lesions , their impact on the stability and structure of the dna double helix , and their biological consequences . as discussed above , some of these epimeric 2-deoxyribose lesions have been observed to be generated in dna from either radiation and/or independently generated radicals in vitro , while their in vivo presence remains elusive . in addition , it has been observed that the presence of an individual epimeric 2-deoxyribose lesion in dna destabilizes the dna double helix and introduces local structural alterations in dna , which has been demonstrated to vary with dna sequence context and architecture . some of these lesions also compromise dna replication , being promutagenic or potentially cytotoxic . existing literature has offered significant precedence for the potential formation of the epimeric 2-deoxyribose lesions in vivo , as well as an initial understanding of the potential biological consequences that they may exert , while much remains to be learned for this family of dna lesions . in particular , determining if these dna lesions form for all four nucleosides and measuring their respective levels of formation in vitro and in vivo will not only broaden our scope of knowledge for these dna lesions but also unveil whether and how the fate of the 2-deoxyribose radicals is influenced by the nature of the nucleobase . moreover , future studies addressing the impact of these lesions on dna replication and transcription and how they are repaired in mammalian cells will offer important knowledge for understanding the implications of these lesions in human diseases .

genomic integrity is constantly challenged by dna damaging agents such as reactive oxygen species ( ros ) . consequently , dna damage can compromise the fidelity and efficiency of essential dna metabolic processes , including replication and transcription , which may contribute significantly to the etiology of many human diseases . here , we review one family of dna lesions , the epimeric 2-deoxyribose lesions , which arise from the improper chemical repair of the 2-deoxyribose radicals . unlike most other dna lesions , the epimeric 2-deoxyribose lesions are indistinguishable from their corresponding unmodified nucleosides in both molecular mass and chemical reactivity . we placed our emphasis of discussion on the formation of these lesions , their impact on the structure and stability of duplex dna , their biological consequences , their potential therapeutic relevance , and future research directions about these modified nucleosides .

Introduction C1-Epimer C3-Epimer C4-Epimer Physiological Relavance Potential Therapeutic Implications Prospects and Limitations Conclusions

reactive oxygen species ( ros ) are essential for many biological processes , including immune responses , cell signaling , neurotransmission , etc . despite the presence of enzymatic and nonenzymatic antioxidant defense mechanisms , oxidative stress may persist in cells as a result of endogenously and exogenously generated ros , which may lead to damage to macromolecules including dna , proteins and lipids . oxidative dna damage is known to be associated with the onset of many human diseases including cancer and neurodegeneration . a variety of dna lesions including modified nucleobases and 2-deoxyribose , dna strand breaks , dna dna cross - links , and dna protein cross - links can be produced . some dna lesions like phosphoglycoaldehydes and abasic sites are chemically reactive , whereas lesions like the 8,5-cyclopurine-2-deoxynucleosides are not chemically reactive and exhibit elevated stabilities compared to the corresponding unmodified nucleosides . unrepaired dna lesions are known to compromise the fidelity and/or efficiency of dna - templated processes , including dna replication and transcription . thus , identifying the dna lesions generated , evaluating their impact on dna replication and transcription , and investigating their repair are fundamental to gaining insights into their implications in human health . pseudorepair or inverted lesions , may arise from the improper chemical repair of 2-deoxyribose radicals generated from ros attack . specifically , the hydroxyl radical can abstract a hydrogen atom from the 2-deoxyribose moiety to yield carbon - centered radicals . in the presence of an h - atom donor , improper repair of these radicals formed at the c1- , c3- , and c4-positions can result in the inversion of stereochemical configuration at these carbons ( scheme 1 ) . in this review , we will summarize the research progress that has been made pertaining to the epimeric lesions of 2-deoxyribose in dna . areas to be addressed include the known formation of these lesions , their impact on stability and structure of duplex dna , and their biological consequences . additionally , we will discuss their potential therapeutic relevance , as well as future research directions about these modified nucleosides . with the exception of the loss of adenine ( which displayed a 3-fold higher level of occurrence relative to -da ) , the formation of this c1-epimer was identified as a major lesion of da in dna under anaerobic conditions . relative to the dna damage induced by rays , exposure of da and 2-deoxyadenosine-5-monophosphate with rays resulted in significantly less amounts of -da relative to odns , indicating that the polynucleotide structure enhances the formation of -da . currently , the formation of -dg , -dc , or -dt in dna remains to be determined . this leads to the generation of the c1-cation ( 10 ) , immediately followed by the reconstitution of the pentofuranosyl moiety in the presence of an h - atom donor , generating the two c1-epimers ( 1 and 7 ) . this mechanism for -da formation is supported by the presence of two other da damage products also proposed to originate from the c4-radical , 9-(2-deoxy--d - erythro - pentopyranosyl)adenine and 9-(2-deoxy--d - erythro - pentopyranosyl)adenine , which require a free 5-hydroxyl group for the pentopyranosyl ring formation . however , selectively generated c4-radicals of da and dt did not give rise to the formation of -da or -dt , respectively , suggesting that the c4-radical is unlikely to be a precursor for the formation of the c1-epimers . instead , the epimerization of the c1-position is more likely attributed to the formation of the c1-radical ( 4 ) ; indeed , both -du and -du were observed to form from the independently generated 2-deoxyurid-1-yl radical . the impact of the -anomer on the thermodynamic stability of duplex dna containing 2-deoxynucleosides with the normal 3-5 phosphodiester linkage has only been characterized for -da . in the case of the incorrect base pairings of -da ( -dada , -dadg , and -dadc ) , significant destabilization was observed , as reflected by a drop in tm values by 7.211.0 c and a rise in g25 c values by 1.672.64 kcal / mol . aside from -da , the impact of -dg , -dt , and -dc on duplex stability was evaluated for duplexes containing two -dns with unconventional phosphodiester linkages ( 3-3 and 5-5 ) . in that study , -dc was found to be significantly more destabilizing ( tm = 10.0 c ) than -da , -dg , and -dt , which all exhibit similar effects ( average tm = 5.3 c ) . circular dichroism ( cd ) experiments demonstrated that the global conformation of duplex dna harboring an individual -da remains in b - form ( figure 1a ) . in addition , the nmr solution structure of duplex dna containing an -dadt pair revealed local changes in duplex structure ( figure 1b ) , where -da participates in both reverse watson crick base - pairing with dt and base - stacking interactions with the neighboring nucleobase ( figure 1c ) . ( d ) structural comparison highlighting the impact of the nearest - neighbor base pair on the minor - groove width and kink angle introduced by -da . in addition , promutagenic properties and bypass efficiency of -da were found to be sequence - dependent , particularly by the nearest neighbor base pair but independent of the exonuclease activity of the polymerase . on the grounds that the -dns are not recognized by dna glycosylases in the base excision repair ( ber ) pathway , it has been proposed that these c1-epimeric lesions are repaired by the nucleotide incision repair ( nir ) pathway , an alternative pathway to ber . the inversion of configuration at the c3-position results in the generation of the c3-epimeric lesions , i.e. these types of lesions were first observed to form from the independently generated c3-radicals of nucleosides in the presence of an h - atom donor . moreover , the formation of the c3-epimeric lesion competes directly with strand break formation under anaerobic conditions ( scheme 3 ) . future investigations about the in vivo formation of dxt and other dxns will provide valuable insights into the physiological relevance of the c3-epimeric lesions . in model dna replication - relevant architectures ( fork , 5-overhang , and 3-overhang ) , dxt was more destabilizing and introduced greater structural alterations when located closer to the 3-terminus of the damaged odn . initial structural characterizations of all four dxns individually , as well as the structural analysis of fully xylose duplex dna , indicate that the sugar pucker of the dxns adopts the c3-endo conformation , resembling that of ribonucleosides ( figure 2 ) . together , these reports suggest that the presence of an isolated dxn may perturb the stability and structure of duplex dna in vivo . 2-deoxyribose sugar puckers observed in duplex dna ( b and a form ) , rna , and for dxns . future investigations seeking to obtain detailed structural data , as well as experiments measuring the bypass efficiencies of the dxn lesions , will significantly expand the current knowledge about the impact of these lesions on dna replication . similar to the c3-epimeric lesion , the formation of the c4-epimer competes with strand break generation ( scheme 4 ) . nevertheless , the levels of 5 were reported not to be affected by either the source of h - atom donor or the identity of the nucleoside ( dt or da ) from which the radical is generated , suggesting that the c4-epimeric lesions may be produced in dna at similar levels for the four canonical 2-deoxynucleosides . currently , the formation of these c4-epimeric lesions in vivo remains to be determined . crick base - pairing interactions based on the fidelity of nucleotide incorporation opposite this lesion , as discussed in section 4.3 below ; nevertheless , it remains to be assessed whether the same findings can be extended to the c4-epimers of other nucleosides . not much is known about the biological consequences of the c4-epimeric lesions with the exception of the studies assessing how this lesion affects dna replication . intriguingly , the levels of incorporation of the incorrect dntps were found to be decreased relative to the undamaged substrate . thus , the presence of the c4-epimeric lesion of da was found not to be mutagenic in vitro but may be cytotoxic as reflected by the stalling of the klenow fragment of dna polymerase i. with respect to dna repair , the ability of the ner pathway to repair the c4-epimer of da was investigated in vitro with the use of human cell extracts . in duplex dna , the lesion was found not to be a substrate for human ner when it is paired with a dt ; however , it could be recognized by the human ner machinery when it is present in mispairs . currently , none of the epimeric 2-deoxyribose lesions have been detected in vivo . given the literature precedence in identifying the formation of these lesions in vitro at all three stereocenters ( c1 , c3 , and c4 ) of 2-deoxyribose , it is highly likely that these purely structural lesions can be generated in vivo from oxidative dna damage . the presence of an h - atom donor source facilitates the formation of these epimeric dna lesions . as observed at both the c1- and c4-positions , the nature of the h - atom donor does not impact lesion formation . upon the formation of the 2-deoxyribose radicals , the presence of o2 readily competes with h - atom donors via generating peroxyl radicals of 2-deoxyribose , which can also be reduced by h - atom donors . thus , it is anticipated that the greatest levels of these epimeric lesions are generated under conditions and/or microenvironments where o2 levels are low and h - atom donors are prevalent . for example , the nucleus is known to contain relatively low concentrations of o2 and adequate concentrations ( 315 mm ) of gsh , which was previously demonstrated to facilitate the formation of these lesions . several anticancer and antiviral drugs , as well as artificial metal nucleases , function by inducing strand breaks via h - atom abstraction from the 2-deoxyribose moiety . the specific 2-deoxyribose radicals generated are dictated by both dna structure and dna - oxidizer - binding interactions . given that these drugs bind to the minor groove of dna , accessibility of the h - atom donor to the face of the c1- and c4-radicals via the minor groove is likely inhibited , potentially facilitating the improper chemical repair of the 2-deoxyribose radicals by cellular thiols . to our knowledge , the formation of the c1- and/or c4-epimers from enediynes or bleomycins in the presence of thiols has not been reported . similarly , major - groove binders known to generate the c3-radical , such as rhodium complexes , may induce the preferential formation of the c3-epimeric lesions ( figure 3b ) . in this case , the h - atom donor may only have access to the face of the c3-radical through the minor groove , while the proper chemical repair from the face may be inhibited by the presence of the dna - binding agent targeting the major groove . potential formation of the epimeric 2-deoxyribose lesions resulting from dna binding agents . ( a ) proposed formation of the c1- and c4-epimers resulting from the h - atom abstraction by minor - groove binders . ( b ) proposed formation of the c3-epimers resulting from the c3-radical generation by major - groove binders . in considering the potential generation of these lesions by chemotherapy or radiation therapy , regard , hypoxia is known to confer resistance to chemotherapeutics and radiation therapy , where depleted o2 levels could decrease directly the toxicity of many therapeutics and serve as a signal for hypoxia - inducible transcription factors . we reason that the drug resistance manifested during hypoxia could also be attributed , in part , to the formation of these epimeric 2-deoxyribose lesions facilitated by cellular thiols , which competes directly with the formation of the intended dna strand breaks . in this regard , the formation of these lesions would prevent the generation of the dna strand breaks , which is a toxic effect of many therapeutic agents targeting cancer cells . consequently , any biological activity associated with the generation of the epimeric 2-deoxyribose lesions may be masked by the already abnormal activity of the cancer cells . as a result , the formation of these epimeric lesions may be generated as an alternative route for cancer cell survival during hypoxia , preventing lethal strand scissions at the expense of the biological consequences introduced by the epimeric lesions . thus , the formation of these epimeric 2-deoxyribose lesions in cells upon exposure to chemotherapeutic agents and ionizing radiation under normoxic and hypoxic conditions merits future investigations . additionally , such studies may unveil the impact of the cellular environment on the generation of these lesions . the potential formation of the aforementioned epimeric dna lesions in vivo is well prefaced with the reported in vitro literature . demonstrating the formation of these lesions in vivo is essential for expanding the current scope of their investigations . this endeavor is challenged by the lack of alterations in chemical functional groups introduced by these lesions , necessitating a structural and/or enzymatic approach for their identification in vivo . this would necessitate optimized methods to facilitate the chromatographic separation and/or mass spectrometric differentiation of these epimeric lesions from their unmodified counterparts . regardless of the approach , method development for assessing the formation of these lesions in vivo will require them to be synthetically available . in the case of the c4-epimers , available synthetic routes are currently limited to da , which is generated utilizing a modified radical precursor . thus , the development of facile synthetic methods for generating the full spectrum of c4-epimeric lesions will directly broaden the experimental scope of future investigations and enable the assessment about the in vivo formation of these epimeric lesions . future studies evaluating the impact of these lesions on processes such as dna replication , transcription , and repair will yield insightful results on the biological consequences for the formation of these lesions . if these lesions are in fact formed in vivo , understanding the repair of these lesions and the effects of these lesions on dna replication and transcription in mammalian cells should be pursued . additionally , it is possible that these lesions are formed in vivo under hypoxic conditions , in which case they may be utilized as biomarkers of hypoxia . in this review , we summarized our current knowledge of the epimeric 2-deoxyribose lesions in dna , which are the improper chemical repair products of the c1- , c3- , and c4-radicals of 2-deoxyribose . particularly , we have reviewed relevant literature highlighting the known formation of these lesions , their impact on the stability and structure of the dna double helix , and their biological consequences . as discussed above , some of these epimeric 2-deoxyribose lesions have been observed to be generated in dna from either radiation and/or independently generated radicals in vitro , while their in vivo presence remains elusive . in addition , it has been observed that the presence of an individual epimeric 2-deoxyribose lesion in dna destabilizes the dna double helix and introduces local structural alterations in dna , which has been demonstrated to vary with dna sequence context and architecture . some of these lesions also compromise dna replication , being promutagenic or potentially cytotoxic . existing literature has offered significant precedence for the potential formation of the epimeric 2-deoxyribose lesions in vivo , as well as an initial understanding of the potential biological consequences that they may exert , while much remains to be learned for this family of dna lesions . in particular , determining if these dna lesions form for all four nucleosides and measuring their respective levels of formation in vitro and in vivo will not only broaden our scope of knowledge for these dna lesions but also unveil whether and how the fate of the 2-deoxyribose radicals is influenced by the nature of the nucleobase . moreover , future studies addressing the impact of these lesions on dna replication and transcription and how they are repaired in mammalian cells will offer important knowledge for understanding the implications of these lesions in human diseases .

endoscopic retrograde cholangiopancreatography ( ercp ) with endoscopic sphincterotomy ( es ) was first introduced in 1974 in germany and japan 1 2 and a few years later in the nordic countries , as an alternative to open surgery with choledochotomy in the treatment of common bile duct stones . it was mainly used in elderly patients or patients with severe comorbidity , in whom mortality after ercp was reduced compared to open surgery 3 , whereas young and healthy patients were still predominantly treated with open surgical procedures . since the introduction of laparoscopic cholecystectomy , ercp with es , with or without cholecystectomy , has become the most common procedure in the treatment of common bile duct stones worldwide . a consequence of this alteration in therapeutic regime is that an increased number of young patients , with a long life expectancy , currently undergo es . interruption of the sphincter of oddi has been reported to cause overgrowth of bacteria 4 in the common bile duct resulting in cholangitis and recurrent stone formation 5 . studies also show an increased incidence of cholangiocarcinoma after es , presumably due to the bacterial overgrowth and chronic inflammation in the bile ducts 5 6 . this is in agreement with other reports concerning the development of malignancies after open bilioenteric anastomoses 7 8 , and transduodenal sphincterotomy 9 . these data prompted us to hypothesize that es increases the long - term risk of cholangiocarcinoma , which we were subsequently able to test in a swedish nationwide population - based study of all 27 708 patients registered for an ercp for benign disease in the swedish inpatient registry , since the introduction of ercp in the 1970s until 2005 10 . our results showed that , among the 12 629 patients who had undergone es , the risk of cancer in the bile ducts was not increased compared to the non - es group , but on the other hand , that there was a tendency toward a decreased long - term risk of bile duct cancer in the es group . however , due to the small number of patients followed for more than 10 years , our data did not have the statistical power to address this question . in this study , we also found a decreased risk of malignancy in patients who had had a cholecystectomy . the primary aim of this population - based cohort study of all patients registered for an ercp in the inpatient registries in finland and sweden was to study the risk of malignancy in the bile ducts in a larger cohort and with a longer follow - up . a secondary aim was to study the relationship between severe common bile duct stone exposure , which is a valid assumption for the entire ercp cohort irrespective of es , and malignancy in the biliary tract . because of expected extensive misclassification between extrahepatic bile duct cancers and pancreatic cancer , as well as between intrahepatic bile duct cancers and liver cancer , both bile duct cancer alone as well as bile duct cancer together with pancreatic and liver cancers , were used as outcome parameters . we used data from the finnish and swedish hospital discharge registers , in which discharge diagnoses and surgical procedures are computerized for each hospitalization . unique national registration numbers for each inhabitant are in use in both finland and sweden , and we used them for identification . the coverage of the registries is nearly 100 % from 1986 and onwards 11 12 . the hospital discharge registries were introduced 1986 in finland and 1965 in sweden , and the first ercp procedure was registered in 1976 . we identified patients from 1986 to 2010 in finland and 1976 to 2010 in sweden with at least one in - hospital episode with a discharge procedure code for ercp or endoscopic sphincterotomy . in the ercp group , patients with a code of ercp with or without biopsy and cholangioscopy were included while the es group included patients with codes of sphincterotomy , extraction of stones , insertion of stent or nasobiliary drainage ( international classification of operations and major procedures , codes 9014 , ujk02 , ujk05 , ujk12 , ujk15 for ercp or 5388 , 5394 , jke 02 , jke 12 , jke 15 , jke 18 , jke 25 , jke 98 for es or procedures for which es is normally a prerequisite ) . using the national registration number , the patients identified with the procedure codes listed above were linked to each country s nationwide cancer registry , population registry , and migration registry for cancer outcome ascertainment and censoring due to death and emigration , respectively . a detailed description of the methods used has been described elsewhere 13 . for further analyses , the total ercp cohort for each country was divided into two subgroups : ( 1 ) patients having at least one procedure code registration for es or any other endoscopic biliary procedures for which an es is normally a prerequisite ( international classification of operations and major procedures , codes 5388 , 5394 , jke 02 , jke 12 , jke 15 , jke 18 , jke 25 , jke 98 ) , and ( 2 ) patients in the cohort without any procedure code registration for es or any other endoscopic biliary procedure implying es . those patients who had a diagnosis of malignant tumor in the bile ducts , liver or pancreas ( icd - o-3 c22 25 in the finnish data and icd7 155 157 in the swedish data or corresponding codes in the later classifications ) at the time of the procedure or within 2 years after it , were excluded from further analyses to avoid selection bias , since the registered ercp in these cases may have been performed because of the tumor or due to symptoms caused by a tumor that was still undiagnosed . considering the poor prognosis of malignancies in the biliary tract , liver and pancreas , it is highly unlikely that a tumor causing symptoms would be diagnosed more than 2 years later . the cohorts were then followed from entry ( 2 years after the procedure ) until diagnosis of an outcome malignancy ( primary malignant tumors in the liver , bile ducts including ampullary region and pancreas , but excluding gallbladder malignancy , icd7 codes : 155 and 157 , but excluding 1551 and icd10 codes c22.1 , c24.0 , c24.1 , c24.8 , and c25 ) , death , emigration or end of follow - up ( 31 december 2010 ) , whichever occurred first . the study was approved by the national institute for health and welfare in finland and the regional research ethics committee of stockholm and helsinki . for each patient , every first - time procedure was regarded as the index procedure for the corresponding cancer analyses . if the first - time procedure included or implied es , the patient s person - time was only included in the es subgroup . if a patient s first procedure was non - es or es - implying followed by a subsequent procedure implying es , this patient had two index procedures : one without es , with person - time counted from 2 years after the procedure until the subsequent es procedure ( i. e. censored at the time of the es procedure ) ; and another one after which person - time was counted in the es subgroup from 2 years after the es procedure . however , for the whole ercp group , person - time was counted from 2 years after entry until the occurrence of cancer , death , emigration or end of follow - up , irrespective of es procedure ( fig . 1 ) . thus the total person - time in the whole ercp cohort was more than the sum of the person - time experienced by the two subgroups . end of follow - up was 31 december 2010 , a diagnosis of malignancy , death , emigration or second procedure . the standardized incidence ratio ( sir ) , the ratio of the observed to the expected number of malignancies , was used to calculate relative risk . the background cancer incidence rates for the general swedish and finish populations were provided by the national cancer registries in the corresponding country , which was the same as the source for linkage to identify cancer cases during follow - up . the expected number of cancers was calculated by using the sex , calendar , age , and country - specific incidence rates . the standardized incidence ratios are inherently adjusted for confounding by age at follow - up , gender , and calendar year of follow - up . to calculate the pooled sir , we first combined the number of observed cancers , o , by summing data from the two countries , and the combined number of expected cancers , e , was determined in a similar way . the 95 % confidence intervals ( cis ) for the pooled sir were determined by assuming a poisson distribution of the observed numbers of cancer . results using the weighted meta - analysis were essentially identical with those using the pooled method , and tests for heterogeneity using the chi - squared test as well as i statistics demonstrated that the inconsistencies across the two studies were minor ( = 0.19 , df = 1 , p = 0.66 ; i = 0 % ) . therefore , we just present the data from the pooled analyses 14 15 16 . further analyses were stratified by duration of follow - up ( 2 4 , 5 9 , 10 years ) . the excess absolute risk , i. e. difference between observed and expected number of cases divided by person - years ( (o - e)/person - years 100 000 ) , was also calculated . a two - sided p value less than 0.05 was considered to be statistically significant . all analyses were conducted with sas statistical software , version 9.4 ( cary , north carolina , united states ) . we used data from the finnish and swedish hospital discharge registers , in which discharge diagnoses and surgical procedures are computerized for each hospitalization . unique national registration numbers for each inhabitant are in use in both finland and sweden , and we used them for identification . the coverage of the registries is nearly 100 % from 1986 and onwards 11 12 . the hospital discharge registries were introduced 1986 in finland and 1965 in sweden , and the first ercp procedure was registered in 1976 . we identified patients from 1986 to 2010 in finland and 1976 to 2010 in sweden with at least one in - hospital episode with a discharge procedure code for ercp or endoscopic sphincterotomy . in the ercp group , patients with a code of ercp with or without biopsy and cholangioscopy were included while the es group included patients with codes of sphincterotomy , extraction of stones , insertion of stent or nasobiliary drainage ( international classification of operations and major procedures , codes 9014 , ujk02 , ujk05 , ujk12 , ujk15 for ercp or 5388 , 5394 , jke 02 , jke 12 , jke 15 , jke 18 , jke 25 , jke 98 for es or procedures for which es is normally a prerequisite ) . using the national registration number , the patients identified with the procedure codes listed above were linked to each country s nationwide cancer registry , population registry , and migration registry for cancer outcome ascertainment and censoring due to death and emigration , respectively . a detailed description of the methods used has been described elsewhere 13 . for further analyses , the total ercp cohort for each country was divided into two subgroups : ( 1 ) patients having at least one procedure code registration for es or any other endoscopic biliary procedures for which an es is normally a prerequisite ( international classification of operations and major procedures , codes 5388 , 5394 , jke 02 , jke 12 , jke 15 , jke 18 , jke 25 , jke 98 ) , and ( 2 ) patients in the cohort without any procedure code registration for es or any other endoscopic biliary procedure implying es . those patients who had a diagnosis of malignant tumor in the bile ducts , liver or pancreas ( icd - o-3 c22 25 in the finnish data and icd7 155 157 in the swedish data or corresponding codes in the later classifications ) at the time of the procedure or within 2 years after it , were excluded from further analyses to avoid selection bias , since the registered ercp in these cases may have been performed because of the tumor or due to symptoms caused by a tumor that was still undiagnosed . considering the poor prognosis of malignancies in the biliary tract , liver and pancreas , it is highly unlikely that a tumor causing symptoms would be diagnosed more than 2 years later . the cohorts were then followed from entry ( 2 years after the procedure ) until diagnosis of an outcome malignancy ( primary malignant tumors in the liver , bile ducts including ampullary region and pancreas , but excluding gallbladder malignancy , icd7 codes : 155 and 157 , but excluding 1551 and icd10 codes c22.1 , c24.0 , c24.1 , c24.8 , and c25 ) , death , emigration or end of follow - up ( 31 december 2010 ) , whichever occurred first . the study was approved by the national institute for health and welfare in finland and the regional research ethics committee of stockholm and helsinki . several patients had ercp or es procedures registered more than once . for each patient , every first - time procedure was regarded as the index procedure for the corresponding cancer analyses . if the first - time procedure included or implied es , the patient s person - time was only included in the es subgroup . if a patient s first procedure was non - es or es - implying followed by a subsequent procedure implying es , this patient had two index procedures : one without es , with person - time counted from 2 years after the procedure until the subsequent es procedure ( i. e. censored at the time of the es procedure ) ; and another one after which person - time was counted in the es subgroup from 2 years after the es procedure . however , for the whole ercp group , person - time was counted from 2 years after entry until the occurrence of cancer , death , emigration or end of follow - up , irrespective of es procedure ( fig . 1 ) . thus the total person - time in the whole ercp cohort was more than the sum of the person - time experienced by the two subgroups . end of follow - up was 31 december 2010 , a diagnosis of malignancy , death , emigration or second procedure . the standardized incidence ratio ( sir ) , the ratio of the observed to the expected number of malignancies , was used to calculate relative risk . the background cancer incidence rates for the general swedish and finish populations were provided by the national cancer registries in the corresponding country , which was the same as the source for linkage to identify cancer cases during follow - up . the expected number of cancers was calculated by using the sex , calendar , age , and country - specific incidence rates . the standardized incidence ratios are inherently adjusted for confounding by age at follow - up , gender , and calendar year of follow - up . to calculate the pooled sir , we first combined the number of observed cancers , o , by summing data from the two countries , and the combined number of expected cancers , e , was determined in a similar way . the 95 % confidence intervals ( cis ) for the pooled sir were determined by assuming a poisson distribution of the observed numbers of cancer . results using the weighted meta - analysis were essentially identical with those using the pooled method , and tests for heterogeneity using the chi - squared test as well as i statistics demonstrated that the inconsistencies across the two studies were minor ( = 0.19 , df = 1 , p = 0.66 ; i = 0 % ) . therefore , we just present the data from the pooled analyses 14 15 16 . further analyses were stratified by duration of follow - up ( 2 4 , 5 9 , 10 years ) . the excess absolute risk , i. e. difference between observed and expected number of cases divided by person - years ( (o - e)/person - years 100 000 ) , was also calculated . a two - sided p value less than 0.05 was considered to be statistically significant . all analyses were conducted with sas statistical software , version 9.4 ( cary , north carolina , united states ) . the final cohorts of patients having undergone ercp , for diagnostics or therapy of non - malignant diseases , included 16 575 patients in finland and 53 350 patients in sweden contributing to a total of 506 998 person - years of follow - up . table 1 shows the steps for selection of patients from the total cohorts including all ercp procedures to the eligible final benign disease ercp cohorts . the sums of the es and non - es groups are larger than the all ercp groups , because a number of patients had an ercp without es before a subsequent ercp with es . of these , 784 did not have a diagnosis of malignancy at the time of the first or second index procedures or within 2 years thereafter , and were thus counted in both subgroups . the sum of the es group and ercp without es group was more than the whole ercp cohort , since some patients contributed person - time to both groups . . the general characteristics of the cohorts and the number of observed malignancies 2 years or more after the index procedure are included in table 2 . the mean age at entry , 2 years after the procedure , was 66 years in sweden and 64 years in finland . the mean follow - up time was 7.3 years in the all ercp cohort , but was shorter among patients who had undergone an es . bile duct malignancy was infrequent and despite the large size of the cohort , the number of bile duct malignancies in the studied population was low . the risk of developing malignancy in the bile ducts , liver , or pancreas , explicitly excluding gallbladder malignancy , was increased in the all ercp cohorts , the es cohorts , and the ercp without es cohorts in both finland and sweden ( data not shown ) . in the pooled all ercp cohort , the risk was more than twofold compared to the general national populations ( sir 2.3 ; 95 % ci 2.1 2.5 ) . likewise , it was increased in the pooled es subgroup ( sir 2.2 ; 95 % ci 2.0 2.5 ) and almost the same in the non - es subgroup ( sir 2.1 ; 95 % ci 1.9 2.3 ) ( table 3 ) . the relative risk of bile duct malignancy alone , was increased nearly four times ( sir 3.9 ; 95 % ci 3.3 4.5 ) in the pooled all ercp cohort , and tended to be higher in the es cohort ( sir 4.3 ; 95 % ci 3.5 5.5 ) than in the cohort without es ( sir 2.7 ; 95 % ci 2.0 3.5 ) ( table 3 ) . ercp , endoscopic retrograde cholangiopancreatography ; es , endoscopic sphincterotomy ; sir , standardized incidence ratio . the sir of malignancy of the bile ducts , liver , or pancreas was much elevated in the first two years after the procedure at 69.2 ( 95 % ci 66.8 71.6 ) , but then decreased gradually with increasing follow - up duration . in the pooled all ercp cohort in the period between 2 and 5 years after the index ercp , 3.5 ) and decreased to 1.9 ( 95 % ci 1.6 2.2 ) for 10 years and more after the index ercp . the excess absolute risks were similarly decreasing with follow - up duration ( table 4 ) . the analyses stratified by es showed a similar pattern , with decreasing point - estimates for relative risks and absolute risks with longer follow - up . the risk of developing cancer in the bile ducts after ercp , with or without es also decreased with length of follow - up , but since cholangiocarcinoma is a rare tumor , the confidence intervals are wide despite the large size of the cohort ( table 5 ) . ercp , endoscopic retrograde cholangiopancreatography ; es , endoscopic sphincterotomy ; sir , standardized incidence ratio . calculated from ( o ercp , endoscopic retrograde cholangiopancreatography ; es , endoscopic sphincterotomy ; sir , standardized incidence ratio . calculated from ( o this large , population - based cohort study was designed primarily to investigate the risk of developing malignancy in the bile ducts after es in a large cohort with a long follow - up , a concern raised in other publications 5 17 18 . we demonstrate here a fourfold significantly increased risk of malignancy in the bile ducts alone after ercp , and not significantly higher after es . among patients who had undergone ercp , with or without es , we found a twofold increased risk of malignancy in the bile ducts , liver , or pancreas . in both the es and non - es groups , the absolute excess risks as well as the sirs of developing a malignancy in the bile ducts , liver , and pancreas together , decreased with increasing length of follow - up . the strengths of our study include the population - based cohort design with follow - up of a vast majority of patients having undergone ercp procedures in finland and sweden during the study - period . another strength is the close to complete follow - up of the patients in the registries used in this study , avoiding differential misclassification and thus ensuring high internal validity . furthermore , the large size of the cohort provided adequate statistical power , which enabled us to analyze the relative risks of the studied malignancies in the bile ducts , liver , and pancreas after stratification for es . a limitation of this study may be that cancer of the bile ducts is a rare malignancy with a low expected incidence even in a large pooled cohort , limiting the subgroup analyses of cancer risk by follow - up duration . the mean follow - up time was shorter in the es subgroup than in the ercp without es group in both the finnish and swedish cohorts , as a result of the fact that es was not performed at the beginning of the period . however , the size of the pooled cohort gives an opportunity to interpret data with a better precision than in previous studies . the risk of misclassification between intrahepatic cholangiocarcinoma and liver cancer , as well as between extrahepatic cholangiocarcinoma and pancreatic cancer must be acknowledged . therefore , both bile duct malignancy alone as well as bile duct malignancy together with liver and pancreatic malignancies , were used as outcomes in spite of the fact that our hypothesis primarily concerned cholangiocarcinoma of the intrahepatic and extrahepatic bile ducts . the influence of this type of misclassification is predictable and thus tolerable given the use of both outcomes described above . this study demonstrates an approximately fourfold increase in the risk of malignancy in the bile ducts alone and a twofold increase in the bile ducts , liver and pancreas together , in a large cohort of patients followed up after es . this increased risk is not likely to be caused by the es procedure in itself . if that were the case , one would expect to identify a non - elevated risk of tumors at the beginning of the follow - up period and then a gradual increase over time . on the contrary , in this cohort , both in the es subgroup and in the non - es subgroup , the relative risk of malignancy was greatest at the beginning of follow - up , i. e. between 2 and 5 years after the index procedure . in a study of the long - term consequences of es by tanaka et al . 5 , 410 patients were followed on average for 10 years after es , and carcinoma in the biliary tract was found in eight patients , three of them late , giving a marked elevated risk of malignancy both in the short and long term , a finding that was attributed to the es . the result of this study differs from ours by showing a much higher risk of development of carcinoma in the biliary tract , but is based on a much smaller cohort with a higher risk of random error . 17 reported a long - term risk of malignancy in the biliary system , but predominantly gallbladder cancer , of 3.1 % after es , a rate they concluded to be comparable with the incidence of gallbladder cancer found in patients with gallstone disease . on the other hand , in a previously published population - based study of 992 patients who underwent es at six different hospitals , karlsson et al . found no increase in the risk of cancer in the liver , bile ducts , or pancreas at follow - up 1 year or more after the procedure 19 this study , also performed in sweden , is based on a regional subgroup of our own study cohort . in a large population - based danish study , 20 compared more than 20 000 patients undergoing ercp with or without es and found a high incidence of cholangiocarcinoma in both groups in the first year after the procedure but a decreasing incidence with length of follow - up , the same result as in the present study , and concluded a lack of causal association between es and cholangiocarcinoma . our study shows an increased risk of malignancy after ercp but since the risk diminishes with length of follow - up , it is likely to be caused by some common exposure before the ercp procedure , a conclusion well in line with the danish findings and the result of our previous study . by far the most likely candidate is gallstone disease , in particular , ductal gallstone disease . in our previous study 10 , based on a subgroup of the present cohort , we found that patients who had ever had a cholecystectomy had a lower risk of malignancy , irrespective of es , a finding that could support this theory . we also calculated the risk of lung cancer as an estimate for potential bias of tobacco smoking , a known risk factor for pancreatic carcinoma , and found no elevated risk compared to the general population . all patients with a diagnosis of malignancy in the biliary tract , liver , or pancreas at the time of the index procedure , or within 2 years after it , were excluded from the cohort . patients having uncharacteristic abdominal discomfort could have been subject to ercp on the assumption of gallstone disease when the symptoms were instead caused by an undiagnosed malignancy explaining the extremely elevated sirs . we also excluded all patients who were diagnosed with gallbladder malignancy since gallbladder carcinoma is known to be strongly associated with gallstone disease 21 , a condition likely to be very common among patients having had an ercp without a malignant diagnosis . it is unlikely to be explained by a preexisting undiagnosed cancer at the time of the index procedure , considering the poor prognosis of symptom - generating malignancies in the area . one possible mechanism by which ductal gallstones could cause the high risk of malignancy observed during this part of the follow - up , could be that they may have started a slow , initially purely inflammatory but subsequently carcinogenic , process before they were removed and that the process continued after removal of the stones . in conclusion , this study shows an elevated risk of malignancy both in the bile ducts alone and in the bile ducts , liver or pancreas together , after ercp . the risk is not influenced by including es so es or any other part of the procedure is unlikely to be the cause . the elevated risk of malignancy is more likely to be caused by some other carcinogenic exposure prior to the ercp procedure , possibly ductal gallstone disease .

background and study aims : elevated long - term risk of cholangiocarcinoma is reported after endoscopic sphincterotomy ( es ) , but in a previous study we found a trend towards a decreased risk . the aim of this study was to evaluate the association in a larger cohort with a longer follow - up . patients and methods : data concerning all patients having had an inpatient endoscopic retrograde cholangiopancreatography ( ercp ) were collected from the hospital discharge registries of finland and sweden . incident cases of malignancy were identified through linkage to the nationwide cancer registries . patients with a diagnosis of malignancy , before or within 2 years of the ercp , were excluded . the cohorts were followed until a diagnosis of malignancy , death or emigration , or end of follow - up ( end of 2010 ) . the relative risk of malignancy was calculated as standardized incidence ratio ( sir ) compared with the general population , inherently adjusting for age , gender , and calendar year of follow - up . results : a total of 69 925 patients undergoing ercp from 1976 through 2008 were included in the pooled cohort . es was performed in 40 193 subjects . the risk of malignancy was elevated in the total cohort ( sir = 2.3 ; 95 % confidence interval [ ci ] 2.1 2.5 ) irrespective of whether es was performed or not . the sirs diminished with duration of follow - up . conclusions : we found an elevated risk of malignancy both in the bile ducts alone and in the bile ducts , liver or pancreas together , after ercp . the risk was the same , regardless of whether es had been performed or not , so es was unlikely to be the cause , and a common carcinogenic exposure previous to the ercp procedure , possibly ductal gallstone disease , was more likely .

Introduction Materials and methods Registries, cohort and follow-up Statistical analyses Results Discussion

endoscopic retrograde cholangiopancreatography ( ercp ) with endoscopic sphincterotomy ( es ) was first introduced in 1974 in germany and japan 1 2 and a few years later in the nordic countries , as an alternative to open surgery with choledochotomy in the treatment of common bile duct stones . studies also show an increased incidence of cholangiocarcinoma after es , presumably due to the bacterial overgrowth and chronic inflammation in the bile ducts 5 6 . these data prompted us to hypothesize that es increases the long - term risk of cholangiocarcinoma , which we were subsequently able to test in a swedish nationwide population - based study of all 27 708 patients registered for an ercp for benign disease in the swedish inpatient registry , since the introduction of ercp in the 1970s until 2005 10 . our results showed that , among the 12 629 patients who had undergone es , the risk of cancer in the bile ducts was not increased compared to the non - es group , but on the other hand , that there was a tendency toward a decreased long - term risk of bile duct cancer in the es group . in this study , we also found a decreased risk of malignancy in patients who had had a cholecystectomy . the primary aim of this population - based cohort study of all patients registered for an ercp in the inpatient registries in finland and sweden was to study the risk of malignancy in the bile ducts in a larger cohort and with a longer follow - up . the hospital discharge registries were introduced 1986 in finland and 1965 in sweden , and the first ercp procedure was registered in 1976 . in the ercp group , patients with a code of ercp with or without biopsy and cholangioscopy were included while the es group included patients with codes of sphincterotomy , extraction of stones , insertion of stent or nasobiliary drainage ( international classification of operations and major procedures , codes 9014 , ujk02 , ujk05 , ujk12 , ujk15 for ercp or 5388 , 5394 , jke 02 , jke 12 , jke 15 , jke 18 , jke 25 , jke 98 for es or procedures for which es is normally a prerequisite ) . using the national registration number , the patients identified with the procedure codes listed above were linked to each country s nationwide cancer registry , population registry , and migration registry for cancer outcome ascertainment and censoring due to death and emigration , respectively . for further analyses , the total ercp cohort for each country was divided into two subgroups : ( 1 ) patients having at least one procedure code registration for es or any other endoscopic biliary procedures for which an es is normally a prerequisite ( international classification of operations and major procedures , codes 5388 , 5394 , jke 02 , jke 12 , jke 15 , jke 18 , jke 25 , jke 98 ) , and ( 2 ) patients in the cohort without any procedure code registration for es or any other endoscopic biliary procedure implying es . those patients who had a diagnosis of malignant tumor in the bile ducts , liver or pancreas ( icd - o-3 c22 25 in the finnish data and icd7 155 157 in the swedish data or corresponding codes in the later classifications ) at the time of the procedure or within 2 years after it , were excluded from further analyses to avoid selection bias , since the registered ercp in these cases may have been performed because of the tumor or due to symptoms caused by a tumor that was still undiagnosed . the cohorts were then followed from entry ( 2 years after the procedure ) until diagnosis of an outcome malignancy ( primary malignant tumors in the liver , bile ducts including ampullary region and pancreas , but excluding gallbladder malignancy , icd7 codes : 155 and 157 , but excluding 1551 and icd10 codes c22.1 , c24.0 , c24.1 , c24.8 , and c25 ) , death , emigration or end of follow - up ( 31 december 2010 ) , whichever occurred first . however , for the whole ercp group , person - time was counted from 2 years after entry until the occurrence of cancer , death , emigration or end of follow - up , irrespective of es procedure ( fig . thus the total person - time in the whole ercp cohort was more than the sum of the person - time experienced by the two subgroups . end of follow - up was 31 december 2010 , a diagnosis of malignancy , death , emigration or second procedure . the standardized incidence ratio ( sir ) , the ratio of the observed to the expected number of malignancies , was used to calculate relative risk . the background cancer incidence rates for the general swedish and finish populations were provided by the national cancer registries in the corresponding country , which was the same as the source for linkage to identify cancer cases during follow - up . the standardized incidence ratios are inherently adjusted for confounding by age at follow - up , gender , and calendar year of follow - up . to calculate the pooled sir , we first combined the number of observed cancers , o , by summing data from the two countries , and the combined number of expected cancers , e , was determined in a similar way . the 95 % confidence intervals ( cis ) for the pooled sir were determined by assuming a poisson distribution of the observed numbers of cancer . further analyses were stratified by duration of follow - up ( 2 4 , 5 9 , 10 years ) . the hospital discharge registries were introduced 1986 in finland and 1965 in sweden , and the first ercp procedure was registered in 1976 . in the ercp group , patients with a code of ercp with or without biopsy and cholangioscopy were included while the es group included patients with codes of sphincterotomy , extraction of stones , insertion of stent or nasobiliary drainage ( international classification of operations and major procedures , codes 9014 , ujk02 , ujk05 , ujk12 , ujk15 for ercp or 5388 , 5394 , jke 02 , jke 12 , jke 15 , jke 18 , jke 25 , jke 98 for es or procedures for which es is normally a prerequisite ) . using the national registration number , the patients identified with the procedure codes listed above were linked to each country s nationwide cancer registry , population registry , and migration registry for cancer outcome ascertainment and censoring due to death and emigration , respectively . for further analyses , the total ercp cohort for each country was divided into two subgroups : ( 1 ) patients having at least one procedure code registration for es or any other endoscopic biliary procedures for which an es is normally a prerequisite ( international classification of operations and major procedures , codes 5388 , 5394 , jke 02 , jke 12 , jke 15 , jke 18 , jke 25 , jke 98 ) , and ( 2 ) patients in the cohort without any procedure code registration for es or any other endoscopic biliary procedure implying es . those patients who had a diagnosis of malignant tumor in the bile ducts , liver or pancreas ( icd - o-3 c22 25 in the finnish data and icd7 155 157 in the swedish data or corresponding codes in the later classifications ) at the time of the procedure or within 2 years after it , were excluded from further analyses to avoid selection bias , since the registered ercp in these cases may have been performed because of the tumor or due to symptoms caused by a tumor that was still undiagnosed . the cohorts were then followed from entry ( 2 years after the procedure ) until diagnosis of an outcome malignancy ( primary malignant tumors in the liver , bile ducts including ampullary region and pancreas , but excluding gallbladder malignancy , icd7 codes : 155 and 157 , but excluding 1551 and icd10 codes c22.1 , c24.0 , c24.1 , c24.8 , and c25 ) , death , emigration or end of follow - up ( 31 december 2010 ) , whichever occurred first . however , for the whole ercp group , person - time was counted from 2 years after entry until the occurrence of cancer , death , emigration or end of follow - up , irrespective of es procedure ( fig . thus the total person - time in the whole ercp cohort was more than the sum of the person - time experienced by the two subgroups . end of follow - up was 31 december 2010 , a diagnosis of malignancy , death , emigration or second procedure . the standardized incidence ratio ( sir ) , the ratio of the observed to the expected number of malignancies , was used to calculate relative risk . the background cancer incidence rates for the general swedish and finish populations were provided by the national cancer registries in the corresponding country , which was the same as the source for linkage to identify cancer cases during follow - up . the standardized incidence ratios are inherently adjusted for confounding by age at follow - up , gender , and calendar year of follow - up . to calculate the pooled sir , we first combined the number of observed cancers , o , by summing data from the two countries , and the combined number of expected cancers , e , was determined in a similar way . the 95 % confidence intervals ( cis ) for the pooled sir were determined by assuming a poisson distribution of the observed numbers of cancer . further analyses were stratified by duration of follow - up ( 2 4 , 5 9 , 10 years ) . the final cohorts of patients having undergone ercp , for diagnostics or therapy of non - malignant diseases , included 16 575 patients in finland and 53 350 patients in sweden contributing to a total of 506 998 person - years of follow - up . of these , 784 did not have a diagnosis of malignancy at the time of the first or second index procedures or within 2 years thereafter , and were thus counted in both subgroups . the general characteristics of the cohorts and the number of observed malignancies 2 years or more after the index procedure are included in table 2 . the mean follow - up time was 7.3 years in the all ercp cohort , but was shorter among patients who had undergone an es . the risk of developing malignancy in the bile ducts , liver , or pancreas , explicitly excluding gallbladder malignancy , was increased in the all ercp cohorts , the es cohorts , and the ercp without es cohorts in both finland and sweden ( data not shown ) . in the pooled all ercp cohort , the risk was more than twofold compared to the general national populations ( sir 2.3 ; 95 % ci 2.1 2.5 ) . likewise , it was increased in the pooled es subgroup ( sir 2.2 ; 95 % ci 2.0 2.5 ) and almost the same in the non - es subgroup ( sir 2.1 ; 95 % ci 1.9 2.3 ) ( table 3 ) . the relative risk of bile duct malignancy alone , was increased nearly four times ( sir 3.9 ; 95 % ci 3.3 4.5 ) in the pooled all ercp cohort , and tended to be higher in the es cohort ( sir 4.3 ; 95 % ci 3.5 5.5 ) than in the cohort without es ( sir 2.7 ; 95 % ci 2.0 3.5 ) ( table 3 ) . ercp , endoscopic retrograde cholangiopancreatography ; es , endoscopic sphincterotomy ; sir , standardized incidence ratio . the sir of malignancy of the bile ducts , liver , or pancreas was much elevated in the first two years after the procedure at 69.2 ( 95 % ci 66.8 71.6 ) , but then decreased gradually with increasing follow - up duration . in the pooled all ercp cohort in the period between 2 and 5 years after the index ercp , 3.5 ) and decreased to 1.9 ( 95 % ci 1.6 2.2 ) for 10 years and more after the index ercp . the risk of developing cancer in the bile ducts after ercp , with or without es also decreased with length of follow - up , but since cholangiocarcinoma is a rare tumor , the confidence intervals are wide despite the large size of the cohort ( table 5 ) . ercp , endoscopic retrograde cholangiopancreatography ; es , endoscopic sphincterotomy ; sir , standardized incidence ratio . calculated from ( o ercp , endoscopic retrograde cholangiopancreatography ; es , endoscopic sphincterotomy ; sir , standardized incidence ratio . calculated from ( o this large , population - based cohort study was designed primarily to investigate the risk of developing malignancy in the bile ducts after es in a large cohort with a long follow - up , a concern raised in other publications 5 17 18 . we demonstrate here a fourfold significantly increased risk of malignancy in the bile ducts alone after ercp , and not significantly higher after es . among patients who had undergone ercp , with or without es , we found a twofold increased risk of malignancy in the bile ducts , liver , or pancreas . in both the es and non - es groups , the absolute excess risks as well as the sirs of developing a malignancy in the bile ducts , liver , and pancreas together , decreased with increasing length of follow - up . the strengths of our study include the population - based cohort design with follow - up of a vast majority of patients having undergone ercp procedures in finland and sweden during the study - period . another strength is the close to complete follow - up of the patients in the registries used in this study , avoiding differential misclassification and thus ensuring high internal validity . furthermore , the large size of the cohort provided adequate statistical power , which enabled us to analyze the relative risks of the studied malignancies in the bile ducts , liver , and pancreas after stratification for es . a limitation of this study may be that cancer of the bile ducts is a rare malignancy with a low expected incidence even in a large pooled cohort , limiting the subgroup analyses of cancer risk by follow - up duration . the mean follow - up time was shorter in the es subgroup than in the ercp without es group in both the finnish and swedish cohorts , as a result of the fact that es was not performed at the beginning of the period . however , the size of the pooled cohort gives an opportunity to interpret data with a better precision than in previous studies . this study demonstrates an approximately fourfold increase in the risk of malignancy in the bile ducts alone and a twofold increase in the bile ducts , liver and pancreas together , in a large cohort of patients followed up after es . if that were the case , one would expect to identify a non - elevated risk of tumors at the beginning of the follow - up period and then a gradual increase over time . on the contrary , in this cohort , both in the es subgroup and in the non - es subgroup , the relative risk of malignancy was greatest at the beginning of follow - up , i. e. between 2 and 5 years after the index procedure . 5 , 410 patients were followed on average for 10 years after es , and carcinoma in the biliary tract was found in eight patients , three of them late , giving a marked elevated risk of malignancy both in the short and long term , a finding that was attributed to the es . the result of this study differs from ours by showing a much higher risk of development of carcinoma in the biliary tract , but is based on a much smaller cohort with a higher risk of random error . 17 reported a long - term risk of malignancy in the biliary system , but predominantly gallbladder cancer , of 3.1 % after es , a rate they concluded to be comparable with the incidence of gallbladder cancer found in patients with gallstone disease . found no increase in the risk of cancer in the liver , bile ducts , or pancreas at follow - up 1 year or more after the procedure 19 this study , also performed in sweden , is based on a regional subgroup of our own study cohort . in a large population - based danish study , 20 compared more than 20 000 patients undergoing ercp with or without es and found a high incidence of cholangiocarcinoma in both groups in the first year after the procedure but a decreasing incidence with length of follow - up , the same result as in the present study , and concluded a lack of causal association between es and cholangiocarcinoma . our study shows an increased risk of malignancy after ercp but since the risk diminishes with length of follow - up , it is likely to be caused by some common exposure before the ercp procedure , a conclusion well in line with the danish findings and the result of our previous study . in our previous study 10 , based on a subgroup of the present cohort , we found that patients who had ever had a cholecystectomy had a lower risk of malignancy , irrespective of es , a finding that could support this theory . we also calculated the risk of lung cancer as an estimate for potential bias of tobacco smoking , a known risk factor for pancreatic carcinoma , and found no elevated risk compared to the general population . all patients with a diagnosis of malignancy in the biliary tract , liver , or pancreas at the time of the index procedure , or within 2 years after it , were excluded from the cohort . we also excluded all patients who were diagnosed with gallbladder malignancy since gallbladder carcinoma is known to be strongly associated with gallstone disease 21 , a condition likely to be very common among patients having had an ercp without a malignant diagnosis . it is unlikely to be explained by a preexisting undiagnosed cancer at the time of the index procedure , considering the poor prognosis of symptom - generating malignancies in the area . one possible mechanism by which ductal gallstones could cause the high risk of malignancy observed during this part of the follow - up , could be that they may have started a slow , initially purely inflammatory but subsequently carcinogenic , process before they were removed and that the process continued after removal of the stones . in conclusion , this study shows an elevated risk of malignancy both in the bile ducts alone and in the bile ducts , liver or pancreas together , after ercp . the risk is not influenced by including es so es or any other part of the procedure is unlikely to be the cause . the elevated risk of malignancy is more likely to be caused by some other carcinogenic exposure prior to the ercp procedure , possibly ductal gallstone disease .

chloroquine ( cq ) and hydroxychloroquine ( hcq ) are widely used to control effects of various rheumatologic diseases , for example , rheumatoid arthritis ( ra ) , systemic lupus erythematosus ( sle ) , systemic sclerosis ( ss ) , or other connective tissue diseases . they are found to inhibit cd4 t - cell stimulation while also promoting cd8 t - cell stimulation , and hence they are a popular choice for controlling the progression of autoimmune diseases without increasing the rate of opportunistic infections.1 hcq is more molecularly polar than cq making it less lipophilic , leading to poorer diffusion through biological membranes and resulting in less toxicity.1 one of the most severe complications of these drugs is retinopathy , which leads to visual field defects and visual loss as a result of damaged photoreceptors . this damage is incurable and may progress even after the drugs are discontinued.2 an ophthalmologic screening examination , therefore , has to be regularly performed to early detect and prevent this destructive morbidity.2 the american academy of ophthalmology ( aao ) established guidelines for cq and hcq retinopathy screening examinations , which was revised in 2011 and 2016.3,4 patients were classified as high risk when they have at least one of the following risk factors : an average daily dose exceeding 2.3 mg / kg real weight for cq and 5 mg / kg real weight for hcq,4 cumulative doses exceeding 460 g for cq and 1,000 g for hcq,3 a duration of treatment exceeding 5 years , being elderly , having concomitant renal disease , or using tamoxifen.4 routinely , one baseline examination within the first year and annual screening beginning 5 years after the first dose are recommended.3,4 since most of studies reported are performed in caucasian populations,58 application of the findings of these studies and recommendations may not be appropriate for asian populations . for the thai patients , there have been some studies on prevalence and risk factors of cq retinopathy , but the results seemed to be paradoxical to the aao recommendations , and the obvious risk factors are still not well defined.911 hence , this study was intended to produce a more inclusive report on incidence of and risk factors for cq and hcq retinopathy in thai rheumatologic patients . this study was conducted in accordance with the tenets of the declaration of helsinki and was approved by the khon kaen university ethics committee for human research ( he571052 ) . the khon kaen university ethics committee for human research waived the requirement for patient consent due to the retrospective nature of the study . a retrospective cohort study of rheumatologic patients at srinagarind hospital , khon kaen university , thailand , was conducted . the patients , aged not less than 18 years old , started receiving either cq or hcq from 2004 to 2014 and regularly underwent eye screening examinations for cq or hcq retinopathy at the kku eye center . patients whose medical records were incomplete or those who had undeterminable records of the cq or hcq dosages were excluded . also , patients who started the drugs before being referred to the hospital or were switched from cq to hcq or vice versa were excluded . moreover , patients with retinal or macular diseases or optic neuropathy , which could possibly interfere with the visual field test , were excluded as well . the medical records of all the patients who fulfilled the criteria mentioned in the subjects and methods section were reviewed and recorded in prepared case report forms . the record form for each patient was labeled with a new number to conceal the real identity of patients . the information collected were : age : the age at the latest eye examination.gender : male or female.body weight : the real body weight at the latest follow - up at the rheumatologic clinics.underlying disease : diabetes mellitus , dyslipidemia , hypertension , or other diseases.history of rheumatologic diseases.duration of drug use : if the patients had no retinopathy and still continued the drug , it was counted from the day of the first dose to the day of the last eye examination. if the patients had no retinopathy and stopped the medication , it was counted from the day of the first dose to the day of the last dose. if the patients had retinopathy , it was counted from the day of the first dose to the day that retinopathy was diagnosed despite whether patients had stopped the medication or not.cumulative dosage ( g ) : for each patient , the dosage , the duration of each dosage , and dose changes throughout treatment were recorded . the summation of products of dosage and duration were recorded as the cumulative dosage.average dosage per real body weight per day ( mg / kg / d ) : cumulative dose divided by real body weight and duration in days each patient received the medication.presence of signs of cq or hcq retinopathy from the eye screening examinations . body weight : the real body weight at the latest follow - up at the rheumatologic clinics . duration of drug use : if the patients had no retinopathy and still continued the drug , it was counted from the day of the first dose to the day of the last eye examination. if the patients had no retinopathy and stopped the medication , it was counted from the day of the first dose to the day of the last dose. if the patients had retinopathy , it was counted from the day of the first dose to the day that retinopathy was diagnosed despite whether patients had stopped the medication or not . if the patients had no retinopathy and still continued the drug , it was counted from the day of the first dose to the day of the last eye examination . if the patients had no retinopathy and stopped the medication , it was counted from the day of the first dose to the day of the last dose . if the patients had retinopathy , it was counted from the day of the first dose to the day that retinopathy was diagnosed despite whether patients had stopped the medication or not . cumulative dosage ( g ) : for each patient , the dosage , the duration of each dosage , and dose changes throughout treatment were recorded . average dosage per real body weight per day ( mg / kg / d ) : cumulative dose divided by real body weight and duration in days each patient received the medication . presence of signs of cq or hcq retinopathy from the eye screening examinations . according to aao recommendations , patients who repeatedly demonstrated these conditions in both eyes for at least 2 consecutive tests were recommended to stop the medications until the cause , other than cq or hcq retinopathy , was identified . these conditions were : parafoveal depigmentation of retinal pigment epithelium ( rpe ) as observed by indirect ophthalmoscopic examination after patients pupils were fully dilated.decreased parafoveal visual field sensitivities at the same area for at least 2 consecutive visits as tested by humphrey visual fields ( hvf ) 10 - 2 using humphrey field analyzer ii ( zeiss meditech inc . all the tests must show reliable test - fixation loss of less than 20% and have false - negative and false - positive response rates of less than 33%.loss of parafoveal inner segment outer segment junction from spectral domain optical coherence tomography ( oct ) using spectralis ( heidelberg engineering , heidelberg , germany).increased autofluorescence at parafoveal area using spectralis ( heidelberg engineering ) . parafoveal depigmentation of retinal pigment epithelium ( rpe ) as observed by indirect ophthalmoscopic examination after patients pupils were fully dilated . decreased parafoveal visual field sensitivities at the same area for at least 2 consecutive visits as tested by humphrey visual fields ( hvf ) 10 - 2 using humphrey field analyzer ii ( zeiss meditech inc . , all the tests must show reliable test - fixation loss of less than 20% and have false - negative and false - positive response rates of less than 33% . loss of parafoveal inner segment outer segment junction from spectral domain optical coherence tomography ( oct ) using spectralis ( heidelberg engineering , heidelberg , germany ) . increased autofluorescence at parafoveal area using spectralis ( heidelberg engineering ) . for probable toxicity , the same findings as in possible toxicity were included , but those defects were shown in the bull s eye pattern and in both eyes . the screening tests were routinely performed and interpreted by retinal specialists at the kku eye center . estimated sample size was calculated with 95% confidence level , 5% absolute precision , and 13.5% estimated prevalence , resulting in a sample size of 171.9 as these data did not match that of normal distribution , continuous data were reported as the median with minimal and maximal values , and comparisons between data of each group were performed using mann whitney tests . the incidence of retinopathy and the duration of the medication use were demonstrated with kaplan meier survival analysis . the risk factors that had a value of p<0.2 were further analyzed with multivariate cox regression analysis . all statistics were performed using spss for windows version 20.0 ( ibm corporation , armonk , ny , usa ) . the medical records of all the patients who fulfilled the criteria mentioned in the subjects and methods section were reviewed and recorded in prepared case report forms . the record form for each patient was labeled with a new number to conceal the real identity of patients . the information collected were : age : the age at the latest eye examination.gender : male or female.body weight : the real body weight at the latest follow - up at the rheumatologic clinics.underlying disease : diabetes mellitus , dyslipidemia , hypertension , or other diseases.history of rheumatologic diseases.duration of drug use : if the patients had no retinopathy and still continued the drug , it was counted from the day of the first dose to the day of the last eye examination. if the patients had no retinopathy and stopped the medication , it was counted from the day of the first dose to the day of the last dose. if the patients had retinopathy , it was counted from the day of the first dose to the day that retinopathy was diagnosed despite whether patients had stopped the medication or not.cumulative dosage ( g ) : for each patient , the dosage , the duration of each dosage , and dose changes throughout treatment were recorded . the summation of products of dosage and duration were recorded as the cumulative dosage.average dosage per real body weight per day ( mg / kg / d ) : cumulative dose divided by real body weight and duration in days each patient received the medication.presence of signs of cq or hcq retinopathy from the eye screening examinations . body weight : the real body weight at the latest follow - up at the rheumatologic clinics . duration of drug use : if the patients had no retinopathy and still continued the drug , it was counted from the day of the first dose to the day of the last eye examination. if the patients had no retinopathy and stopped the medication , it was counted from the day of the first dose to the day of the last dose. if the patients had retinopathy , it was counted from the day of the first dose to the day that retinopathy was diagnosed despite whether patients had stopped the medication or not . if the patients had no retinopathy and still continued the drug , it was counted from the day of the first dose to the day of the last eye examination . if the patients had no retinopathy and stopped the medication , it was counted from the day of the first dose to the day of the last dose . if the patients had retinopathy , it was counted from the day of the first dose to the day that retinopathy was diagnosed despite whether patients had stopped the medication or not . cumulative dosage ( g ) : for each patient , the dosage , the duration of each dosage , and dose changes throughout treatment were recorded . average dosage per real body weight per day ( mg / kg / d ) : cumulative dose divided by real body weight and duration in days each patient received the medication . patients who repeatedly demonstrated these conditions in both eyes for at least 2 consecutive tests were recommended to stop the medications until the cause , other than cq or hcq retinopathy , was identified . these conditions were : parafoveal depigmentation of retinal pigment epithelium ( rpe ) as observed by indirect ophthalmoscopic examination after patients pupils were fully dilated.decreased parafoveal visual field sensitivities at the same area for at least 2 consecutive visits as tested by humphrey visual fields ( hvf ) 10 - 2 using humphrey field analyzer ii ( zeiss meditech inc . , all the tests must show reliable test - fixation loss of less than 20% and have false - negative and false - positive response rates of less than 33%.loss of parafoveal inner segment outer segment junction from spectral domain optical coherence tomography ( oct ) using spectralis ( heidelberg engineering , heidelberg , germany).increased autofluorescence at parafoveal area using spectralis ( heidelberg engineering ) . parafoveal depigmentation of retinal pigment epithelium ( rpe ) as observed by indirect ophthalmoscopic examination after patients pupils were fully dilated . decreased parafoveal visual field sensitivities at the same area for at least 2 consecutive visits as tested by humphrey visual fields ( hvf ) 10 - 2 using humphrey field analyzer ii ( zeiss meditech inc . , all the tests must show reliable test - fixation loss of less than 20% and have false - negative and false - positive response rates of less than 33% . loss of parafoveal inner segment outer segment junction from spectral domain optical coherence tomography ( oct ) using spectralis ( heidelberg engineering , heidelberg , germany ) . increased autofluorescence at parafoveal area using spectralis ( heidelberg engineering ) . for probable toxicity , the same findings as in possible toxicity were included , but those defects were shown in the bull s eye pattern and in both eyes . the screening tests were routinely performed and interpreted by retinal specialists at the kku eye center . estimated sample size was calculated with 95% confidence level , 5% absolute precision , and 13.5% estimated prevalence , resulting in a sample size of 171.9 as these data did not match that of normal distribution , continuous data were reported as the median with minimal and maximal values , and comparisons between data of each group were performed using mann whitney tests . the incidence of retinopathy and the duration of the medication use were demonstrated with kaplan meier survival analysis . the risk factors that had a value of p<0.2 were further analyzed with multivariate cox regression analysis . all statistics were performed using spss for windows version 20.0 ( ibm corporation , armonk , ny , usa ) . two hundred and thirty - four patients were enrolled in the study according to the criteria established . there were 173 patients who received only cq ( cq group ) and 61 patients who received only hcq ( hcq group ) . in the cq group , there were 63 ra patients , 63 sle patients , 23 ss patients , and 24 patients with other connective tissue diseases . in the hcq group , there were 11 ra patients , 31 sle patients , 4 ss patients , and 15 patients with other connective tissue diseases . in the cq positive group , possible toxicity as evidenced by indirect ophthalmoscopic examination , hvf test , and oct findings . the incidence of cq retinopathy was 8.09% of the patients who used cq over the 10 years considered . it was noted that only age , real body weight , and cumulative dose showed statistical significance at p<0.05 . moreover , other coexisting diseases , thyroid disease , heart disease , and anemia , in both the retinopathy - positive and the retinopathy - negative groups were recorded . also , hepatitis b virus infection , nasopharyngeal carcinoma , pulmonary tuberculosis , parkinson s disease , and renal disease in the retinopathy - negative group were recorded . unfortunately , the numbers of these diseases were too small to statistically compare the data . in the hcq group , 2 of 61 patients developed retinopathy ; therefore , the incidence of hcq retinopathy was 3.28% over the 10 years of the study . possible toxicity as evidenced by indirect ophthalmoscopic examination , hvf test , and oct findings . other underlying diseases such as thyroid disease , bulimia nervosa , anemia , hepatitis , rheumatic heart disease , and ischemic stroke were also noted in patients in the retinopathy - negative group . in contrast to cq , no statistically significant hazard ratios of any items studied in the hcq group were found . the incidence of cq and hcq retinopathy versus duration of medication use were analyzed via kaplan meier survival analysis and are illustrated in figures 1 and 2 . it is noted that cq retinopathy can occur as early as 4 months after drug use . moreover according to results of this study , the incidence of cq retinopathy was 8.09% over 10 years . back in the 19th century , cq retinopathy was diagnosed by the appearance of bull s eye maculopathy in patients taking cq . , the studies conducted on the disease yielded quite a very low prevalence of disease , 1 in 270.12 currently , physicians tend to detect cq retinopathy at an earlier stage on the basis of the revised aao recommendations ; therefore , most of recent studies showed higher prevalence rates ranging from 7.3% to 26.6%.912 the incidence from this study could not be compared directly with previous studies that reported the prevalence . according to the report of life expectancy in rheumatologic patients by mok et al13 and understanding that the normal thai population has an average life expectancy of 74.72 years,14 the prevalence of retinopathy in this study can be calculated from the incidence as being 14.9% , which was in agreement with the previous studies.912 the revised cq and hcq retinopathy screening recommendations of aao stated that high - risk patients were those who used cq longer than 5 years , or those who had a cumulative dose > 460 g or a daily dose > 250 mg / d or 2.3 mg / kg real weight / d , or those who were elderly or had concurrent liver or renal dysfunction.3,4 this current study seemed to agree with the recommendations that the elderly are at higher risk than younger patients , but the median age was less than 60 years . chiowchanwisawakit et al , also demonstrated that the elderly ( > 60 years ) had a significant association with cq retinopathy.9 this was because their studied populations were only ra patients who were basically older than patients with other connective tissue diseases such as sle or ss . however , puavilai et al and leecharoen et al reported no relationship between old age and cq retinopathy.10,11 this might be because they included pediatric patients in their studies . table 2 also shows that real body weight was one of the statistically significant risk factors for cq retinopathy . regardless of the effect of dose per unit of body weight , no previous studies mentioned the relationship between body weight and occurrence of cq retinopathy . this study found no relationship between duration of cq use and cq retinopathy , whereas the revised recommendations of aao concluded the opposite and recommended screening tests starting not earlier than 5 years of cq use . moreover , those patients in the cq retinopathy - positive group developed the disease at a median time of 724 days ( 1.98 years ) , ranging from 139 to 2,033 days ( 0.385.57 years ) , and only one of them had used cq for more than 5 years . similar results were reported by chiowchanwisawakit et al , leecharoen et al , and puavilai et al.911 the latter recommended screening tests to be done every 6 months after the baseline test . meier analysis of cq presented in figure 1 revealed that cq retinopathy mostly developed within the first 3 years after the patients started the medication and that no retinopathy was detected after 6 years . despite the fact that the present study showed a statistically significant relationship between the occurrence of cq retinopathy and cumulative dose , the results were too small to be clinically significant . in addition , the cumulative dose these patients with cq retinopathy received , 101.3 g ( ranging from 14.3 to 325.1 g ) , was far less than 460 g recommendation of the aao for the high - risk group . the daily dose of cq retinopathy group in this study was 250 mg / d ( averaged > since the available cq was manufactured as 250 mg tablets , the rheumatologist had to prescribe cq at multiplicands of 250 mg doses , and therefore the dose was usually 250 mg / d . this is the reason no significant differences of daily doses in retinopathy in the retinopathy - negative group were found . unfortunately , with a daily dose of 250 mg / d , most of the patients received cq doses exceeding 2.3 mg / kg / d , which is the safe dose according to the aao recommendations . this could be the reason cq retinopathy in the thai patients developed earlier and with a less cumulative dose than the previous studies conducted in caucasian populations . also , cq binds to the melanin pigment of the rpe , which , perhaps , leads to toxicity and damage to rpe . since thai populations have more melanin pigment than caucasians , cq might accumulate more in the rpe than it would in the caucasian eyes . the role of rpe and cq binding to melanin pigment is still unclear and should be further investigated . according to the results of the present study , the incidence of hcq retinopathy was 3.28% over 10 years , which was consistent with the results of a meta - analysis conducted in 2006 , which concluded that the incidence of hcq retinopathy ranged from 0% to 4%.15 a recent prospective study reported an incidence of only 0.65%.16 none of the risk factors showed statistical significance in the present study , as opposed to the revised aao recommendations . a large prospective study , however , has shown consistent results with the current study of no significant risk factors.16 aging showed no relation to the hcq retinopathy in the patients . the participants diagnosed with hcq retinopathy were 36 and 39 years old ( median 37.5 years ) . as compared to the revised aao recommendations , retinopathy developed at a younger age . wolfe and marmor,16 in a recent , large prospective study , showed no relation between aging and hcq retinopathy . hcq retinopathy developed in 2 patients who received hcq for 660828 days ( 1.812.27 years ) , a median of 744 days ( 2.04 years ) ; not nearly reaching 5 years . these 2 patients received the drug cumulatively from 80 to 130 g ( median 105.1 g ) , which was far less than the 1,000 g of cumulative dose recommended by aao . it can actually be seen that the there is a possible difference between the duration and daily dose of hcq retinopathy and the retinopathy - negative patients , but the statistics fail to show the significance because of the small number of participants . similar to the results of cq , the patients seemed not to tolerate as high a hcq dose as the aao recommendations . patients who developed hcq retinopathy used hcq at an average dose of 129.4200 mg / d ( median 164.7 mg / d ) . for the same reasons as with the cq group , the daily dosages of not only patients who were retinopathy positive , but also those who were retinopathy negative tended to be approximately equal to the available dose of the hcq tablet 200 mg . nevertheless , unlike the cq group , the daily dose per real body weight of the patients who were hcq retinopathy positive was only 1.94.4 mg / kg / d ( median : 3.2 mg / kg / d ) , which was less than the daily dose recommended by the aao . in this case , the lower body weight would not be a reasonable explanation for the patients developing retinopathy with less treatment duration and less cumulative dosages compared to caucasians . the theory of correlation between hcq binding to melanin pigment in rpe and its toxicity could be a possible explanation ; hence , further studies on pathophysiology of the disease should be done in the future in order to understand these findings . one of the important aims of this study was to assess incidence of and risk factors for cq and hcq in thai people . surprisingly , many results that diverged from the aao recommendations were found , highlighting the risk factors . these results could lead to further investigation and renewed guidelines for asian people in the future . an obvious limitation of the study was that we used possible toxicity for diagnosis of cq and hcq retinopathy . since bulls - eye retinopathy in patients with definite toxicity is a late irreversible finding , we had to report early toxicity and suggest all patients with possible toxicity to stop the medication before they reached the full - blown disease state . however , we improved the accuracy of diagnosis by using a combination of 3 diagnostic procedures and demonstrated both functional ( hvf ) and structural ( oct ) changes . another limitation was that this study was a retrospective study ; also , the number of patients was too small in the hcq group . it is recommended that further prospective studies on the same issue with larger sample sizes be pursued . there was a statistically significant relationship between age , real body weight , and cumulative dose in patients with retinopathy . it was found that the retinopathy could develop before 5 years and with a lower cumulative dose than 460 g. the daily dose exceeding 2.3 mg / kg / d could be the reason . the study found no statistically significant relation between age , real body weight , duration of hcq use , cumulative dose , and daily dose . a lack of sufficient patients in the study may have failed to prove the significance of risk factors . since cq and hcq are essential drugs to control the effects of connective tissue diseases and the patients sometime need higher doses to control the diseases , it is suggested that patients taking long - term cq or hcq should undergo one baseline ophthalmologic screening examination at the first visit and annual screening thereafter . moreover , the routine 250 mg cq and 200 mg hcq tablets should be used cautiously , or divided , especially in patients with low body weight .

objectiveto study the incidences of and risk factors for chloroquine ( cq ) and hydroxychloroquine ( hcq ) retinopathy in thai rheumatologic patients.methodsa retrospective cohort study of 234 rheumatologic patients receiving either cq or hcq was conducted . patients were divided based on whether or not they developed retinopathy as retinopathy - positive or negative groups . medical records giving details regarding age , gender , body weight , underlying diseases , daily doses , and cumulative doses of cq or hcq were reviewed . cq and hcq retinopathy were diagnosed by indirect ophthalmoscopy , humphrey visual field test , and optical coherence tomography . the main outcome measures were incidences and risk factors of cq and hcq retinopathy.resultsthe cq retinopathy was detected in 14 of 173 patients ( 8.09% ) who received cq for 1392,033 days , cumulative doses from 14.3 to 325.1 g , and daily doses from 0.8 to 18.5 mg / kg / d . their ages ranged from 27 to 65 years . when compared to the cq retinopathy - negative group , only age , body weight , and cumulative dose showed statistically significant differences . the hcq retinopathy - positive group was comprised of 2 of 61 patients ( 3.28% ) who received hcq for 660828 days , cumulative doses from 80 to 130 g , and daily dose from 1.9 to 4.4 mg / kg / d . their ages were 36 and 39 years . compared to the hcq retinopathy - negative group , there were no statistically significant differences in studied risk factors.conclusionincidences of and risk factors for cq and hcq retinopathy were reported . since most patients developed retinopathy earlier than 5 years , it is suggested that patients taking long - term cq or hcq should undergo ophthalmologic screening annually after the baseline examination .

Introduction Subjects and methods Data collections Determination of CQ and HCQ retinopathy Sample size calculation Statistical analysis Results Discussion Strength and limitations Conclusion

chloroquine ( cq ) and hydroxychloroquine ( hcq ) are widely used to control effects of various rheumatologic diseases , for example , rheumatoid arthritis ( ra ) , systemic lupus erythematosus ( sle ) , systemic sclerosis ( ss ) , or other connective tissue diseases . they are found to inhibit cd4 t - cell stimulation while also promoting cd8 t - cell stimulation , and hence they are a popular choice for controlling the progression of autoimmune diseases without increasing the rate of opportunistic infections.1 hcq is more molecularly polar than cq making it less lipophilic , leading to poorer diffusion through biological membranes and resulting in less toxicity.1 one of the most severe complications of these drugs is retinopathy , which leads to visual field defects and visual loss as a result of damaged photoreceptors . this damage is incurable and may progress even after the drugs are discontinued.2 an ophthalmologic screening examination , therefore , has to be regularly performed to early detect and prevent this destructive morbidity.2 the american academy of ophthalmology ( aao ) established guidelines for cq and hcq retinopathy screening examinations , which was revised in 2011 and 2016.3,4 patients were classified as high risk when they have at least one of the following risk factors : an average daily dose exceeding 2.3 mg / kg real weight for cq and 5 mg / kg real weight for hcq,4 cumulative doses exceeding 460 g for cq and 1,000 g for hcq,3 a duration of treatment exceeding 5 years , being elderly , having concomitant renal disease , or using tamoxifen.4 routinely , one baseline examination within the first year and annual screening beginning 5 years after the first dose are recommended.3,4 since most of studies reported are performed in caucasian populations,58 application of the findings of these studies and recommendations may not be appropriate for asian populations . for the thai patients , there have been some studies on prevalence and risk factors of cq retinopathy , but the results seemed to be paradoxical to the aao recommendations , and the obvious risk factors are still not well defined.911 hence , this study was intended to produce a more inclusive report on incidence of and risk factors for cq and hcq retinopathy in thai rheumatologic patients . a retrospective cohort study of rheumatologic patients at srinagarind hospital , khon kaen university , thailand , was conducted . the patients , aged not less than 18 years old , started receiving either cq or hcq from 2004 to 2014 and regularly underwent eye screening examinations for cq or hcq retinopathy at the kku eye center . patients whose medical records were incomplete or those who had undeterminable records of the cq or hcq dosages were excluded . moreover , patients with retinal or macular diseases or optic neuropathy , which could possibly interfere with the visual field test , were excluded as well . the information collected were : age : the age at the latest eye examination.gender : male or female.body weight : the real body weight at the latest follow - up at the rheumatologic clinics.underlying disease : diabetes mellitus , dyslipidemia , hypertension , or other diseases.history of rheumatologic diseases.duration of drug use : if the patients had no retinopathy and still continued the drug , it was counted from the day of the first dose to the day of the last eye examination. if the patients had no retinopathy and stopped the medication , it was counted from the day of the first dose to the day of the last dose. if the patients had retinopathy , it was counted from the day of the first dose to the day that retinopathy was diagnosed despite whether patients had stopped the medication or not.cumulative dosage ( g ) : for each patient , the dosage , the duration of each dosage , and dose changes throughout treatment were recorded . the summation of products of dosage and duration were recorded as the cumulative dosage.average dosage per real body weight per day ( mg / kg / d ) : cumulative dose divided by real body weight and duration in days each patient received the medication.presence of signs of cq or hcq retinopathy from the eye screening examinations . if the patients had no retinopathy and stopped the medication , it was counted from the day of the first dose to the day of the last dose. if the patients had retinopathy , it was counted from the day of the first dose to the day that retinopathy was diagnosed despite whether patients had stopped the medication or not . if the patients had no retinopathy and still continued the drug , it was counted from the day of the first dose to the day of the last eye examination . if the patients had retinopathy , it was counted from the day of the first dose to the day that retinopathy was diagnosed despite whether patients had stopped the medication or not . average dosage per real body weight per day ( mg / kg / d ) : cumulative dose divided by real body weight and duration in days each patient received the medication . presence of signs of cq or hcq retinopathy from the eye screening examinations . according to aao recommendations , patients who repeatedly demonstrated these conditions in both eyes for at least 2 consecutive tests were recommended to stop the medications until the cause , other than cq or hcq retinopathy , was identified . these conditions were : parafoveal depigmentation of retinal pigment epithelium ( rpe ) as observed by indirect ophthalmoscopic examination after patients pupils were fully dilated.decreased parafoveal visual field sensitivities at the same area for at least 2 consecutive visits as tested by humphrey visual fields ( hvf ) 10 - 2 using humphrey field analyzer ii ( zeiss meditech inc . all the tests must show reliable test - fixation loss of less than 20% and have false - negative and false - positive response rates of less than 33%.loss of parafoveal inner segment outer segment junction from spectral domain optical coherence tomography ( oct ) using spectralis ( heidelberg engineering , heidelberg , germany).increased autofluorescence at parafoveal area using spectralis ( heidelberg engineering ) . , all the tests must show reliable test - fixation loss of less than 20% and have false - negative and false - positive response rates of less than 33% . the information collected were : age : the age at the latest eye examination.gender : male or female.body weight : the real body weight at the latest follow - up at the rheumatologic clinics.underlying disease : diabetes mellitus , dyslipidemia , hypertension , or other diseases.history of rheumatologic diseases.duration of drug use : if the patients had no retinopathy and still continued the drug , it was counted from the day of the first dose to the day of the last eye examination. if the patients had retinopathy , it was counted from the day of the first dose to the day that retinopathy was diagnosed despite whether patients had stopped the medication or not.cumulative dosage ( g ) : for each patient , the dosage , the duration of each dosage , and dose changes throughout treatment were recorded . the summation of products of dosage and duration were recorded as the cumulative dosage.average dosage per real body weight per day ( mg / kg / d ) : cumulative dose divided by real body weight and duration in days each patient received the medication.presence of signs of cq or hcq retinopathy from the eye screening examinations . if the patients had retinopathy , it was counted from the day of the first dose to the day that retinopathy was diagnosed despite whether patients had stopped the medication or not . if the patients had no retinopathy and still continued the drug , it was counted from the day of the first dose to the day of the last eye examination . if the patients had retinopathy , it was counted from the day of the first dose to the day that retinopathy was diagnosed despite whether patients had stopped the medication or not . average dosage per real body weight per day ( mg / kg / d ) : cumulative dose divided by real body weight and duration in days each patient received the medication . patients who repeatedly demonstrated these conditions in both eyes for at least 2 consecutive tests were recommended to stop the medications until the cause , other than cq or hcq retinopathy , was identified . these conditions were : parafoveal depigmentation of retinal pigment epithelium ( rpe ) as observed by indirect ophthalmoscopic examination after patients pupils were fully dilated.decreased parafoveal visual field sensitivities at the same area for at least 2 consecutive visits as tested by humphrey visual fields ( hvf ) 10 - 2 using humphrey field analyzer ii ( zeiss meditech inc . , all the tests must show reliable test - fixation loss of less than 20% and have false - negative and false - positive response rates of less than 33%.loss of parafoveal inner segment outer segment junction from spectral domain optical coherence tomography ( oct ) using spectralis ( heidelberg engineering , heidelberg , germany).increased autofluorescence at parafoveal area using spectralis ( heidelberg engineering ) . decreased parafoveal visual field sensitivities at the same area for at least 2 consecutive visits as tested by humphrey visual fields ( hvf ) 10 - 2 using humphrey field analyzer ii ( zeiss meditech inc . , all the tests must show reliable test - fixation loss of less than 20% and have false - negative and false - positive response rates of less than 33% . loss of parafoveal inner segment outer segment junction from spectral domain optical coherence tomography ( oct ) using spectralis ( heidelberg engineering , heidelberg , germany ) . estimated sample size was calculated with 95% confidence level , 5% absolute precision , and 13.5% estimated prevalence , resulting in a sample size of 171.9 as these data did not match that of normal distribution , continuous data were reported as the median with minimal and maximal values , and comparisons between data of each group were performed using mann whitney tests . there were 173 patients who received only cq ( cq group ) and 61 patients who received only hcq ( hcq group ) . in the cq group , there were 63 ra patients , 63 sle patients , 23 ss patients , and 24 patients with other connective tissue diseases . in the hcq group , there were 11 ra patients , 31 sle patients , 4 ss patients , and 15 patients with other connective tissue diseases . in the cq positive group , possible toxicity as evidenced by indirect ophthalmoscopic examination , hvf test , and oct findings . the incidence of cq retinopathy was 8.09% of the patients who used cq over the 10 years considered . it was noted that only age , real body weight , and cumulative dose showed statistical significance at p<0.05 . moreover , other coexisting diseases , thyroid disease , heart disease , and anemia , in both the retinopathy - positive and the retinopathy - negative groups were recorded . also , hepatitis b virus infection , nasopharyngeal carcinoma , pulmonary tuberculosis , parkinson s disease , and renal disease in the retinopathy - negative group were recorded . in the hcq group , 2 of 61 patients developed retinopathy ; therefore , the incidence of hcq retinopathy was 3.28% over the 10 years of the study . possible toxicity as evidenced by indirect ophthalmoscopic examination , hvf test , and oct findings . other underlying diseases such as thyroid disease , bulimia nervosa , anemia , hepatitis , rheumatic heart disease , and ischemic stroke were also noted in patients in the retinopathy - negative group . in contrast to cq , no statistically significant hazard ratios of any items studied in the hcq group were found . the incidence of cq and hcq retinopathy versus duration of medication use were analyzed via kaplan meier survival analysis and are illustrated in figures 1 and 2 . it is noted that cq retinopathy can occur as early as 4 months after drug use . moreover according to results of this study , the incidence of cq retinopathy was 8.09% over 10 years . back in the 19th century , cq retinopathy was diagnosed by the appearance of bull s eye maculopathy in patients taking cq . according to the report of life expectancy in rheumatologic patients by mok et al13 and understanding that the normal thai population has an average life expectancy of 74.72 years,14 the prevalence of retinopathy in this study can be calculated from the incidence as being 14.9% , which was in agreement with the previous studies.912 the revised cq and hcq retinopathy screening recommendations of aao stated that high - risk patients were those who used cq longer than 5 years , or those who had a cumulative dose > 460 g or a daily dose > 250 mg / d or 2.3 mg / kg real weight / d , or those who were elderly or had concurrent liver or renal dysfunction.3,4 this current study seemed to agree with the recommendations that the elderly are at higher risk than younger patients , but the median age was less than 60 years . table 2 also shows that real body weight was one of the statistically significant risk factors for cq retinopathy . regardless of the effect of dose per unit of body weight , no previous studies mentioned the relationship between body weight and occurrence of cq retinopathy . this study found no relationship between duration of cq use and cq retinopathy , whereas the revised recommendations of aao concluded the opposite and recommended screening tests starting not earlier than 5 years of cq use . moreover , those patients in the cq retinopathy - positive group developed the disease at a median time of 724 days ( 1.98 years ) , ranging from 139 to 2,033 days ( 0.385.57 years ) , and only one of them had used cq for more than 5 years . similar results were reported by chiowchanwisawakit et al , leecharoen et al , and puavilai et al.911 the latter recommended screening tests to be done every 6 months after the baseline test . meier analysis of cq presented in figure 1 revealed that cq retinopathy mostly developed within the first 3 years after the patients started the medication and that no retinopathy was detected after 6 years . despite the fact that the present study showed a statistically significant relationship between the occurrence of cq retinopathy and cumulative dose , the results were too small to be clinically significant . in addition , the cumulative dose these patients with cq retinopathy received , 101.3 g ( ranging from 14.3 to 325.1 g ) , was far less than 460 g recommendation of the aao for the high - risk group . the daily dose of cq retinopathy group in this study was 250 mg / d ( averaged > since the available cq was manufactured as 250 mg tablets , the rheumatologist had to prescribe cq at multiplicands of 250 mg doses , and therefore the dose was usually 250 mg / d . this is the reason no significant differences of daily doses in retinopathy in the retinopathy - negative group were found . unfortunately , with a daily dose of 250 mg / d , most of the patients received cq doses exceeding 2.3 mg / kg / d , which is the safe dose according to the aao recommendations . this could be the reason cq retinopathy in the thai patients developed earlier and with a less cumulative dose than the previous studies conducted in caucasian populations . according to the results of the present study , the incidence of hcq retinopathy was 3.28% over 10 years , which was consistent with the results of a meta - analysis conducted in 2006 , which concluded that the incidence of hcq retinopathy ranged from 0% to 4%.15 a recent prospective study reported an incidence of only 0.65%.16 none of the risk factors showed statistical significance in the present study , as opposed to the revised aao recommendations . a large prospective study , however , has shown consistent results with the current study of no significant risk factors.16 aging showed no relation to the hcq retinopathy in the patients . the participants diagnosed with hcq retinopathy were 36 and 39 years old ( median 37.5 years ) . as compared to the revised aao recommendations , retinopathy developed at a younger age . wolfe and marmor,16 in a recent , large prospective study , showed no relation between aging and hcq retinopathy . hcq retinopathy developed in 2 patients who received hcq for 660828 days ( 1.812.27 years ) , a median of 744 days ( 2.04 years ) ; not nearly reaching 5 years . these 2 patients received the drug cumulatively from 80 to 130 g ( median 105.1 g ) , which was far less than the 1,000 g of cumulative dose recommended by aao . it can actually be seen that the there is a possible difference between the duration and daily dose of hcq retinopathy and the retinopathy - negative patients , but the statistics fail to show the significance because of the small number of participants . similar to the results of cq , the patients seemed not to tolerate as high a hcq dose as the aao recommendations . patients who developed hcq retinopathy used hcq at an average dose of 129.4200 mg / d ( median 164.7 mg / d ) . for the same reasons as with the cq group , the daily dosages of not only patients who were retinopathy positive , but also those who were retinopathy negative tended to be approximately equal to the available dose of the hcq tablet 200 mg . nevertheless , unlike the cq group , the daily dose per real body weight of the patients who were hcq retinopathy positive was only 1.94.4 mg / kg / d ( median : 3.2 mg / kg / d ) , which was less than the daily dose recommended by the aao . one of the important aims of this study was to assess incidence of and risk factors for cq and hcq in thai people . an obvious limitation of the study was that we used possible toxicity for diagnosis of cq and hcq retinopathy . there was a statistically significant relationship between age , real body weight , and cumulative dose in patients with retinopathy . it was found that the retinopathy could develop before 5 years and with a lower cumulative dose than 460 g. the daily dose exceeding 2.3 mg / kg / d could be the reason . the study found no statistically significant relation between age , real body weight , duration of hcq use , cumulative dose , and daily dose . since cq and hcq are essential drugs to control the effects of connective tissue diseases and the patients sometime need higher doses to control the diseases , it is suggested that patients taking long - term cq or hcq should undergo one baseline ophthalmologic screening examination at the first visit and annual screening thereafter .

angiogenesis is a key early step in cancer development that permits tumor expansion , growth , progression , and dissemination ( 3436 ) , as well as being a key target in prevention ( 37 ) . several studies have shown that microvascular density is associated with prognosis and vascular endothelial growth factor ( vegf ) , the major angiogenic factor , has become a target for several cancers ( 36 ) . however , inhibiting vegf alone has had limited success in some cancers , suggesting that other approaches are needed ( 38,39 ) . immune cells infiltrating tumors often show a skewed phenotype that reflects attenuation of antitumor activity and enhancement of protumor activities , including angiogenesis , in a process defined as tumor - induced polarization ( 40,41 ) . by this process tumor cells are able to reprogram the host immune system components in favor of neoplastic progression , including angiogenesis , an essential process for tumor survival , and metastasis . the classic example of tumor - induced polarization occurs in macrophages : m1 macrophages produce th1 cytokines ( such as il-12 and tnf ) and promote anti - tumor responses , while m2-polarized macrophages produce th2 cytokines , induce tissue reconstruction , growth promotion , and angiogenesis ( 42 ) . several subsets of m2 macrophages are generated by diverse stimuli ( il-4 m2a ; immune complexes / il-1 m2b ; il10/tgf m2c ) . tumor - associated macrophages ( tams ) show an m2-like profile ( 42 ) ; other tumor - associated m2-like polarizations include myeloid - derived suppressor cells ( mdscs ) ( 43,44 ) and tie2 expressing macrophages closely associated with the vasculature ( 45,46 ) . several studies show a protumor and proangiogenic polarization for most immune cells in the tumor microenvironment , including macrophages , dendritic , mast , t and b cells , as well as polymorfonuclear cells ( pmn ) ( 4042,46,47 ) . we had previously hypothesized that this phenotypic polarization might be extended to nk cells ( 41 ) . a peculiar behavior of nk cells is found in the developing decidua ( table 1 ) , which converts them from killers to builders ( 48 ) . early on in pregnancy , decidual nk cells ( dnk ) accumulate to become approximately 70% of the local lymphocytes and 3040% of all decidual cells ( 49 ) . dnk cells display a cd56cd16 phenotype ( 49 ) and , although they express kirs as well as perforin and granzymes , are poorly cytotoxic ( 4952 ) . further , they have been found to be associated with induction of cd4 t regulatory ( treg ) cells ( 3 ) , and to produce il-10 ( 51 ) , thus potentially contributing to the immunosuppressive environment of the placenta . dnk express most of the nk cell hallmarks , including the activating receptors nkp46 , nkg2d , and 2b4 , as well as kir and cd94/nkg2a inhibitory receptors , along with markers not found on peripheral blood nk cell subsets , such as cd9 and cd49a ( 53 ) . interestingly , the usually activating coreceptor 2b4 acts as an inhibitory receptor in dnk cells ( 54 ) , suggesting extensive rewiring of the cytoplasmic signals in response to external stimuli . another notable feature of dnk cells is their close link with vascularization of the decidua and spiral artery formation ( 51,52 ) in both humans and mice . dnk cells produce substantial quantities of angiogenic factors ( table 1 ) , including vegf , plgf , as well as il-8 ( cxcl8 ) , and show potent angiogenic activity in vitro and in vivo ( 51 ) . this activity has been linked to tissue construction ( 49 ) that is critical for implantation and fetal development . low levels of dnk cells are associated with miscarriage ( 49 ) . in a different microenvironment , such as that of tumors , it has been shown that addition of angiogenic dnk cells to tumor cell xenografts markedly enhanced growth of tumors ( 51 ) . the origin of dnk cells is controversial ; expansion in loco from nk cell precursors ( 55 ) and conversion of peripheral blood nk cells to dnk upon exposure to decidual endothelial and stromal cells ( 56,57 ) have been proposed . decidual stromal cells produce high levels of tgf that has been reported to convert isolated peripheral blood cd56cd16 nk cells into cd56cd16 cells displaying poor cytotoxicity and expressing other markers , such as cd9 , in common with dnks ( 58,59 ) . a combination of tgf , hypoxia , and a demethylating agent has been found to convert sorted peripheral blood cd56cd16nk cells into a dnk - like phenotype ( 59 ) . these cells show low cytotoxicity ( 59 ) , high levels of vegf expression ( induced by hypoxia ) , expression of the dnk marker cd9 ( induced by tgf ) , and of kirs ( maintained by the demethylating agent ) . dnk cells tend to preferentially express cxcr3 and cxcr4 ( 6062 ) , in part in response to hormones ( 57,60 ) , and the respective ligands of these receptors are able to preferentially induce chemotaxis of cd56cd16 nk cells ( 57,60 ) . list of the key studies concerning the pro - angiogenic and pro - tumor role of natural killer cells * * the table lists selected references concerning the role of natural killer cells ( nk ) in tumor progression and angiogenesis and summarizes the principal findings grouped as : decidual nk ( dnk ) cells , which represent the most investigated subset in relation to angiogenesis . since there are numerous articles , only a few are cited . nk cells during angiogenesis and repair in murine ocular models , where the pro - angiogenic function exerted by nk cells is associated with macrophages . tumor infiltrating / associated nk cells , most of the references regard the impairment of nk cytotoxic activities with only one reference demonstrating a direct association between tumor infiltrating / associated nk cells and angiogenesis . scid - severe combined immunodeficiency ; nod - non obese diabetic . nk cells have also been linked with angiogenesis during tissue repair ( table 1 ) . mice deficient in c - kit showed defective myocardial repair , and gene expression profiling and immunohistochemistry indicated a role for nk cells ( 63 ) . further analyses showed that nk cells were required for angiogenesis in the damaged murine myocardium ( 64 ) and that engagement of klrg1 ( killer cell lectin - like receptor 1 ) by cadherins and 47 integrin by vcams ( vascular cell adhesion proteins ) were important for nk activation . in models of bfgf ( basic - fibroblast growth factor)-induced corneal and laser - induced choroidal angiogenesis , nk cell depletion led to a substantial reduction of neovascularization ( 65 ) . in nk - depleted animals , reduced macrophage infiltration into the cornea and lower levels of vegf - a , vegf - c , and ifn mrnas were observed . in vitro , a coculture of nk , macrophages , and endothelial cells showed an ifn-dependent increase in vegf ( 65 ) . given the angiogenic tissue construction activity of dnk and several similarities between the decidual , tissue repair , and tumor microenvironments , a working hypothesis ( 41 ) is that tinks or tanks could have a proangiogenic activity , resulting in tumor promotion rather than tumor inhibition ( table 1 ) . our recent data in the context of non - small cell lung cancer ( nsclc ) supports this hypothesis ( 66 ) . lung tissues are relatively rich in nk cells ( 6668 ) , where the predominant subset in normal parenchyma is cd56cd16 ( 66 , 69 ) . in contrast , the major nk subset in nsclc tissues appears to be cytokine - producing cd56cd16 nk cells ( 66,69 ) . the nsclc tinks are localized in the tumor stroma ( 69 ) and at the invasive margin ( 70 ) of the tumor . these tinks express activating receptors and some of the cd56perforin nk cells display a discrete expression of kirs ( 69,70 ) . in patients with pleural effusions , including some lung cancer patients , the cd56cd16 nk subset predominated ; these cells expressed high levels of ifn upon activation with il-2 and became efficient in killing suitable target cells ( 71 ) . when examined for angiogenic cytokine production , nk cells of nsclc patients were found to produce relatively high levels of vegf , placental - derived growth factor ( plgf ) , and il-8 ( 66 ) . interestingly , this was not limited to the tink cells , as pro - angiogenic factor production was also observed in adjacent tissues and even peripheral blood ( tanks ) . further , nk cells in nsclc patients with squamous cell carcinoma produced substantially higher levels of vegf and plgf as compared with those with adenocarcinomas ( 66 ) . the levels of vegf and plgf in tanks from adenocarcinoma patients were comparable with those from healthy controls , while that of tanks from squamous cell carcinoma patients was markedly higher . further , supernatants of nsclc tinks showed angiogenesis - associated activities in vitro , in particular those of squamous cell carcinomas ( 66 ) . most nsclc tinks show low levels of the nk maturation markers cd11b and cd27 ( 72 ) , while the increased numbers of fully mature nk cells indicated by the cd57 marker were a positive prognostic indicator in nsclc squamous cell carcinomas ( 73 ) . these data imply that like dnk cells , tinks and tanks have some markers of mature nk cells , including granzyme and perforin , yet display low cytotoxicity , while producing angiogenesis - promoting factors . in the case of squamous nsclc , a potent effect of the tumor on the systemic nk phenotype was observed , since nk polarization was found even in peripheral blood . transcriptome profiling studies indicate that nsclc has a distinct effect on peripheral blood mononuclear cell gene expression ( 74,75 ) . this was true in particular for squamous cell nsclc , and many genes making up a prognostic signature using these profiles were nk associated ( 74,75 ) . the data show that even a squamous cell carcinoma of small ( operable ) size had a substantial systemic effect on the immune system , in particular nk cells . a similar systemic alteration of tanks has been observed for colorectal ( 76 ) and breast ( 77 ) cancers , where these cells showed repression of nk functions . in breast cancer , the level of functional repression correlated with disease stage ( 77 ) and was reverted in long - term survivors . the mechanisms leading to the systemic effect creating tanks are as yet unknown , and the overall consequences of this remain unclear . nk trafficking through the tumor with local polarization or a systemic release of factors affecting nk cell polarization could result in generation of tanks . the variable reports of ratios between cd56cd16 to cd56cd16 within the tink / tank in nsclc surgical samples ( 66,69,70 ) may relate to nontumor to tumor tissue ratio of the biopsies . like the dnk cells of the decidua , the origin of the angiogenic cytokine - producing tinks is not clear ( figure 1 ) . cytotoxic nk cells have a stable and apparently terminal phenotype ( 10 ) , although cd56 nk cells can become cd56 upon in vitro activation and , at the same time , cd16 may be downregulated ( 10 ) . the low expression of perforin and granzyme on a large part of tinks , similar to that of cd16 peripheral blood nk cells , has suggested that tinks are derived from cd56cd16 nk cells , and that factors within the tumor microenvironment might induce expression of some kirs on these cells ( 69 ) . microarray studies ( 78 ) have suggested that dnk cells express a very different molecular profile as compared with cd56cd16 and cd56cd16 nk cells . a microarray analysis on seven adenocarcinoma and five squamous nsclc - derived nks ( sorted as cd3cd56 cells ) indicated distinct profiles between patient and control samples ( 79 ) . interestingly , as compared with other nk cells , both dnk and nsclc tinks showed upregulation of several activation markers and in particular granzyme a ( 7879 ) . dnk cells also showed increased expression levels of granzyme b , while tinks showed high production of granzyme k and fas . hypothesis of natural killer ( nk ) cell subset differentiation and functions in decidual and tumoral tissues . nk precursors are generated in the bone marrow and undergo several steps toward maturation into the major peripheral blood ( pb ) nk cell subsets . the main pb subsets are the cd56cd16 ( 90%95% of circulating nk cells ) and cd56cd16 nk cells ( 510% of circulating nk cells ) associated with lower cytotoxic activity and production of ifn and other cytokines , expressing the cxcr3 and ccr7 chemokine receptors . these cells also appear to be a precursor to the cytotoxic cd56cd16 nk , showing high levels of perforin and granzyme and expression of cx3cr1 , cxcr1 and cxcr2 . the developing decidua contain high levels of dnk cells that are recruited into this tissue . within the decidua ( shown as a pink area ) trophoblast cells release transforming growth factor ( tgf ) and express hla - g molecules associated with reduction of nk cytotoxicity and promotion of pro - angiogenic activity . the dnk cells express cx3cr1 , cxcr3 and cxcr4 , and secrete angiogenic factors including vegf , plgf , cxcl8 , cxcl12 . within many tumor microenvironments ( shown as a grey area ) these elements have been associated with conversion of pb nk cells into poorly cytolytic dnk - like tumor infiltrating natural killer cells ( tinks ) cells able to release vascular endothelial ( vegf ) , placental ( plgf ) growth factors and il8 ( cxcl-8 ) , thus sustaining angiogenesis . it is not clear if tumor associated natural killer cells ( tanks ) are the result of emigration of tinks from the tumor microenvironment or due to systemic effects on nk cells of cancer - related products . the tumor microenvironment likely affects the polarization of cd56cd16 nk cells toward a proangiogenic phenotype , inducing the production of cytokines such as vegf , plgf , and il-8 . high levels of tgf and hypoxia are also features typical of the tumor microenvironment ( figure 1 ) that could impact the nature and function of cd56cd16 tinks . in cancer , tgf1 acts as a janus - like cytokine : in the initial phases of cancer formation , it behaves as a tumor suppressor , inhibiting tumor cell replication and favoring apoptosis ( 80,81 ) . in contrast , in later stages of tumor progression , tgf1 exerts a protumorigenic role , promoting survival and epithelial - mesenchymal transition and invasion , as well as acting within the tumor microenvironment as an immune - suppressive and angiogenic agent ( 80,81 ) . tgf appears to be a strong immune - cell polarizing agent , active on most immune cells ( 81 ) . as mentioned above , several studies from the strominger / kopcow groups have indicated that tgf , eventually together with other factors , can induce a dnk - like differentiation from peripheral blood cd56cd16 nk cells ( 58,59,82 ) . we found that tgf produces polarization of peripheral blood nk cells from healthy subjects toward vegf- , plgf- , and il-8-producing nk cells ( 66 ) , again indicating a parallel with the dnk - like phenotype . these data suggest that , within the decidua and the tumor microenvironment , tgf , along with other cues such as hypoxia , might result in the polarization of nk cell precursors or even mature nk cells toward angiogenic dnk - like cells . this would be in keeping with the immunomodulatory role of tgf in several tumors and tumor models ( 80,83 ) . tumor - derived tgf has been shown to upregulate cxcr3 and cxcr4 in nk cells ( 84 ) , similar to what occurs for dnk cells ( 6062 ) , and to exert an immunosuppressive activity ( 85 ) . blocking of tgf has been shown to overcome immune suppression ( 85 ) and enhance the effects of nk cell therapy ( 86 ) . plasma tgf levels have been shown to be substantially upregulated in lung and colon cancer patient immunosuppression linked to downregulation of nkg2d ( 8790 ) . it is also possible that tumor - microenvironment associated factors induce a further modulation of cd56cd16 cells into the tink phenotype . hla - g , a unique immunoregulatory class i mhc molecule expressed by decidual trophoblasts ( 91 ) and in diverse tumor tissues ( 91,92 ) might play a role in tink and , through release of soluble forms , tank polarization ( figure 1 ) . hla - g is expressed by trophoblasts in the decidua ( 91 ) and is upregulated in several tumor tissues ( 70,91,92 ) . tumor and serum hla - g expression levels have been found to be an independent marker of poor prognosis in nsclc , ovarian , breast , colorectal , esophageal , and gastric cancers , as well as hepatocellular and endometrial carcinoma ( 9194 ) . hla - g expression can be quite heterogeneous within the tumor ; however , a mechanism involving exosome release and trogocytosis by neighboring tumor and immune cells , including nk cells , has been evoked to explain the apparent bystander effect of hla - g mediated immune suppression ( 91,92 ) . hla - g seems to exert its immunosuppressive effects through interactions with three key inhibitory receptors : immunoglobulin - like transcripts ilt-2 ( found on macrophages , dendritic , decidual and peripheral nk cells , as well as b and t lymphocytes ) , ilt-4 ( expressed by macrophages and dendritic cells ) , and kir2dl4 ( in decidual and peripheral blood nk cells ) ( 91,92 ) . through these receptors , hla - g inhibits cd8 t cells and nk cytotoxicity , alters dendritic cell ( dc ) maturation , trafficking , antigen presentation , and cross talk with t and nk cells . hla - g interaction with the nonclassical kir2dl4 ( cd158d ) has been shown to stimulate resting nk cells to secrete proinflammatory and proangiogenic factors ( 95 ) through induction of a senescence - associated secretory phenotype ( 96 ) . frequently strongly expressed in ovarian cancer , hla - g in a metastasis model was associated with increased metastases and reduced nk cell cytotoxicity ( 97 ) . among other effects , soluble hla - g differentially modulated chemokine receptor expression between cd56 and cd56 populations , downmodulating ccr2 in the cd56 cells and cxcr3 in both populations , while having little effect on cxcr4 ( 98 ) , suggesting modulation of nk cell recruitment by microenvironments expressing hla - g ( figure 1 ) . other nk suppressive factors in common between the decidua and tumor microenvironment are indoleamine 2,3-dioxygenase ( ido ) and prostaglandin e2 ( 99 ) . in terms of angiogenic cytokine production within the tumor microenvironment , the relative contribution of nk cells remains to be determined , which likely depends on the tissue of origin . unlike other innate cells , within the tumor microenvironment there is a strong presence of nk - activating ligands that could enhance the cytokine - producing activity of tinks , as has been found in the decidua ( 100 ) . nk cells also interact with other immune cells , modulating both their presence and activation state . as examples , in the cornea and choroid , nk depletion was associated with reduced macrophage recruitment and loss of angiogenic potential ( 65 ) ; in the premetastatic niche , nk cells are associated with mdsc accumulation ( 101 ) . interestingly , nk cells have also been reported to convert into mdscs within the tumor microenvironment ( 102 ) . tumor cells and the tumor microenvironment substantially suppress the antitumor function of tinks through a variety of mechanisms ( 103 ) . in nsclc , tinks have been shown to be highly enriched in the cd56perforin nk cell subset ( 66,69 ) , a phenotype quite similar to that of tinks from colorectal ( 76 ) and breast ( 77 ) cancers . consistent with the lower cytotoxicity of the cd56perforin nk cell subset , tinks uniformly show poor cytotoxicity ( 66,69,76,77,103 ) . in terms of total lymphocytes , nk cell frequency varies depending on the tissue , ranging from relatively high ( approximately 25% in kidney ; 15% in liver and subcutaneous adipose tissues ) to quite low ( 14% in colon , stomach , adrenal gland ) ( 104 ) . we observed that in cancers , the frequency of cd56perforin nk cells among the total nk cell compartment may be substantially higher than in matched normal tissues , as in the case of lung and breast cancers ( 54% vs 9.2% and 30% vs 4.1% , respectively ) ( 104 ) . the comparative analyses of the chemokine expression patterns of tinks with those derived from matched healthy tissues showed that the tumor microenvironment might account for this specific accumulation of nk with the tink phenotype . neoplastic lung tissues showed a marked downregulation of cxcl2 , a chemokine able to specifically attract cd56 nk cells , since its cognate receptor , cxcr2 , is expressed by cd56 cytotoxic nk cells but not on the cd56 counterpart . at the same time , the upregulation of ccl19 , cxcl9 , and cxcl10 , chemokines more specific for cd56 nk cells , occur in lung tissues following neoplastic transformation . a similar trend is observed in breast cancers , where the cd56-associated chemokines , cxcl2 , cx3cl1 , cxcl1 , and cxcl8 , are downregulated in the tumor tissue , and the upregulation of ccl5 and ccl19 is simultaneously observed ( 104 ) . remarkably , the frequency of tinks among the total tumor - infiltrating lymphocytes did not show statistically significant differences when compared to the percent of nk cells as a function of total lymphocytes from normal matched tissues ( 66,69,104,105 ) . these recent data suggest that neoplastic transformation did not determine an increased migration of nk cells within the tissues . rather , as a consequence of switches in chemokine expression patterns , cytotoxic nk cells are substantially decreased compared with healthy tissues , at least in some frequently occurring cancers such as breast and lung carcinomas . this new evidence now emerges as a further mechanism of cancer immunoediting with implications for both immunosurveillance and tumor escape from immune attack . in the context of tumor cell heterogeneity , relapse and metastasis , substantial interest has arisen regarding the concept of cancer - stem or cancer - initiating cells ( cscs / cics ) . current concepts are that these cells , at least for some tumor subtypes , show slow replication and thus are resistant to most chemotherapeutics and radiotherapy , while through asymmetric replication cscs / cics give rise to rapidly proliferating cells that make up the bulk of the tumor ( 106109 ) . by definition , cscs / cics are those cells able to give rise to new tumors , clinically leading to tumor recurrence and metastatic disease . targeting cscs / cics has therefore become of paramount importance , and properly activated nk cells may have the capacity to eliminate this insidious cell population . glioblastoma stem cells , which express low levels of mhc - class i molecules and high levels of the activating dnam-1 ligands pvr and nectin-2 , were susceptible to lysis by nk cells following il-2 or il-15 activation ( 110 ) . nk cells have been found to be able to kill oral squamous carcinoma stem cells as well as bona fide embryonic , mesenchymal , and dental pulp stem cells ( 111 ) . again , however , microenvironmental effects may modulate and compromise this activity ( 112 ) . cell lines bearing the ovarian cancer stem cell marker cd24 are resistant to chemotherapy yet preferentially targeted by nk cells ( 113 ) . a detailed study on human colorectal carcinoma confirmed that the cics express lower mhc - class i and higher levels of nk - activating ligands and that cics were preferentially targeted over bulk tumor by allogenic and autologus nk cells ( 114 ) . increasing experimental ( 86,115121 ) and clinical ( 86,117,122 ) evidence indicate that nk cells modulate metastatic dissemination , suggesting that nk cells are able to kill the cscs / cics that give rise to metastatic disease . however , most of the experimental studies ( 86,115121 ) used tumor cells injected directly into healthy mice , rather than conditioning the host . attenuation of nk cell activity is associated with generation of the premetastatic niche and metastasis efficiency in murine models ( 101 ) . hypoxic tumor - cell conditioned media increased nk and mdcs recruitment into the premetastatic niche and reduced nk cytolytic activity ( 101 ) . mdscs appear to play a role in modulating nk cell function ( 101 ) , which in part is overcome upon il-2 cytokine intervention ( 123 ) . a murine in vivo study showed that tam family tyrosine kinases are involved in tumor - induced nk attenuation ( 121 ) . inhibition of the tam family tyrosine kinase activity resulted in nk licensing to kill tumor and metastatic cells in murine models of both hematogenous and lymphatic routes , resulting in slower tumor growth and substantial reduction in metastases ( 121 ) . these data indicate that murine nk cells can be switched from a poorly antitumor or even protumor phenotype to antitumor activities . it becomes clear that the compromised cytotoxic activity of nk cells is also implicated in the lack of csc / cic killing in cancer patients . the potential tumor - cell killing capacity of nk cells suggests that these cells could be useful in therapeutic approaches ( 124 ) . several chemotherapies and targeted agents can enhance nk function , including metronomic cyclophosphamide ( 125 ) , imatinib ( 126,127 ) , gefitinib ( 128 ) , and sorafenib ( 129 ) ( although this latter point is controversial ) . in the case of metronomic cyclophosphamide , a role for vegf receptors in activating nk cells has been suggested ( 131 ) . immunomodulatory drugs such as lenalidomide are associated with nk activation in vivo and reversion of tumor microenvironment ( il-6 , tgf ) effects on nk cells in vitro through inhibition of stat3 ( 132 ) . a key consideration is that nk cells infiltrating cancer tissues display low levels of cd16 on their surface . cd16 ( also known as fcriii ) is an fc receptor able to recognize igg that is bound to the surface of target cells ( 133 ) , and it is expressed at high levels on peripheral blood cd56 cytotoxic nk cells . activation of cytotoxic nk cell fcriii by igg causes the release of cytotoxic mediators like perforin and granzymes that enter the target cell and promote cell death by triggering apoptosis . the adcc process is currently recognized as a major mechanism of action of several targeted therapies for the treatment of neoplastic diseases : these therapies aim to exploit nk cell adcc by the administration of tumor - specific monoclonal antibodies ( mab ) ( 134 ) . tinks of those cancer tissues studied to date express little or no cd16 ( 66,69,104 ) . in addition , cd16 appears to be downregulated in cd56perforin nk cells infiltrating nsclc ( 69,70 ) . in view of these recent results , the limited expression of fcriii on nk cells infiltrating the tumors should be considered when mab - mediated target therapies are proposed for the treatment of solid tumors , including nsclc . cd56perforin nk cells upon cytokine - induced activation ( exposure to il-2 , il-15 , or both ) , can convert into perforin nk cells able to efficiently kill cancer cells ( 9,10 ) . a hypothesis is that sufficient triggering of activating receptors might induce maturation to a perforin nk , even in the absence of specific cytokines . although further studies are warranted to better clarify the functions and the possible differentiation pathways of tumor - infiltrating nk cell subsets , the use of il-2 or il-15 , currently available as pharmaceutical grade compounds , can be envisaged in association with current tumor - specific , mab - mediated target therapies ( 124 ) . this therapeutic association should be advantageous for inducing tumor - cell killing capability in this cd56cd16 nk cell subset , which abundantly infiltrates cancer tissues . taken together , these data suggest that , with proper activation , nk cells could become effective weapons against tumor cell recurrence following radio- and/or chemotherapy . nk cells are currently considered in cancer immunotherapy but most studies have focused on the single goal of exploiting their direct antitumor cytotoxic potential . for example , nk cells can rapidly expand in vitro and in vivo , in particular following haploallogenic bone marrow transplantation , where they appear to serve in both host defense as well as inhibition of graft - vs - host reactions ( 135 ) . it is now well established that interactions occurring between nk cells and dendritic cells ( dcs ) play a major role in the priming of antitumor responses ( figure 2 ) . bidirectional activations occur during the nk / dc cross talk ( 136138 ) , which substantially modulates the immune responses of both cells and the adaptive as well as innate arms of immunity . in agreement with the experimental results , a statistically significant association between clinical responses and the presence of activated nk cells postvaccination has been reported in cancer patients who were treated with peptide - loaded , dc - based vaccines ( 139141 ) . natural killer ( nk ) cell editing of dendritic cells ( dcs ) plays a crucial role in the regulation of both innate and adaptive immunity . in normal tissues ( light blue area ) , plasmacytoid and classical myeloid dcs can activate nk cells via the release of several cytokines . activated nk cells can edit dcs , either inducing their maturation or eliminating the immature , allegedly tolerogenic dcs ( idcs ) . nk cells also release ifn and can eliminate cancer stem cells ( cscs ) . in the normal context / microenvironment , nk cells sustain th1 polarization , necessary for an effective adaptive immune response against tumors . in the tumor microenvironment ( grey area ) nk cells are characterized by poor cytotoxicity , leading to idc accumulation , block of cytolytic t cell responses and inducing immune tolerance of tumor cells . several studies have shown that nk cells can be efficiently activated by dcs to release cytokines , to proliferate , and to enhance their cytotoxicity ( 136,142,143 ) . activated nk cells can then kill tumor cells , but , at the same time , play a relevant immunoregulatory role by editing dcs ( 144 ) ( figure 2 ) . nk cell - mediated dc editing may occur by eliminating the less immature , allegedly tolerogenic dcs ( 136,144,145 ) . the relevance of this phenomenon for the generation of an effective tumor - specific adaptive immune response has recently been demonstrated also in vivo ( 146,147 ) . the decreased cytotoxicity of tumor - infiltrating nk cells could substantially alter dc - nk interactions ( figure 2 ) , where poorly cytotoxic nk cells would not only have reduced capacity to kill tumor cells , but also reduced editing of immature dendritic cells ( 2 ) . in the tumor microenvironment , nk editing of dc might be compromised , leading to tolerogenic dc ( figure 2 ) , similar to that found in hiv infection , where lack of nk editing leads to increased immature dcs and likely subsequent immune suppression ( 148 ) . on the other hand , nk cells can also shape adaptive immune responses by causing dc maturation and influencing the polarization of primary t - cell responses ( 149152 ) . this helper role of nk cells for the development of adaptive immunity takes place via the release of soluble factors , such as tnf and ifn , ( 138,143,153 ) . these cytokines are released following nk recognition of target tumor cells and represent an alternative mode for dc activation apart from direct recognition of pathogen constituents , particularly relevant in the tumor microenvironment , where there are no signals from pathogens and other danger signs , thus impairing proper dc maturation . taken together , these data show that nk cells are not only activated by dcs but , in turn , can also influence the emerging adaptive immune responses that are initiated by these antigen - presenting cells ( figure 2 ) . this reciprocal activation of nk cells and dcs provides a strong rationale for the combined use of nk cells and dcs in cancer immunotherapy . in view of the most recent knowledge on nk - dc liaisons , it is possible to envisage use of adoptive immunotherapy with nk cells ( figure 3 ) for obtaining also an optimal dc maturation ( 154,155 ) . at the same time , dc - based vaccines should be reconsidered with the aim of activating and recruiting nk cells . in this regard , it is noteworthy that vaccination with unpulsed exogenous dcs ( ie , dcs carrying no tumor antigens ) has been shown to induce tumor - specific t cells in a tumor - bearing host via the activation of nk cells . in this cellular network , cytokines released by activated nk cells cause the maturation of endogenous dcs ( figure 2 ) that can eventually present tumor antigens derived by nk cell - mediated tumor cell lysis ( 142,156 ) . nk cell interactions with antigen - presenting cells need to be considered in future immunotherapies of cancer , aiming to activate nk cells not only for their direct cytotoxic potential but also for enhancing the effectiveness of antitumor t - cell response , including tumor - specific t - cell memory , as required in cancer vaccine approaches ( figure 3 ) . tumor infiltrating natural killer cells ( tinks ) are characterized by a substantial pro - angiogenic subset able to produce vascular endothelial ( vegf ) , placental ( plgf ) growth factors and il8 ( cxcl-8 ) . several strategies to revert the normal anti - tumor activity are indicated : the cytotoxic activity in response to activating receptors ( ar ) and tumor necrosis factors ( tnfs ) and interferon ( ifn ) production of perforin - low ( perf ) tinks could be restored by exposure to il-2 , il-15 and/or il-12 , leading to a perf phenotype . because these cytokines also induce up - regulation of fc receptor riii ( cd16 ) , antibody - dependent cell cytotoxicity ( adcc ) could be restored by association of il-2/-15/-12 with monoclonal antibody ( mabs ) therapeutic approaches . cytokine treated cd56 tinks could regain the ability of dendritic cell ( dc ) editing , leading to a th1 polarization and promotion of an adaptive anti - tumor response . in addition , chemotherapy , alone or in association with il-2 and/or il-12 could be used to functionally switch tinks to an anti - tumor phenotype by down - regulating pro - angiogenic factors ( vegf , plgf , il-8 ) and promoting production of ifns and tnfs .

tumor - infiltrating leukocytes are often induced by the cancer microenvironment to display a protumor , proangiogenic phenotype . this polarization has been described for several myeloid cells , in particular macrophages . natural killer ( nk ) cells represent another population of innate immune cells able to infiltrate tumors . the role of nk in tumor progression and angiogenesis has not yet been fully investigated . several studies have shown that tumor - infiltrating nk ( here referred to as tinks ) and tumor - associated nk ( altered peripheral nk cells , which here we call tanks ) are compromised in their ability to lysew tumor cells . recent data have suggested that they are potentially protumorigenic and can also acquire a proangiogenic phenotype . here we review the properties of tinks and tanks and compare their activities to that of nk cells endowed with a physiological proangiogenic phenotype , in particular decidual nk cells . we speculate on the potential origins of tinks and tanks and on the immune signals involved in their differentiation and polarization . the tink and tank phenotype has broad implications in the immune response to tumors , ranging from a deficient control of cancer and cancer stem cells to an altered crosstalk with other relevant players of the immune response , such as dendritic cells , to induction of cancer angiogenesis . with this recently acquired knowledge that has not yet been put into perspective , we point out new potential avenues for therapeutic intervention involving nk cells as a target or an ally in oncology .

Angiogenesis and Immune Cells in Tumor Progression Physiological Proangiogenic Activity of NK Cells Similarities and Diversities Between Angiogenic NKs and TINKs/TANKs TINKs Have a Reduced Cytotoxic Potential Toward Cancer and Cancer Stem Cells Implications of TINK Polarization for Cancer Therapy The Natural Killer-Dendritic Cell Interface in the Development of Antitumor Immune Responses Implications for Novel Strategies of Cancer Immunotherapy Supplementary Material

several studies have shown that microvascular density is associated with prognosis and vascular endothelial growth factor ( vegf ) , the major angiogenic factor , has become a target for several cancers ( 36 ) . immune cells infiltrating tumors often show a skewed phenotype that reflects attenuation of antitumor activity and enhancement of protumor activities , including angiogenesis , in a process defined as tumor - induced polarization ( 40,41 ) . the classic example of tumor - induced polarization occurs in macrophages : m1 macrophages produce th1 cytokines ( such as il-12 and tnf ) and promote anti - tumor responses , while m2-polarized macrophages produce th2 cytokines , induce tissue reconstruction , growth promotion , and angiogenesis ( 42 ) . tumor - associated macrophages ( tams ) show an m2-like profile ( 42 ) ; other tumor - associated m2-like polarizations include myeloid - derived suppressor cells ( mdscs ) ( 43,44 ) and tie2 expressing macrophages closely associated with the vasculature ( 45,46 ) . several studies show a protumor and proangiogenic polarization for most immune cells in the tumor microenvironment , including macrophages , dendritic , mast , t and b cells , as well as polymorfonuclear cells ( pmn ) ( 4042,46,47 ) . a peculiar behavior of nk cells is found in the developing decidua ( table 1 ) , which converts them from killers to builders ( 48 ) . early on in pregnancy , decidual nk cells ( dnk ) accumulate to become approximately 70% of the local lymphocytes and 3040% of all decidual cells ( 49 ) . further , they have been found to be associated with induction of cd4 t regulatory ( treg ) cells ( 3 ) , and to produce il-10 ( 51 ) , thus potentially contributing to the immunosuppressive environment of the placenta . dnk express most of the nk cell hallmarks , including the activating receptors nkp46 , nkg2d , and 2b4 , as well as kir and cd94/nkg2a inhibitory receptors , along with markers not found on peripheral blood nk cell subsets , such as cd9 and cd49a ( 53 ) . in a different microenvironment , such as that of tumors , it has been shown that addition of angiogenic dnk cells to tumor cell xenografts markedly enhanced growth of tumors ( 51 ) . the origin of dnk cells is controversial ; expansion in loco from nk cell precursors ( 55 ) and conversion of peripheral blood nk cells to dnk upon exposure to decidual endothelial and stromal cells ( 56,57 ) have been proposed . decidual stromal cells produce high levels of tgf that has been reported to convert isolated peripheral blood cd56cd16 nk cells into cd56cd16 cells displaying poor cytotoxicity and expressing other markers , such as cd9 , in common with dnks ( 58,59 ) . these cells show low cytotoxicity ( 59 ) , high levels of vegf expression ( induced by hypoxia ) , expression of the dnk marker cd9 ( induced by tgf ) , and of kirs ( maintained by the demethylating agent ) . dnk cells tend to preferentially express cxcr3 and cxcr4 ( 6062 ) , in part in response to hormones ( 57,60 ) , and the respective ligands of these receptors are able to preferentially induce chemotaxis of cd56cd16 nk cells ( 57,60 ) . list of the key studies concerning the pro - angiogenic and pro - tumor role of natural killer cells * * the table lists selected references concerning the role of natural killer cells ( nk ) in tumor progression and angiogenesis and summarizes the principal findings grouped as : decidual nk ( dnk ) cells , which represent the most investigated subset in relation to angiogenesis . tumor infiltrating / associated nk cells , most of the references regard the impairment of nk cytotoxic activities with only one reference demonstrating a direct association between tumor infiltrating / associated nk cells and angiogenesis . further analyses showed that nk cells were required for angiogenesis in the damaged murine myocardium ( 64 ) and that engagement of klrg1 ( killer cell lectin - like receptor 1 ) by cadherins and 47 integrin by vcams ( vascular cell adhesion proteins ) were important for nk activation . given the angiogenic tissue construction activity of dnk and several similarities between the decidual , tissue repair , and tumor microenvironments , a working hypothesis ( 41 ) is that tinks or tanks could have a proangiogenic activity , resulting in tumor promotion rather than tumor inhibition ( table 1 ) . the nsclc tinks are localized in the tumor stroma ( 69 ) and at the invasive margin ( 70 ) of the tumor . these tinks express activating receptors and some of the cd56perforin nk cells display a discrete expression of kirs ( 69,70 ) . interestingly , this was not limited to the tink cells , as pro - angiogenic factor production was also observed in adjacent tissues and even peripheral blood ( tanks ) . further , supernatants of nsclc tinks showed angiogenesis - associated activities in vitro , in particular those of squamous cell carcinomas ( 66 ) . most nsclc tinks show low levels of the nk maturation markers cd11b and cd27 ( 72 ) , while the increased numbers of fully mature nk cells indicated by the cd57 marker were a positive prognostic indicator in nsclc squamous cell carcinomas ( 73 ) . these data imply that like dnk cells , tinks and tanks have some markers of mature nk cells , including granzyme and perforin , yet display low cytotoxicity , while producing angiogenesis - promoting factors . in the case of squamous nsclc , a potent effect of the tumor on the systemic nk phenotype was observed , since nk polarization was found even in peripheral blood . the data show that even a squamous cell carcinoma of small ( operable ) size had a substantial systemic effect on the immune system , in particular nk cells . a similar systemic alteration of tanks has been observed for colorectal ( 76 ) and breast ( 77 ) cancers , where these cells showed repression of nk functions . the low expression of perforin and granzyme on a large part of tinks , similar to that of cd16 peripheral blood nk cells , has suggested that tinks are derived from cd56cd16 nk cells , and that factors within the tumor microenvironment might induce expression of some kirs on these cells ( 69 ) . microarray studies ( 78 ) have suggested that dnk cells express a very different molecular profile as compared with cd56cd16 and cd56cd16 nk cells . interestingly , as compared with other nk cells , both dnk and nsclc tinks showed upregulation of several activation markers and in particular granzyme a ( 7879 ) . hypothesis of natural killer ( nk ) cell subset differentiation and functions in decidual and tumoral tissues . within the decidua ( shown as a pink area ) trophoblast cells release transforming growth factor ( tgf ) and express hla - g molecules associated with reduction of nk cytotoxicity and promotion of pro - angiogenic activity . within many tumor microenvironments ( shown as a grey area ) these elements have been associated with conversion of pb nk cells into poorly cytolytic dnk - like tumor infiltrating natural killer cells ( tinks ) cells able to release vascular endothelial ( vegf ) , placental ( plgf ) growth factors and il8 ( cxcl-8 ) , thus sustaining angiogenesis . it is not clear if tumor associated natural killer cells ( tanks ) are the result of emigration of tinks from the tumor microenvironment or due to systemic effects on nk cells of cancer - related products . the tumor microenvironment likely affects the polarization of cd56cd16 nk cells toward a proangiogenic phenotype , inducing the production of cytokines such as vegf , plgf , and il-8 . in cancer , tgf1 acts as a janus - like cytokine : in the initial phases of cancer formation , it behaves as a tumor suppressor , inhibiting tumor cell replication and favoring apoptosis ( 80,81 ) . as mentioned above , several studies from the strominger / kopcow groups have indicated that tgf , eventually together with other factors , can induce a dnk - like differentiation from peripheral blood cd56cd16 nk cells ( 58,59,82 ) . these data suggest that , within the decidua and the tumor microenvironment , tgf , along with other cues such as hypoxia , might result in the polarization of nk cell precursors or even mature nk cells toward angiogenic dnk - like cells . tumor - derived tgf has been shown to upregulate cxcr3 and cxcr4 in nk cells ( 84 ) , similar to what occurs for dnk cells ( 6062 ) , and to exert an immunosuppressive activity ( 85 ) . blocking of tgf has been shown to overcome immune suppression ( 85 ) and enhance the effects of nk cell therapy ( 86 ) . it is also possible that tumor - microenvironment associated factors induce a further modulation of cd56cd16 cells into the tink phenotype . hla - g expression can be quite heterogeneous within the tumor ; however , a mechanism involving exosome release and trogocytosis by neighboring tumor and immune cells , including nk cells , has been evoked to explain the apparent bystander effect of hla - g mediated immune suppression ( 91,92 ) . hla - g seems to exert its immunosuppressive effects through interactions with three key inhibitory receptors : immunoglobulin - like transcripts ilt-2 ( found on macrophages , dendritic , decidual and peripheral nk cells , as well as b and t lymphocytes ) , ilt-4 ( expressed by macrophages and dendritic cells ) , and kir2dl4 ( in decidual and peripheral blood nk cells ) ( 91,92 ) . hla - g interaction with the nonclassical kir2dl4 ( cd158d ) has been shown to stimulate resting nk cells to secrete proinflammatory and proangiogenic factors ( 95 ) through induction of a senescence - associated secretory phenotype ( 96 ) . in terms of angiogenic cytokine production within the tumor microenvironment , the relative contribution of nk cells remains to be determined , which likely depends on the tissue of origin . unlike other innate cells , within the tumor microenvironment there is a strong presence of nk - activating ligands that could enhance the cytokine - producing activity of tinks , as has been found in the decidua ( 100 ) . nk cells also interact with other immune cells , modulating both their presence and activation state . as examples , in the cornea and choroid , nk depletion was associated with reduced macrophage recruitment and loss of angiogenic potential ( 65 ) ; in the premetastatic niche , nk cells are associated with mdsc accumulation ( 101 ) . in nsclc , tinks have been shown to be highly enriched in the cd56perforin nk cell subset ( 66,69 ) , a phenotype quite similar to that of tinks from colorectal ( 76 ) and breast ( 77 ) cancers . in terms of total lymphocytes , nk cell frequency varies depending on the tissue , ranging from relatively high ( approximately 25% in kidney ; 15% in liver and subcutaneous adipose tissues ) to quite low ( 14% in colon , stomach , adrenal gland ) ( 104 ) . the comparative analyses of the chemokine expression patterns of tinks with those derived from matched healthy tissues showed that the tumor microenvironment might account for this specific accumulation of nk with the tink phenotype . neoplastic lung tissues showed a marked downregulation of cxcl2 , a chemokine able to specifically attract cd56 nk cells , since its cognate receptor , cxcr2 , is expressed by cd56 cytotoxic nk cells but not on the cd56 counterpart . remarkably , the frequency of tinks among the total tumor - infiltrating lymphocytes did not show statistically significant differences when compared to the percent of nk cells as a function of total lymphocytes from normal matched tissues ( 66,69,104,105 ) . these recent data suggest that neoplastic transformation did not determine an increased migration of nk cells within the tissues . rather , as a consequence of switches in chemokine expression patterns , cytotoxic nk cells are substantially decreased compared with healthy tissues , at least in some frequently occurring cancers such as breast and lung carcinomas . this new evidence now emerges as a further mechanism of cancer immunoediting with implications for both immunosurveillance and tumor escape from immune attack . by definition , cscs / cics are those cells able to give rise to new tumors , clinically leading to tumor recurrence and metastatic disease . glioblastoma stem cells , which express low levels of mhc - class i molecules and high levels of the activating dnam-1 ligands pvr and nectin-2 , were susceptible to lysis by nk cells following il-2 or il-15 activation ( 110 ) . nk cells have been found to be able to kill oral squamous carcinoma stem cells as well as bona fide embryonic , mesenchymal , and dental pulp stem cells ( 111 ) . increasing experimental ( 86,115121 ) and clinical ( 86,117,122 ) evidence indicate that nk cells modulate metastatic dissemination , suggesting that nk cells are able to kill the cscs / cics that give rise to metastatic disease . a murine in vivo study showed that tam family tyrosine kinases are involved in tumor - induced nk attenuation ( 121 ) . it becomes clear that the compromised cytotoxic activity of nk cells is also implicated in the lack of csc / cic killing in cancer patients . the potential tumor - cell killing capacity of nk cells suggests that these cells could be useful in therapeutic approaches ( 124 ) . in the case of metronomic cyclophosphamide , a role for vegf receptors in activating nk cells has been suggested ( 131 ) . the adcc process is currently recognized as a major mechanism of action of several targeted therapies for the treatment of neoplastic diseases : these therapies aim to exploit nk cell adcc by the administration of tumor - specific monoclonal antibodies ( mab ) ( 134 ) . cd56perforin nk cells upon cytokine - induced activation ( exposure to il-2 , il-15 , or both ) , can convert into perforin nk cells able to efficiently kill cancer cells ( 9,10 ) . although further studies are warranted to better clarify the functions and the possible differentiation pathways of tumor - infiltrating nk cell subsets , the use of il-2 or il-15 , currently available as pharmaceutical grade compounds , can be envisaged in association with current tumor - specific , mab - mediated target therapies ( 124 ) . nk cells are currently considered in cancer immunotherapy but most studies have focused on the single goal of exploiting their direct antitumor cytotoxic potential . for example , nk cells can rapidly expand in vitro and in vivo , in particular following haploallogenic bone marrow transplantation , where they appear to serve in both host defense as well as inhibition of graft - vs - host reactions ( 135 ) . it is now well established that interactions occurring between nk cells and dendritic cells ( dcs ) play a major role in the priming of antitumor responses ( figure 2 ) . natural killer ( nk ) cell editing of dendritic cells ( dcs ) plays a crucial role in the regulation of both innate and adaptive immunity . nk cells also release ifn and can eliminate cancer stem cells ( cscs ) . in the normal context / microenvironment , nk cells sustain th1 polarization , necessary for an effective adaptive immune response against tumors . in the tumor microenvironment ( grey area ) nk cells are characterized by poor cytotoxicity , leading to idc accumulation , block of cytolytic t cell responses and inducing immune tolerance of tumor cells . several studies have shown that nk cells can be efficiently activated by dcs to release cytokines , to proliferate , and to enhance their cytotoxicity ( 136,142,143 ) . activated nk cells can then kill tumor cells , but , at the same time , play a relevant immunoregulatory role by editing dcs ( 144 ) ( figure 2 ) . the decreased cytotoxicity of tumor - infiltrating nk cells could substantially alter dc - nk interactions ( figure 2 ) , where poorly cytotoxic nk cells would not only have reduced capacity to kill tumor cells , but also reduced editing of immature dendritic cells ( 2 ) . in the tumor microenvironment , nk editing of dc might be compromised , leading to tolerogenic dc ( figure 2 ) , similar to that found in hiv infection , where lack of nk editing leads to increased immature dcs and likely subsequent immune suppression ( 148 ) . on the other hand , nk cells can also shape adaptive immune responses by causing dc maturation and influencing the polarization of primary t - cell responses ( 149152 ) . this helper role of nk cells for the development of adaptive immunity takes place via the release of soluble factors , such as tnf and ifn , ( 138,143,153 ) . taken together , these data show that nk cells are not only activated by dcs but , in turn , can also influence the emerging adaptive immune responses that are initiated by these antigen - presenting cells ( figure 2 ) . in this regard , it is noteworthy that vaccination with unpulsed exogenous dcs ( ie , dcs carrying no tumor antigens ) has been shown to induce tumor - specific t cells in a tumor - bearing host via the activation of nk cells . nk cell interactions with antigen - presenting cells need to be considered in future immunotherapies of cancer , aiming to activate nk cells not only for their direct cytotoxic potential but also for enhancing the effectiveness of antitumor t - cell response , including tumor - specific t - cell memory , as required in cancer vaccine approaches ( figure 3 ) . tumor infiltrating natural killer cells ( tinks ) are characterized by a substantial pro - angiogenic subset able to produce vascular endothelial ( vegf ) , placental ( plgf ) growth factors and il8 ( cxcl-8 ) . several strategies to revert the normal anti - tumor activity are indicated : the cytotoxic activity in response to activating receptors ( ar ) and tumor necrosis factors ( tnfs ) and interferon ( ifn ) production of perforin - low ( perf ) tinks could be restored by exposure to il-2 , il-15 and/or il-12 , leading to a perf phenotype .

calcineurin inhibitor ( cni-)-based immunosuppressive therapy is mainly used to prevent acute rejection episodes ( ares ) in patients after heart transplantation ( htx).1,2 referring to current international society for heart and lung transplantation registry data , the use of cyclosporine a ( csa ) decreased in recent years , and accordingly , tacrolimus ( tac ) has become the predominant cni in patients after htx , as tac is more effective in avoiding ares.1,37 given the improved patient mortality during the last decades , possible toxic effects of immunosuppression , especially nephrotoxicity , have to be considered more carefully.8 in addition , prevention of severe renal dysfunction becomes all the more important , as heart transplanted patients receiving long - term dialysis have adverse survival rates.9,10 for this reason , this study was initiated to compare both cni immunosuppressive regimens regarding renal function within the first 2 years after htx in patients continuously receiving the primarily applied cni regimen . primary endpoint was change in renal function assessed by modification of diet in renal disease ( mdrd ) equation during the first 2 years after htx . in addition , we compared patients receiving conventional tacrolimus ( ctac ) with patients receiving extended - release tacrolimus ( etac ) regarding renal function . all human studies were reviewed by the ethics committee of the university of heidelberg and have been performed in accordance with the ethical standards laid down in the 2008 declaration of helsinki . a total of 150 patients who were continuously receiving the primary applied cni within first 2 years after htx were retrospectively analyzed . inclusion criteria included cni - based immunosuppression without change of primarily applied cni within the first 2 years after htx ( per protocol analysis ) . exclusion criteria were external follow - up and death within first 2 years after transplantation . patients underwent htx between may 1998 and october 2010 at the heidelberg heart transplantation center in germany . all patients received routine follow - up visits according to center standard , including physical examination , and routine laboratory analysis , including immunosuppressive drug monitoring . routine follow - up visits were performed monthly after primary discharge after htx until month 6 , bimonthly from month 6 to month 12 , and every 3 months in the second year after transplantation . renal function was analyzed by mdrd equation and serum creatinine levels.11 for the purpose of the present study , renal function tests immediately after htx as baseline value and at months 6 , 12 , and 24 after htx were analyzed . referring to the national kidney foundation , patients were divided into three groups , based on estimated glomerular filtration rate ( egfr ) , assessed by mdrd equation.12 in group 1 , egfr was < 30 ml / minute/1.73 m , in group 2 , egfr was 30 and 60 ml / minute/1.73 m , and in group 3 , egfr was > 60 ml / minute/1.73 m. in subgroup analysis , patients renal function was analyzed according to primarily applied tac formulation . laboratory analysis was performed during routine follow - up visits , including complete blood count , liver function tests , blood glucose levels , and blood lipid profile . immunosuppressive medication was applied and monitored according to the center s routine protocol . in line with the center s routine protocol , target levels of immunosuppressive drugs were reduced during the study period.13 in general , cni therapy was part of a dual immunosuppressive regimen . in 2001 , steroids were routinely applied during the first half year after htx and were tapered whenever possible . numerical data were expressed as mean value standard deviation or were described as absolute numbers or percentages . analyses of differences in groups were detected using t - test for continuous data and chi - square - test for categorical data . to detect significant deterioration of renal function within groups between study visits , mcnemar s test was used . a total of 150 patients who were continuously receiving the primary applied cni within first 2 years after htx were retrospectively analyzed . inclusion criteria included cni - based immunosuppression without change of primarily applied cni within the first 2 years after htx ( per protocol analysis ) . exclusion criteria were external follow - up and death within first 2 years after transplantation . patients underwent htx between may 1998 and october 2010 at the heidelberg heart transplantation center in germany . all patients received routine follow - up visits according to center standard , including physical examination , and routine laboratory analysis , including immunosuppressive drug monitoring . routine follow - up visits were performed monthly after primary discharge after htx until month 6 , bimonthly from month 6 to month 12 , and every 3 months in the second year after transplantation . renal function was analyzed by mdrd equation and serum creatinine levels.11 for the purpose of the present study , renal function tests immediately after htx as baseline value and at months 6 , 12 , and 24 after htx were analyzed . referring to the national kidney foundation , patients were divided into three groups , based on estimated glomerular filtration rate ( egfr ) , assessed by mdrd equation.12 in group 1 , egfr was < 30 ml / minute/1.73 m , in group 2 , egfr was 30 and 60 ml / minute/1.73 m , and in group 3 , egfr was > 60 ml / minute/1.73 m. in subgroup analysis , patients renal function was analyzed according to primarily applied tac formulation . laboratory analysis was performed during routine follow - up visits , including complete blood count , liver function tests , blood glucose levels , and blood lipid profile . immunosuppressive medication was applied and monitored according to the center s routine protocol . in line with the center s routine protocol , target levels of immunosuppressive drugs were reduced during the study period.13 in general , cni therapy was part of a dual immunosuppressive regimen . in 2001 , steroids were routinely applied during the first half year after htx and were tapered whenever possible . numerical data were expressed as mean value standard deviation or were described as absolute numbers or percentages . analyses of differences in groups were detected using t - test for continuous data and chi - square - test for categorical data . to detect significant deterioration of renal function within groups between study visits , mcnemar s test was used . seventy - eight patients ( 52.0% ) received a csa - based immunosuppressive regimen , and 72 patients ( 48.0% ) received a tac - based immunosuppressive therapy continuously during the first 2 years after htx . within the tac group , 29 patients ( 40.3% ) received a ctac - based de novo regimen , and 43 ( 59.7% ) patients received an etac immunosuppressive regimen ( as soon as oral uptake was possible ) . in each subgroup , five patients changed their primarily applied immunosuppressive regimen from ctac to etac , respectively etac to ctac . regarding age at htx , no statistically significant differences between the groups were detectable ( p = not statistically significant [ ns ] ) . however , in patients receiving a tac - based immunosuppressive regimen , ischemic time was significantly longer ( p<0.0001 ) , donors were older ( p<0.0001 ) , percentage of male donors was lower ( p=0.0037 ) , and sex mismatch was higher ( p=0.038 ) . further analysis of diagnoses leading to htx detected a significantly higher percentage of patients transplanted due to coronary heart disease in the csa group ( p=0.0134 ) ; all other principal diagnoses leading to htx did not differ statistically significantly between both groups ( all p = ns ) . regarding concomitant medication , no statistically significant differences regarding use of statins was seen between groups 2 years after htx ( p = ns ) . analysis of renal function referring to egfr by mdrd equation is shown in figure 1 . renal function during the study period according to serum creatinine levels is described in figure 2 . analysis of renal function at each visit with regard to egfr detected no statistically significant differences between patients receiving tac and patients receiving csa ( all p = ns ; table 2 ) . a significant decrease of egfr during the study period was detectable in both groups ( csa group , 2 years after htx vs baseline , p<0.0001 ; tac group , 2 years after htx vs baseline , p=0.0094 ) . mcnemar s test detected a significant accumulation of patients with deterioration of renal function in the first half year after htx among patients receiving csa ( p=0.0004 ) . in patients receiving tac , no significant accumulation of patients with deterioration of renal function during the first 2 years after htx was detectable ( all p = ns ) . figures 3 and 4 show the composition of both cni groups referring to egfr at performed study visits . within the first 2 years after transplantation , serum creatinine increased significantly in csa patients ( 2 years after htx vs baseline p<0.0001 ) , whereas in patients receiving tac , serum creatinine did not differ significantly from baseline values ( p = ns ) . at baseline , serum creatinine in patients receiving csa was significantly lower ( csa , 1.120.53 mg / dl ; tac , 1.470.94 mg / dl ; p=0.0066 ) . starting from month 6 after htx , serum creatinine levels in patients receiving csa and patients receiving tac did not differ statistically significantly ( all p = ns ) . analysis of serum electrolytes , that is , serum sodium and serum potassium , detected no statistically significant differences between both cni regimens ( all p = ns ) . changes within both study groups regarding egfr , serum creatinine , and electrolytes versus baseline values are given in table 2 . analyzing egfr in tac subgroups detected no statistically significant differences between patients primarily receiving an etac or patients receiving a ctac immunosuppressive regimen at all performed study visits ( all p = ns ) . at baseline , egfr in patients receiving ctac treatment was 71.6241.17 ml / minute/1.73 m versus 70.1639.28 ml / minute/1.73 m after the study period , egfr was 57.4627.64 ml / minute/1.73 m in patients receiving ctac versus 57.7123.25 ml / minute/1.73 m in patients receiving etac ( p = ns ) . analysis of serum creatinine detected no statistically significant differences between patients receiving ctac and patients receiving etac at performed study visits ( all p = ns ) . no statistically significant differences between patients receiving csa- and tac - based therapy were found regarding serum triglycerides and high - density lipoprotein at baseline and during the study period ( all p = ns ) . in contrast to patients receiving csa , within the patients receiving tac , a significant reduction of serum triglyceride levels during the study period was observed ( 2 years after htx vs baseline , p=0.0031 ) . analysis of serum cholesterol levels detected no significant differences within the first year after htx . beginning at month 12 after transplantation , statistically significant differences regarding blood cholesterol levels were seen between both groups ( month 12 after htx , p=0.0002 ; month 24 after htx , p=0.0252 ) . however , a significantly lower blood cholesterol level was detected in both cni groups 2 years after htx versus baseline values ( patients receiving csa : 2 years after htx vs baseline , p=0.0010 ; patients receiving tac : 2 years after htx vs baseline , p=0.0003 ) . two years after htx , high - density lipoprotein levels were significantly lower in both groups ( csa : 2 years vs baseline , p=0.0004 ; tac : 2 years vs baseline , p=0.0002 ) , low - density lipoprotein was continuously higher in patients receiving csa at all performed study visits . except from month 6 after htx , serum glucose did not differ significantly between patients receiving csa and patients receiving tac ( p=0.0169 ) . analyzing liver function enzymes detected continuously higher values of aspartate aminotransferase and alanine aminotransferase at all performed study visits in patients receiving tac . in addition , gamma - glutamyl - transferase was significantly higher in patients receiving a tac - based immunosuppressive regimen , and a higher serum bilirubin level was detected in patients with de novo csa therapy during study period ( all p<0.0001 ) . with regard to blood pressure profile , no statistically significant differences between groups were seen during the first year after htx ( all p = ns ) . beginning in month 12 after htx , systolic blood pressure was significantly higher in patients receiving csa . immunosuppressive drug trough levels are described in table 4 , and applied doses of immunosuppression are described in table 5 . no statistically significant differences regarding immunosuppressive drug trough levels were found between the ctac and etac groups ( all p = ns ) . only at baseline , patients receiving etac received significantly higher tac doses ( etac : 11.714.71 mg ; ctac : 8.143.13 mg ; p=0.0002 ) . seventy - eight patients ( 52.0% ) received a csa - based immunosuppressive regimen , and 72 patients ( 48.0% ) received a tac - based immunosuppressive therapy continuously during the first 2 years after htx . within the tac group , 29 patients ( 40.3% ) received a ctac - based de novo regimen , and 43 ( 59.7% ) patients received an etac immunosuppressive regimen ( as soon as oral uptake was possible ) . in each subgroup , five patients changed their primarily applied immunosuppressive regimen from ctac to etac , respectively etac to ctac . regarding age at htx , no statistically significant differences between the groups were detectable ( p = not statistically significant [ ns ] ) . however , in patients receiving a tac - based immunosuppressive regimen , ischemic time was significantly longer ( p<0.0001 ) , donors were older ( p<0.0001 ) , percentage of male donors was lower ( p=0.0037 ) , and sex mismatch was higher ( p=0.038 ) . further analysis of diagnoses leading to htx detected a significantly higher percentage of patients transplanted due to coronary heart disease in the csa group ( p=0.0134 ) ; all other principal diagnoses leading to htx did not differ statistically significantly between both groups ( all p = ns ) . regarding concomitant medication , no statistically significant differences regarding use of statins was seen between groups 2 years after htx ( p = ns ) . analysis of renal function referring to egfr by mdrd equation is shown in figure 1 . renal function during the study period according to serum creatinine levels is described in figure 2 . analysis of renal function at each visit with regard to egfr detected no statistically significant differences between patients receiving tac and patients receiving csa ( all p = ns ; table 2 ) . a significant decrease of egfr during the study period was detectable in both groups ( csa group , 2 years after htx vs baseline , p<0.0001 ; tac group , 2 years after htx vs baseline , p=0.0094 ) . mcnemar s test detected a significant accumulation of patients with deterioration of renal function in the first half year after htx among patients receiving csa ( p=0.0004 ) . in patients receiving tac , no significant accumulation of patients with deterioration of renal function during the first 2 years after htx was detectable ( all p = ns ) . figures 3 and 4 show the composition of both cni groups referring to egfr at performed study visits . within the first 2 years after transplantation , serum creatinine increased significantly in csa patients ( 2 years after htx vs baseline p<0.0001 ) , whereas in patients receiving tac , serum creatinine did not differ significantly from baseline values ( p = ns ) . at baseline , serum creatinine in patients receiving csa was significantly lower ( csa , 1.120.53 mg / dl ; tac , 1.470.94 mg / dl ; p=0.0066 ) . starting from month 6 after htx , serum creatinine levels in patients receiving csa and patients receiving tac did not differ statistically significantly ( all p = ns ) . analysis of serum electrolytes , that is , serum sodium and serum potassium , detected no statistically significant differences between both cni regimens ( all p = ns ) . changes within both study groups regarding egfr , serum creatinine , and electrolytes versus baseline values are given in table 2 . analyzing egfr in tac subgroups detected no statistically significant differences between patients primarily receiving an etac or patients receiving a ctac immunosuppressive regimen at all performed study visits ( all p = ns ) . at baseline , egfr in patients receiving ctac treatment was 71.6241.17 ml / minute/1.73 m versus 70.1639.28 ml / minute/1.73 m after the study period , egfr was 57.4627.64 ml / minute/1.73 m in patients receiving ctac versus 57.7123.25 ml / minute/1.73 m in patients receiving etac ( p = ns ) . analysis of serum creatinine detected no statistically significant differences between patients receiving ctac and patients receiving etac at performed study visits ( all p = ns ) . no statistically significant differences between patients receiving csa- and tac - based therapy were found regarding serum triglycerides and high - density lipoprotein at baseline and during the study period ( all p = ns ) . in contrast to patients receiving csa , within the patients receiving tac , a significant reduction of serum triglyceride levels during the study period was observed ( 2 years after htx vs baseline , p=0.0031 ) . analysis of serum cholesterol levels detected no significant differences within the first year after htx . beginning at month 12 after transplantation , statistically significant differences regarding blood cholesterol levels were seen between both groups ( month 12 after htx , p=0.0002 ; month 24 after htx , p=0.0252 ) . however , a significantly lower blood cholesterol level was detected in both cni groups 2 years after htx versus baseline values ( patients receiving csa : 2 years after htx vs baseline , p=0.0010 ; patients receiving tac : 2 years after htx vs baseline , p=0.0003 ) . two years after htx , high - density lipoprotein levels were significantly lower in both groups ( csa : 2 years vs baseline , p=0.0004 ; tac : 2 years vs baseline , p=0.0002 ) , low - density lipoprotein was continuously higher in patients receiving csa at all performed study visits . except from month 6 after htx , serum glucose did not differ significantly between patients receiving csa and patients receiving tac ( p=0.0169 ) . analyzing liver function enzymes detected continuously higher values of aspartate aminotransferase and alanine aminotransferase at all performed study visits in patients receiving tac . in addition , gamma - glutamyl - transferase was significantly higher in patients receiving a tac - based immunosuppressive regimen , and a higher serum bilirubin level was detected in patients with de novo csa therapy during study period ( all p<0.0001 ) . with regard to blood pressure profile , no statistically significant differences between groups were seen during the first year after htx ( all p = ns ) . beginning in month 12 after htx , systolic blood pressure was significantly higher in patients receiving csa . immunosuppressive drug trough levels are described in table 4 , and applied doses of immunosuppression are described in table 5 . no statistically significant differences regarding immunosuppressive drug trough levels were found between the ctac and etac groups ( all p = ns ) . only at baseline , patients receiving etac received significantly higher tac doses ( etac : 11.714.71 mg ; ctac : 8.143.13 mg ; p=0.0002 ) . the primary endpoint of our study was to evaluate the nephrotoxic effects of a tac- or csa - based immunosuppressive regimen in patients after htx . to detect possible effects caused by cni , we only analyzed the data of patients who received the same cni , that is , csa or tac , for 2 years after htx . analyzing renal function by means of serum creatinine detected a significant deterioration of renal function in patients receiving csa as their baseline immunosuppressive regimen within the first 2 years after htx ( 2 years after htx vs baseline , p<0.0001 ) . in patients receiving tac , no statistically significant deterioration of renal function measured by serum creatinine was detectable ( 2 years after htx vs baseline , p = ns ) . as the mdrd equation is a more appropriate marker to assess renal function than serum creatinine , we used the mdrd equation to analyze renal function more precisely in our study cohort.11 analyzing renal function according to the mdrd equation detected a significant decrease of egfr , both in patients receiving csa and in patients receiving tac . on the basis of egfr , we divided both cni groups into three subgroups to analyze the composition of both groups at performed study visits . to detect a significant shift of patients with deterioration of renal function between study visits in contrast to recently published data reporting similar nephrotoxic effects of both cnis , we detected significant deterioration of renal function in patients receiving csa.6,1416 our data underline data reporting better renal function of tac - based immunosuppression , as a significant accumulation of patients with deterioration of renal function was only seen in patients receiving csa immunosuppressive treatment within the first 6 months after htx.4,17 between all other study visits , a significant shift of patients receiving csa with deterioration of renal function was not seen ( all p = ns ) . analyzing patients receiving tac by mcnemar data comparing etac and ctac are rarely published ; however , in contrast to recently published data in patients after renal transplantation , no significant differences regarding renal function in patients receiving either etac or ctac therapy were found in our study cohort.18 to clarify the effects on renal function of both tac regimes , randomized controlled studies should be initiated . the importance of local renal factors , for example , variability in p - glycoprotein and cyp3a4/5 expression or activity , older kidney age , salt depletion , the use of nonsteroidal anti - inflammatory drugs , and genetic polymorphisms in genes such as tgf- and ace , in comparison with systemic exposure to cnis , for susceptibility to cni - induced nephrotoxicity has been previously published.19 in addition , the effects of comedication ( eg , ace - inhibitors , amlodipine ) , cni avoidance and dose minimization , and simultaneous injection of anti - tgf- antibodies have to be taken into account.2026 cholestasis and hepatotoxic effects in patients receiving a tac - based medication have already been detected in previously published case reports ; however , both cnis are suspected to cause cholestasis.2729 in line with previously published data , blood cholesterol levels and systolic blood pressure values were significantly higher in patients receiving csa.7,14,15,30 higher systolic blood pressure may partly be explained by a higher sympathetic tone caused by vasoconstriction of afferent and efferent glomerular arterioles.31,32 our retrospective study was performed as a single - center study , and patients were followed up according to the center s specific routine follow - up care . moreover , we only considered patients without change of cni immunosuppressive regimen to analyze specific cni effects on renal function . a possible era effect can not be ruled out , given the long follow - up period . our retrospective study was performed as a single - center study , and patients were followed up according to the center s specific routine follow - up care . moreover , we only considered patients without change of cni immunosuppressive regimen to analyze specific cni effects on renal function . a possible era effect can not be ruled out , given the long follow - up period . the superior rejection profile of the tac - based immunosuppressive regimen had already been demonstrated previously.7 in addition , our data detect the favorable effects of a tac - based immunosuppression with respect to nephrotoxicity , as csa immunosuppressive therapy was associated with a significant deterioration of renal function ( mcnemar s test ) , especially in the first 6 months after htx , when high immunosuppressive drug trough levels are needed to prevent ares . given the increasingly older htx recipients , with a multitude of comorbidities , preservation of renal function is of eminent interest . therefore , in our opinion , a tac - based immunosuppressive regimen is favorable after htx .

backgroundnephrotoxicity is a serious adverse effect of calcineurin inhibitor therapy in patients after heart transplantation ( htx).aimin this retrospective registry study , renal function within the first 2 years after htx in patients receiving de novo calcineurin inhibitor treatment , that is , cyclosporine a ( csa ) or tacrolimus ( tac ) , was analyzed . in a consecutive subgroup analysis , renal function in patients receiving conventional tacrolimus ( ctac ) was compared with that of patients receiving extended - release tacrolimus ( etac).methodsdata from 150 htx patients at heidelberg heart transplantation center were retrospectively analyzed . all patients were continuously receiving the primarily applied calcineurin inhibitor during the first 2 years after htx and received follow - up care according to center practice.resultswithin the first 2 years after htx , serum creatinine increased significantly in patients receiving csa ( p<0.0001 ) , whereas in patients receiving tac , change of serum creatinine was not statistically significant ( p = not statistically significant [ ns ] ) . mcnemar s test detected a significant accumulation of patients with deterioration of renal function in the first half year after htx among patients receiving csa ( p=0.0004 ) . in patients receiving tac , no significant accumulation of patients with deterioration of renal function during the first 2 years after htx was detectable ( all p = ns ) . direct comparison of patients receiving ctac versus those receiving etac detected no significant differences regarding renal function between patients primarily receiving ctac or etac treatment during study period ( all p = ns).conclusioncsa is associated with a more pronounced deterioration of renal function , especially in the first 6 months after htx , in comparison with patients receiving tac as baseline immunosuppressive therapy .

Introduction Patients and methods Patients Renal function Routine laboratory testing and immunosuppressive drug-level monitoring Statistical analysis Results Patient demographic and baseline characteristics Renal function and serum electrolytes Laboratory evaluation and physical data Immunosuppressive regimen and measured drug levels Discussion Limitations Conclusion

calcineurin inhibitor ( cni-)-based immunosuppressive therapy is mainly used to prevent acute rejection episodes ( ares ) in patients after heart transplantation ( htx).1,2 referring to current international society for heart and lung transplantation registry data , the use of cyclosporine a ( csa ) decreased in recent years , and accordingly , tacrolimus ( tac ) has become the predominant cni in patients after htx , as tac is more effective in avoiding ares.1,37 given the improved patient mortality during the last decades , possible toxic effects of immunosuppression , especially nephrotoxicity , have to be considered more carefully.8 in addition , prevention of severe renal dysfunction becomes all the more important , as heart transplanted patients receiving long - term dialysis have adverse survival rates.9,10 for this reason , this study was initiated to compare both cni immunosuppressive regimens regarding renal function within the first 2 years after htx in patients continuously receiving the primarily applied cni regimen . primary endpoint was change in renal function assessed by modification of diet in renal disease ( mdrd ) equation during the first 2 years after htx . in addition , we compared patients receiving conventional tacrolimus ( ctac ) with patients receiving extended - release tacrolimus ( etac ) regarding renal function . a total of 150 patients who were continuously receiving the primary applied cni within first 2 years after htx were retrospectively analyzed . inclusion criteria included cni - based immunosuppression without change of primarily applied cni within the first 2 years after htx ( per protocol analysis ) . all patients received routine follow - up visits according to center standard , including physical examination , and routine laboratory analysis , including immunosuppressive drug monitoring . routine follow - up visits were performed monthly after primary discharge after htx until month 6 , bimonthly from month 6 to month 12 , and every 3 months in the second year after transplantation . renal function was analyzed by mdrd equation and serum creatinine levels.11 for the purpose of the present study , renal function tests immediately after htx as baseline value and at months 6 , 12 , and 24 after htx were analyzed . referring to the national kidney foundation , patients were divided into three groups , based on estimated glomerular filtration rate ( egfr ) , assessed by mdrd equation.12 in group 1 , egfr was < 30 ml / minute/1.73 m , in group 2 , egfr was 30 and 60 ml / minute/1.73 m , and in group 3 , egfr was > 60 ml / minute/1.73 m. in subgroup analysis , patients renal function was analyzed according to primarily applied tac formulation . in 2001 , steroids were routinely applied during the first half year after htx and were tapered whenever possible . to detect significant deterioration of renal function within groups between study visits , mcnemar s test was used . a total of 150 patients who were continuously receiving the primary applied cni within first 2 years after htx were retrospectively analyzed . inclusion criteria included cni - based immunosuppression without change of primarily applied cni within the first 2 years after htx ( per protocol analysis ) . all patients received routine follow - up visits according to center standard , including physical examination , and routine laboratory analysis , including immunosuppressive drug monitoring . renal function was analyzed by mdrd equation and serum creatinine levels.11 for the purpose of the present study , renal function tests immediately after htx as baseline value and at months 6 , 12 , and 24 after htx were analyzed . referring to the national kidney foundation , patients were divided into three groups , based on estimated glomerular filtration rate ( egfr ) , assessed by mdrd equation.12 in group 1 , egfr was < 30 ml / minute/1.73 m , in group 2 , egfr was 30 and 60 ml / minute/1.73 m , and in group 3 , egfr was > 60 ml / minute/1.73 m. in subgroup analysis , patients renal function was analyzed according to primarily applied tac formulation . in 2001 , steroids were routinely applied during the first half year after htx and were tapered whenever possible . to detect significant deterioration of renal function within groups between study visits , mcnemar s test was used . seventy - eight patients ( 52.0% ) received a csa - based immunosuppressive regimen , and 72 patients ( 48.0% ) received a tac - based immunosuppressive therapy continuously during the first 2 years after htx . regarding age at htx , no statistically significant differences between the groups were detectable ( p = not statistically significant [ ns ] ) . however , in patients receiving a tac - based immunosuppressive regimen , ischemic time was significantly longer ( p<0.0001 ) , donors were older ( p<0.0001 ) , percentage of male donors was lower ( p=0.0037 ) , and sex mismatch was higher ( p=0.038 ) . further analysis of diagnoses leading to htx detected a significantly higher percentage of patients transplanted due to coronary heart disease in the csa group ( p=0.0134 ) ; all other principal diagnoses leading to htx did not differ statistically significantly between both groups ( all p = ns ) . regarding concomitant medication , no statistically significant differences regarding use of statins was seen between groups 2 years after htx ( p = ns ) . renal function during the study period according to serum creatinine levels is described in figure 2 . analysis of renal function at each visit with regard to egfr detected no statistically significant differences between patients receiving tac and patients receiving csa ( all p = ns ; table 2 ) . a significant decrease of egfr during the study period was detectable in both groups ( csa group , 2 years after htx vs baseline , p<0.0001 ; tac group , 2 years after htx vs baseline , p=0.0094 ) . mcnemar s test detected a significant accumulation of patients with deterioration of renal function in the first half year after htx among patients receiving csa ( p=0.0004 ) . in patients receiving tac , no significant accumulation of patients with deterioration of renal function during the first 2 years after htx was detectable ( all p = ns ) . within the first 2 years after transplantation , serum creatinine increased significantly in csa patients ( 2 years after htx vs baseline p<0.0001 ) , whereas in patients receiving tac , serum creatinine did not differ significantly from baseline values ( p = ns ) . at baseline , serum creatinine in patients receiving csa was significantly lower ( csa , 1.120.53 mg / dl ; tac , 1.470.94 mg / dl ; p=0.0066 ) . starting from month 6 after htx , serum creatinine levels in patients receiving csa and patients receiving tac did not differ statistically significantly ( all p = ns ) . analysis of serum electrolytes , that is , serum sodium and serum potassium , detected no statistically significant differences between both cni regimens ( all p = ns ) . analyzing egfr in tac subgroups detected no statistically significant differences between patients primarily receiving an etac or patients receiving a ctac immunosuppressive regimen at all performed study visits ( all p = ns ) . at baseline , egfr in patients receiving ctac treatment was 71.6241.17 ml / minute/1.73 m versus 70.1639.28 ml / minute/1.73 m after the study period , egfr was 57.4627.64 ml / minute/1.73 m in patients receiving ctac versus 57.7123.25 ml / minute/1.73 m in patients receiving etac ( p = ns ) . analysis of serum creatinine detected no statistically significant differences between patients receiving ctac and patients receiving etac at performed study visits ( all p = ns ) . no statistically significant differences between patients receiving csa- and tac - based therapy were found regarding serum triglycerides and high - density lipoprotein at baseline and during the study period ( all p = ns ) . in contrast to patients receiving csa , within the patients receiving tac , a significant reduction of serum triglyceride levels during the study period was observed ( 2 years after htx vs baseline , p=0.0031 ) . analysis of serum cholesterol levels detected no significant differences within the first year after htx . however , a significantly lower blood cholesterol level was detected in both cni groups 2 years after htx versus baseline values ( patients receiving csa : 2 years after htx vs baseline , p=0.0010 ; patients receiving tac : 2 years after htx vs baseline , p=0.0003 ) . two years after htx , high - density lipoprotein levels were significantly lower in both groups ( csa : 2 years vs baseline , p=0.0004 ; tac : 2 years vs baseline , p=0.0002 ) , low - density lipoprotein was continuously higher in patients receiving csa at all performed study visits . except from month 6 after htx , serum glucose did not differ significantly between patients receiving csa and patients receiving tac ( p=0.0169 ) . in addition , gamma - glutamyl - transferase was significantly higher in patients receiving a tac - based immunosuppressive regimen , and a higher serum bilirubin level was detected in patients with de novo csa therapy during study period ( all p<0.0001 ) . with regard to blood pressure profile , no statistically significant differences between groups were seen during the first year after htx ( all p = ns ) . beginning in month 12 after htx , systolic blood pressure was significantly higher in patients receiving csa . no statistically significant differences regarding immunosuppressive drug trough levels were found between the ctac and etac groups ( all p = ns ) . seventy - eight patients ( 52.0% ) received a csa - based immunosuppressive regimen , and 72 patients ( 48.0% ) received a tac - based immunosuppressive therapy continuously during the first 2 years after htx . regarding age at htx , no statistically significant differences between the groups were detectable ( p = not statistically significant [ ns ] ) . however , in patients receiving a tac - based immunosuppressive regimen , ischemic time was significantly longer ( p<0.0001 ) , donors were older ( p<0.0001 ) , percentage of male donors was lower ( p=0.0037 ) , and sex mismatch was higher ( p=0.038 ) . further analysis of diagnoses leading to htx detected a significantly higher percentage of patients transplanted due to coronary heart disease in the csa group ( p=0.0134 ) ; all other principal diagnoses leading to htx did not differ statistically significantly between both groups ( all p = ns ) . regarding concomitant medication , no statistically significant differences regarding use of statins was seen between groups 2 years after htx ( p = ns ) . renal function during the study period according to serum creatinine levels is described in figure 2 . analysis of renal function at each visit with regard to egfr detected no statistically significant differences between patients receiving tac and patients receiving csa ( all p = ns ; table 2 ) . a significant decrease of egfr during the study period was detectable in both groups ( csa group , 2 years after htx vs baseline , p<0.0001 ; tac group , 2 years after htx vs baseline , p=0.0094 ) . mcnemar s test detected a significant accumulation of patients with deterioration of renal function in the first half year after htx among patients receiving csa ( p=0.0004 ) . in patients receiving tac , no significant accumulation of patients with deterioration of renal function during the first 2 years after htx was detectable ( all p = ns ) . within the first 2 years after transplantation , serum creatinine increased significantly in csa patients ( 2 years after htx vs baseline p<0.0001 ) , whereas in patients receiving tac , serum creatinine did not differ significantly from baseline values ( p = ns ) . at baseline , serum creatinine in patients receiving csa was significantly lower ( csa , 1.120.53 mg / dl ; tac , 1.470.94 mg / dl ; p=0.0066 ) . starting from month 6 after htx , serum creatinine levels in patients receiving csa and patients receiving tac did not differ statistically significantly ( all p = ns ) . analysis of serum electrolytes , that is , serum sodium and serum potassium , detected no statistically significant differences between both cni regimens ( all p = ns ) . analyzing egfr in tac subgroups detected no statistically significant differences between patients primarily receiving an etac or patients receiving a ctac immunosuppressive regimen at all performed study visits ( all p = ns ) . at baseline , egfr in patients receiving ctac treatment was 71.6241.17 ml / minute/1.73 m versus 70.1639.28 ml / minute/1.73 m after the study period , egfr was 57.4627.64 ml / minute/1.73 m in patients receiving ctac versus 57.7123.25 ml / minute/1.73 m in patients receiving etac ( p = ns ) . analysis of serum creatinine detected no statistically significant differences between patients receiving ctac and patients receiving etac at performed study visits ( all p = ns ) . no statistically significant differences between patients receiving csa- and tac - based therapy were found regarding serum triglycerides and high - density lipoprotein at baseline and during the study period ( all p = ns ) . in contrast to patients receiving csa , within the patients receiving tac , a significant reduction of serum triglyceride levels during the study period was observed ( 2 years after htx vs baseline , p=0.0031 ) . analysis of serum cholesterol levels detected no significant differences within the first year after htx . however , a significantly lower blood cholesterol level was detected in both cni groups 2 years after htx versus baseline values ( patients receiving csa : 2 years after htx vs baseline , p=0.0010 ; patients receiving tac : 2 years after htx vs baseline , p=0.0003 ) . two years after htx , high - density lipoprotein levels were significantly lower in both groups ( csa : 2 years vs baseline , p=0.0004 ; tac : 2 years vs baseline , p=0.0002 ) , low - density lipoprotein was continuously higher in patients receiving csa at all performed study visits . except from month 6 after htx , serum glucose did not differ significantly between patients receiving csa and patients receiving tac ( p=0.0169 ) . in addition , gamma - glutamyl - transferase was significantly higher in patients receiving a tac - based immunosuppressive regimen , and a higher serum bilirubin level was detected in patients with de novo csa therapy during study period ( all p<0.0001 ) . with regard to blood pressure profile , no statistically significant differences between groups were seen during the first year after htx ( all p = ns ) . beginning in month 12 after htx , systolic blood pressure was significantly higher in patients receiving csa . no statistically significant differences regarding immunosuppressive drug trough levels were found between the ctac and etac groups ( all p = ns ) . to detect possible effects caused by cni , we only analyzed the data of patients who received the same cni , that is , csa or tac , for 2 years after htx . analyzing renal function by means of serum creatinine detected a significant deterioration of renal function in patients receiving csa as their baseline immunosuppressive regimen within the first 2 years after htx ( 2 years after htx vs baseline , p<0.0001 ) . in patients receiving tac , no statistically significant deterioration of renal function measured by serum creatinine was detectable ( 2 years after htx vs baseline , p = ns ) . as the mdrd equation is a more appropriate marker to assess renal function than serum creatinine , we used the mdrd equation to analyze renal function more precisely in our study cohort.11 analyzing renal function according to the mdrd equation detected a significant decrease of egfr , both in patients receiving csa and in patients receiving tac . to detect a significant shift of patients with deterioration of renal function between study visits in contrast to recently published data reporting similar nephrotoxic effects of both cnis , we detected significant deterioration of renal function in patients receiving csa.6,1416 our data underline data reporting better renal function of tac - based immunosuppression , as a significant accumulation of patients with deterioration of renal function was only seen in patients receiving csa immunosuppressive treatment within the first 6 months after htx.4,17 between all other study visits , a significant shift of patients receiving csa with deterioration of renal function was not seen ( all p = ns ) . analyzing patients receiving tac by mcnemar data comparing etac and ctac are rarely published ; however , in contrast to recently published data in patients after renal transplantation , no significant differences regarding renal function in patients receiving either etac or ctac therapy were found in our study cohort.18 to clarify the effects on renal function of both tac regimes , randomized controlled studies should be initiated . the importance of local renal factors , for example , variability in p - glycoprotein and cyp3a4/5 expression or activity , older kidney age , salt depletion , the use of nonsteroidal anti - inflammatory drugs , and genetic polymorphisms in genes such as tgf- and ace , in comparison with systemic exposure to cnis , for susceptibility to cni - induced nephrotoxicity has been previously published.19 in addition , the effects of comedication ( eg , ace - inhibitors , amlodipine ) , cni avoidance and dose minimization , and simultaneous injection of anti - tgf- antibodies have to be taken into account.2026 cholestasis and hepatotoxic effects in patients receiving a tac - based medication have already been detected in previously published case reports ; however , both cnis are suspected to cause cholestasis.2729 in line with previously published data , blood cholesterol levels and systolic blood pressure values were significantly higher in patients receiving csa.7,14,15,30 higher systolic blood pressure may partly be explained by a higher sympathetic tone caused by vasoconstriction of afferent and efferent glomerular arterioles.31,32 our retrospective study was performed as a single - center study , and patients were followed up according to the center s specific routine follow - up care . our retrospective study was performed as a single - center study , and patients were followed up according to the center s specific routine follow - up care . the superior rejection profile of the tac - based immunosuppressive regimen had already been demonstrated previously.7 in addition , our data detect the favorable effects of a tac - based immunosuppression with respect to nephrotoxicity , as csa immunosuppressive therapy was associated with a significant deterioration of renal function ( mcnemar s test ) , especially in the first 6 months after htx , when high immunosuppressive drug trough levels are needed to prevent ares .

highly active antiretroviral ( arv ) therapy ( haart ) has decreased hiv mother - to - child transmission ( mtct ) to < 2% in the us and other countries where arvs are readily available [ 15 ] . scheduled cesarean section decreases the risk of mtct [ 1 , 6 , 7 ] . recent studies , however , suggest that cesarean sections are not of significant benefit when plasma hiv rna is undetectable at delivery [ 2 , 3 ] . since cesarean sections increase the cost of deliveries and risk of maternal complications , understanding the kinetics of the virologic response to haart during pregnancy may avoid performing unnecessary cesarean sections . the dominant risk factor for mtct is high maternal viral load at delivery . in nonpregnant individuals , this relationship has prompted the use of cd4 + counts as a surrogate measure of response to therapy when plasma hiv rna measurements are not readily available . in pregnancy , however , this approach may not be valid , as pregnancy is associated with lower cd4 + numbers . the kinetics of plasma hiv rna and cd4 + cells in response to haart during pregnancy have been reported in separate studies [ 9 , 10 ] . the information provided by these studies suggested that there may be a disconnect between changes in cd4 + cells and plasma hiv rna during pregnancy . in this study another important issue for prevention of mtct ( pmtct ) is optimization of drug regimens during pregnancy , which typically include 2 nucleoside reverse transcriptase inhibitors ( nrti ) and a protease inhibitor ( pi ) . pharmacokinetics and therapeutic drug monitoring ( tdm ) studies during pregnancy showed that absorption and distribution of pis differ in 3rd trimester pregnant women compared with nonpregnant adults , such that dose adjustments may be warranted [ 1113 ] . however , there is a lack of information regarding pi pharmacokinetics during early pregnancy . to address these issues , we conducted a retrospective chart review of pregnancies managed by the children 's human immunodeficiency program ( chip ) in denver . in august 1997 , chip became the reference center for the care of hiv - infected pregnant women in colorado and neighboring states . these women come from a relatively broad patient population that is highly representative of the hiv epidemic in the southwestern us . patients were cared for by a multidisciplinary team , comprising specialists in adult and pediatric infectious diseases , obstetrics and maternal - fetal medicine , nursing , social work , mental health , and nutrition . hiv genotype was determined before treatment initiation in patients with plasma hiv rna 1000 copies / ml . adherence was facilitated by distributing pill boxes , watches , and pagers , office counseling or home visits , and direct observed therapy . pi plasma concentrations were assessed using high - pressure liquid chromatography assays performed at the national jewish hospital pharmacology laboratory , certified by the aids clinical trials group and compliant with the clinical laboratory improvements amendments . for tdm , drugs were administered in clinic with or without food , as indicated . peak concentrations of lopinavir ( lpv ) were measured 4 hours after administration of lpv / ritonavir ( lpvr ) and of other pis 2 hours after administration of the drug . treatment was modified based on drug levels , virologic response , safety , and tolerability . centralized treatment decisions were made by the physician in charge of the maternal care of the pmtct program in consultation with the primary infectious diseases provider of the patient , if different from the pmtct physician . the hiv - specific obstetrical plan included intravenous zidovudine ( azt ) administration during delivery except for patients on stavudine , who continued their oral medications . cesarean sections were scheduled at 38 weeks of gestation for consenting patients with vl > 1000 copies / ml at 34 weeks gestation , as indicated by other medical obstetrical conditions or patients ' choice . when the maternal vl at the last visit prior to delivery was 5,000 copies / ml or > 1000 copies / ml with vaginal delivery , the neonate received 2 additional drugs from 2 arv classes for the first 4 weeks of life . this study was reviewed by the local irb and deemed exempt from irb approval due to the retrospective nature of the study and absence of disclosure of any personal information . information was abstracted for pregnancies with 16-week duration and 2 visits at chip , using study - specific forms . wilcoxon rank - sum test was used for comparisons of continuous variables between two groups . changes in vl and cd4 + t - cell counts were tested using a signed - rank test . to determine the kinetics of the virologic response , log10 vl was modeled as smooth functions of time on treatment using a natural cubic b - spline transformation on time . this analysis was run in the sas mixed procedure ( sas institute , inc . ) and the b - splines were calculated using the ns ( ) command in splus v8.0 ( insightful corp ) . a random intercept was included in all models and a random slope was modeled using natural cubic b - splines . we allowed a maximum of 5 and 3 degrees of freedom ( df ) for the fixed and random effect b - splines , respectively , and the final model included the maximum , resulting in the most flexible model . the same flexible analysis was used to model cd4 + t - cell counts over time . the final model included 5 and 1 df for the fixed and random effects , respectively . six limits exist in our data : < 20 , 2050 , < 50 , < 200 , 200400 , and < 400 . in preparation for the b - spline analyses , we imputed plasma hiv rna copies / ml such that those with ( a ) < 20 were set to 10 , ( b ) 2050 to 35 , ( c ) < 50 to the mean of all values < 50 ( real and imputed ) , ( d ) < 200 to the mean of all values below 200 ( real and imputed ) , ( e ) 200400 to 300 , and ( f ) < 400 to the mean of all values < 400 ( real and imputed ) . thirty - one patients ( 30% ) were black and 41 ( 39% ) were hispanic . the most common risk factor for hiv acquisition was heterosexual intercourse , reported by 97 patients ( 92% ) . in 70 patients ( 67% ) , of the 35 patients diagnosed with hiv during pregnancy , 11 ( 34% ) were diagnosed during the first trimester , 16 ( 50% ) during the second trimester , and 5 ( 16% ) during the third trimester . at the first pregnancy visit , the median vl was 2657 copies / ml including 78 ( 72% ) with > 400 copies / ml . the median cd4 + t - cells was 450 cells/l ; 13 women ( 12% ) had < 200 cells/l . in 51 pregnancies ( 44% ) , the women were arv nave and in 59 ( 50% ) arv experienced , including 29 patients who became pregnant on therapy . median gestational age at delivery was 38 weeks ( interquartile range ( iqr ) = 3740 weeks ) . thirty - six women ( 31% ) delivered by scheduled cesarean section . the 114 single and 3 twin pregnancies resulted in , 117 infants without hiv infection , 1 fetal demise , 1 stillbirth and 1 neonate who died of sepsis at 1 day of life . of the 117 neonates who lived > 1 day , 98 ( 84% ) received azt and 19 ( 16% ) haart . the incidence of mtct was 0 with a 95% confidence interval = 0% to 3% . at delivery , 106/114 ( 94% ) pregnant women were on haart ( 3 drugs from 2 classes ) ; 7 women ( 4% ) , who did not tolerate pis or nnrtis , were on 2 or 3 nrtis ; and 1 woman , who delivered shortly after referral precluding a change in therapy , was on azt monotherapy . the most common drugs at delivery were azt ( n = 89 ) , lamivudine ( n = 108 ) , stavudine ( n = 22 ) , nevirapine ( nvp ; n = 19 ) , nelfinavir ( nfv ; n = 55 ) , and lpvr ( n = 25 ) . the median duration of continuous therapy up to delivery was 22 weeks ( iqr of 15 to 35 weeks ) . among 84 women who started arv during pregnancy after being off therapy for several months or never being on therapy , the median duration of therapy during pregnancy was 20 weeks ( iqr of 11 to 25 weeks ) . of these , 77 ( 92% ) received arv for 4 weeks and 61 ( 73% ) for 12 weeks . adherence was measured by pill counts , pharmacy refill histories , mems caps , and detailed interviews in 44 pregnancies . of these , 36 women ( 82% ) took 95% doses of the prescribed medication , 7 ( 16% ) took 50% doses95% , and 1 took < 50% doses . the analysis of adherence by arv regimen ( table 2 ) did not reveal any differences across regimens . a median of 7 support interventions per pregnancy took place ( iqr = 4 to 13 ) . these included case management visits ( n = 59 ) , referrals to housing agencies ( n = 31 ) and peer advocacy groups ( n = 30 ) , food ( n = 59 ) and financial ( n = 56 ) assistance , and psychoeducational group activities ( n = 37 ) . for all study participants , the median vl at delivery was 51 copies / ml ( iqr < 20 to 292 ; n = 95 ) , which represented a significant decrease compared with the first pregnancy visit median of 2657 copies / ml ( p < .0001 ) . at the last visit before delivery , 80 women ( 82% ) had plasma hiv rna < 400 copies / ml and 53 women ( 56% ) had < 50 hiv rna copies / ml . to determine the kinetics of the virologic response to haart during pregnancy , we applied a mixed - model analysis to data from 76 of 84 patients who initiated or reinitiated haart during pregnancy and had multiple tests during pregnancy ( figure 1 ) . the model , which uses all available data to estimate the mean vl trajectory over time , estimated that after 4 weeks of haart the log10 vl decreased by ~2 logs from baseline to a back - transformed mean < 100 copies / ml . both at 12 and 24 weeks of haart , the estimated back - transformed mean vl was < 50 copies / ml . we verified the predictions made by the model by calculating the proportions of subjects with vl < 100 copies / ml at 4 weeks and < 50 copies / ml at 12 and 24 weeks of haart . of 39 women with vl measurements at 4 weeks of haart , 15 ( 42% ) had < 100 copies / ml ; at 12 weeks of haart , 11 of 26 women ( 42% ) had < 50 copies / ml ; and at 24 weeks of haart , 9 of 16 women ( 56% ) had < 50 copies / ml . these data confirmed the predictions of the model . at delivery , the median cd4 + t - cell number was 540 cells/l ( iqr of 416 to 678 ; n = 92 ) : 92% of values > 200 cells/l . the median cd4 + t - cell increase between first pregnancy visit and delivery was 88 cells/l ( p < .0001 by signed - rank test ; n = 92 ) . in the mixed - model analysis used to elucidate the kinetics of cd4 + t - cells during pregnancy , we set the reference time at delivery to take into account the upward effect of arv and the downward effect of pregnancy on the cd4 + t - cells . the model indicated that the cd4 + t - cells started to increase at 6 weeks prior to delivery and peaked at approximately 8 weeks after delivery in all subjects ( figure 2(a ) ) and in those who initiated or reinitiated haart during pregnancy ( figure 2(b ) ) . among 54 women with postpartum data , there were no appreciable differences in postpartum cd4 + t - cell numbers of 36 women who continued therapy for 12 weeks , compared with those of 18 women who discontinued therapy immediately after delivery . an additional mixed - model analysis allowed for separate cd4 + t - cell trajectories postdelivery for the 18 women who discontinued therapy immediately after delivery and the 36 women who continued therapy for 12 weeks . from this model , the estimated cd4 + t - cell count at 12 weeks postpartum was 763 and 679 , for women who stopped therapy and continued therapy for 12 weeks , respectively , a difference of 84 ( p = .29 ) . thirty - two pregnant women had 1 pi tdm for a total of 92 measurements . tdm was performed at a median of 5.5 weeks after initiation or change in therapy ( iqr of 3.5 to 30 weeks ) . eighty - two tdm assays were performed in women taking pi doses recommended for nonpregnant adults ( regular ) and 10 in women taking increased doses . of these , 4 ( 57% ) were within the targeted range ( normal ) , 2 ( 29% ) were low , and 1 ( 14% ) was high . the 57 tdm assays from the second trimester included 34 ( 60% ) normal , 20 ( 35% ) low , and 3 ( 5% ) high results . the 18 tdm assays from the 3rd trimester included 9 ( 50% ) normal , 8 ( 44% ) low , and 1 ( 6% ) high results . across all gestational ages , the distribution of tdm results did not significantly differ across trimesters ( fisher 's exact test , p = .70 ) . the analysis of the plasma concentrations achieved by the most commonly used pi in this study , lpvr , nfv , and saquinavir ( saqr ) , showed that 75% of all lpv measurements were normal and 25% were low across all trimesters ; 20% to 59% of the nfv levels were normal with a trend of decreasing concentrations from 1st to 3rd trimester ; and 30 to 50% of saq concentrations were normal . there were 10 pi tdm results during the 2nd or 3rd trimester in women who received increased pi doses ( 6 received lpvr 533/133 mg bid and 3 nfv 1500 mg bid ) . of these , 3 measurements ( 30% ) were below target . to evaluate potential differences between haart regimens with respect to vl decay during pregnancy , the mixed - model analysis of log10 vl described above ( virologic response to therapy ) was used to compare nfv- ( n = 49 ) versus lpvr - containing haart ( n = 20 ) . differences in estimated log10 vl between the regimens , calculated as nfv estimate minus lpvr estimate , were 0.01 ( p = .96 ) , 0.44 ( p = .10 ) , 0.39 ( p = .12 ) , and 0.09 ( p = .78 ) at 4 , 8 , 12 , and 24 weeks on treatment , respectively . an analysis of the factors that could potentially affect the response to therapy defined by undetectable vl at delivery , including race , ethnicity , country of origin ( as a surrogate of hiv-1 clade ) , magnitude of vl before initiation of therapy , arv experience , hiv drug resistance ( data presented elsewhere ) , adherence , tdm , and utilization of psychosocial support services , was performed ( table 4 ) . the data revealed significant associations only for adherence ( p = .006 ) and psychosocial support ( p = .010 ) . to determine what length of haart administration during pregnancy was needed to achieve undetectable vl , we analyzed the kinetics of the virologic response in women who started haart during pregnancy . the time to undetectable vl did not differ from that previously reported in hiv - infected nonpregnant adults [ 17 , 18 ] , with a majority of pregnant women achieving vl < 50 copies / ml after 12 to 24 weeks of therapy . the corollary of this observation is that pregnant women who start haart during or before the 2nd trimester are likely to achieve vl < 50 copies / ml at delivery . in contrast , women starting haart less than 4 weeks before the anticipated date of delivery are likely candidates to scheduled cesarean sections . in our patient population , the rate of cesarean section was similar to that reported for other areas in the us and lower than that reported in europe , where the use of scheduled cesarean section is twice as common as in the us [ 1 , 19 ] . however , mtct in this study was lower than that documented in contemporaneous studies in the us [ 4 , 19 ] and similar to that reported in europe [ 1 , 3 ] . these differences may be explained by the fact that 94% of hiv - infected pregnant women in this study received haart for pmtct , which is a higher proportion than the rate of 50% to 70% documented in other studies [ 25 ] . taken together , these observations suggest that high uptake of haart may decrease the need of scheduled cesarean sections for pmtct [ 2 , 3 ] without compromising protection of the neonate against hiv infection . the analysis of the kinetics of cd4 + t - cells in pregnancy revealed a delay in the response to haart compared with the kinetics of vl . in pregnancy , the most significant increases in cd4 + t - cell numbers occurred in the last 6 weeks of pregnancy and first 6 weeks of postpartum . this pattern was observed in women who started haart before or during pregnancy and was independent of continuing haart post - partum . this pattern is most likely due to physiologic changes of pregnancy , including the increased volume of distribution of white blood cells and/or hormonal changes depressing cd4 + t - cells . the implication of this observation is that a lack of increase of cd4 + t - cells in pregnant women may not indicate virologic failure . an important question in the care of hiv - infected pregnant women is whether there is an arv regimen more likely than others to rapidly decrease the vl in women diagnosed with hiv late in pregnancy . we did not find any significant differences in the kfinetics of vl as a function of the pi used in haart . previous studies showed that nonpregnant hiv - infected adults on nfv - containing regimens were less likely to achieve or maintain viral suppression compared with individuals receiving lpvr - containing regimens . however , during the limited duration of therapy for pmtct , this difference may not be relevant . these data suggest that although haart is critical in order to achieve a satisfactory virologic response during pregnancy , the choice of pi has secondary importance with respect to virologic response . patel et al . showed that nvp - based regimens are associated with faster decay of vl compared with nfv - based regimens . in this study , the number of women who intitiated nvp during pregnancy was too low for a formal analysis . further studies using nnrtis that do not carry the same high risk of serious adverse events as nvp are warranted to determine whether an nnrti - based regimen may be advantageous when therapy is initiated late in pregnancy . with respect to plasma concentrations achieved by various pis during pregnancy , we found that lpvr levels were below target in 20% to 25% of pregnant women on adult recommended doses across all trimesters , whereas for another pi , 35% to 80% of the levels were below target . others have shown that up to 72% of women on lpvr do not reach target concentrations of lpv during the 3rd trimester [ 11 , 12 , 21 ] . acosta et al . found saq plasma levels uniformly above target during pregnancy , whereas in our study 50% of subjects had levels below target . our findings on nfv plasma concentrations are in accordance with those of nellen et al . who also found that 51% of pregnant women did not reach target concentrations . differences in drug concentrations across studies may be accounted by technical differences , effects of race and ethnicity on drug metabolism , and compliance with therapy . a novel contribution of our study is to reveal that plasma pi concentrations are often below target not only in the last trimester of pregnancy but also earlier in pregnancy . this suggests that other factors , in addition to the increased volume of distribution of the drugs , may contribute to this effect . the practical implication of our findings is to underscore the importance of performing tdm or other pharmacokinetic studies during the 1st and 2nd trimesters of pregnancy and also after each dose modification , since 30% of the pregnant women on increased pi doses had plasma drug levels below target . the use of tdm did not significantly affect the virologic response to therapy in this study . some studies in nonpregnant hiv - infected adults showed improved virologic response to pi - containing regimens in subjects whose doses were adjusted based on tdm results [ 2426 ] , but one study failed to show any benefit . likewise , it may be difficult to assess the benefit of tdm in pregnancy due to the limited duration of therapy . nevertheless , it is reasonable to assume that it would be undesirable to administer drugs at subtherapeutic levels to control a virus that has the ability to quickly develop drug resistance mutations , particularly if arv continues to be administered after pregnancy . further studies are warranted to determine the effect of tdm on the development of resistance to arv . among multiple factors that could potentially affect virologic response to therapy , suppression of viral replication at delivery we did not find significant differences in adherence between patients receiving once daily versus twice daily regimens or pi - containing versus sparing regimens , which is a novel finding , perhaps specific of pregnancy and has important implications for the clinical practice . overall , 95% adherence was recorded in 82% of the women in this study , which is consistent with other reports . utilization of psychosocial support services was significantly associated with virologic response to therapy during pregnancy , which makes intuitive sense , but has not been previously demonstrated in a formal analysis . although the association between utilization of psychosocial services and virologic success may indicate that women who are committed to medical treatment for pmtct are also likely to seek resources to improve other aspects of their lives , it may also indicate that support services facilitate adherence to therapy [ 3032 ] . in summary , undetectable vl at delivery was associated with lack of vertical transmission of hiv in this group of women who predominantly delivered by vaginal route . undetectable vl was achieved as soon as 4 weeks after initiation of haart in 50% of the patients and at 12 weeks in the vast majority . achieving undetectable vl at delivery adherence , which was important to reach undetectable vl at delivery , did not vary with the drugs included in the treatment regimen , either . the pattern that emerges is that critical factors for pmtct without high utilization of cesarean sections are initiating therapy 12 weeks prior to delivery with a regimen that is well tolerated and conducive to 95% adherence .

hiv - infected pregnant women with undetectable plasma hiv rna concentrations at delivery pose a minimal risk of vertical transmission . we studied the kinetics and the determinants of the virologic response to antiretroviral therapy in 117 consecutive pregnancies . patients who initiated therapy during pregnancy had a vl decrease of 2 and 2.5 log10 after 4 and 24 weeks , respectively . therapeutic drug monitoring ( tdm ) of the protease inhibitors administered in doses recommended for nonpregnant adults resulted in below - target concentrations in 29% , 35% , and 44% of 1st , 2nd , and 3rd trimester measurements , respectively , but low drug concentrations did not correlate with virologic failure . demographic characteristics , antiretroviral experience prior to pregnancy , baseline vl , or use of specific antiretrovirals did not affect the virologic response . adherence to 95% of prescribed doses and utilization of psychosocial services were associated with undetectable plasma hiv rna at delivery . in conclusion , the virologic responses of pregnant and nonpregnant adults share similar charactersitics .

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

scheduled cesarean section decreases the risk of mtct [ 1 , 6 , 7 ] . recent studies , however , suggest that cesarean sections are not of significant benefit when plasma hiv rna is undetectable at delivery [ 2 , 3 ] . since cesarean sections increase the cost of deliveries and risk of maternal complications , understanding the kinetics of the virologic response to haart during pregnancy may avoid performing unnecessary cesarean sections . the dominant risk factor for mtct is high maternal viral load at delivery . in nonpregnant individuals , this relationship has prompted the use of cd4 + counts as a surrogate measure of response to therapy when plasma hiv rna measurements are not readily available . in pregnancy , however , this approach may not be valid , as pregnancy is associated with lower cd4 + numbers . the kinetics of plasma hiv rna and cd4 + cells in response to haart during pregnancy have been reported in separate studies [ 9 , 10 ] . the information provided by these studies suggested that there may be a disconnect between changes in cd4 + cells and plasma hiv rna during pregnancy . in this study another important issue for prevention of mtct ( pmtct ) is optimization of drug regimens during pregnancy , which typically include 2 nucleoside reverse transcriptase inhibitors ( nrti ) and a protease inhibitor ( pi ) . pharmacokinetics and therapeutic drug monitoring ( tdm ) studies during pregnancy showed that absorption and distribution of pis differ in 3rd trimester pregnant women compared with nonpregnant adults , such that dose adjustments may be warranted [ 1113 ] . in august 1997 , chip became the reference center for the care of hiv - infected pregnant women in colorado and neighboring states . these women come from a relatively broad patient population that is highly representative of the hiv epidemic in the southwestern us . patients were cared for by a multidisciplinary team , comprising specialists in adult and pediatric infectious diseases , obstetrics and maternal - fetal medicine , nursing , social work , mental health , and nutrition . hiv genotype was determined before treatment initiation in patients with plasma hiv rna 1000 copies / ml . adherence was facilitated by distributing pill boxes , watches , and pagers , office counseling or home visits , and direct observed therapy . for tdm , drugs were administered in clinic with or without food , as indicated . peak concentrations of lopinavir ( lpv ) were measured 4 hours after administration of lpv / ritonavir ( lpvr ) and of other pis 2 hours after administration of the drug . treatment was modified based on drug levels , virologic response , safety , and tolerability . centralized treatment decisions were made by the physician in charge of the maternal care of the pmtct program in consultation with the primary infectious diseases provider of the patient , if different from the pmtct physician . the hiv - specific obstetrical plan included intravenous zidovudine ( azt ) administration during delivery except for patients on stavudine , who continued their oral medications . when the maternal vl at the last visit prior to delivery was 5,000 copies / ml or > 1000 copies / ml with vaginal delivery , the neonate received 2 additional drugs from 2 arv classes for the first 4 weeks of life . this study was reviewed by the local irb and deemed exempt from irb approval due to the retrospective nature of the study and absence of disclosure of any personal information . information was abstracted for pregnancies with 16-week duration and 2 visits at chip , using study - specific forms . to determine the kinetics of the virologic response , log10 vl was modeled as smooth functions of time on treatment using a natural cubic b - spline transformation on time . and the b - splines were calculated using the ns ( ) command in splus v8.0 ( insightful corp ) . we allowed a maximum of 5 and 3 degrees of freedom ( df ) for the fixed and random effect b - splines , respectively , and the final model included the maximum , resulting in the most flexible model . the final model included 5 and 1 df for the fixed and random effects , respectively . six limits exist in our data : < 20 , 2050 , < 50 , < 200 , 200400 , and < 400 . in preparation for the b - spline analyses , we imputed plasma hiv rna copies / ml such that those with ( a ) < 20 were set to 10 , ( b ) 2050 to 35 , ( c ) < 50 to the mean of all values < 50 ( real and imputed ) , ( d ) < 200 to the mean of all values below 200 ( real and imputed ) , ( e ) 200400 to 300 , and ( f ) < 400 to the mean of all values < 400 ( real and imputed ) . in 70 patients ( 67% ) , of the 35 patients diagnosed with hiv during pregnancy , 11 ( 34% ) were diagnosed during the first trimester , 16 ( 50% ) during the second trimester , and 5 ( 16% ) during the third trimester . at the first pregnancy visit , the median vl was 2657 copies / ml including 78 ( 72% ) with > 400 copies / ml . in 51 pregnancies ( 44% ) , the women were arv nave and in 59 ( 50% ) arv experienced , including 29 patients who became pregnant on therapy . median gestational age at delivery was 38 weeks ( interquartile range ( iqr ) = 3740 weeks ) . the 114 single and 3 twin pregnancies resulted in , 117 infants without hiv infection , 1 fetal demise , 1 stillbirth and 1 neonate who died of sepsis at 1 day of life . of the 117 neonates who lived > 1 day , 98 ( 84% ) received azt and 19 ( 16% ) haart . at delivery , 106/114 ( 94% ) pregnant women were on haart ( 3 drugs from 2 classes ) ; 7 women ( 4% ) , who did not tolerate pis or nnrtis , were on 2 or 3 nrtis ; and 1 woman , who delivered shortly after referral precluding a change in therapy , was on azt monotherapy . the most common drugs at delivery were azt ( n = 89 ) , lamivudine ( n = 108 ) , stavudine ( n = 22 ) , nevirapine ( nvp ; n = 19 ) , nelfinavir ( nfv ; n = 55 ) , and lpvr ( n = 25 ) . among 84 women who started arv during pregnancy after being off therapy for several months or never being on therapy , the median duration of therapy during pregnancy was 20 weeks ( iqr of 11 to 25 weeks ) . adherence was measured by pill counts , pharmacy refill histories , mems caps , and detailed interviews in 44 pregnancies . of these , 36 women ( 82% ) took 95% doses of the prescribed medication , 7 ( 16% ) took 50% doses95% , and 1 took < 50% doses . the analysis of adherence by arv regimen ( table 2 ) did not reveal any differences across regimens . these included case management visits ( n = 59 ) , referrals to housing agencies ( n = 31 ) and peer advocacy groups ( n = 30 ) , food ( n = 59 ) and financial ( n = 56 ) assistance , and psychoeducational group activities ( n = 37 ) . for all study participants , the median vl at delivery was 51 copies / ml ( iqr < 20 to 292 ; n = 95 ) , which represented a significant decrease compared with the first pregnancy visit median of 2657 copies / ml ( p < .0001 ) . at the last visit before delivery , 80 women ( 82% ) had plasma hiv rna < 400 copies / ml and 53 women ( 56% ) had < 50 hiv rna copies / ml . to determine the kinetics of the virologic response to haart during pregnancy , we applied a mixed - model analysis to data from 76 of 84 patients who initiated or reinitiated haart during pregnancy and had multiple tests during pregnancy ( figure 1 ) . the model , which uses all available data to estimate the mean vl trajectory over time , estimated that after 4 weeks of haart the log10 vl decreased by ~2 logs from baseline to a back - transformed mean < 100 copies / ml . both at 12 and 24 weeks of haart , the estimated back - transformed mean vl was < 50 copies / ml . we verified the predictions made by the model by calculating the proportions of subjects with vl < 100 copies / ml at 4 weeks and < 50 copies / ml at 12 and 24 weeks of haart . of 39 women with vl measurements at 4 weeks of haart , 15 ( 42% ) had < 100 copies / ml ; at 12 weeks of haart , 11 of 26 women ( 42% ) had < 50 copies / ml ; and at 24 weeks of haart , 9 of 16 women ( 56% ) had < 50 copies / ml . these data confirmed the predictions of the model . at delivery , the median cd4 + t - cell number was 540 cells/l ( iqr of 416 to 678 ; n = 92 ) : 92% of values > 200 cells/l . in the mixed - model analysis used to elucidate the kinetics of cd4 + t - cells during pregnancy , we set the reference time at delivery to take into account the upward effect of arv and the downward effect of pregnancy on the cd4 + t - cells . the model indicated that the cd4 + t - cells started to increase at 6 weeks prior to delivery and peaked at approximately 8 weeks after delivery in all subjects ( figure 2(a ) ) and in those who initiated or reinitiated haart during pregnancy ( figure 2(b ) ) . among 54 women with postpartum data , there were no appreciable differences in postpartum cd4 + t - cell numbers of 36 women who continued therapy for 12 weeks , compared with those of 18 women who discontinued therapy immediately after delivery . an additional mixed - model analysis allowed for separate cd4 + t - cell trajectories postdelivery for the 18 women who discontinued therapy immediately after delivery and the 36 women who continued therapy for 12 weeks . from this model , the estimated cd4 + t - cell count at 12 weeks postpartum was 763 and 679 , for women who stopped therapy and continued therapy for 12 weeks , respectively , a difference of 84 ( p = .29 ) . eighty - two tdm assays were performed in women taking pi doses recommended for nonpregnant adults ( regular ) and 10 in women taking increased doses . of these , 4 ( 57% ) were within the targeted range ( normal ) , 2 ( 29% ) were low , and 1 ( 14% ) was high . the 57 tdm assays from the second trimester included 34 ( 60% ) normal , 20 ( 35% ) low , and 3 ( 5% ) high results . the 18 tdm assays from the 3rd trimester included 9 ( 50% ) normal , 8 ( 44% ) low , and 1 ( 6% ) high results . across all gestational ages , the distribution of tdm results did not significantly differ across trimesters ( fisher 's exact test , p = .70 ) . the analysis of the plasma concentrations achieved by the most commonly used pi in this study , lpvr , nfv , and saquinavir ( saqr ) , showed that 75% of all lpv measurements were normal and 25% were low across all trimesters ; 20% to 59% of the nfv levels were normal with a trend of decreasing concentrations from 1st to 3rd trimester ; and 30 to 50% of saq concentrations were normal . there were 10 pi tdm results during the 2nd or 3rd trimester in women who received increased pi doses ( 6 received lpvr 533/133 mg bid and 3 nfv 1500 mg bid ) . to evaluate potential differences between haart regimens with respect to vl decay during pregnancy , the mixed - model analysis of log10 vl described above ( virologic response to therapy ) was used to compare nfv- ( n = 49 ) versus lpvr - containing haart ( n = 20 ) . differences in estimated log10 vl between the regimens , calculated as nfv estimate minus lpvr estimate , were 0.01 ( p = .96 ) , 0.44 ( p = .10 ) , 0.39 ( p = .12 ) , and 0.09 ( p = .78 ) at 4 , 8 , 12 , and 24 weeks on treatment , respectively . an analysis of the factors that could potentially affect the response to therapy defined by undetectable vl at delivery , including race , ethnicity , country of origin ( as a surrogate of hiv-1 clade ) , magnitude of vl before initiation of therapy , arv experience , hiv drug resistance ( data presented elsewhere ) , adherence , tdm , and utilization of psychosocial support services , was performed ( table 4 ) . to determine what length of haart administration during pregnancy was needed to achieve undetectable vl , we analyzed the kinetics of the virologic response in women who started haart during pregnancy . the time to undetectable vl did not differ from that previously reported in hiv - infected nonpregnant adults [ 17 , 18 ] , with a majority of pregnant women achieving vl < 50 copies / ml after 12 to 24 weeks of therapy . the corollary of this observation is that pregnant women who start haart during or before the 2nd trimester are likely to achieve vl < 50 copies / ml at delivery . in our patient population , the rate of cesarean section was similar to that reported for other areas in the us and lower than that reported in europe , where the use of scheduled cesarean section is twice as common as in the us [ 1 , 19 ] . these differences may be explained by the fact that 94% of hiv - infected pregnant women in this study received haart for pmtct , which is a higher proportion than the rate of 50% to 70% documented in other studies [ 25 ] . taken together , these observations suggest that high uptake of haart may decrease the need of scheduled cesarean sections for pmtct [ 2 , 3 ] without compromising protection of the neonate against hiv infection . the analysis of the kinetics of cd4 + t - cells in pregnancy revealed a delay in the response to haart compared with the kinetics of vl . in pregnancy , the most significant increases in cd4 + t - cell numbers occurred in the last 6 weeks of pregnancy and first 6 weeks of postpartum . this pattern was observed in women who started haart before or during pregnancy and was independent of continuing haart post - partum . this pattern is most likely due to physiologic changes of pregnancy , including the increased volume of distribution of white blood cells and/or hormonal changes depressing cd4 + t - cells . the implication of this observation is that a lack of increase of cd4 + t - cells in pregnant women may not indicate virologic failure . an important question in the care of hiv - infected pregnant women is whether there is an arv regimen more likely than others to rapidly decrease the vl in women diagnosed with hiv late in pregnancy . we did not find any significant differences in the kfinetics of vl as a function of the pi used in haart . previous studies showed that nonpregnant hiv - infected adults on nfv - containing regimens were less likely to achieve or maintain viral suppression compared with individuals receiving lpvr - containing regimens . these data suggest that although haart is critical in order to achieve a satisfactory virologic response during pregnancy , the choice of pi has secondary importance with respect to virologic response . showed that nvp - based regimens are associated with faster decay of vl compared with nfv - based regimens . in this study , the number of women who intitiated nvp during pregnancy was too low for a formal analysis . further studies using nnrtis that do not carry the same high risk of serious adverse events as nvp are warranted to determine whether an nnrti - based regimen may be advantageous when therapy is initiated late in pregnancy . with respect to plasma concentrations achieved by various pis during pregnancy , we found that lpvr levels were below target in 20% to 25% of pregnant women on adult recommended doses across all trimesters , whereas for another pi , 35% to 80% of the levels were below target . others have shown that up to 72% of women on lpvr do not reach target concentrations of lpv during the 3rd trimester [ 11 , 12 , 21 ] . found saq plasma levels uniformly above target during pregnancy , whereas in our study 50% of subjects had levels below target . who also found that 51% of pregnant women did not reach target concentrations . differences in drug concentrations across studies may be accounted by technical differences , effects of race and ethnicity on drug metabolism , and compliance with therapy . this suggests that other factors , in addition to the increased volume of distribution of the drugs , may contribute to this effect . the practical implication of our findings is to underscore the importance of performing tdm or other pharmacokinetic studies during the 1st and 2nd trimesters of pregnancy and also after each dose modification , since 30% of the pregnant women on increased pi doses had plasma drug levels below target . the use of tdm did not significantly affect the virologic response to therapy in this study . some studies in nonpregnant hiv - infected adults showed improved virologic response to pi - containing regimens in subjects whose doses were adjusted based on tdm results [ 2426 ] , but one study failed to show any benefit . among multiple factors that could potentially affect virologic response to therapy , suppression of viral replication at delivery we did not find significant differences in adherence between patients receiving once daily versus twice daily regimens or pi - containing versus sparing regimens , which is a novel finding , perhaps specific of pregnancy and has important implications for the clinical practice . overall , 95% adherence was recorded in 82% of the women in this study , which is consistent with other reports . utilization of psychosocial support services was significantly associated with virologic response to therapy during pregnancy , which makes intuitive sense , but has not been previously demonstrated in a formal analysis . although the association between utilization of psychosocial services and virologic success may indicate that women who are committed to medical treatment for pmtct are also likely to seek resources to improve other aspects of their lives , it may also indicate that support services facilitate adherence to therapy [ 3032 ] . in summary , undetectable vl at delivery was associated with lack of vertical transmission of hiv in this group of women who predominantly delivered by vaginal route . undetectable vl was achieved as soon as 4 weeks after initiation of haart in 50% of the patients and at 12 weeks in the vast majority . achieving undetectable vl at delivery adherence , which was important to reach undetectable vl at delivery , did not vary with the drugs included in the treatment regimen , either . the pattern that emerges is that critical factors for pmtct without high utilization of cesarean sections are initiating therapy 12 weeks prior to delivery with a regimen that is well tolerated and conducive to 95% adherence .

thyroid associated orbitopathy ( tao ) comprises a spectrum of ophthalmic and extraocular manifestations including eyelid retraction , exophthalmos , eyelid and conjunctival edema , extraocular muscle fibrosis and compressive optic neuropathy , resulting from autoimmune stimulation of orbit epitopes , including the adipose tissue and the extraocular muscles.1,2 the constellation of these findings may not always be present3 and atypical modes of presentation , especially among euthyroid patients , may obviate the diagnosis . a number of less common features of tao have been reported in the literature,47 including superior limbic keratoconjunctivitis and prominent premalar and cheek swelling ( table 1 ) . in this study we report on the finding of a prominent subconjunctival protrusion noted in the lateral canthal area of six patients with tao and comment on the diagnostic significance of this , not previously reported to our knowledge,1,4,5 clinical association . we recorded the demographic and clinical characteristics of six consecutive patients who presented with the finding of a temporally located subconjunctival swelling and were diagnosed with tao . the study has followed the tenets of the declaration of helsinki and was approved by the institutional review board of athens general hospital g. gennimatas . in accordance to irb requirements the sex , age at presentation of the specific sign under study as well as the age at onset of tao , the age at onset of autoimmune dysthyroidism ( when applicable ) , thyroid and autoimmune status as well as the therapeutic modalities employed were recorded . a complete ophthalmic examination , including visual acuity testing , an orthoptic evaluation , hertel exophthalmometry and assessment of the clinical activity score,8 applanation tonometry and fundoscopy ( in selected time points ) were performed at the initial and follow - up examinations . magnetic resonance imaging ( mri ) of the orbit was obtained in four patients . a comparison with the demographic characteristics ( age and sex ) for the total pool of 192 consecutive patients with tao not bearing this sign who were examined in the corresponding time period in the same tertiary care referral center was performed using descriptive statistics ( independent samples t - test , fisher s exact test ) . the first patient who drew our attention to the diagnostic significance of the superotemporal subconjunctival protrusion was a euthyroid 68-year - old man who was referred by his primary physician with the clinical suspicion of lacrimal gland enlargement in his right eye ( patient # 1 ) . five more patients , three men and two women , with a similar clinical picture and an eventual or preexisting diagnosis of tao , were prospectively enrolled . the lesion was located in the temporal or superior temporal quadrant in all six patients and shared the morphology of a convex smooth anterior surface , with the posterior surface disappearing within the fornix and the orbit . it was yellowish to purple - yellow , movable and could be easily repositioned into the orbit by applying direct pressure on it . the protrusion became more easily seen when the upper eyelids were elevated or the eye was rotated medially or downward and increased in size with retropulsion of the globe . the yellowish hue of the lesion was indicative of fat composition with occasionally visible fat globules . an orbit mri was performed in four patients and demonstrated fat density in the area of protrusion in continuity with the intraconal fat ( figures 1b , 2d , 3b ) . the incidence of this finding in a series of 198 patients with tao examined by one of us ( kic ) in the same tertiary care referral center in the corresponding 7-year time period is 3.03% . it developed unilaterally in two patients and bilaterally , albeit asymmetrically , in the other four cases . the mean age at presentation with tao symptomatology was 64.8 ( 6.6 ) years ( range 52.576 ) for these six patients compared to 51.8 ( 14.3 ) for the pool of 192 patients not bearing this sign ( p=0.003 , independent sample t - test ) . a male to female preponderance of 4:2 was noted in this series compared to approximately 6:10 ( 73/119 ) male to female ratio for the remaining 192 patients with tao . the difference in the percentage of male patients ( 66.7% versus 38.02% , respectively ) was not statistically significant ( p=0.210 , fisher s exact test ) . one of the female patients ( case # 2 ) who developed this sign at the age of 58 had a markedly obese somatotype . a history of diplopia and marked ipsilateral proptosis was reported in four patients ( cases # 1 , 2 , 4 and 5 ) . three patients ( cases # 2 , 4 , 5 ) developed a severe form of tao . it is noteworthy that apart from impairing cosmesis , the clinical course of the specific lesion was benign , there was no need for additional therapy other than lubrication of the globe to prevent exacerbation of dry eye symptoms . a comprehensive outline of tao findings for each patient is provided in table 2 . a 68-year - old male presented in may 2006 complaining of a visible mass protruding under the lateral part of his mildly swollen right upper eyelid and intermittent diplopia . the mass had a yellowish purple hue and was soft and non tender upon palpation . upper eyelid retraction and restriction of elevation was noted in the right eye ( figure 1a ) . a systemic work - up for autoimmune dysthyroidism revealed euthyroid status and borderline elevation of anti - tsh receptor antibodies : 8.8 ( nl < 9 , sensitivity 0.8 ) . the diagnosis of euthyroid graves orbitopathy was established based on the typical constellation of ipsilateral proptosis , eyelid retraction , lid and conjunctival edema as well as tendon sparing enlargement of right inferior and medial rectus muscles . an orbit mri outlined anterior herniation of intraorbital fat ( figure 1b d ) with slight displacement of the lacrimal gland temporally in the involved right orbit as opposed to normal appearance of the lacrimal gland on the left side . he was treated with intravenous and oral steroids with partial resolution of exophthalmos and diplopia . the prolapse did not cause any symptoms and remained unchanged after 44 months of follow - up time . a 55-year - old obese woman was first examined in march 2006 complaining of diplopia and unilateral exophthalmos in the left eye ( figure 2a and b ) . she reported a 2.5-year long history of tao in the context of graves disease , poorly responsive to intravenous methylprednisolone regimens . she returned 3 years later with a recent onset of symptoms in the previously not involved right eye . she was particularly concerned about the emergence of a soft lump in the outer part of the recently involved right eye . exophthalmos , eyelid retraction and the presence of a soft lump under the eyelid protruding above the lateral canthus were noted in the right eye ( figure 2c ) . the lesion was soft upon palpation and persisted unchanged after a 7-month follow - up time despite gradual stabilization of thyroid levels . an orbit mri showed orbital fat prolapsing anteriorly , anterior bowing of the orbital septum and lateral displacement of the lacrimal gland which looked smaller in the involved orbit as opposed to normal size and appearance of the lacrimal gland in the contralateral left orbit ( figure 2d ) . a 72-year - old man presented in october 2008 complaining of a yellowish bulge protruding under the upper eyelid of both eyes for the past 6 months and a gradually worsening swelling of his eyes . the lacrimal gland on either side was identified in place and was of normal size . he was followed for 15 months , during that time thyroid levels stabilized , without an improvement in the size of the lesion . a 72-year - old man presented in november 2008 with pronounced ocular misalignment and a conjunctival protrusion under both upper eyelids . orbit mri showed anterior herniation of orbital fat with the lacrimal gland identified at the normal position and size ( figure 4a ) . the patient declined strabismus surgery and has been followed for 20 months with no change in the clinical picture . mass like protrusion under the lateral part of both upper eyelids for the last several months ( figure 4b ) . he had previously undergone orbit decompression and strabismus surgery in both eyes elsewhere for tao diagnosed 5 years before , confirmed with past orbit mri scans . the lesion had a light purple - yellow hue ( figure 4c and d ) and was soft and non tender upon palpation . a 65-year - old woman presented in october 2010 for evaluation of a melanocytic lesion on her right upper eyelid . she reported a long history of hypothyroidism and bilateral exophthalmos , upon remission at the time of presentation . asymmetric bilateral subconjunctival temporal protrusions were noted ( figure 4e ) which by history had emerged shortly after the onset of tao . there was no change in the clinical picture after 5 months a 68-year - old male presented in may 2006 complaining of a visible mass protruding under the lateral part of his mildly swollen right upper eyelid and intermittent diplopia . the mass had a yellowish purple hue and was soft and non tender upon palpation . upper eyelid retraction and restriction of elevation was noted in the right eye ( figure 1a ) . a systemic work - up for autoimmune dysthyroidism revealed euthyroid status and borderline elevation of anti - tsh receptor antibodies : 8.8 ( nl < 9 , sensitivity 0.8 ) . the diagnosis of euthyroid graves orbitopathy was established based on the typical constellation of ipsilateral proptosis , eyelid retraction , lid and conjunctival edema as well as tendon sparing enlargement of right inferior and medial rectus muscles . an orbit mri outlined anterior herniation of intraorbital fat ( figure 1b d ) with slight displacement of the lacrimal gland temporally in the involved right orbit as opposed to normal appearance of the lacrimal gland on the left side . he was treated with intravenous and oral steroids with partial resolution of exophthalmos and diplopia . the prolapse did not cause any symptoms and remained unchanged after 44 months of follow - up time . a 55-year - old obese woman was first examined in march 2006 complaining of diplopia and unilateral exophthalmos in the left eye ( figure 2a and b ) . she reported a 2.5-year long history of tao in the context of graves disease , poorly responsive to intravenous methylprednisolone regimens . she returned 3 years later with a recent onset of symptoms in the previously not involved right eye . she was particularly concerned about the emergence of a soft lump in the outer part of the recently involved right eye . exophthalmos , eyelid retraction and the presence of a soft lump under the eyelid protruding above the lateral canthus were noted in the right eye ( figure 2c ) . the lesion was soft upon palpation and persisted unchanged after a 7-month follow - up time despite gradual stabilization of thyroid levels . an orbit mri showed orbital fat prolapsing anteriorly , anterior bowing of the orbital septum and lateral displacement of the lacrimal gland which looked smaller in the involved orbit as opposed to normal size and appearance of the lacrimal gland in the contralateral left orbit ( figure 2d ) . a 72-year - old man presented in october 2008 complaining of a yellowish bulge protruding under the upper eyelid of both eyes for the past 6 months and a gradually worsening swelling of his eyes . the lacrimal gland on either side was identified in place and was of normal size . he was followed for 15 months , during that time thyroid levels stabilized , without an improvement in the size of the lesion . a 72-year - old man presented in november 2008 with pronounced ocular misalignment and a conjunctival protrusion under both upper eyelids . orbit mri showed anterior herniation of orbital fat with the lacrimal gland identified at the normal position and size ( figure 4a ) . the patient declined strabismus surgery and has been followed for 20 months with no change in the clinical picture . a 71-year - old man presented in november 2007 complaining of a mass like protrusion under the lateral part of both upper eyelids for the last several months ( figure 4b ) . he had previously undergone orbit decompression and strabismus surgery in both eyes elsewhere for tao diagnosed 5 years before , confirmed with past orbit mri scans . the lesion had a light purple - yellow hue ( figure 4c and d ) and was soft and non tender upon palpation . a 65-year - old woman presented in october 2010 for evaluation of a melanocytic lesion on her right upper eyelid . she reported a long history of hypothyroidism and bilateral exophthalmos , upon remission at the time of presentation . asymmetric bilateral subconjunctival temporal protrusions were noted ( figure 4e ) which by history had emerged shortly after the onset of tao . there was no change in the clinical picture after 5 months a 68-year - old male presented in may 2006 complaining of a visible mass protruding under the lateral part of his mildly swollen right upper eyelid and intermittent diplopia . the mass had a yellowish purple hue and was soft and non tender upon palpation . upper eyelid retraction and restriction of elevation was noted in the right eye ( figure 1a ) . a systemic work - up for autoimmune dysthyroidism revealed euthyroid status and borderline elevation of anti - tsh receptor antibodies : 8.8 ( nl < 9 , sensitivity 0.8 ) . the diagnosis of euthyroid graves orbitopathy was established based on the typical constellation of ipsilateral proptosis , eyelid retraction , lid and conjunctival edema as well as tendon sparing enlargement of right inferior and medial rectus muscles . an orbit mri outlined anterior herniation of intraorbital fat ( figure 1b d ) with slight displacement of the lacrimal gland temporally in the involved right orbit as opposed to normal appearance of the lacrimal gland on the left side . he was treated with intravenous and oral steroids with partial resolution of exophthalmos and diplopia . the prolapse did not cause any symptoms and remained unchanged after 44 months of follow - up time . a 55-year - old obese woman was first examined in march 2006 complaining of diplopia and unilateral exophthalmos in the left eye ( figure 2a and b ) . she reported a 2.5-year long history of tao in the context of graves disease , poorly responsive to intravenous methylprednisolone regimens . she returned 3 years later with a recent onset of symptoms in the previously not involved right eye . she was particularly concerned about the emergence of a soft lump in the outer part of the recently involved right eye . exophthalmos , eyelid retraction and the presence of a soft lump under the eyelid protruding above the lateral canthus were noted in the right eye ( figure 2c ) . the lesion was soft upon palpation and persisted unchanged after a 7-month follow - up time despite gradual stabilization of thyroid levels . an orbit mri showed orbital fat prolapsing anteriorly , anterior bowing of the orbital septum and lateral displacement of the lacrimal gland which looked smaller in the involved orbit as opposed to normal size and appearance of the lacrimal gland in the contralateral left orbit ( figure 2d ) . a 72-year - old man presented in october 2008 complaining of a yellowish bulge protruding under the upper eyelid of both eyes for the past 6 months and a gradually worsening swelling of his eyes . the lacrimal gland on either side was identified in place and was of normal size . he was followed for 15 months , during that time thyroid levels stabilized , without an improvement in the size of the lesion . a 72-year - old man presented in november 2008 with pronounced ocular misalignment and a conjunctival protrusion under both upper eyelids . orbit mri showed anterior herniation of orbital fat with the lacrimal gland identified at the normal position and size ( figure 4a ) . the patient declined strabismus surgery and has been followed for 20 months with no change in the clinical picture . a 71-year - old man presented in november 2007 complaining of a mass like protrusion under the lateral part of both upper eyelids for the last several months ( figure 4b ) . he had previously undergone orbit decompression and strabismus surgery in both eyes elsewhere for tao diagnosed 5 years before , confirmed with past orbit mri scans . the lesion had a light purple - yellow hue ( figure 4c and d ) and was soft and non tender upon palpation . a 65-year - old woman presented in october 2010 for evaluation of a melanocytic lesion on her right upper eyelid . she reported a long history of hypothyroidism and bilateral exophthalmos , upon remission at the time of presentation . asymmetric bilateral subconjunctival temporal protrusions were noted ( figure 4e ) which by history had emerged shortly after the onset of tao . there was no change in the clinical picture after 5 months schmack et al9 in their comprehensive study of histopatho - logical findings of subconjunctival herniated orbital fat noted that 2 of 17 patients in their series had a history of endocrine orbitopathy . saban et al10 noted that 2 of 27 patients submitted to surgical treatment for subconjunctival fat prolapse had a history of graves ophthalmopathy . the high prevalence derived from these reports ( 11.7% and 7.4% , respectively ) may suggest an association between subconjunctival fat prolapse and tao . from the clinical standpoint , subconjunctival prolapse of orbital fat accompanied the presentation of active thyroid eye disease in four of the ten eyes reported in this series ( cases # 1 , # 2 , right eye and # 3 ) and was by simple inspection an impressive enough finding to draw the internist s and ophthalmologist s attention to initiate the work - up that led to the diagnosis of tao and autoimmune dysthyroidism . the prolapse developed subsequently in the course of autoimmune orbitopathy in the other three cases . the patients presented with concern about the new finding of a mass like lesion in the outer part of the eye . orbital fat is located within the intraconal and extraconal space and is contained by tenon s capsule , periorbita and orbital septum . tenon s capsule is a thin membrane that envelops the eyeball , separating it from fat . it extends from the limbus of the cornea anteriorly , to the optic nerve posteriorly and is pierced by the tendons of the extraocular muscles.9,11 despite its support , orbital fat has the tendency to migrate under the influence of gravity and pressure . any mechanism ( eg , aging , surgery , trauma , disease ) resulting in breaching of the surrounding connective tissue allows a forward movement of the orbital fat into either the subconjunctival space ( intraconal fat ) or the eyelids ( extraconal fat).1215 in contrast to prolapsed extraconal fat causing the well - known palpebral bags which is a common finding in elderly patients , subconjunctival intraorbital fat herniation is a rare clinical condition attributed to trauma or surgery16 or occurring spontaneously,17 typically in elderly male patients with a mean age of 6572 years ( range , 2894 years).12 it typically occurs in the lateral canthal area beneath the temporal or superotemporal bulbar conjunctiva as a soft yellowish mass with a convex anterior margin and faint superficial blood vessels.14,15,1720 it is usually bilateral , but may also be unilateral . as the extension of prolapsed fat is often asymmetric , minor lesions in the contralateral eye may be sometimes overlooked.11 the pathogenesis of spontaneous subconjunctival fat prolapse is believed to be associated with an age - related acquired local attenuation of tenon s capsule,14 allowing the fat from the intraconal space to move forward and appear clinically as a subconjunctival mass.13,18 the relative contribution of raised intraorbital pressure in facilitating anterior prolapse of orbital fat has not been appreciated so far . we postulate that the raised intraorbital pressure and increase in orbital fat characteristic of tao may predispose to anterior herniation of intraorbital fat.21 it is reasonable that this mechanism may underlie the concurrent presentation of active tao and subconjunctival fat prolapse in four patients in this series as well as an increased prevalence of this lesion among tao patients in general . it is of note that exophthalmos and lid swelling , to mention only two of the most common signs of graves ophthalmopathy , are not specific for this disease either . although subconjunctival fat prolapse is reported in case reports13,20 or small case series12,14,15,17,18 as a rare entity , there are no data in the literature accessible to us on its incidence in the general population to compare with the 3.03% rate we noted among our tao patients . the clinical manifestations of tao may be encountered at variable frequencies ranging from 92% of patients developing upper eyelid retraction during the course of their disease to 3.3% developing superior limbic keratoconjunctivitis in the large prospective study by bartley.1 less common signs , such as prominent malar and cheek swelling have been recognized in 1.8% of tao patients in another series7 ( table 1 ) . bartley and gorman3 noted that only 5% of patients with tao have the complete constellation of the so - called classic findings : eyelid retraction , exophthalmos , optic nerve dysfunction , extraocular muscle involvement and hyperthyroidism . the diagnosis may be difficult in the absence of thyroid dysfunction and should rely on the combination of certain clinical features . subconjunctival fat prolapse brought two of our patients ( case # 1 and # 3 ) to medical attention initiating the work - up that led to the diagnosis of tao ; this underscores its potential value in increasing the index of suspicion for the diagnosis of tao and hyperthyroidism . it is of note that despite the fact that it proved to be a quite impressive , and sometimes presenting clinical manifestation of tao , it was not an isolated sign of tao in any of our patients . based on these remarks , we suggest that if a substantial amount of orbital fat prolapse is noted , the clinician should check carefully for other signs of tao and the diagnosis of autoimmune dysthyroidism should be kept in mind . from the ophthalmologist s perspective , the differential diagnosis may include lacrimal gland prolapse or enlargement , dermolipoma , subconjunctival and orbital lipomas and lymphoma.14,16,2124 the differential diagnosis with a lacrimal gland mass which may also be associated with exophthalmos , inferomedial globe displacement and fullness of the eyelid ( common signs of tao , see patient # 1 in this series ) is of particular importance given the morbidity of lacrimal gland tumors . enlargement of the palpebral lobe of the lacrimal gland by inflammation , infiltration or tumor produces a palpable solid mass in the superotemporal portion of the orbit and imaging findings of lacrimal gland enlargement sometimes in association with bone molding or destruction . orbit mri identified the lacrimal gland in a posterior and laterally displaced position ( figure 2d ) in our patient . overall subconjunctival fat prolapse may easily be distinguished25 by clinical inspection and palpation alone based on its characteristic softness , being readily indented or moved by a cotton - tip applicator . computed tomography and mri are definitively helpful when in doubt , showing the continuity between the prolapsed fat and the intraconal fat . except for the cosmetic blemish , the lesion caused no major symptomatology , hence required no treatment and no histopathological examination in our patients . whenever judged necessary , surgical excision of fat under local anesthesia has been suggested in the literature as a safe and effective means for treating these lesions1214,16,21,2427 with a low recurrence rate after local excision.9,10,25 based on data from this series , subconjunctival fat prolapse may be an uncommon , not isolated , but clinically impressive part of the clinical picture of tao developing primarily among male patients with an advanced age at presentation . awareness of this association may alert to the diagnosis of thyroid dysfunction and reassure the patient and physician as to the benign character of the lesion .

backgroundthyroid associated orbitopathy ( tao ) comprises a spectrum of well - recognized clinical signs including exophthalmos , eyelid retraction , soft tissue swelling , ocular misalignment , keratopathy as well as a number of less common manifestations . subconjunctival fat prolapse is a rare clinical condition occurring typically spontaneously in elderly patients with a mean age of 6572 years . we describe subconjunctival prolapse of orbital fat as an uncommon clinical association of tao.materials and methodsobservational study of six patients presenting with a subconjunctival protrusion under the lateral canthus in a series of 198 consecutive cases with tao examined at a tertiary care referral center.resultsa superotemporally located yellowish , very soft , freely mobile subconjunctival protrusion developed unilaterally in two and bilaterally in four patients with tao ( incidence 3.03% ) . it was one of the presenting manifestations of tao in four of ten eyes studied and incited the diagnostic work - up for tao in two of six patients in this series . magnetic resonance imaging of the orbit indicated fat density in continuity with intraorbital fat in the area of protrusion . a male to female preponderance of 4:2 and an advanced mean age at onset of tao is noteworthy for these six patients compared to the pool of 192 patients ( 64.8 versus 51.8 years , respectively , p=0.003 ) not bearing this sign.conclusionsubconjunctival orbital fat prolapse , a clinically impressive age - related ocular lesion , may occasionally predominate amid other clinical manifestations of tao . it is a nonspecific sign developing most commonly among patients with a relatively advanced age at presentation . awareness of this association may alert to the diagnosis of thyroid orbitopathy and reassure the patient and physician as to the benign character of the lesion .

Introduction Materials and methods Results Report of cases Patient 1 Patient 2 Patient 3 Patient 4 Patient 5 Patient 6 Discussion Conclusion

thyroid associated orbitopathy ( tao ) comprises a spectrum of ophthalmic and extraocular manifestations including eyelid retraction , exophthalmos , eyelid and conjunctival edema , extraocular muscle fibrosis and compressive optic neuropathy , resulting from autoimmune stimulation of orbit epitopes , including the adipose tissue and the extraocular muscles.1,2 the constellation of these findings may not always be present3 and atypical modes of presentation , especially among euthyroid patients , may obviate the diagnosis . a number of less common features of tao have been reported in the literature,47 including superior limbic keratoconjunctivitis and prominent premalar and cheek swelling ( table 1 ) . in this study we report on the finding of a prominent subconjunctival protrusion noted in the lateral canthal area of six patients with tao and comment on the diagnostic significance of this , not previously reported to our knowledge,1,4,5 clinical association . in accordance to irb requirements the sex , age at presentation of the specific sign under study as well as the age at onset of tao , the age at onset of autoimmune dysthyroidism ( when applicable ) , thyroid and autoimmune status as well as the therapeutic modalities employed were recorded . a complete ophthalmic examination , including visual acuity testing , an orthoptic evaluation , hertel exophthalmometry and assessment of the clinical activity score,8 applanation tonometry and fundoscopy ( in selected time points ) were performed at the initial and follow - up examinations . magnetic resonance imaging ( mri ) of the orbit was obtained in four patients . a comparison with the demographic characteristics ( age and sex ) for the total pool of 192 consecutive patients with tao not bearing this sign who were examined in the corresponding time period in the same tertiary care referral center was performed using descriptive statistics ( independent samples t - test , fisher s exact test ) . the first patient who drew our attention to the diagnostic significance of the superotemporal subconjunctival protrusion was a euthyroid 68-year - old man who was referred by his primary physician with the clinical suspicion of lacrimal gland enlargement in his right eye ( patient # 1 ) . five more patients , three men and two women , with a similar clinical picture and an eventual or preexisting diagnosis of tao , were prospectively enrolled . the lesion was located in the temporal or superior temporal quadrant in all six patients and shared the morphology of a convex smooth anterior surface , with the posterior surface disappearing within the fornix and the orbit . it was yellowish to purple - yellow , movable and could be easily repositioned into the orbit by applying direct pressure on it . the yellowish hue of the lesion was indicative of fat composition with occasionally visible fat globules . an orbit mri was performed in four patients and demonstrated fat density in the area of protrusion in continuity with the intraconal fat ( figures 1b , 2d , 3b ) . the incidence of this finding in a series of 198 patients with tao examined by one of us ( kic ) in the same tertiary care referral center in the corresponding 7-year time period is 3.03% . it developed unilaterally in two patients and bilaterally , albeit asymmetrically , in the other four cases . the mean age at presentation with tao symptomatology was 64.8 ( 6.6 ) years ( range 52.576 ) for these six patients compared to 51.8 ( 14.3 ) for the pool of 192 patients not bearing this sign ( p=0.003 , independent sample t - test ) . a male to female preponderance of 4:2 was noted in this series compared to approximately 6:10 ( 73/119 ) male to female ratio for the remaining 192 patients with tao . the difference in the percentage of male patients ( 66.7% versus 38.02% , respectively ) was not statistically significant ( p=0.210 , fisher s exact test ) . one of the female patients ( case # 2 ) who developed this sign at the age of 58 had a markedly obese somatotype . a history of diplopia and marked ipsilateral proptosis was reported in four patients ( cases # 1 , 2 , 4 and 5 ) . three patients ( cases # 2 , 4 , 5 ) developed a severe form of tao . it is noteworthy that apart from impairing cosmesis , the clinical course of the specific lesion was benign , there was no need for additional therapy other than lubrication of the globe to prevent exacerbation of dry eye symptoms . a 68-year - old male presented in may 2006 complaining of a visible mass protruding under the lateral part of his mildly swollen right upper eyelid and intermittent diplopia . upper eyelid retraction and restriction of elevation was noted in the right eye ( figure 1a ) . a systemic work - up for autoimmune dysthyroidism revealed euthyroid status and borderline elevation of anti - tsh receptor antibodies : 8.8 ( nl < 9 , sensitivity 0.8 ) . the diagnosis of euthyroid graves orbitopathy was established based on the typical constellation of ipsilateral proptosis , eyelid retraction , lid and conjunctival edema as well as tendon sparing enlargement of right inferior and medial rectus muscles . an orbit mri outlined anterior herniation of intraorbital fat ( figure 1b d ) with slight displacement of the lacrimal gland temporally in the involved right orbit as opposed to normal appearance of the lacrimal gland on the left side . she reported a 2.5-year long history of tao in the context of graves disease , poorly responsive to intravenous methylprednisolone regimens . she returned 3 years later with a recent onset of symptoms in the previously not involved right eye . she was particularly concerned about the emergence of a soft lump in the outer part of the recently involved right eye . exophthalmos , eyelid retraction and the presence of a soft lump under the eyelid protruding above the lateral canthus were noted in the right eye ( figure 2c ) . the lesion was soft upon palpation and persisted unchanged after a 7-month follow - up time despite gradual stabilization of thyroid levels . an orbit mri showed orbital fat prolapsing anteriorly , anterior bowing of the orbital septum and lateral displacement of the lacrimal gland which looked smaller in the involved orbit as opposed to normal size and appearance of the lacrimal gland in the contralateral left orbit ( figure 2d ) . he was followed for 15 months , during that time thyroid levels stabilized , without an improvement in the size of the lesion . a 72-year - old man presented in november 2008 with pronounced ocular misalignment and a conjunctival protrusion under both upper eyelids . mass like protrusion under the lateral part of both upper eyelids for the last several months ( figure 4b ) . asymmetric bilateral subconjunctival temporal protrusions were noted ( figure 4e ) which by history had emerged shortly after the onset of tao . there was no change in the clinical picture after 5 months a 68-year - old male presented in may 2006 complaining of a visible mass protruding under the lateral part of his mildly swollen right upper eyelid and intermittent diplopia . upper eyelid retraction and restriction of elevation was noted in the right eye ( figure 1a ) . a systemic work - up for autoimmune dysthyroidism revealed euthyroid status and borderline elevation of anti - tsh receptor antibodies : 8.8 ( nl < 9 , sensitivity 0.8 ) . the diagnosis of euthyroid graves orbitopathy was established based on the typical constellation of ipsilateral proptosis , eyelid retraction , lid and conjunctival edema as well as tendon sparing enlargement of right inferior and medial rectus muscles . an orbit mri outlined anterior herniation of intraorbital fat ( figure 1b d ) with slight displacement of the lacrimal gland temporally in the involved right orbit as opposed to normal appearance of the lacrimal gland on the left side . she reported a 2.5-year long history of tao in the context of graves disease , poorly responsive to intravenous methylprednisolone regimens . she returned 3 years later with a recent onset of symptoms in the previously not involved right eye . she was particularly concerned about the emergence of a soft lump in the outer part of the recently involved right eye . exophthalmos , eyelid retraction and the presence of a soft lump under the eyelid protruding above the lateral canthus were noted in the right eye ( figure 2c ) . the lesion was soft upon palpation and persisted unchanged after a 7-month follow - up time despite gradual stabilization of thyroid levels . an orbit mri showed orbital fat prolapsing anteriorly , anterior bowing of the orbital septum and lateral displacement of the lacrimal gland which looked smaller in the involved orbit as opposed to normal size and appearance of the lacrimal gland in the contralateral left orbit ( figure 2d ) . he was followed for 15 months , during that time thyroid levels stabilized , without an improvement in the size of the lesion . a 72-year - old man presented in november 2008 with pronounced ocular misalignment and a conjunctival protrusion under both upper eyelids . orbit mri showed anterior herniation of orbital fat with the lacrimal gland identified at the normal position and size ( figure 4a ) . a 71-year - old man presented in november 2007 complaining of a mass like protrusion under the lateral part of both upper eyelids for the last several months ( figure 4b ) . asymmetric bilateral subconjunctival temporal protrusions were noted ( figure 4e ) which by history had emerged shortly after the onset of tao . there was no change in the clinical picture after 5 months a 68-year - old male presented in may 2006 complaining of a visible mass protruding under the lateral part of his mildly swollen right upper eyelid and intermittent diplopia . upper eyelid retraction and restriction of elevation was noted in the right eye ( figure 1a ) . a systemic work - up for autoimmune dysthyroidism revealed euthyroid status and borderline elevation of anti - tsh receptor antibodies : 8.8 ( nl < 9 , sensitivity 0.8 ) . the diagnosis of euthyroid graves orbitopathy was established based on the typical constellation of ipsilateral proptosis , eyelid retraction , lid and conjunctival edema as well as tendon sparing enlargement of right inferior and medial rectus muscles . an orbit mri outlined anterior herniation of intraorbital fat ( figure 1b d ) with slight displacement of the lacrimal gland temporally in the involved right orbit as opposed to normal appearance of the lacrimal gland on the left side . she reported a 2.5-year long history of tao in the context of graves disease , poorly responsive to intravenous methylprednisolone regimens . she returned 3 years later with a recent onset of symptoms in the previously not involved right eye . she was particularly concerned about the emergence of a soft lump in the outer part of the recently involved right eye . exophthalmos , eyelid retraction and the presence of a soft lump under the eyelid protruding above the lateral canthus were noted in the right eye ( figure 2c ) . the lesion was soft upon palpation and persisted unchanged after a 7-month follow - up time despite gradual stabilization of thyroid levels . an orbit mri showed orbital fat prolapsing anteriorly , anterior bowing of the orbital septum and lateral displacement of the lacrimal gland which looked smaller in the involved orbit as opposed to normal size and appearance of the lacrimal gland in the contralateral left orbit ( figure 2d ) . he was followed for 15 months , during that time thyroid levels stabilized , without an improvement in the size of the lesion . a 72-year - old man presented in november 2008 with pronounced ocular misalignment and a conjunctival protrusion under both upper eyelids . orbit mri showed anterior herniation of orbital fat with the lacrimal gland identified at the normal position and size ( figure 4a ) . a 71-year - old man presented in november 2007 complaining of a mass like protrusion under the lateral part of both upper eyelids for the last several months ( figure 4b ) . asymmetric bilateral subconjunctival temporal protrusions were noted ( figure 4e ) which by history had emerged shortly after the onset of tao . there was no change in the clinical picture after 5 months schmack et al9 in their comprehensive study of histopatho - logical findings of subconjunctival herniated orbital fat noted that 2 of 17 patients in their series had a history of endocrine orbitopathy . saban et al10 noted that 2 of 27 patients submitted to surgical treatment for subconjunctival fat prolapse had a history of graves ophthalmopathy . the high prevalence derived from these reports ( 11.7% and 7.4% , respectively ) may suggest an association between subconjunctival fat prolapse and tao . from the clinical standpoint , subconjunctival prolapse of orbital fat accompanied the presentation of active thyroid eye disease in four of the ten eyes reported in this series ( cases # 1 , # 2 , right eye and # 3 ) and was by simple inspection an impressive enough finding to draw the internist s and ophthalmologist s attention to initiate the work - up that led to the diagnosis of tao and autoimmune dysthyroidism . the patients presented with concern about the new finding of a mass like lesion in the outer part of the eye . it extends from the limbus of the cornea anteriorly , to the optic nerve posteriorly and is pierced by the tendons of the extraocular muscles.9,11 despite its support , orbital fat has the tendency to migrate under the influence of gravity and pressure . any mechanism ( eg , aging , surgery , trauma , disease ) resulting in breaching of the surrounding connective tissue allows a forward movement of the orbital fat into either the subconjunctival space ( intraconal fat ) or the eyelids ( extraconal fat).1215 in contrast to prolapsed extraconal fat causing the well - known palpebral bags which is a common finding in elderly patients , subconjunctival intraorbital fat herniation is a rare clinical condition attributed to trauma or surgery16 or occurring spontaneously,17 typically in elderly male patients with a mean age of 6572 years ( range , 2894 years).12 it typically occurs in the lateral canthal area beneath the temporal or superotemporal bulbar conjunctiva as a soft yellowish mass with a convex anterior margin and faint superficial blood vessels.14,15,1720 it is usually bilateral , but may also be unilateral . as the extension of prolapsed fat is often asymmetric , minor lesions in the contralateral eye may be sometimes overlooked.11 the pathogenesis of spontaneous subconjunctival fat prolapse is believed to be associated with an age - related acquired local attenuation of tenon s capsule,14 allowing the fat from the intraconal space to move forward and appear clinically as a subconjunctival mass.13,18 the relative contribution of raised intraorbital pressure in facilitating anterior prolapse of orbital fat has not been appreciated so far . we postulate that the raised intraorbital pressure and increase in orbital fat characteristic of tao may predispose to anterior herniation of intraorbital fat.21 it is reasonable that this mechanism may underlie the concurrent presentation of active tao and subconjunctival fat prolapse in four patients in this series as well as an increased prevalence of this lesion among tao patients in general . it is of note that exophthalmos and lid swelling , to mention only two of the most common signs of graves ophthalmopathy , are not specific for this disease either . although subconjunctival fat prolapse is reported in case reports13,20 or small case series12,14,15,17,18 as a rare entity , there are no data in the literature accessible to us on its incidence in the general population to compare with the 3.03% rate we noted among our tao patients . the clinical manifestations of tao may be encountered at variable frequencies ranging from 92% of patients developing upper eyelid retraction during the course of their disease to 3.3% developing superior limbic keratoconjunctivitis in the large prospective study by bartley.1 less common signs , such as prominent malar and cheek swelling have been recognized in 1.8% of tao patients in another series7 ( table 1 ) . bartley and gorman3 noted that only 5% of patients with tao have the complete constellation of the so - called classic findings : eyelid retraction , exophthalmos , optic nerve dysfunction , extraocular muscle involvement and hyperthyroidism . the diagnosis may be difficult in the absence of thyroid dysfunction and should rely on the combination of certain clinical features . subconjunctival fat prolapse brought two of our patients ( case # 1 and # 3 ) to medical attention initiating the work - up that led to the diagnosis of tao ; this underscores its potential value in increasing the index of suspicion for the diagnosis of tao and hyperthyroidism . it is of note that despite the fact that it proved to be a quite impressive , and sometimes presenting clinical manifestation of tao , it was not an isolated sign of tao in any of our patients . based on these remarks , we suggest that if a substantial amount of orbital fat prolapse is noted , the clinician should check carefully for other signs of tao and the diagnosis of autoimmune dysthyroidism should be kept in mind . from the ophthalmologist s perspective , the differential diagnosis may include lacrimal gland prolapse or enlargement , dermolipoma , subconjunctival and orbital lipomas and lymphoma.14,16,2124 the differential diagnosis with a lacrimal gland mass which may also be associated with exophthalmos , inferomedial globe displacement and fullness of the eyelid ( common signs of tao , see patient # 1 in this series ) is of particular importance given the morbidity of lacrimal gland tumors . enlargement of the palpebral lobe of the lacrimal gland by inflammation , infiltration or tumor produces a palpable solid mass in the superotemporal portion of the orbit and imaging findings of lacrimal gland enlargement sometimes in association with bone molding or destruction . overall subconjunctival fat prolapse may easily be distinguished25 by clinical inspection and palpation alone based on its characteristic softness , being readily indented or moved by a cotton - tip applicator . whenever judged necessary , surgical excision of fat under local anesthesia has been suggested in the literature as a safe and effective means for treating these lesions1214,16,21,2427 with a low recurrence rate after local excision.9,10,25 based on data from this series , subconjunctival fat prolapse may be an uncommon , not isolated , but clinically impressive part of the clinical picture of tao developing primarily among male patients with an advanced age at presentation . awareness of this association may alert to the diagnosis of thyroid dysfunction and reassure the patient and physician as to the benign character of the lesion .

definitive rt includes external beam radiation therapy ( ebrt ) to the pelvis and intracavitary brachytherapy ( bt ) . rt in combination with surgery has been used for treatment of local disease , where rt has been added postoperatively to cases , showing unfavorable prognostic feature at surgery or has been given preoperatively [ 14 ] . comparisons between treatment regimens with different fractionations , dose rates and treatment time as well as the evaluation of their effectiveness on tumor and normal tissues is possible through the use of bioeffect dose models that take into account the variation of the response with all the above - mentioned factors . the linear quadratic ( lq ) model is currently the most used model for this purpose as it can account for differences in time , dose and fractionation with a relatively small number of parameters [ 57 ] . several studies have investigated the predictive value of the lq model in treatment of cervical cancer using bt [ 911 ] , but there are very few reports on biological effective dose ( bed ) and the outcome of the patients treated with a combination of radiotherapy and surgery . one recent study on cervical cancer patients treated with mri - guided preoperative brachytherapy has investigated dose volume histogram parameters and outcome . however , to the best of our knowledge , there is no study investigating the relationship between the biological effective dose ( bed ) and the outcome of patients treated with a combination of radiotherapy and surgery . it is therefore the aim of this paper to explore the relationship between bed for various treatment schedules and local control , overall survival and the risk for bladder and rectal complications for patients treated with rt combined with surgery or rt alone . a retrospective study was performed on 171 patients with cervical carcinoma figo stage ib - iib treated with curative intent using rt combined with surgery or rt alone , between january 1989 and december 1991 . one - hundred - and - thirteen patients were diagnosed in stage ib , 44 patients in stage iia and 14 cases in stage iib . squamous cell carcinoma was diagnosed in 126 patients and adenocarcinoma in 34 cases ( table 1 ) . age at diagnosis varied between 22 and 87 years with a median of 43 years for patients treated with combination rt and surgery and 72 years for patients treated with rt only . outcome , in terms of survival and local control , were defined at five years . late complications in bladder and rectum were recorded retrospectively according to the glossary of chassagne and were defined as complications persistent or occurring more than three months after radiotherapy . complications grade 2 included symptoms resulting in intermittent or persistent interference with normal activity and/or requiring investigation such as rectoscopy or cystoscopy ; grade 3 complications were defined as symptoms that affect the performance status of the patient and that require surgery or invasive procedures . patient characteristics bt brachytherapy , ebrt external beam radiotherapy during the period of this study , the treatment of choice at the department of gynecologic oncology , radiumhemmet , karolinska university hospital , for patients with cervical cancer stage ib and iia was intracervical pre - operative brachytherapy . if lymph node metastases or unradical resection margins were diagnosed at surgery , post - operative external beam radiotherapy ( ebrt ) was added . patients in stage iib were treated with radiotherapy ( rt ) alone , as well as patients in stage ib and iia not suitable for surgery due to medical reasons . for 128 patients , treatment included two bt fractions with a three - week interval , followed by radical surgery according to the wertheim - meig procedure four weeks after the second bt . postoperative ebrt over a pelvic and a para - aortic field was added to cases with lymph node metastases , while patients with non - radical surgery received ebrt over the pelvis . forty - three patients were treated with radiotherapy only , including two bt fractions with a three week interval , followed by ebrt over the pelvis four weeks later ( table 1 ) . ebrt was delivered by 6 - 21 mv linear accelerators using a - p fields with a daily fractionation of 1.6 gy . the prescribed dose to the pelvis was 45 gy ( median 44.2 gy ; range 40 - 50 gy ) and to the para - aortic field 40 gy ( median 40 gy ; range 10.6 - 45.6 gy ) . central shielding was performed from the start of ebrt with an individualized shielding to the bladder and rectum depending on the dose already given by bt , and the dose to the organs at risk under the shielding was determined based on the computerized treatment planning system . summary of the patient data on overall survival and bed to the tumour for the stage ib , iia and iib patients treated with radiotherapy and surgery or with radiotherapy alone all patients received low dose rate brachytherapy . however , during the studied period , brachytherapy technique changed from manual radium technique to remote after - loading technique with cesium-137 ( selectron ) . the manual technique was used in 39 cases ( 91% ) in the group of patients treated by rt only and in 52 cases ( 41% ) among patients treated by rt and surgery . in manual technique , the dose to bladder and rectum was estimated by measuring the dose rate by a gammameter at multiple points and the clinical dose to point a was retrospectively estimated to a median of 56 gy ( range 22 - 66 gy ) . in the after - loading technique the rectal point dose was calculated at the anterior rectal wall five mm below the posterior level of the ring - applicator and the dose to bladder was estimated at a point on the posterior surface of a catheter balloon according to the international commission on radiation units and measurements report 38 recommendations . the median dose to point a was 45 gy ( range 28 - 52 gy ) . comparison between the various treatment techniques was done based on biologically effective dose ( bed ) [ 6 , 7 ] , which allows easy comparison and addition of the effects of various complete or partial treatment regimens , irrespective of method or pattern of delivery employed . for ebrt 1bedebrt = nd{1+d/ } where n is the number of fractions , d is the dose per fraction and / is the ratio of the lq parameters of the tissue investigated . when central shielding was used with an individualized shielding to the bladder and rectum , the dose to organs at risk determined under the shielding was used in the above formula for calculating the corresponding biologically effective dose . for low dose rate bt where significant repair of damage takes place during the treatment duration , bed for each session is given by equation 2.2bedbt = d{1 + 2d(/)t[1 - 1t(1-e-t ) ] } where d is the radiation dose , t is the duration of the bt session and is a parameter characterizing the repair of sublethal damage in the irradiated tissues ; = ln(2)/t 1/2 , where t1/2 is half - life of sublethal damage repair . for combined treatment schedules , one could calculate the total bed as:3bedtot=ibedi where bed i are individual biologically effective doses from each session of rt . equation 3 gives the general bed expression that does not take into account the proliferation during treatment . however , tumor cell kinetic studies showed that proliferation of the cells after the start of treatment could be significantly higher than the proliferation before the initiation of irradiation , and therefore the biologically effective dose to the tumor could decrease to as much as about 1 gy / day . the effect of proliferation could be expressed through a supplementary term subtracted from bedtot ( equation 4).4bedprolif=ibedi - ln(2)ttreat - tktp where t treat is the overall treatment time , t k is the time for the onset of proliferation , t p is the effective doubling time during proliferation and is the linear parameter of lq model . thus , the repopulation time t p describes the proliferation of tumors after damage has been inflicted through treatment . it is thought to describe the proliferation without inhibiting factors such as nutrient supply or even as the result of active processes as appear in the healing of normal tissues . t k and therefore no correction for proliferation is needed if the overall treatment time is shorter than t k. the expressions in equations 1 - 4 were used to calculate bed for tumors and normal tissues . the parameters used for calculations have been chosen in agreement with existing literature [ 8 , 12 , 17 ] . generic values of 10 gy and 3 gy have been assumed for the fractionation sensitivities of tumors and normal tissues , respectively . the corresponding biologically effective dose for tumor was denoted bed10 and for the organs at risk bed3 , respectively . the repair half - life for sublethal damage was 1.5 h resulting in a repair parameter = 0.46 h , the time for the onset of proliferation t k = 21 days , the effective proliferation doubling time t p = 5 days and = 0.3 gy . the biological effective dose is further reported as bed calculated as described the above section , but also as equivalent dose in 2 gy per fraction , eqd2 , calculated as:5eqd2=bed1 + 2/ in this way the comparison of the results with previously reported studies using the bed concept could be easily performed , but also further comparison with studies following the new recommendations of the european gynecological brachytherapy group / european society of therapeutic radiology and oncology ( gec - estro ) for dose reporting , which recommends the use of the eqd2-formalism . a retrospective study was performed on 171 patients with cervical carcinoma figo stage ib - iib treated with curative intent using rt combined with surgery or rt alone , between january 1989 and december 1991 . one - hundred - and - thirteen patients were diagnosed in stage ib , 44 patients in stage iia and 14 cases in stage iib . squamous cell carcinoma was diagnosed in 126 patients and adenocarcinoma in 34 cases ( table 1 ) . age at diagnosis varied between 22 and 87 years with a median of 43 years for patients treated with combination rt and surgery and 72 years for patients treated with rt only . outcome , in terms of survival and local control , were defined at five years . late complications in bladder and rectum were recorded retrospectively according to the glossary of chassagne and were defined as complications persistent or occurring more than three months after radiotherapy . complications grade 2 included symptoms resulting in intermittent or persistent interference with normal activity and/or requiring investigation such as rectoscopy or cystoscopy ; grade 3 complications were defined as symptoms that affect the performance status of the patient and that require surgery or invasive procedures . during the period of this study , the treatment of choice at the department of gynecologic oncology , radiumhemmet , karolinska university hospital , for patients with cervical cancer stage ib and iia was intracervical pre - operative brachytherapy . if lymph node metastases or unradical resection margins were diagnosed at surgery , post - operative external beam radiotherapy ( ebrt ) was added . patients in stage iib were treated with radiotherapy ( rt ) alone , as well as patients in stage ib and iia not suitable for surgery due to medical reasons . for 128 patients , treatment included two bt fractions with a three - week interval , followed by radical surgery according to the wertheim - meig procedure four weeks after the second bt . postoperative ebrt over a pelvic and a para - aortic field was added to cases with lymph node metastases , while patients with non - radical surgery received ebrt over the pelvis . forty - three patients were treated with radiotherapy only , including two bt fractions with a three week interval , followed by ebrt over the pelvis four weeks later ( table 1 ) . ebrt was delivered by 6 - 21 mv linear accelerators using a - p fields with a daily fractionation of 1.6 gy . the prescribed dose to the pelvis was 45 gy ( median 44.2 gy ; range 40 - 50 gy ) and to the para - aortic field 40 gy ( median 40 gy ; range 10.6 - 45.6 gy ) . central shielding was performed from the start of ebrt with an individualized shielding to the bladder and rectum depending on the dose already given by bt , and the dose to the organs at risk under the shielding was determined based on the computerized treatment planning system . summary of the patient data on overall survival and bed to the tumour for the stage ib , iia and iib patients treated with radiotherapy and surgery or with radiotherapy alone all patients received low dose rate brachytherapy . however , during the studied period , brachytherapy technique changed from manual radium technique to remote after - loading technique with cesium-137 ( selectron ) . the manual technique was used in 39 cases ( 91% ) in the group of patients treated by rt only and in 52 cases ( 41% ) among patients treated by rt and surgery . in manual technique , the dose to bladder and rectum was estimated by measuring the dose rate by a gammameter at multiple points and the clinical dose to point a was retrospectively estimated to a median of 56 gy ( range 22 - 66 gy ) . in the after - loading technique the rectal point dose was calculated at the anterior rectal wall five mm below the posterior level of the ring - applicator and the dose to bladder was estimated at a point on the posterior surface of a catheter balloon according to the international commission on radiation units and measurements report 38 recommendations . the median dose to point a was 45 gy ( range 28 - 52 gy ) . a detailed description of the two bt techniques used has been reported elsewhere . comparison between the various treatment techniques was done based on biologically effective dose ( bed ) [ 6 , 7 ] , which allows easy comparison and addition of the effects of various complete or partial treatment regimens , irrespective of method or pattern of delivery employed . for ebrt , 1bedebrt = nd{1+d/ } where n is the number of fractions , d is the dose per fraction and / is the ratio of the lq parameters of the tissue investigated . when central shielding was used with an individualized shielding to the bladder and rectum , the dose to organs at risk determined under the shielding was used in the above formula for calculating the corresponding biologically effective dose . for low dose rate bt where significant repair of damage takes place during the treatment duration , bed for each session is given by equation 2.2bedbt = d{1 + 2d(/)t[1 - 1t(1-e-t ) ] } where d is the radiation dose , t is the duration of the bt session and is a parameter characterizing the repair of sublethal damage in the irradiated tissues ; = ln(2)/t 1/2 , where t1/2 is half - life of sublethal damage repair . for combined treatment schedules , one could calculate the total bed as:3bedtot=ibedi where bed i are individual biologically effective doses from each session of rt . equation 3 gives the general bed expression that does not take into account the proliferation during treatment . however , tumor cell kinetic studies showed that proliferation of the cells after the start of treatment could be significantly higher than the proliferation before the initiation of irradiation , and therefore the biologically effective dose to the tumor could decrease to as much as about 1 gy / day . the effect of proliferation could be expressed through a supplementary term subtracted from bedtot ( equation 4).4bedprolif=ibedi - ln(2)ttreat - tktp where t treat is the overall treatment time , t k is the time for the onset of proliferation , t p is the effective doubling time during proliferation and is the linear parameter of lq model . thus , the repopulation time t p describes the proliferation of tumors after damage has been inflicted through treatment . it is thought to describe the proliferation without inhibiting factors such as nutrient supply or even as the result of active processes as appear in the healing of normal tissues . t k and therefore no correction for proliferation is needed if the overall treatment time is shorter than t k. the expressions in equations 1 - 4 were used to calculate bed for tumors and normal tissues . the parameters used for calculations have been chosen in agreement with existing literature [ 8 , 12 , 17 ] . generic values of 10 gy and 3 gy have been assumed for the fractionation sensitivities of tumors and normal tissues , respectively . the corresponding biologically effective dose for tumor was denoted bed10 and for the organs at risk bed3 , respectively . the repair half - life for sublethal damage was 1.5 h resulting in a repair parameter = 0.46 h , the time for the onset of proliferation t k = 21 days , the effective proliferation doubling time t p = 5 days and = 0.3 gy . the biological effective dose is further reported as bed calculated as described the above section , but also as equivalent dose in 2 gy per fraction , eqd2 , calculated as:5eqd2=bed1 + 2/ in this way the comparison of the results with previously reported studies using the bed concept could be easily performed , but also further comparison with studies following the new recommendations of the european gynecological brachytherapy group / european society of therapeutic radiology and oncology ( gec - estro ) for dose reporting , which recommends the use of the eqd2-formalism . the disease - specific 5-year survival rate ( dss ) was 87% for stage ib , 75% for stage iia and 54% for stage iib . the corresponding overall 5-year survival rates ( oas ) were 84% , 68% and 43% , respectively . patients treated with radiotherapy and surgery had a local control rate at five years of 78% , while for patients treated with rt alone the five - year local control rate was 77% . table 2 presents a summary of the overall survival data for all the patients in the study divided according to stage of disease and treatment type , together with the mean and median bed10 without considering proliferation for each group . the data in the table shows that for the group of patients treated only with radiation therapy , the outcome appears to correlate with radiation dose . for the group of patients treated with bt and surgery , differences in irradiation dose calculated as bed10 did not seem to influence overall survival . among patients receiving a combination of both bt and ebrt together with surgery , the results diverge with an inverted correlation between bed10 and survival for patients in stage ib , suggesting more complicated pattern of correlations . the remaining analysis will therefore focus on the group of patients receiving only rt . figure 1 shows the distribution of patients treated with rt only , according to bed10 and their overall survival status at the follow - up time . the data show that the surviving patients had higher mean and median bed10 than the patients that were deceased at the time of analysis . this indicates a positive correlation between bed10 and treatment outcome for the patients receiving only rt . 2 where bed10 above the median value of 94 gy10 , corresponding to an equivalent dose in 2 gy per fraction ( eqd2 ) of 81 gy , correlates with better overall survival ( p = 0.0075 ) . the same tendency is maintained if proliferation is taken into account for bed10 calculation . number of patients with stage ib , iia and iib cervical carcinoma as a function of bed10 to the tumour calculated with ( right panels ) and without proliferation ( left panels ) upper panels no . of surviving patients ( 27 ) lower panel no . of deceased patients ( 16 ) dashed lines indicate the mean bed10 and solid lines correspond to the median bed10 overall survival in patients treated with rt only , correlated to bed10 above median ( number of patients = 21 ) and below median ( number of patients = 22 ) . median bed10 = 94 gy10 the dose dependence of overall survival for the 43 patients treated with rt alone is represented in fig . 3 . a logit fit of the data yields a d50 of 85.2 gy10 , the mean biological effective dose to the tumor that corresponds to a surviving fraction of 50% of the patients , corresponding to an eqd2 of 71 gy . a similar analysis was performed with bed10 that includes the potential effect of proliferation , yielding d50 = 55.7 gy10 ( eqd2 = 46 gy ) and = 1.04 . dose response curve illustrating the distribution of the surviving fraction of patients with respect to the mean bed10 for the 43 patients with stage ib , iia and iib cervical carcinoma . the data point marked with * has been excluded from the fitting figure 4 shows the correlation between bed10 and local control among patients receiving rt only . as for overall survival the dose response relationship yielded d50 = 61.9 gy10 ( eqd2 = 52 gy ) and = 0.92 for bed10 without proliferation and d50 = 36.0 gy10 ( eqd2 = 30 gy ) and = 0.80 for bed10 that accounts for proliferation . number of patients with stage ib , iia and iib cervical carcinoma as a function of bed10 to the tumour calculated with ( right panels ) and without proliferation ( left panels ) upper panel no . of patients with local control ( 36 ) lower panel no . of patients with local failure ( 7 ) dashed lines indicate the mean bed10 and solid lines correspond to the median bed10 late toxicity from bladder and rectum were recorded in 25 out of 171 patients ( 15% ) , of which five patients showed complications from both organs . grade 2 and grade 3 complications were found in 23 and 2 patients , respectively . figure 5 shows the distribution of patients on the dose intervals from the point of view of the complications to the rectum and the bladder for the patients undergoing rt only . the bed3 has been calculated from the dose to the organ determined as described in the materials and methods section . the mean bed3 was 86 gy3 ( eqd2 = 51 gy ) for rectum and 76 gy3 ( eqd2 = 46 gy ) for bladder . late rectal complications were recorded in 30.2% ( 13/43 ) for a mean bed3 of 83 gy3 ( eqd2 = 50 gy ) and bladder complications in 14% ( 6/43 ) for a mean bed3 of 88 gy3 ( eqd2 = 53 gy ) . distribution of rectal and bladder complications with respect to bed3 for all 43 patients for stage ib , iia and iib cervical carcinoma expressed as number of patients ( upper panels ) and probability ( lower panels ) the disease - specific 5-year survival rate ( dss ) was 87% for stage ib , 75% for stage iia and 54% for stage iib . the corresponding overall 5-year survival rates ( oas ) were 84% , 68% and 43% , respectively . patients treated with radiotherapy and surgery had a local control rate at five years of 78% , while for patients treated with rt alone the five - year local control rate was 77% . table 2 presents a summary of the overall survival data for all the patients in the study divided according to stage of disease and treatment type , together with the mean and median bed10 without considering proliferation for each group . the data in the table shows that for the group of patients treated only with radiation therapy , the outcome appears to correlate with radiation dose . for the group of patients treated with bt and surgery , differences in irradiation dose calculated as bed10 did not seem to influence overall survival . among patients receiving a combination of both bt and ebrt together with surgery , the results diverge with an inverted correlation between bed10 and survival for patients in stage ib , suggesting more complicated pattern of correlations . the remaining analysis will therefore focus on the group of patients receiving only rt . figure 1 shows the distribution of patients treated with rt only , according to bed10 and their overall survival status at the follow - up time . the data show that the surviving patients had higher mean and median bed10 than the patients that were deceased at the time of analysis . this indicates a positive correlation between bed10 and treatment outcome for the patients receiving only rt . 2 where bed10 above the median value of 94 gy10 , corresponding to an equivalent dose in 2 gy per fraction ( eqd2 ) of 81 gy , correlates with better overall survival ( p = 0.0075 ) . the same tendency is maintained if proliferation is taken into account for bed10 calculation . number of patients with stage ib , iia and iib cervical carcinoma as a function of bed10 to the tumour calculated with ( right panels ) and without proliferation ( left panels ) upper panels no . of surviving patients ( 27 ) lower panel no . of deceased patients ( 16 ) dashed lines indicate the mean bed10 and solid lines correspond to the median bed10 overall survival in patients treated with rt only , correlated to bed10 above median ( number of patients = 21 ) and below median ( number of patients = 22 ) . median bed10 = 94 gy10 the dose dependence of overall survival for the 43 patients treated with rt alone is represented in fig . 3 . a logit fit of the data yields a d50 of 85.2 gy10 , the mean biological effective dose to the tumor that corresponds to a surviving fraction of 50% of the patients , corresponding to an eqd2 of 71 gy . a similar analysis was performed with bed10 that includes the potential effect of proliferation , yielding d50 = 55.7 gy10 ( eqd2 = 46 gy ) and = 1.04 . dose response curve illustrating the distribution of the surviving fraction of patients with respect to the mean bed10 for the 43 patients with stage ib , iia and iib cervical carcinoma . the data point marked with * has been excluded from the fitting figure 4 shows the correlation between bed10 and local control among patients receiving rt only . as for overall survival the dose response relationship yielded d50 = 61.9 gy10 ( eqd2 = 52 gy ) and = 0.92 for bed10 without proliferation and d50 = 36.0 gy10 ( eqd2 = 30 gy ) and = 0.80 for bed10 that accounts for proliferation . number of patients with stage ib , iia and iib cervical carcinoma as a function of bed10 to the tumour calculated with ( right panels ) and without proliferation ( left panels ) upper panel no . of patients with local control ( 36 ) lower panel no . of patients with local failure ( 7 ) dashed lines indicate the mean bed10 and solid lines correspond to the median bed10 late toxicity from bladder and rectum were recorded in 25 out of 171 patients ( 15% ) , of which five patients showed complications from both organs . grade 2 and grade 3 complications were found in 23 and 2 patients , respectively . figure 5 shows the distribution of patients on the dose intervals from the point of view of the complications to the rectum and the bladder for the patients undergoing rt only . the bed3 has been calculated from the dose to the organ determined as described in the materials and methods section . the mean bed3 was 86 gy3 ( eqd2 = 51 gy ) for rectum and 76 gy3 ( eqd2 = 46 gy ) for bladder . late rectal complications were recorded in 30.2% ( 13/43 ) for a mean bed3 of 83 gy3 ( eqd2 = 50 gy ) and bladder complications in 14% ( 6/43 ) for a mean bed3 of 88 gy3 ( eqd2 = 53 gy ) . distribution of rectal and bladder complications with respect to bed3 for all 43 patients for stage ib , iia and iib cervical carcinoma expressed as number of patients ( upper panels ) and probability ( lower panels ) radiation therapy is an important modality for the treatment of cervical cancers , alone or in combination with surgery . several studies have investigated the outcome of rt in relation to bed , but to our knowledge there are no reports on bed and outcome for patients treated with a combination of rt and surgery . for treatment schedules including ebrt and bt it has been difficult to obtain a dose response relationship due to the diversity of combinations of treatment modalities and the composition of the analyzed patient populations . in a large patterns of care study lanciano et al . reported no dose response relationship in the results from 1558 patients treated with doses ranging from less than 75 gy to more than 85 gy in the clinical dose to point a. similarly , a review of 24 studies performed by petereit and pearcey failed to find a correlation between bed10 in point a and local control or survival in patients with cervical cancer stage i - iii . evaluation of individual studies reporting various beds is very difficult due to the variation in the patient composition of the analyzed populations . in an analysis by petereit et al . comparing the outcome for stage i - iii patients treated with hdr or ldr brachytherapy in combination with ebrt , bed10 varied in the range of 96 - 109 gy10 and no dose - response relationship for pelvic control or relapse - free survival and bed10 sood et al . reported a median bed10 of 87.3 gy10 for patients with local control and 87.7 gy10 for patients with local failure treated with rt alone . however , it should be noted , that sood et al . reported better local control for patients receiving treatments with bed10 above median than for patients with bed10 below the median . our results in terms of mean bed10 for the patients treated only with rt compare well with these reported studies . furthermore , we also found a positive correlation between the overall survival and bed in patients treated only with radiotherapy . the mean bed10 corresponding to a surviving fraction of 50% of the patients was d50 = 85.2 gy10 and = 1.62 . a similar analysis was performed with bed10 that includes the potential effect of proliferation , yielding d50 = 55.7 gy10 and = 1.04 . the latter analysis showed increased heterogeneity of the data , as supported by the lower , suggesting that proliferation may not be as important as recommended by the parameters used . nevertheless , it has to be mentioned , that the observed trends were maintained even after the inclusion of a proliferation factor with generic parameters in the calculation of bed10 . the size of the patient population available for this study does not however allow a more detailed analysis of the influence of proliferation . figure 4 shows the correlation between bed10 and local control in the investigated population . as for overall survival the dose response relationship yielded d50 = 61.9 gy10 and = 0.92 for bed10 without proliferation and d50 = 36.0 gy10 and = 0.80 for bed10 that accounts for proliferation . as for overall survival , the analysis of the data that included proliferation showed increased heterogeneity , supporting the hypothesis that the proliferation in the investigated population might have other characteristics than predicted by the parameters used . the late rectal complication rate we have observed was 30% ( 13/43 ) and for the bladder complication rate was 14% ( 6/43 ) calculated as the rate of complications for rt patients only . these compare well with the findings of chen et al . who reported 30% and 5% late complications from rectum and bladder , respectively . however , lanciano et al . reported a late complication rate of 9.5% , but in that study only major complications were recorded . in contrast reported only one late rectal complication among 26 patients treated with rt alone , but the definition of late complications it is not well described . we found mean bed3 of 86 gy3 for rectum and 76 gy3 for bladder , with mean bed3 of 83 gy3 and 88 gy3 for patients with rectal and bladder complications , respectively . in a study of stage ib patients treated with rt by lee et al . mean rectal bed3 was 125.6 gy3 for patients without rectal complications compared to 142.7 gy3 for patients with late complications , and the rectal complications increased when mean bed3 was 131 gy3 . in our study we had only two patients with mean bed3 131 gy3 and none of them were recorded with any late rectal complications . in this study no correlation has been observed between the rate of complications and bed3 to the organs at risk . this is in agreement with the studies by clark et al . and chen et al . who reported 125 gy3 and 110 gy3 , respectively threshold bed3 for the correlation between the dose and the rectal complications . our median bed3 for the rectum of 85 gy3 was lower than the reported threshold which could be one explanation to the lack of correlation . the low median bed3 in or study might lead to low level of complication probability , which would not totally agree to our results . this may be explained by the rather high age among patients treated with rt only . another factor of importance could be the large fraction of patients treated with manual technique generally associated with higher dosimetric uncertainties . this stresses the difficulties in comparing studies from different institutions , from different time periods or with different stage compositions of the patient populations . it has to be mentioned that the predictions of complication rates based on correlations with the biological effective dose for the organs at risk are subject of discussion due to the intrinsic limitations of the bed concept , which does not take into account the volume effects and the dose heterogeneity within the organ . while for the tumors , these are not a critical issues since they are typical parallel structures and their treatment outcome would correlate with the minimum dose , for rectum or bladder the volume effect and the distribution of the dose within the organ might be of high importance . this study shows that the outcome of the treatment correlates with biological effective dose for patients treated only with radiation therapy , but not for patients treated with radiotherapy and surgery . the dose response parameters found based on bed10 calculations were d50 = 85.2 gy10 ( eqd2 = 71 gy ) and = 1.62 for overall survival and d50 = 61.6 gy10 ( eqd2 = 52 gy ) and = 0.92 for local control .

purposethis study aims to retrospectively evaluate dosimetric parameters calculated as biological effective dose in relation to outcome in patients with cervical cancer treated with various treatment approaches , including radiotherapy with and without surgery.material and methodscalculations of biological effective dose ( bed ) were performed on data from a retrospective analysis of 171 patients with cervical carcinoma stages ib - iib treated with curative intent , between january 1989 and december 1991 . 43 patients were treated only with radiotherapy and 128 patients were treated with a combination of radiotherapy and surgery . external beam radiotherapy was delivered with 6 - 21 mv photons from linear accelerators . brachytherapy was delivered either with a manual radium technique or with a remote afterloading technique . the treatment outcome was evaluated at 5 years.resultsthe disease - specific survival rate was 87% for stage ib , 75% for stage iia and 54% for stage iib , while the overall survival rates were 84% for stage ib , 68% for stage iia and 43% for stage iib . patients treated only with radiotherapy had a local control rate of 77% which was comparable to that for radiotherapy and surgery patients ( 78% ) . late complications were recorded in 25 patients ( 15% ) . among patients treated with radiotherapy and surgery , differences in radiation dose calculated as bed10 did not seem to influence survival . for patients treated with radiotherapy only , a higher bed10 was correlated to a higher overall survival ( p = 0.0075 ) . the dose response parameters found based on biological effective dose calculations were d50 = 85.2 gy10 and the normalized to total dose slope of the dose response curve = 1.62 for survival and d50 = 61.6 gy10 and = 0.92 , respectively for local control.conclusionsthe outcome correlates with biological effective dose for patients treated with radiation therapy alone , but not for patients treated with radiotherapy and surgery . no correlations were found between bed and late toxicity from bladder and rectum .

Purpose Material and methods Patients Treatment Bioeffect dose calculations Results Tumor response Late toxicity Discussion Conclusions

several studies have investigated the predictive value of the lq model in treatment of cervical cancer using bt [ 911 ] , but there are very few reports on biological effective dose ( bed ) and the outcome of the patients treated with a combination of radiotherapy and surgery . however , to the best of our knowledge , there is no study investigating the relationship between the biological effective dose ( bed ) and the outcome of patients treated with a combination of radiotherapy and surgery . it is therefore the aim of this paper to explore the relationship between bed for various treatment schedules and local control , overall survival and the risk for bladder and rectal complications for patients treated with rt combined with surgery or rt alone . a retrospective study was performed on 171 patients with cervical carcinoma figo stage ib - iib treated with curative intent using rt combined with surgery or rt alone , between january 1989 and december 1991 . one - hundred - and - thirteen patients were diagnosed in stage ib , 44 patients in stage iia and 14 cases in stage iib . age at diagnosis varied between 22 and 87 years with a median of 43 years for patients treated with combination rt and surgery and 72 years for patients treated with rt only . late complications in bladder and rectum were recorded retrospectively according to the glossary of chassagne and were defined as complications persistent or occurring more than three months after radiotherapy . patient characteristics bt brachytherapy , ebrt external beam radiotherapy during the period of this study , the treatment of choice at the department of gynecologic oncology , radiumhemmet , karolinska university hospital , for patients with cervical cancer stage ib and iia was intracervical pre - operative brachytherapy . patients in stage iib were treated with radiotherapy ( rt ) alone , as well as patients in stage ib and iia not suitable for surgery due to medical reasons . forty - three patients were treated with radiotherapy only , including two bt fractions with a three week interval , followed by ebrt over the pelvis four weeks later ( table 1 ) . ebrt was delivered by 6 - 21 mv linear accelerators using a - p fields with a daily fractionation of 1.6 gy . central shielding was performed from the start of ebrt with an individualized shielding to the bladder and rectum depending on the dose already given by bt , and the dose to the organs at risk under the shielding was determined based on the computerized treatment planning system . summary of the patient data on overall survival and bed to the tumour for the stage ib , iia and iib patients treated with radiotherapy and surgery or with radiotherapy alone all patients received low dose rate brachytherapy . the manual technique was used in 39 cases ( 91% ) in the group of patients treated by rt only and in 52 cases ( 41% ) among patients treated by rt and surgery . in manual technique , the dose to bladder and rectum was estimated by measuring the dose rate by a gammameter at multiple points and the clinical dose to point a was retrospectively estimated to a median of 56 gy ( range 22 - 66 gy ) . in the after - loading technique the rectal point dose was calculated at the anterior rectal wall five mm below the posterior level of the ring - applicator and the dose to bladder was estimated at a point on the posterior surface of a catheter balloon according to the international commission on radiation units and measurements report 38 recommendations . comparison between the various treatment techniques was done based on biologically effective dose ( bed ) [ 6 , 7 ] , which allows easy comparison and addition of the effects of various complete or partial treatment regimens , irrespective of method or pattern of delivery employed . when central shielding was used with an individualized shielding to the bladder and rectum , the dose to organs at risk determined under the shielding was used in the above formula for calculating the corresponding biologically effective dose . the biological effective dose is further reported as bed calculated as described the above section , but also as equivalent dose in 2 gy per fraction , eqd2 , calculated as:5eqd2=bed1 + 2/ in this way the comparison of the results with previously reported studies using the bed concept could be easily performed , but also further comparison with studies following the new recommendations of the european gynecological brachytherapy group / european society of therapeutic radiology and oncology ( gec - estro ) for dose reporting , which recommends the use of the eqd2-formalism . a retrospective study was performed on 171 patients with cervical carcinoma figo stage ib - iib treated with curative intent using rt combined with surgery or rt alone , between january 1989 and december 1991 . one - hundred - and - thirteen patients were diagnosed in stage ib , 44 patients in stage iia and 14 cases in stage iib . age at diagnosis varied between 22 and 87 years with a median of 43 years for patients treated with combination rt and surgery and 72 years for patients treated with rt only . late complications in bladder and rectum were recorded retrospectively according to the glossary of chassagne and were defined as complications persistent or occurring more than three months after radiotherapy . during the period of this study , the treatment of choice at the department of gynecologic oncology , radiumhemmet , karolinska university hospital , for patients with cervical cancer stage ib and iia was intracervical pre - operative brachytherapy . patients in stage iib were treated with radiotherapy ( rt ) alone , as well as patients in stage ib and iia not suitable for surgery due to medical reasons . forty - three patients were treated with radiotherapy only , including two bt fractions with a three week interval , followed by ebrt over the pelvis four weeks later ( table 1 ) . ebrt was delivered by 6 - 21 mv linear accelerators using a - p fields with a daily fractionation of 1.6 gy . central shielding was performed from the start of ebrt with an individualized shielding to the bladder and rectum depending on the dose already given by bt , and the dose to the organs at risk under the shielding was determined based on the computerized treatment planning system . summary of the patient data on overall survival and bed to the tumour for the stage ib , iia and iib patients treated with radiotherapy and surgery or with radiotherapy alone all patients received low dose rate brachytherapy . the manual technique was used in 39 cases ( 91% ) in the group of patients treated by rt only and in 52 cases ( 41% ) among patients treated by rt and surgery . in manual technique , the dose to bladder and rectum was estimated by measuring the dose rate by a gammameter at multiple points and the clinical dose to point a was retrospectively estimated to a median of 56 gy ( range 22 - 66 gy ) . in the after - loading technique the rectal point dose was calculated at the anterior rectal wall five mm below the posterior level of the ring - applicator and the dose to bladder was estimated at a point on the posterior surface of a catheter balloon according to the international commission on radiation units and measurements report 38 recommendations . comparison between the various treatment techniques was done based on biologically effective dose ( bed ) [ 6 , 7 ] , which allows easy comparison and addition of the effects of various complete or partial treatment regimens , irrespective of method or pattern of delivery employed . when central shielding was used with an individualized shielding to the bladder and rectum , the dose to organs at risk determined under the shielding was used in the above formula for calculating the corresponding biologically effective dose . the biological effective dose is further reported as bed calculated as described the above section , but also as equivalent dose in 2 gy per fraction , eqd2 , calculated as:5eqd2=bed1 + 2/ in this way the comparison of the results with previously reported studies using the bed concept could be easily performed , but also further comparison with studies following the new recommendations of the european gynecological brachytherapy group / european society of therapeutic radiology and oncology ( gec - estro ) for dose reporting , which recommends the use of the eqd2-formalism . the disease - specific 5-year survival rate ( dss ) was 87% for stage ib , 75% for stage iia and 54% for stage iib . the corresponding overall 5-year survival rates ( oas ) were 84% , 68% and 43% , respectively . patients treated with radiotherapy and surgery had a local control rate at five years of 78% , while for patients treated with rt alone the five - year local control rate was 77% . table 2 presents a summary of the overall survival data for all the patients in the study divided according to stage of disease and treatment type , together with the mean and median bed10 without considering proliferation for each group . the data in the table shows that for the group of patients treated only with radiation therapy , the outcome appears to correlate with radiation dose . for the group of patients treated with bt and surgery , differences in irradiation dose calculated as bed10 did not seem to influence overall survival . among patients receiving a combination of both bt and ebrt together with surgery , the results diverge with an inverted correlation between bed10 and survival for patients in stage ib , suggesting more complicated pattern of correlations . figure 1 shows the distribution of patients treated with rt only , according to bed10 and their overall survival status at the follow - up time . 2 where bed10 above the median value of 94 gy10 , corresponding to an equivalent dose in 2 gy per fraction ( eqd2 ) of 81 gy , correlates with better overall survival ( p = 0.0075 ) . number of patients with stage ib , iia and iib cervical carcinoma as a function of bed10 to the tumour calculated with ( right panels ) and without proliferation ( left panels ) upper panels no . of deceased patients ( 16 ) dashed lines indicate the mean bed10 and solid lines correspond to the median bed10 overall survival in patients treated with rt only , correlated to bed10 above median ( number of patients = 21 ) and below median ( number of patients = 22 ) . median bed10 = 94 gy10 the dose dependence of overall survival for the 43 patients treated with rt alone is represented in fig . a logit fit of the data yields a d50 of 85.2 gy10 , the mean biological effective dose to the tumor that corresponds to a surviving fraction of 50% of the patients , corresponding to an eqd2 of 71 gy . a similar analysis was performed with bed10 that includes the potential effect of proliferation , yielding d50 = 55.7 gy10 ( eqd2 = 46 gy ) and = 1.04 . dose response curve illustrating the distribution of the surviving fraction of patients with respect to the mean bed10 for the 43 patients with stage ib , iia and iib cervical carcinoma . as for overall survival the dose response relationship yielded d50 = 61.9 gy10 ( eqd2 = 52 gy ) and = 0.92 for bed10 without proliferation and d50 = 36.0 gy10 ( eqd2 = 30 gy ) and = 0.80 for bed10 that accounts for proliferation . number of patients with stage ib , iia and iib cervical carcinoma as a function of bed10 to the tumour calculated with ( right panels ) and without proliferation ( left panels ) upper panel no . of patients with local failure ( 7 ) dashed lines indicate the mean bed10 and solid lines correspond to the median bed10 late toxicity from bladder and rectum were recorded in 25 out of 171 patients ( 15% ) , of which five patients showed complications from both organs . distribution of rectal and bladder complications with respect to bed3 for all 43 patients for stage ib , iia and iib cervical carcinoma expressed as number of patients ( upper panels ) and probability ( lower panels ) the disease - specific 5-year survival rate ( dss ) was 87% for stage ib , 75% for stage iia and 54% for stage iib . the corresponding overall 5-year survival rates ( oas ) were 84% , 68% and 43% , respectively . patients treated with radiotherapy and surgery had a local control rate at five years of 78% , while for patients treated with rt alone the five - year local control rate was 77% . table 2 presents a summary of the overall survival data for all the patients in the study divided according to stage of disease and treatment type , together with the mean and median bed10 without considering proliferation for each group . the data in the table shows that for the group of patients treated only with radiation therapy , the outcome appears to correlate with radiation dose . for the group of patients treated with bt and surgery , differences in irradiation dose calculated as bed10 did not seem to influence overall survival . among patients receiving a combination of both bt and ebrt together with surgery , the results diverge with an inverted correlation between bed10 and survival for patients in stage ib , suggesting more complicated pattern of correlations . figure 1 shows the distribution of patients treated with rt only , according to bed10 and their overall survival status at the follow - up time . 2 where bed10 above the median value of 94 gy10 , corresponding to an equivalent dose in 2 gy per fraction ( eqd2 ) of 81 gy , correlates with better overall survival ( p = 0.0075 ) . number of patients with stage ib , iia and iib cervical carcinoma as a function of bed10 to the tumour calculated with ( right panels ) and without proliferation ( left panels ) upper panels no . of deceased patients ( 16 ) dashed lines indicate the mean bed10 and solid lines correspond to the median bed10 overall survival in patients treated with rt only , correlated to bed10 above median ( number of patients = 21 ) and below median ( number of patients = 22 ) . median bed10 = 94 gy10 the dose dependence of overall survival for the 43 patients treated with rt alone is represented in fig . a logit fit of the data yields a d50 of 85.2 gy10 , the mean biological effective dose to the tumor that corresponds to a surviving fraction of 50% of the patients , corresponding to an eqd2 of 71 gy . dose response curve illustrating the distribution of the surviving fraction of patients with respect to the mean bed10 for the 43 patients with stage ib , iia and iib cervical carcinoma . as for overall survival the dose response relationship yielded d50 = 61.9 gy10 ( eqd2 = 52 gy ) and = 0.92 for bed10 without proliferation and d50 = 36.0 gy10 ( eqd2 = 30 gy ) and = 0.80 for bed10 that accounts for proliferation . number of patients with stage ib , iia and iib cervical carcinoma as a function of bed10 to the tumour calculated with ( right panels ) and without proliferation ( left panels ) upper panel no . of patients with local failure ( 7 ) dashed lines indicate the mean bed10 and solid lines correspond to the median bed10 late toxicity from bladder and rectum were recorded in 25 out of 171 patients ( 15% ) , of which five patients showed complications from both organs . figure 5 shows the distribution of patients on the dose intervals from the point of view of the complications to the rectum and the bladder for the patients undergoing rt only . late rectal complications were recorded in 30.2% ( 13/43 ) for a mean bed3 of 83 gy3 ( eqd2 = 50 gy ) and bladder complications in 14% ( 6/43 ) for a mean bed3 of 88 gy3 ( eqd2 = 53 gy ) . distribution of rectal and bladder complications with respect to bed3 for all 43 patients for stage ib , iia and iib cervical carcinoma expressed as number of patients ( upper panels ) and probability ( lower panels ) radiation therapy is an important modality for the treatment of cervical cancers , alone or in combination with surgery . several studies have investigated the outcome of rt in relation to bed , but to our knowledge there are no reports on bed and outcome for patients treated with a combination of rt and surgery . reported no dose response relationship in the results from 1558 patients treated with doses ranging from less than 75 gy to more than 85 gy in the clinical dose to point a. similarly , a review of 24 studies performed by petereit and pearcey failed to find a correlation between bed10 in point a and local control or survival in patients with cervical cancer stage i - iii . comparing the outcome for stage i - iii patients treated with hdr or ldr brachytherapy in combination with ebrt , bed10 varied in the range of 96 - 109 gy10 and no dose - response relationship for pelvic control or relapse - free survival and bed10 sood et al . reported a median bed10 of 87.3 gy10 for patients with local control and 87.7 gy10 for patients with local failure treated with rt alone . furthermore , we also found a positive correlation between the overall survival and bed in patients treated only with radiotherapy . the mean bed10 corresponding to a surviving fraction of 50% of the patients was d50 = 85.2 gy10 and = 1.62 . a similar analysis was performed with bed10 that includes the potential effect of proliferation , yielding d50 = 55.7 gy10 and = 1.04 . as for overall survival the dose response relationship yielded d50 = 61.9 gy10 and = 0.92 for bed10 without proliferation and d50 = 36.0 gy10 and = 0.80 for bed10 that accounts for proliferation . as for overall survival , the analysis of the data that included proliferation showed increased heterogeneity , supporting the hypothesis that the proliferation in the investigated population might have other characteristics than predicted by the parameters used . the late rectal complication rate we have observed was 30% ( 13/43 ) and for the bladder complication rate was 14% ( 6/43 ) calculated as the rate of complications for rt patients only . reported a late complication rate of 9.5% , but in that study only major complications were recorded . in contrast reported only one late rectal complication among 26 patients treated with rt alone , but the definition of late complications it is not well described . mean rectal bed3 was 125.6 gy3 for patients without rectal complications compared to 142.7 gy3 for patients with late complications , and the rectal complications increased when mean bed3 was 131 gy3 . it has to be mentioned that the predictions of complication rates based on correlations with the biological effective dose for the organs at risk are subject of discussion due to the intrinsic limitations of the bed concept , which does not take into account the volume effects and the dose heterogeneity within the organ . while for the tumors , these are not a critical issues since they are typical parallel structures and their treatment outcome would correlate with the minimum dose , for rectum or bladder the volume effect and the distribution of the dose within the organ might be of high importance . this study shows that the outcome of the treatment correlates with biological effective dose for patients treated only with radiation therapy , but not for patients treated with radiotherapy and surgery . the dose response parameters found based on bed10 calculations were d50 = 85.2 gy10 ( eqd2 = 71 gy ) and = 1.62 for overall survival and d50 = 61.6 gy10 ( eqd2 = 52 gy ) and = 0.92 for local control .

as part of the health literacy screening study,21 we assembled a retrospective cohort of patients who were discharged from an ahf hospitalization at a quaternary care hospital between november 1 , 2010 and june 30 , 2013 . all health literacy and patient outcome data were extracted from the medical center s enterprise data warehouse and research derivative databases , where data from the electronic health record ( ehr ) are stored . the medical center s institutional review board approved this study with a waiver of informed consent . hospitalization for ahf was defined as a primary discharge diagnosis of heart failure , indicated by icd-9 billing codes 402.01 , 402.11 , 402.91 , 404.01 , 404.03 , 404.11 , 404.13 , 404.91 , 404.93 , or 428.*. this algorithm was developed for use in all - payer claims datasets22 and has been utilized in multiple prior publications to identify ahf hospitalizations.2325 patients were included if they were age 18 years or older ; race and gender were available in the ehr ; the 3 brief health literacy screen ( bhls ) items were recorded during the index hospitalization ; and the patient was discharged from the hospital to home . patients transferred to other facilities such as rehabilitation or postacute care facilities were excluded because these patients would not typically manage their own medications and diet . patients who were discharged with home hospice were excluded because they often had end - stage heart failure . the first hospitalization for ahf during the study period was used as the index hospitalization . health literacy was measured by the bhls,26,27 which consists of 3 items , each on a 5-point scale ( how often do you have problems learning about your medical condition because of difficulty understanding written information ? ; how confident are you filling out medical forms by yourself ? ; and how often do you have someone ( like a family member , friend , hospital / clinic worker , or caregiver ) help you read hospital materials ? ) . since 2010 , these items have been routinely administered by nursing staff as part of hospital admission intake.21 language preference and use of an interpreter were recorded in the nursing intake assessment . implementation and widespread use of the bhls as part of nursing intake was accomplished prior to this study using a quality - improvement framework , with a focus on acceptability , adoption , appropriateness , feasibility , fidelity , and sustainability . the bhls was implemented across all adult hospital units , the ed , and 3 primary care practices between november 2010 and april 2012 . completion rate was 91.8% for the hospital among 55 611 adult inpatient admissions between november 2010 and september 2011.21 nurses orally administered the items and recorded patient responses as well as highest level of education , generally within the first 24 hours of hospitalization . continued compliance with documentation of these items has been high ( 85% to 90% ) , though nurses could opt out of asking the bhls questions if the patient was unable to answer due to medical reasons such as intubation or altered mental status . the nurse - administered bhls has been shown to be a valid measure of health literacy in our health system when compared to the bhls and short test of functional health literacy in adults administered by research personnel.21,28 the bhls item addressing confidence with forms was reverse scored , and the 3 responses were summed to generate a score between 3 and 15 , where higher scores indicated higher health literacy . this threshold is based on our prior work and was chosen to maximize sensitivity and specificity for correctly classifying patients with low health literacy compared to the short test of functional health literacy in adults.29 the primary outcome was the time from hospital discharge to death , which was censored on december 31 , 2013 . date of death was obtained from the social security death index ( ssdi ) and by extraction from the institution s ehr , which also contains clinical documentation of deaths . of 403 identified deaths , 286 ( 71.0% ) were identified in both social security death index and ehr ; 64 ( 15.9% ) were identified by social security death index , and 53 ( 13.2% ) were identified in the ehr ( and confirmed by chart review ) . planned secondary outcomes included time to the first rehospitalization and time to the first ed visit within 90 days of index hospital discharge . covariates were chosen based on expected relationships with health literacy , death , or healthcare utilization . the following covariates were recorded during the index hospitalization and extracted from the ehr : age , sex , race ( white , black , other ; by self - report),30 insurance ( private , state / federal , uninsured ) , and highest level of education . hospital length of stay was computed by administrative time stamps , and comorbid conditions31 were measured by the elixhauser comorbidity index . the elixhauser index includes 30 comorbid conditions and is associated with hospital length of stay , hospital charges , and mortality.32 the primary analysis tested the a priori hypothesis that patients with low health literacy had a shorter time / greater risk of the outcome of death after hospitalization for ahf . cumulative incidence plots were constructed to visualize the relationship between health literacy level ( bhls 9 versus bhls > 9 ) and outcomes , and the log rank test was used to compare the survival distributions . adjusted analyses used a cox proportional hazards model to examine the relationships between health literacy and the primary outcome of time - to - death , adjusting for covariates age , sex , race , insurance status , education , index hospital length of stay , and the elixhauser comorbidity index . the proportional hazards assumption was evaluated by log - log plots and tested using schoenfeld residuals.33 the same modeling approach was applied to the planned secondary outcomes of time - to - first rehospitalization and time - to - first ed visit within 90 days , in separate models . first , we examined the relationship between health literacy and death across the range of health literacy thresholds . adjusted cox regression was repeated for bhls dichotomized at a score of 14 ( bhls=15 , bhls < 15 ) and for continuous bhls with restricted cubic splines with 4 knots ( at 13 , 10 , 7 , and 4 ) . second , we conducted stratified analyses by age ( < 65 , 65 ) , race ( white , nonwhite ) , education ( high school , less than high school ) , and length of stay for the index hospitalization ( 5 days , > 5 days ) . third , patients could have multiple hospitalizations over the course of the study duration ; therefore , we evaluated rehospitalizations and ed visits using robust standard errors . no adjustment was made for multiple analyses.34 analyses were conducted using stata 12.0 ( statacorp , college station , tx ) . as part of the health literacy screening study,21 we assembled a retrospective cohort of patients who were discharged from an ahf hospitalization at a quaternary care hospital between november 1 , 2010 and june 30 , 2013 . all health literacy and patient outcome data were extracted from the medical center s enterprise data warehouse and research derivative databases , where data from the electronic health record ( ehr ) are stored . the medical center s institutional review board approved this study with a waiver of informed consent . hospitalization for ahf was defined as a primary discharge diagnosis of heart failure , indicated by icd-9 billing codes 402.01 , 402.11 , 402.91 , 404.01 , 404.03 , 404.11 , 404.13 , 404.91 , 404.93 , or 428.*. this algorithm was developed for use in all - payer claims datasets22 and has been utilized in multiple prior publications to identify ahf hospitalizations.2325 patients were included if they were age 18 years or older ; race and gender were available in the ehr ; the 3 brief health literacy screen ( bhls ) items were recorded during the index hospitalization ; and the patient was discharged from the hospital to home . patients transferred to other facilities such as rehabilitation or postacute care facilities were excluded because these patients would not typically manage their own medications and diet . patients who were discharged with home hospice were excluded because they often had end - stage heart failure . the first hospitalization for ahf during the study period was used as the index hospitalization . health literacy was measured by the bhls,26,27 which consists of 3 items , each on a 5-point scale ( how often do you have problems learning about your medical condition because of difficulty understanding written information ? ; how confident are you filling out medical forms by yourself ? ; and how often do you have someone ( like a family member , friend , hospital / clinic worker , or caregiver ) help you read hospital materials ? ) . since 2010 , these items have been routinely administered by nursing staff as part of hospital admission intake.21 language preference and use of an interpreter were recorded in the nursing intake assessment . implementation and widespread use of the bhls as part of nursing intake was accomplished prior to this study using a quality - improvement framework , with a focus on acceptability , adoption , appropriateness , feasibility , fidelity , and sustainability . the bhls was implemented across all adult hospital units , the ed , and 3 primary care practices between november 2010 and april 2012 . completion rate was 91.8% for the hospital among 55 611 adult inpatient admissions between november 2010 and september 2011.21 nurses orally administered the items and recorded patient responses as well as highest level of education , generally within the first 24 hours of hospitalization . continued compliance with documentation of these items has been high ( 85% to 90% ) , though nurses could opt out of asking the bhls questions if the patient was unable to answer due to medical reasons such as intubation or altered mental status . the nurse - administered bhls has been shown to be a valid measure of health literacy in our health system when compared to the bhls and short test of functional health literacy in adults administered by research personnel.21,28 the bhls item addressing confidence with forms was reverse scored , and the 3 responses were summed to generate a score between 3 and 15 , where higher scores indicated higher health literacy . this threshold is based on our prior work and was chosen to maximize sensitivity and specificity for correctly classifying patients with low health literacy compared to the short test of functional health literacy in adults.29 the primary outcome was the time from hospital discharge to death , which was censored on december 31 , 2013 . date of death was obtained from the social security death index ( ssdi ) and by extraction from the institution s ehr , which also contains clinical documentation of deaths . of 403 identified deaths , 286 ( 71.0% ) were identified in both social security death index and ehr ; 64 ( 15.9% ) were identified by social security death index , and 53 ( 13.2% ) were identified in the ehr ( and confirmed by chart review ) . planned secondary outcomes included time to the first rehospitalization and time to the first ed visit within 90 days of index hospital discharge . date of rehospitalization and ed visits were identified by extraction from the ehr . only rehospitalizations and ed visits at the index medical center were identified . covariates were chosen based on expected relationships with health literacy , death , or healthcare utilization . the following covariates were recorded during the index hospitalization and extracted from the ehr : age , sex , race ( white , black , other ; by self - report),30 insurance ( private , state / federal , uninsured ) , and highest level of education . hospital length of stay was computed by administrative time stamps , and comorbid conditions31 were measured by the elixhauser comorbidity index . the elixhauser index includes 30 comorbid conditions and is associated with hospital length of stay , hospital charges , and mortality.32 the primary analysis tested the a priori hypothesis that patients with low health literacy had a shorter time / greater risk of the outcome of death after hospitalization for ahf . cumulative incidence plots were constructed to visualize the relationship between health literacy level ( bhls 9 versus bhls > 9 ) and outcomes , and the log rank test was used to compare the survival distributions . adjusted analyses used a cox proportional hazards model to examine the relationships between health literacy and the primary outcome of time - to - death , adjusting for covariates age , sex , race , insurance status , education , index hospital length of stay , and the elixhauser comorbidity index . the proportional hazards assumption was evaluated by log - log plots and tested using schoenfeld residuals.33 the same modeling approach was applied to the planned secondary outcomes of time - to - first rehospitalization and time - to - first ed visit within 90 days , in separate models . first , we examined the relationship between health literacy and death across the range of health literacy thresholds . adjusted cox regression was repeated for bhls dichotomized at a score of 14 ( bhls=15 , bhls < 15 ) and for continuous bhls with restricted cubic splines with 4 knots ( at 13 , 10 , 7 , and 4 ) . second , we conducted stratified analyses by age ( < 65 , 65 ) , race ( white , nonwhite ) , education ( high school , less than high school ) , and length of stay for the index hospitalization ( 5 days , > 5 days ) . third , patients could have multiple hospitalizations over the course of the study duration ; therefore , we evaluated rehospitalizations and ed visits using robust standard errors . no adjustment was made for multiple analyses.34 analyses were conducted using stata 12.0 ( statacorp , college station , tx ) . between november 1 , 2010 and june 30 , 2013 , there were 2132 patients hospitalized for ahf ; of these , 162 died in the hospital or had unknown hospital disposition and 251 were transferred to a care facility or hospice . of the remaining 1719 patients , 245 had incomplete bhls and 95 were missing covariates , leaving 1379 who were discharged home and had complete bhls and demographic information ( figure 1 ) . of these , 1055 ( 76.5% ) had a bhls > 9 , and 324 ( 23.5% ) had low health literacy ( bhls 9 ) . use of an interpreter was recorded for 33 patients ( 2.3% ) . patient characteristics by health literacy status are found in table 1 ; analysis results are found in table 2 and table 3 . patients with low health literacy were older and more likely to be male , insured by federal or state health insurance , and to have not completed high school . characteristics of patients excluded from the analyses for missing bhls or covariates are shown in tabletable 4 . characteristics of patients by bhls score bhls indicates brief health literacy screen ; iqr , interquartile range ; los , length of stay . adjusted cox regression analyses for outcomes : death , 90-day rehospitalization , and 90-day ed visit by bhls bhls indicates brief health literacy screen ; ed , emergency department ; hr , hazard ratio . cox regression adjusted for age , gender , race , insurance , education , index hospitalization length of stay , and disease severity . cox model demonstrates the hazard for the risk of the outcome among those with a bhls score 9 compared to those with a bhls score > 9 . stratified analyses for mortality by subgroups bhls indicates brief health literacy screen ; hr , hazard ratio ; los , length of stay . cox regression adjusted for age , gender , race , insurance , education , index hospitalization length of stay , and disease severity . cox model demonstrates the hazard for the risk of the outcome among those with a bhls score 9 compared to those with a bhls score > 9 . patient characteristics among those with incomplete bhls , bhls 9 , and bhls > 9 bhls indicates brief health literacy screen ; hr , hazard ratio ; los , length of stay . kruskal wallis or nonparametric test for trend , as appropriate . cox regression for death by bhls > 9 , bhls 9 , incomplete bhls ; adjusted for age , gender , race , insurance , education , index hospitalization length of stay , and disease severity . there were 403 ( 29.2% ) deaths during study follow - up ( table2 ) . median follow - up was 20.7 months ( interquartile range 12.8 , 29.6 months ) . of the 324 patients with bhls 9 , 124 ( 38.3% ) died , compared to 279 ( 26.5% ) deaths among the 1055 patients with bhls > 9 . the median time to death was shorter for patients with bhls 9 than for patients with bhls > 9 ( log rank p<0.001 ) . after adjusting for age , gender , race , insurance , highest level of education , hospital length of stay , and comorbid conditions , the risk of death for patients with bhls 9 was 32% higher than for patients with bhls > 9 ( ahr 1.32 ; 95% ci 1.05 , 1.66 , p=0.02 ) . bhls indicates brief health literacy screen . during the 90-day follow - up after each hospitalization for ahf , 415 ( 30.1% ) patients were rehospitalized and 201 ( 14.6% ) had an ed visit and were discharged home . the average number of ed visits within 90 days per patient was 1.84 ( median 1 , interquartile range 0 , 2 ) . there was no evident association of health literacy with time to first rehospitalization or ed visit ( table2 ) . when we varied the threshold for low health literacy to a bhls score of < 15 , results were consistent with the main findings ( ahr=1.35 , 95% ci 1.08 , 1.69 ) . when bhls was modeled using restricted cubic splines , the hazard of death increased as bhls decreased ( figure 3 ) . thus , the risk of death for each decreasing level of health literacy was as follows : ahr 1.23 ( 95% ci 1.03 , 1.47 ) , 1.43 ( 95% ci 1.11 , 1.85 ) , 1.48 ( 95% ci 1.14 , 1.91 ) , and 1.50 ( 95% ci 1.05 , 2.13 ) for bhls scores of 13 , 10 , 7 , and 4 , respectively . health literacy and association with mortality restricted cubic splines . adjusted for age , sex , race , insurance , education , index hospitalization length of stay , and disease severity . there was no evidence for effect modification by age , race , education , and hospital length - of - stay , with interaction term p - values all larger than 0.05 , although relationships between literacy and mortality were statistically significant only among subgroups for subjects with age 65 years , nonwhite race , less than high school education , and index hospital length - of - stay 5 days ( table3 ) . allowing multiple hospitalizations did not change the results of analyses for rehospitalizations or ed visits . compared to patients who completed all 3 bhls survey items , those with incomplete bhls were younger , more likely to be female , white , have private insurance , and have had a shorter hospital length of stay . education level was missing for 220 ( 89.8% ) of these patients ; of the 25 patients with education available , none had completed high school ( table4 ) . after adjustment for disease severity and comorbid conditions , there was no difference in mortality risk between those with incomplete and complete bhls ( ahr of death 0.73 , 95% ci 0.30 , 1.76 , table4 ) . there were 403 ( 29.2% ) deaths during study follow - up ( table2 ) . median follow - up was 20.7 months ( interquartile range 12.8 , 29.6 months ) . of the 324 patients with bhls 9 , 124 ( 38.3% ) died , compared to 279 ( 26.5% ) deaths among the 1055 patients with bhls > 9 . the median time to death was shorter for patients with bhls 9 than for patients with bhls > 9 ( log rank p<0.001 ) . after adjusting for age , gender , race , insurance , highest level of education , hospital length of stay , and comorbid conditions , the risk of death for patients with bhls 9 was 32% higher than for patients with bhls > 9 ( ahr 1.32 ; 95% ci 1.05 , 1.66 , p=0.02 ) . during the 90-day follow - up after each hospitalization for ahf , 415 ( 30.1% ) patients were rehospitalized and 201 ( 14.6% ) had an ed visit and were discharged home . the average number of ed visits within 90 days per patient was 1.84 ( median 1 , interquartile range 0 , 2 ) . there was no evident association of health literacy with time to first rehospitalization or ed visit ( table2 ) . when we varied the threshold for low health literacy to a bhls score of < 15 , results were consistent with the main findings ( ahr=1.35 , 95% ci 1.08 , 1.69 ) . when bhls was modeled using restricted cubic splines , the hazard of death increased as bhls decreased ( figure 3 ) . thus , the risk of death for each decreasing level of health literacy was as follows : ahr 1.23 ( 95% ci 1.03 , 1.47 ) , 1.43 ( 95% ci 1.11 , 1.85 ) , 1.48 ( 95% ci 1.14 , 1.91 ) , and 1.50 ( 95% ci 1.05 , 2.13 ) for bhls scores of 13 , 10 , 7 , and 4 , respectively . adjusted for age , sex , race , insurance , education , index hospitalization length of stay , and disease severity . there was no evidence for effect modification by age , race , education , and hospital length - of - stay , with interaction term p - values all larger than 0.05 , although relationships between literacy and mortality were statistically significant only among subgroups for subjects with age 65 years , nonwhite race , less than high school education , and index hospital length - of - stay 5 days ( table3 ) . allowing multiple hospitalizations there were 245 subjects excluded for incomplete bhls . compared to patients who completed all 3 bhls survey items , those with incomplete bhls were younger , more likely to be female , white , have private insurance , and have had a shorter hospital length of stay . education level was missing for 220 ( 89.8% ) of these patients ; of the 25 patients with education available , none had completed high school ( table4 ) . after adjustment for disease severity and comorbid conditions , there was no difference in mortality risk between those with incomplete and complete bhls ( ahr of death 0.73 , 95% ci 0.30 , 1.76 , table4 ) . in this study of 1379 patients hospitalized for ahf , low health literacy was associated with increased risk of death ( ahr 1.32 , 95% ci 1.05 , 1.66 , p=0.02 ) after adjustment for education , age , sex , race , health insurance status , hospital length of stay , and comorbidities . low health literacy , defined as bhls < 15 , bhls < 13 , or bhls 9 was associated with increased risk of mortality . taken together , these results suggest that patients with anything less than optimal health literacy who are hospitalized for ahf may warrant additional health resources , assistance , discharge planning , or coordination of care to facilitate the transition from hospital to outpatient care in order to optimize long - term self - care of heart failure . in this study , the relationship between health literacy and mortality was not modified by age , race , or education . our findings lend additional weight to the importance of health literacy and provide guidance for healthcare providers as they risk stratify patients hospitalized for ahf . in our healthcare system , results of the health literacy measure are available to all healthcare providers through the ehr , overcoming previous difficulties identifying patients with low health literacy16,17 ; in the future , this easy access to a reliable measure of health literacy may facilitate more accurate identification of and intervention for patients with heart failure to reduce the risk of death . in general , low health literacy has been associated with multiple health outcomes , including death and healthcare utilization.19,35 self - care is recognized as a vital component of chronic heart failure management,10 which includes monitoring weight , salt intake , and fluid intake , and titrating diuretics ; these in turn may be related to health literacy and mortality . however , the direct relationships among health literacy , self - care , and outcomes including mortality and healthcare utilization are not well understood.14,15 investigation regarding effective interventions to improve heart failure chronic self - care and patient - centered outcomes is ongoing.36 patient education alone has generally not been enough to improve outcomes.37 health literacy can influence chronic disease management . healthcare providers often overestimate the health literacy skills of their patients.16,17 as a result , patients with unrecognized low health literacy may receive healthcare instructions and prescription regimens that are complex , difficult to understand and implement , limiting their ability to successfully achieve outpatient disease control . these patients may also have difficulty communicating with healthcare providers , navigating the health care system , recognizing signs of health decline , and knowing when and who to contact when they do become ill . low health literacy may influence mortality via disease self - management as well as unrecognized impact on outpatient disease management by healthcare providers . in analyses of 90-day rehospitalization and ed visits these findings are consistent with work by peterson et al,19 which was conducted among outpatients with heart failure in which the bhls was administered via mail ; in their study , low health literacy ( defined as bhls < 6 ) was found to predict all - cause mortality but not hospitalization . we expand on these findings by showing that this association with mortality is also found among patients hospitalized for ahf and that the risk of death is borne by patients with bhls < 15 , not only those with bhls 9 or even bhls < 6 . although we did not find evidence for a relationship between low health literacy and ed visits or with rehospitalizations within 90 days , the proportion of patients who were rehospitalized or had an ed visit is similar to those reported elsewhere.38 ed visits and rehospitalizations after ahf are multifactorial39,40 ; the relationship between health literacy and mortality among patients with heart failure may be independent of markers of healthcare utilization such as ed visits and rehospitalization . in addition , a weak relationship with ed visits or rehospitalization may not have been detectable in the 90-day follow - up period , compared to the multiyear follow - up for mortality . finally , we can not conclusively exclude a relationship between health literacy and rehospitalizations or ed visits because we were unable to include all rehospitalizations and ed visits to other hospitals . health literacy was measured by the bhls , a 3-item survey administered by nursing staff at the time of hospitalization . we have shown this method of administration to be a valid measure of health literacy compared to the short test of functional health literacy in adults.41 while patient self - report may increase the risk of social desirability bias , nondifferential misclassification of health literacy would underestimate the true risk of death.42 in a highly health - literate population , the bhls may be subject to a ceiling effect . however , even in our population , in which 23.5% had low health literacy , we found that low health literacy was associated with increased risk of death . analyses included adjustment for age , sex , race , health insurance , highest level of education , hospital length of stay , and comorbidities . despite this , it is possible that type of heart disease or other difficult - to - measure factors such as depression and patient frailty may have a residual influence on the relationship between health literacy and mortality . we did not include medication changes and management decisions made after discharge from the hospital , as these may be part of the path between patient s health literacy and outcomes . hospitalized patients with incomplete education or bhls may differ in their characteristics from those who with complete bhls information.43 we did not perform multiple imputation of the bhls for the 245 subjects with 1 or more bhls items missing due to the possibility that missingness may not have been random . however , after adjusting for patient characteristics , we did not find a difference in the risk of death between patients with complete and those with incomplete bhls ( table4 ) . within the available data , we are unable to account for potential prior hospitalizations , cardiac events , or other indicators of disease severity such as ejection fraction , which have been shown to be associated with death8,44 and may be related to health literacy . finally , we selected this population from patients hospitalized at a quaternary medical center for ahf . while this population may not be reflective of all patients hospitalized with ahf , the methods for identifying patients hospitalized for ahf are standardized and widely used for quality metric reporting . as a result , our findings are likely to be generalizable to many patients hospitalized for ahf who have low health literacy . in this retrospective study of patients discharged home after hospitalization for ahf , death , rehospitalization , and ed visits were common . patients with a lower level of health literacy as measured by the bhls had an increased risk of all - cause death ; this risk rose with decreasing health literacy . this suggests that additional measures to overcome barriers to effective long - term heart failure self - care may be needed among patients with any degree of low health literacy . this research was supported by funding from the national heart , lung , and blood institute ( r21hl096581 , k12hl109109 , and ul1tr000445 ) . dr storrow is a current consultant for roche diagnostics ; novartis pharmaceuticals corp , usa ; alere diagnostics ; trevena ; and beckman coulter .

backgroundmore than 30% of patients hospitalized for heart failure are rehospitalized or die within 90 days of discharge . lower health literacy is associated with mortality among outpatients with chronic heart failure ; little is known about this relationship after hospitalization for acute heart failure.methods and resultspatients hospitalized for acute heart failure and discharged home between november 2010 and june 2013 were followed through december 31 , 2013 . nurses administered the brief health literacy screen at admission ; low health literacy was defined as brief health literacy screen 9 . the primary outcome was all - cause mortality . secondary outcomes were time to first rehospitalization and , separately , time to first emergency department visit within 90 days of discharge . cox proportional hazards models determined their relationships with health literacy , adjusting for age , gender , race , insurance , education , comorbidity , and hospital length of stay . for the 1379 patients , average age was 63.1 years , 566 ( 41.0% ) were female , and 324 ( 23.5% ) had low health literacy . median follow - up was 20.7 months ( interquartile range 12.8 to 29.6 months ) , and 403 ( 29.2% ) patients died . adjusted hazard ratio for death among patients with low health literacy was 1.34 ( 95% ci 1.04 , 1.73 , p=0.02 ) compared to brief health literacy screen > 9 . within 90 days of discharge , there were 415 ( 30.1% ) rehospitalizations and 201 ( 14.6% ) emergency department visits , with no evident association with health literacy.conclusionslower health literacy was associated with increased risk of death after hospitalization for acute heart failure . there was no evident relationship between health literacy and 90-day rehospitalization or emergency department visits .

Methods Study Design, Setting, and Data Sources Population Patient-Reported Health Literacy Outcomes: Death, 90-Day Rehospitalization, ED Visit Covariates Statistical Analysis Sensitivity and Subgroup Analyses Results Primary Outcome: Hazard of Mortality by Health Literacy Secondary Outcomes: Rehospitalization, ED Visits Within 90Days Sensitivity and Subgroup Analysis Incomplete BHLS Discussion Conclusions Sources of Funding Disclosures

as part of the health literacy screening study,21 we assembled a retrospective cohort of patients who were discharged from an ahf hospitalization at a quaternary care hospital between november 1 , 2010 and june 30 , 2013 . hospitalization for ahf was defined as a primary discharge diagnosis of heart failure , indicated by icd-9 billing codes 402.01 , 402.11 , 402.91 , 404.01 , 404.03 , 404.11 , 404.13 , 404.91 , 404.93 , or 428.*. this algorithm was developed for use in all - payer claims datasets22 and has been utilized in multiple prior publications to identify ahf hospitalizations.2325 patients were included if they were age 18 years or older ; race and gender were available in the ehr ; the 3 brief health literacy screen ( bhls ) items were recorded during the index hospitalization ; and the patient was discharged from the hospital to home . the bhls was implemented across all adult hospital units , the ed , and 3 primary care practices between november 2010 and april 2012 . completion rate was 91.8% for the hospital among 55 611 adult inpatient admissions between november 2010 and september 2011.21 nurses orally administered the items and recorded patient responses as well as highest level of education , generally within the first 24 hours of hospitalization . this threshold is based on our prior work and was chosen to maximize sensitivity and specificity for correctly classifying patients with low health literacy compared to the short test of functional health literacy in adults.29 the primary outcome was the time from hospital discharge to death , which was censored on december 31 , 2013 . planned secondary outcomes included time to the first rehospitalization and time to the first ed visit within 90 days of index hospital discharge . covariates were chosen based on expected relationships with health literacy , death , or healthcare utilization . the following covariates were recorded during the index hospitalization and extracted from the ehr : age , sex , race ( white , black , other ; by self - report),30 insurance ( private , state / federal , uninsured ) , and highest level of education . hospital length of stay was computed by administrative time stamps , and comorbid conditions31 were measured by the elixhauser comorbidity index . the elixhauser index includes 30 comorbid conditions and is associated with hospital length of stay , hospital charges , and mortality.32 the primary analysis tested the a priori hypothesis that patients with low health literacy had a shorter time / greater risk of the outcome of death after hospitalization for ahf . cumulative incidence plots were constructed to visualize the relationship between health literacy level ( bhls 9 versus bhls > 9 ) and outcomes , and the log rank test was used to compare the survival distributions . adjusted analyses used a cox proportional hazards model to examine the relationships between health literacy and the primary outcome of time - to - death , adjusting for covariates age , sex , race , insurance status , education , index hospital length of stay , and the elixhauser comorbidity index . the proportional hazards assumption was evaluated by log - log plots and tested using schoenfeld residuals.33 the same modeling approach was applied to the planned secondary outcomes of time - to - first rehospitalization and time - to - first ed visit within 90 days , in separate models . first , we examined the relationship between health literacy and death across the range of health literacy thresholds . second , we conducted stratified analyses by age ( < 65 , 65 ) , race ( white , nonwhite ) , education ( high school , less than high school ) , and length of stay for the index hospitalization ( 5 days , > 5 days ) . as part of the health literacy screening study,21 we assembled a retrospective cohort of patients who were discharged from an ahf hospitalization at a quaternary care hospital between november 1 , 2010 and june 30 , 2013 . hospitalization for ahf was defined as a primary discharge diagnosis of heart failure , indicated by icd-9 billing codes 402.01 , 402.11 , 402.91 , 404.01 , 404.03 , 404.11 , 404.13 , 404.91 , 404.93 , or 428.*. completion rate was 91.8% for the hospital among 55 611 adult inpatient admissions between november 2010 and september 2011.21 nurses orally administered the items and recorded patient responses as well as highest level of education , generally within the first 24 hours of hospitalization . this threshold is based on our prior work and was chosen to maximize sensitivity and specificity for correctly classifying patients with low health literacy compared to the short test of functional health literacy in adults.29 the primary outcome was the time from hospital discharge to death , which was censored on december 31 , 2013 . of 403 identified deaths , 286 ( 71.0% ) were identified in both social security death index and ehr ; 64 ( 15.9% ) were identified by social security death index , and 53 ( 13.2% ) were identified in the ehr ( and confirmed by chart review ) . planned secondary outcomes included time to the first rehospitalization and time to the first ed visit within 90 days of index hospital discharge . covariates were chosen based on expected relationships with health literacy , death , or healthcare utilization . the following covariates were recorded during the index hospitalization and extracted from the ehr : age , sex , race ( white , black , other ; by self - report),30 insurance ( private , state / federal , uninsured ) , and highest level of education . hospital length of stay was computed by administrative time stamps , and comorbid conditions31 were measured by the elixhauser comorbidity index . the elixhauser index includes 30 comorbid conditions and is associated with hospital length of stay , hospital charges , and mortality.32 the primary analysis tested the a priori hypothesis that patients with low health literacy had a shorter time / greater risk of the outcome of death after hospitalization for ahf . cumulative incidence plots were constructed to visualize the relationship between health literacy level ( bhls 9 versus bhls > 9 ) and outcomes , and the log rank test was used to compare the survival distributions . adjusted analyses used a cox proportional hazards model to examine the relationships between health literacy and the primary outcome of time - to - death , adjusting for covariates age , sex , race , insurance status , education , index hospital length of stay , and the elixhauser comorbidity index . the proportional hazards assumption was evaluated by log - log plots and tested using schoenfeld residuals.33 the same modeling approach was applied to the planned secondary outcomes of time - to - first rehospitalization and time - to - first ed visit within 90 days , in separate models . second , we conducted stratified analyses by age ( < 65 , 65 ) , race ( white , nonwhite ) , education ( high school , less than high school ) , and length of stay for the index hospitalization ( 5 days , > 5 days ) . between november 1 , 2010 and june 30 , 2013 , there were 2132 patients hospitalized for ahf ; of these , 162 died in the hospital or had unknown hospital disposition and 251 were transferred to a care facility or hospice . of these , 1055 ( 76.5% ) had a bhls > 9 , and 324 ( 23.5% ) had low health literacy ( bhls 9 ) . patients with low health literacy were older and more likely to be male , insured by federal or state health insurance , and to have not completed high school . characteristics of patients by bhls score bhls indicates brief health literacy screen ; iqr , interquartile range ; los , length of stay . adjusted cox regression analyses for outcomes : death , 90-day rehospitalization , and 90-day ed visit by bhls bhls indicates brief health literacy screen ; ed , emergency department ; hr , hazard ratio . cox regression adjusted for age , gender , race , insurance , education , index hospitalization length of stay , and disease severity . cox model demonstrates the hazard for the risk of the outcome among those with a bhls score 9 compared to those with a bhls score > 9 . stratified analyses for mortality by subgroups bhls indicates brief health literacy screen ; hr , hazard ratio ; los , length of stay . cox regression adjusted for age , gender , race , insurance , education , index hospitalization length of stay , and disease severity . cox model demonstrates the hazard for the risk of the outcome among those with a bhls score 9 compared to those with a bhls score > 9 . patient characteristics among those with incomplete bhls , bhls 9 , and bhls > 9 bhls indicates brief health literacy screen ; hr , hazard ratio ; los , length of stay . cox regression for death by bhls > 9 , bhls 9 , incomplete bhls ; adjusted for age , gender , race , insurance , education , index hospitalization length of stay , and disease severity . there were 403 ( 29.2% ) deaths during study follow - up ( table2 ) . median follow - up was 20.7 months ( interquartile range 12.8 , 29.6 months ) . after adjusting for age , gender , race , insurance , highest level of education , hospital length of stay , and comorbid conditions , the risk of death for patients with bhls 9 was 32% higher than for patients with bhls > 9 ( ahr 1.32 ; 95% ci 1.05 , 1.66 , p=0.02 ) . during the 90-day follow - up after each hospitalization for ahf , 415 ( 30.1% ) patients were rehospitalized and 201 ( 14.6% ) had an ed visit and were discharged home . the average number of ed visits within 90 days per patient was 1.84 ( median 1 , interquartile range 0 , 2 ) . there was no evident association of health literacy with time to first rehospitalization or ed visit ( table2 ) . when we varied the threshold for low health literacy to a bhls score of < 15 , results were consistent with the main findings ( ahr=1.35 , 95% ci 1.08 , 1.69 ) . thus , the risk of death for each decreasing level of health literacy was as follows : ahr 1.23 ( 95% ci 1.03 , 1.47 ) , 1.43 ( 95% ci 1.11 , 1.85 ) , 1.48 ( 95% ci 1.14 , 1.91 ) , and 1.50 ( 95% ci 1.05 , 2.13 ) for bhls scores of 13 , 10 , 7 , and 4 , respectively . health literacy and association with mortality restricted cubic splines . adjusted for age , sex , race , insurance , education , index hospitalization length of stay , and disease severity . there was no evidence for effect modification by age , race , education , and hospital length - of - stay , with interaction term p - values all larger than 0.05 , although relationships between literacy and mortality were statistically significant only among subgroups for subjects with age 65 years , nonwhite race , less than high school education , and index hospital length - of - stay 5 days ( table3 ) . compared to patients who completed all 3 bhls survey items , those with incomplete bhls were younger , more likely to be female , white , have private insurance , and have had a shorter hospital length of stay . after adjustment for disease severity and comorbid conditions , there was no difference in mortality risk between those with incomplete and complete bhls ( ahr of death 0.73 , 95% ci 0.30 , 1.76 , table4 ) . there were 403 ( 29.2% ) deaths during study follow - up ( table2 ) . median follow - up was 20.7 months ( interquartile range 12.8 , 29.6 months ) . of the 324 patients with bhls 9 , 124 ( 38.3% ) died , compared to 279 ( 26.5% ) deaths among the 1055 patients with bhls > 9 . after adjusting for age , gender , race , insurance , highest level of education , hospital length of stay , and comorbid conditions , the risk of death for patients with bhls 9 was 32% higher than for patients with bhls > 9 ( ahr 1.32 ; 95% ci 1.05 , 1.66 , p=0.02 ) . during the 90-day follow - up after each hospitalization for ahf , 415 ( 30.1% ) patients were rehospitalized and 201 ( 14.6% ) had an ed visit and were discharged home . the average number of ed visits within 90 days per patient was 1.84 ( median 1 , interquartile range 0 , 2 ) . there was no evident association of health literacy with time to first rehospitalization or ed visit ( table2 ) . when we varied the threshold for low health literacy to a bhls score of < 15 , results were consistent with the main findings ( ahr=1.35 , 95% ci 1.08 , 1.69 ) . thus , the risk of death for each decreasing level of health literacy was as follows : ahr 1.23 ( 95% ci 1.03 , 1.47 ) , 1.43 ( 95% ci 1.11 , 1.85 ) , 1.48 ( 95% ci 1.14 , 1.91 ) , and 1.50 ( 95% ci 1.05 , 2.13 ) for bhls scores of 13 , 10 , 7 , and 4 , respectively . adjusted for age , sex , race , insurance , education , index hospitalization length of stay , and disease severity . there was no evidence for effect modification by age , race , education , and hospital length - of - stay , with interaction term p - values all larger than 0.05 , although relationships between literacy and mortality were statistically significant only among subgroups for subjects with age 65 years , nonwhite race , less than high school education , and index hospital length - of - stay 5 days ( table3 ) . compared to patients who completed all 3 bhls survey items , those with incomplete bhls were younger , more likely to be female , white , have private insurance , and have had a shorter hospital length of stay . after adjustment for disease severity and comorbid conditions , there was no difference in mortality risk between those with incomplete and complete bhls ( ahr of death 0.73 , 95% ci 0.30 , 1.76 , table4 ) . in this study of 1379 patients hospitalized for ahf , low health literacy was associated with increased risk of death ( ahr 1.32 , 95% ci 1.05 , 1.66 , p=0.02 ) after adjustment for education , age , sex , race , health insurance status , hospital length of stay , and comorbidities . low health literacy , defined as bhls < 15 , bhls < 13 , or bhls 9 was associated with increased risk of mortality . taken together , these results suggest that patients with anything less than optimal health literacy who are hospitalized for ahf may warrant additional health resources , assistance , discharge planning , or coordination of care to facilitate the transition from hospital to outpatient care in order to optimize long - term self - care of heart failure . in this study , the relationship between health literacy and mortality was not modified by age , race , or education . our findings lend additional weight to the importance of health literacy and provide guidance for healthcare providers as they risk stratify patients hospitalized for ahf . in our healthcare system , results of the health literacy measure are available to all healthcare providers through the ehr , overcoming previous difficulties identifying patients with low health literacy16,17 ; in the future , this easy access to a reliable measure of health literacy may facilitate more accurate identification of and intervention for patients with heart failure to reduce the risk of death . in general , low health literacy has been associated with multiple health outcomes , including death and healthcare utilization.19,35 self - care is recognized as a vital component of chronic heart failure management,10 which includes monitoring weight , salt intake , and fluid intake , and titrating diuretics ; these in turn may be related to health literacy and mortality . however , the direct relationships among health literacy , self - care , and outcomes including mortality and healthcare utilization are not well understood.14,15 investigation regarding effective interventions to improve heart failure chronic self - care and patient - centered outcomes is ongoing.36 patient education alone has generally not been enough to improve outcomes.37 health literacy can influence chronic disease management . in analyses of 90-day rehospitalization and ed visits these findings are consistent with work by peterson et al,19 which was conducted among outpatients with heart failure in which the bhls was administered via mail ; in their study , low health literacy ( defined as bhls < 6 ) was found to predict all - cause mortality but not hospitalization . we expand on these findings by showing that this association with mortality is also found among patients hospitalized for ahf and that the risk of death is borne by patients with bhls < 15 , not only those with bhls 9 or even bhls < 6 . although we did not find evidence for a relationship between low health literacy and ed visits or with rehospitalizations within 90 days , the proportion of patients who were rehospitalized or had an ed visit is similar to those reported elsewhere.38 ed visits and rehospitalizations after ahf are multifactorial39,40 ; the relationship between health literacy and mortality among patients with heart failure may be independent of markers of healthcare utilization such as ed visits and rehospitalization . finally , we can not conclusively exclude a relationship between health literacy and rehospitalizations or ed visits because we were unable to include all rehospitalizations and ed visits to other hospitals . we have shown this method of administration to be a valid measure of health literacy compared to the short test of functional health literacy in adults.41 while patient self - report may increase the risk of social desirability bias , nondifferential misclassification of health literacy would underestimate the true risk of death.42 in a highly health - literate population , the bhls may be subject to a ceiling effect . however , even in our population , in which 23.5% had low health literacy , we found that low health literacy was associated with increased risk of death . analyses included adjustment for age , sex , race , health insurance , highest level of education , hospital length of stay , and comorbidities . despite this , it is possible that type of heart disease or other difficult - to - measure factors such as depression and patient frailty may have a residual influence on the relationship between health literacy and mortality . however , after adjusting for patient characteristics , we did not find a difference in the risk of death between patients with complete and those with incomplete bhls ( table4 ) . as a result , our findings are likely to be generalizable to many patients hospitalized for ahf who have low health literacy . in this retrospective study of patients discharged home after hospitalization for ahf , death , rehospitalization , and ed visits were common . patients with a lower level of health literacy as measured by the bhls had an increased risk of all - cause death ; this risk rose with decreasing health literacy . this suggests that additional measures to overcome barriers to effective long - term heart failure self - care may be needed among patients with any degree of low health literacy .

these peptides appear to be part of the plant defense system , based on observations that certain cyclotides exhibit insecticidal or antimicrobial activities . their characteristic structure comprises a head - to - tail cyclized cystine - knot motif , which confers them great stability and resistance to enzymatic , thermal , and chemical degradation . hence , cyclotides offer an interesting scaffold for peptide drug development , and they have been utilized as templates for grafting of bioactive epitopes onto the stabilized framework . for example , they are ligands of the human oxytocin and vasopressin v1a receptors , both members of the g protein - coupled receptor family , and they inhibit the activity of human prolyl oligopeptidase , a serine - protease involved in the processing of neuropeptides . these properties were discovered in bioassay - guided fractionation approaches using plant - derived peptide extracts as starting materials ; needless to say , a robust and efficient analytical workflow is crucial to determine the molecular structure of each bioactive cyclotide . therefore , advances in the discovery and identification of cyclotides are of great interest to provide novel peptide candidates for drug discovery and pharmacological applications . cyclotide distribution within flowering plants has been explored extensively , and today members of the angiosperm families cucurbitaceae , fabaceae , solanaceae , poaceae , rubiaceae , and violaceae are known to express these circular peptides . all species of violaceae examined hitherto are well - known as a rich source of cyclotides . the violets are the only plant family that ubiquitously contain cyclotides in every species sampled to date . within the violaceae mnage the species viola tricolor l. is well - known as a traditional medicinal plant . in particular , herbal extracts of v. tricolor have been described to treat inflammatory diseases such as inflammatory atopic dermatitis or psoriasis . although plants contain a number of different bioactive substances , a recent study highlighted that a cyclotide - enriched v. tricolor extract inhibited the proliferation of activated peripheral blood mononuclear cells . earlier it was demonstrated that cyclotides isolated from v. tricolor are antiproliferative agents against cancer cell lines . cyclotides have also been discovered in other members of violaceae , including v. odorata l. , v. yedoensis mak . in addition , cyclotides isolated from species of violaceae have been intensively investigated by mass spectrometry ( ms ) and nuclear magnetic resonance ( nmr ) spectroscopy to provide structural data and insight into their stability and chemical folding . as a consequence of the cyclic nature of cyclotides and therefore , the lack of c- and n - termini , enzymatic processing is required to open the backbone ring . given that an enzymatic digest of a peptide / protein - enriched plant extract yields thousands of peptides , the analysis of cyclotides in nonpurified preparations is extremely challenging , in particular due to the lack of appropriate nucleic acid / protein data sets available for proteomics analysis . to date , only laborious peptide purification combined with manual de novo sequencing appears to be suitable for precise sequence elucidation . more recently , next generation sequencing and bioinformatics have emerged as potential approaches for cyclotide discovery . in this study we combined transcriptome mining and ms to identify and characterize novel vitri cyclotides from v. tricolor . transcriptome analysis provided new insights about the structure of cyclotide precursors , processing sites , and mature peptide sequences . furthermore , we characterized the cyclotide peptidome by malditof / tof and lc ms / ms . systematic analysis and comparison of cyclotide identification at the nucleic acid level with application of different proteomics techniques provided the first comprehensive picture about cyclotide diversity and their post - translational modifications ( ptm ) in a single plant species . the outcome of this work may serve as a starting point for a routine high - throughput proteomics identification of cyclotides and other ribosomally synthesized and post - translationally modified natural peptide products . acetonitrile ( acn ) , methanol ( meoh ) , dichlormethane , trifluoroacetic acid ( tfa ) , and h2o were purchased as hplc or lc - ms grade from carl roth ( karlsruhe , germany ) . dithiothreitol ( dtt ) , -cyano - hydroxy cinnamic acid , trypsin ( sequencing grade ) , and iodoacetamide ( iaa ) were purchased as bioultra grade from sigma - aldrich ( sigma - aldrich , vienna , austria ) . endoproteinase glu c ( sequencing grade ) was purchased from new england biolabs ( ipswich , ma ) . v. tricolor was cultivated in a planting area near the entrance of brywood , 16th avenue and nw 27th terrace , gainesville , florida 32605 ( coordinates 294000.6n 822148.2w ) . fresh leaves of v. tricolor were identified by d.s . and colleagues and collected directly into liquid nitrogen . a voucher specimen ( soltis and miles 2930 ) was deposited at the university of florida herbarium ( flas ) . rna was isolated using a trizol / rnaqueous - midi kit ( ambion , paisley , uk ) . samples were sequenced as an indexed rnaseq library on an illumina ga ii machine ( illumina , san diego , ca ) . sequencing was paired - end ( 73 bp + 75 bp ) and produced a total of over 2.3 gbp . the sequences were assembled into scaffolds using soapdenovo - trans ( beijing genomics institute , china ) . transcriptome data of v. tricolor were accessed via the 1kp - project ( www.onekp.com ) . the v. tricolor data were accessed via tblastn using a set of published cyclotide precursor sequences as queries ( www.cybase.org.au ) . resulting hits and contig sequences were translated and annotated based on homology to known cyclotide precursors according to a similar methodology as described recently . briefly , hits were collected and translated into their respective amino acid sequence ( six - frame translation ) . all transcriptome contig sequence ids , nucleotide sequence data of precursors vitri 1 to vitri 99 , as well as protein translations are listed in supplementary data s-1 . novel full - length precursor sequences are available in the genbank database under the accession numbers kt203800kt203810 . any ambiguous or erroneous assembly was analyzed by transrate to identify mis - assembled contigs or assembly artifacts ( http://www.stevekellylab.com/software/transrate ) . dried v. tricolor total herbaceous plant material ( herba violae tricoloris plv briefly , plant material was extracted in a dichloromethane / meoh 1:1 ( v / v ) solution for 24 h. afterward the addition of one - half volume of ddh2o allowed liquid liquid phase extraction . the meoh / water phase encompasses all moderately hydrophobic plant constituents such as peptides , whereas lipophilic plant pigments are enriched in the dichloromethane phase . the meoh / water phase was further treated in batch by solid phase extraction . zeoprep 60 , c18 irregular 4063 m material ( zeochem , uetikon , switzerland ) was used according to the manufacturer s recommendation with surface activation by meoh , equilibration with 0.1% tfa and peptide elution with acn / ddh2o / tfa 80/20/0.1% ( v / v / v ) . fractionation of vitri extract was performed using a dionex ultimate 3000 hplc system ( thermo fisher scientific , waltham , ma ) equipped with a binary pump , autosampler , multiwavelength detector , and a fraction collector . chromatography was performed using a preparative dichrom kromasil column ( dichrom gmbh , marl , germany ; 250 20 mm , spherical c18 modified particles , 10 m , 110 ) . mobile phase eluent a consisted of 0.1% tfa ( aqueous ) and mobile phase eluent b consisted of acn / ddh2o / tfa 90/10/0.08% ( v / v / v ) . peptide separation on column was achieved with linear stepwise gradients ( 0-5% b , 5-5% b , 12.5-20% b , 62.5-70% b , 63.5-99% b , 65-99% b , 66-5% b and 72-5% b ) . fractions of 40 ml ( 5 min ) each , starting at 20% eluent b , were collected and lyophilized . the dried samples were kept at 20 c until further analysis , e.g. , mass deconvolution of cyclotide mass signals or automated ms / ms sequencing and database search of cyclotides for peptide identification in the proteomics workflow . for ms - based analysis via malditof or lcms vitri extract was dissolved in 0.1% tfa ( aqueous ) at a concentration of 3 mg / ml . for proteolytic digest the extract was dissolved in 0.1 m nh4hco3 ( ph 7.8 ) at a concentration of 3 mg / ml . any insoluble material was removed by centrifugation at 12000 g for 10 min , and the supernatant was used for further analysis . for proteomics analysis by ms / ms an aliquot of peptide solution ( 2040 l ) was used for reduction , alkylation , and proteolytic digest . briefly , disulfides were reduced with 20 mm dithiothreitol ( dtt ) for 1 h at 37 c . free thiols were carbamidomethylated using 100 mm iodoacetamide for 20 min at 23 c in the darkness . this reaction was quenched with 1 l of 100 mm dtt for 10 min at 37 c . the reduced and alkylated samples were digested with trypsin ( 0.3 g ) or endoproteinase gluc ( 0.6 g ) for 1216 h at 37 c . double digests were performed accordingly , starting with trypsin digestion overnight , followed by transient trypsin deactivation with dtt and addition of endoproteinase gluc . for all samples , proteolytic digests were stopped by adding tfa to a final concentration of 0.5% ( v / v ) . vitri extracts and fractions were analyzed via maldi - tof to evaluate the samples for peaks in the cyclotide specific mass range of 25004000 da . maldi - tof analysis was performed on a 4800 reflector tof / tof ( time - of - flight ) analyzer from absciex ( framingham , ma ) . samples were mixed with a saturated matrix solution of -cyano - hydroxy cinnamic acid dissolved in acn / ddh2o / tfa 50/50/0.1% ( v / v / v ) at a ratio of 1:6 , and 0.5 l of this mixture was transferred onto the target plate to allow air - drying in the darkness . mass accuracy was ensured by daily calibration using peptide mix 1 [ ( bradykinin fragment 839 ( 572.7 da ) , angiotensin ii ( 1046.2 da ) , neurotensin ( 1672.9 da ) , adrenocorticotropin fragment 1839 ( 2465.7 da ) and bovine insulin chain b ( 3495.9 da ) ] ( laserbiolabs , sophia - antipolis , france ) . ms spectra were recorded using the reflector in positive ionization mode in a spectral range between 2500 and 4500 da . vitri extracts were evaluated via lc ms scan and mass signal deconvolution of ms scans . ms scans were processed with deconvolution to evaluate mass signals in the cyclotide typical mass range of 25004000 da . sample desalting and preconcentration were performed with a trap column , and peptides were separated with a pepmap acclaim capillary column ( thermo fisher scientific ; 150 0.3 mm , 2 m 100 ) using a dionex ultimate 3000 uplc system . the mobile phase for the loading pump consisted of 0.1% tfa ( aqueous ) ; 0.1% formic acid ( aqueous ) and acn / ddh2o / formic acid 80/20/0.08% ( v / v / v ) were used as mobile phases for chromatographic separation . the flow rates were set to 25 l / min for the loading pump and 6 l / min for the separation pump . the gradient for peptide separation was as follows : 0-4% b , 10-4% b , 120-45% b , 125-99% b , 132-99% b , 135-4% b and 150-4% b. the lc qqtof compact system was equipped with a captive nanoflow ionization source , and sample ionization was achieved in the positive electron ionization mode . ion transfer parameters were kept constant for the duration of one run : prepulse storage 5 s , collision energy transfer 9 ev , quadrupole energy 4 ev , funnel 1 400 vpp , funnel 2 600 vpp transfer time , and hexapole rf 400 vpp . for collision energy rf and for ion transfer time stepping from 200 to 1500 vpp and 30150 s external mass calibration was achieved via direct infusion of the low concentration tuning mix from agilent technologies ( santa clara , ca ) . furthermore , internal calibration via lock mass was applied for each analytical run using calibrant hexakis ( 1h , 1h , 4h - hexafluorobutyloxy)phosphazine ( agilent technologies ) . base peak chromatograms in a signal window of 8002200 da were processed with the compound find tool of the data analysis software 4.21 ( build 393 ) ( bruker daltonics ) . tool was applied to each identified compound using the following settings : deconvolution of masses between 25004000 da , abundance cutoff 5% , maximum charge 4 , only consider [ m + h ] ions of proteins or peptides . evidence by charge and/or isotope pattern was recorded for each signal . for ms / ms experiments of vitri extracts or vitri fractions f1f9 the chromatographic conditions were identical as described for the mass signal deconvolution . peptide sequencing and automated identification by subsequent database search were performed by ms / ms experiments on the qqtof mass spectrometer using a bottom - up shotgun proteomics approach . the mass spectrometer was tuned and preoptimized to achieve best sequence coverage for precursors up to 2200 da , as commonly observed from proteolysis of cyclotides . ms / ms sequencing was performed with a cycle time of 3 s and exclusion criteria to reduce triggering of background signals . scan range for precursor recording was set to 502200 da with a minimal precursor mass of 200 m / z . peptides carrying charges from 2 to 5 were set to trigger ms / ms . high energy collision induced dissociation was performed under the regime of a set energy set . this set applies optimized collision energy for precursor signals based on the parameters : isolation m / z , isolation mass range width , and charge state of the ion . the set followed the manufacturer s recommendations , and it was not further modified by the experimenter . the proteinscape software package v. 3.1.5 474 ( build 20140711 - 1459 ) from bruker daltonics together with the mascot algorithm ( matrixscience , ma ) was used for data analysis and database search . the available cyclotide sequence database era was updated and customized with the most recent data set of circular peptide sequences ( www.cybase.org.au ) and from recent cyclotide discovery studies to yield in total 376 individual peptide sequences , including 55 novel precursors from the vitri transcriptome analysis of this study . additionally , contaminants were implemented into the custom database , e.g. , proteolytic fragments of keratins , bovine serum albumins and proteases . the publicly available contaminant database ( http://maxquant.org/contaminants.zip ) was provided by the max planck institute of biochemistry ( martinsried , germany ) . peptide search parameters were set to peptide mass tolerance 10 ppm , ms / ms tolerance 0.05 da , number of possible c atoms one , charge states 2 5 and considered up to one missed cleavage in the peptide search . fixed modification was carbamidomethylation of cysteine side chains , and variable modifications included methionine oxidation , tryptophan modification to kynurenine and 3-hydroxykurenine , deamidation of asparagine and glutamine and ethylation as well as methylation of glutamate or aspartate residues , respectively . all peptides with more than one possible precursor were assigned to the rank one candidate . identified proteins with a probability score > 50 of two independent analyses of at least one digest ( as described in the section sample preparation for mass spectrometry ) were considered to be valid ; this threshold score has been found by manual ms / ms validation to yield acceptable peptide assignment . nevertheless , ion score cutoff was set to > 15 to consider and exclude low quality peptides from multiple ms / ms triggering events . cyclotides with a partial sequence and those giving rise to more than one candidate peptide were evaluated manually for assignment of one hit , and they were cross validated with the vitri extract for native peptide mass signals of the reported ms / ms precursor peak . the ms proteomics data have been deposited at the proteomexchange consortium ( http://proteomecentral.proteomexchange.org ) via the pride partner repository with the data set identifier pxd002867 ( project doi : 10.6019/pxd002867 ) . since the discovery of kalata plant peptides in oldenlandia affinis ( r&s ) dc . by lorentz gran in the 1970s and the appreciation of cyclotides as active substances in medical preparations for uterotonic purpose , these ultrastable peptides have been the focus of numerous investigations to delineate their biological diversity , structural arrangement , and phylogenetic distribution within the plant kingdom . with an estimated 50 000 cyclotides present in rubiaceae alone , the current rate and performance of standard techniques of cyclotide discovery are clearly behind expectations , and it needs further optimization . so far laborious purification of peptides has been necessary to obtain accurate sequence information . hence , not surprisingly , the number of novel cyclotides reported by peptide sequencing in single studies to date is 18 and 24 , respectively , and the overall number of cyclotides deposited in cybase from > 20 years of research is currently at 922 . in the era of high - throughput nucleic acid sequencing , new tools have become available to examine cyclotide diversity on a genome / transcriptome level . therefore , we aimed to discover as many cyclotides as possible expressed in a single plant species and to examine their molecular diversity by a combination of sequence analysis at the nucleic acid level and ms studies at the peptide level . in addition , we performed a state - of - the - art bottom - up proteomics approach using plant extracts as well as hplc fractionated samples to identify the sequences and ptms of many novel cyclotides present in v. tricolor and to establish a robust workflow for cyclotide analysis . to date , only two precursor sequences have been reported for v. tricolor , and these were identified by pcr - based cdna sequencing . little is known about the variety of precursor sequences and how many cyclotides one plant is capable of expressing . therefore , it is of interest to gain insight into the diversity of cyclotide precursors . v. tricolor transcriptome data were available via the 1kp - project ( www.onekp.com ) and identified by tblastn searches using known cyclotide precursor sequences as queries ( www.cybase.org.au ) . contigs from resulting homologous sequences have been translated and annotated based on published cyclotide precursors . this approach led to the identification of 98 unique precursor proteins encoding 108 peptide sequences ( supplementary table s-1 and supplementary data s-1 ) . these findings nearly double the number of precursors compared to a very recent report suggesting that a single viola species contains 53 cyclotide precursor sequences . most cyclotide precursors that have been discovered only contain one cyclotide domain , but it is well - known that precursor molecules may contain multiple cyclotide domains . in fact we identified eight precursor proteins with two cyclotide domains ( vitri precursor 18 , 22 , 27 , 42 , 72 , 85 , 91 , and 94 ) and one precursor , vitri precursor 9 , encoding for three cyclotide domains ( figure 1a ) . four cyclotide sequences ( vitri 24/28 , 36/37 , 43/44 and 9a/53 ) have been found to be encoded by two different precursor proteins . analysis of precursor architecture as well as putative processing sites provides further insight into cyclotide biosynthesis in v. tricolor ( figure 1b , supplementary data s-2 ) and may also help to distinguish cyclotides from other cysteine - rich plant peptides . the majority of identified precursor proteins , namely , 54 precursors contain an asparagine ( or aspartic acid ) at the c - terminus of the cyclotide domain followed by a highly conserved sl , or rarely si , gl or ai in the positions p1/p2 . surprisingly , 10 precursors differ significantly in these common processing sites ; vitri precursors 15 , 16 , 17 , 19 , 27a , 35 , 40 , 66 , 76 , and 79 lack the common asparagine ( aspartic acid ) in their cyclotide domain vital for cyclization ( supplementary data s-1 ) , and another seven peptides completely lack a c - terminal tail sequence altogether ( vitri precursors 8 , 25 , 32 , 33 , 46 , 51 and 52 ) . hence this raises the question of whether these peptides are being cyclized via different mechanisms or whether they represent acyclic homologues ( supplementary table s-1 , supplementary data s-1 ) . the occurrence of linear cyclotides has infrequently been reported for species of the rubiaceae , violaceae , and poaceae plant families . in fact , it was possible to confirm occurrence of acyclic cyclotide homologues in v. tricolor ( see section ms / ms peptide sequencing of cyclotides ) . these acyclic peptides not only lack the n - terminal glycine and/or the c - terminal asparagine , but the precursor proteins may also be significantly shortened . besides containing unusual processing motifs , vitri precursors 24 and 28 exhibit a long c - tail region , which has not been observed previously in a cyclotide precursor . the biosynthesis of cyclotides is still not fully understood ; however research has addressed the role of processing enzymes and the involvement of single residues at the processing sites within the peptide precursors ( supplementary text s-1 ) . ( a ) a generic cyclotide precursor is ribosomally synthesized and contains an er signal , followed by a n - pro - region , n - terminal repeats ( ntr ) , the cyclotide domain , and a c - terminal tail region . cyclotide precursors may encode up to three peptide domains as it has been demonstrated for the v. tricolor tricyclon a precursor . novel vitri precursors discovered by transcriptome mining contain one , two , or three cyclotide domains . as indicated each precursor domain may vary in length . for instance , vitri precursor 24 comprises an extended c - tail , whereas vitri precursor 52 completely lacks this domain . ( b ) sequence comparison of vitri precursor processing site has been provided by a sequence logo . three adjacent residues downstream of the c - terminus of the mature cyclotide domain in positions p1 , p2 , and p3 , and upstream of the n - terminus of the mature cyclotide domain in positions p1 , p2 , and p3 have been considered for being essential for precursor processing and cyclization in planta . loop six comprises the highly conserved ligation sites , namely , glycine and asparagine or aspartic acid as highlighted in green . these novel vitri precursor sequences encode up to 108 putative cyclotides of v. tricolor , and many of them exhibit novel or very rare features ( supplementary table s-1 ) . they have been discussed in the supporting information as extended discussion of the sequence information provided by the presented transcriptome analysis ( supplementary figure s-1 , supplementary text s-1 ) . these new and rare sequence properties underline the structural plasticity of cyclotides , and they may be of future interest for studies utilizing cyclotides in peptide engineering and drug development . although transcriptome data analysis revealed 108 peptide sequences , only a single known v. tricolor cyclotide ( vitri d ) has been identified at the transcript level . moreover , the reported vitri precursor tricyclon precursor a could not be verified by transcriptome mining . it is known that cyclotide expression levels appear to be strongly determined by environmental conditions or seasonal changes . in fact , certain cyclotide precursors are exclusively expressed in certain plant tissues , and biotic and abiotic factors may influence cyclotide transcription . most importantly , the major limitation of in silico sequence mining is the erroneous prediction of processing sites by homology , and hence certain cyclotides may be assigned alternatively ( see footnotes supplementary table s-1 ) . only peptide analysis at the proteome level using ms will be able to verify and confirm the data obtained from transcriptome analysis . having established a comprehensive view of the expression of cyclotide peptides at the nucleic acid level , we were interested in cyclotides present at the protein level . to elucidate the cyclotide proteome of v. tricolor ms was used to collect evidence of the expression of cyclotides in v. tricolor . starting with powdered herb material , hydrophobic compounds were extracted and purified using well established protocols . capillary liquid chromatography coupled to a qqtof mass spectrometer was used to record base peak chromatograms of all specimens in a signal range between 8002200 da ( figure 2a ) . lc ms survey scans were evaluated for putative cyclotide masses using deconvolution tools ( supplementary table s-2 ) . lc ms resolved 126 peptide signals by the detection of the isotopic pattern , charge state deconvolution , or both . most notably , the lc ms system takes advantage of its high resolution power by chromatographic separation and hence in total higher capacity in the scan mode . in contrast malditof ms analysis of vitri plant extract using the 4800 ms analyzer suffers resolution problems due to multiple overlapping signals . this is why maldims only yielded 36 peptide signals ( figure 2b ) . on the other hand , co28 was only detected by maldims , but not by lc lc ms revealed mass signals of vitri peptides kalata s , varv peptide d , e , f , h , he , hm , vitri a , d , e , f , tricyclon a and cycloviolacin o2 . mass signals for vitri b , c and tricyclon b were not found with either technique . obviously the lc ms scan provided better resolution , which resulted in a higher number of identified cyclotide masses . nevertheless , the combination of qqtof and malditof ms appeared to be more powerful for increasing the number of identified cyclotides . to gain deeper insight into cyclotide diversity of v. tricolor , in particular with regard to molecular structure and ptms , we utilized ms - based peptide fragmentation and database searching to identify cyclotides by an automated proteomics workflow . ( a ) plant peptide extract from v. tricolor was analyzed via lc ms . cyclotide masses were found to carry + 2 , + 3 , and rarely + 4 h - ions . signals > 5.0% intensity threshold were assigned to mass and charge deconvolution tool from data analysis software ( bruker daltonics ) . ( b ) accordingly a malditof spectrum is shown between 2750 and 3500 da . for cyclotide names , retention times and molecular weight ( monoisotopic masses ) refer to supplementary table s-2 . ms / ms fragmentation data of vitri extract were recorded with a capillary lc - qqtof instrument and queried against the customized vitri database . for this purpose , the existing era database was updated to contain all vitri precursors or mature peptide sequences that were discovered by transcriptome mining ( supplementary table s-1 and supplementary data s-1 ) . the lack of a c- or n - terminus requires enzymatic processing for opening the cyclotide backbone , and linearization facilitates sufficient fragmentation using collision induced dissociation since intact cyclotides exhibit poor fragmentation . the most advanced strategy is to utilize a combination of multiple endopeptidases , i.e. , trypsin and endoproteinase glu c. this methodology resulted in multiple precursor peptides for one cyclotide with significant hits using the database analysis . identification of a unique full or partial sequence in at least one of the digested samples with a mascot score > 50 was considered valid , and each cyclotide hit was further supported by peptide identification using malditof or lc 38 cyclotides were determined , 26 with full and 12 with partial sequence coverage ( table 1 ) . using this proteomics workflow , it was possible to confirm the expression of vitri cyclotides varv peptide a , d , e , f , h , vitri a , d , e , cycloviolacin o2 and tricyclon a. five cyclotides ( vitri 18b , 22a , 39 , 42a , and 94b ) were detected from the novel transcriptome data set , and tricyclon b as well as oak6 cyclotide 1 were verified at protein level for the first time . further , this methodology yielded identification of the cyclotides co13 , co22 , and co28 , kalata b1 , varv peptide b , c and g , vigno 4 and 5 , viba 30 and 32 , vodo m , vodo n. identified in trypsin digest . identified in endoproteinase gluc digest . best score out of two independent analysis of each of the three digest experiments . modifications recorded in at least one peptide of assigned peptides of in total six independent analysis . one advantage of using an automated proteomics workflow is the possibility to determine ptms . it was possible to confirm many of them occur in cyclotides from v. tricolor . the most commonly observed modifications of vitri cyclotides have been illustrated , such as aspartate methylation , glutamate ethylation , tryptophan oxidation to kynurenine and 3-hydroxykynurenine , as well as deamidation of asparagine and glutamine ( figure 3a d ) . it is noteworthy that some peptides differ only by one pivotal residue , for instance , vitri e ( n29 ) and cycloviolacin o28 ( d29 ) . in this case deamidation of the n29 residue of vitri e would yield cycloviolacin o28 with a d29 residue . judging from the ms / ms data , both cyclotides were found side - by - side in v. tricolor , but nevertheless it was necessary to manually reanalyze the automated assignments . besides identification of common ptms of amino acid side chains , proteomics is a powerful tool to determine cyclization of the n- and c - termini . analysis of endoproteinase gluc digested samples revealed evidence for the presence of acyclic peptides in v. tricolor , namely , acyclo - kalata s , -tricyclon a , -vigno 5 -viba 30 , -varv peptide c , d , and e and -viba 30 . here , the peptides from the c - terminal end to the first cleavage site of the protease served as indicative fragments ( table 1 and figure 3e ) . the acyclic peptide species have been additionally confirmed by ms / ms analysis of reduced and alkylated samples . since cyclized peptides emerge stabilized during collision - induced dissociation , only linear peptide species yielded ms / ms spectra with sufficient fragmentation for database searching . evaluation of post - translational modifications in cyclotides identified by ms / ms sequencing and database analysis . database searching of ms / ms spectra of cyclotides revealed a number of peptides with posttranslational modifications . the most abundant ones have been illustrated exemplarily : ( a ) aspartate methylation in vitri peptide 94b ; ( b ) tryptophan degradation product kynurenine in varv peptide e ; ( c ) tryptophan oxidation to 3-hydroxy - tryptophan in kalata b1 ; and ( d ) glutamate ethylation and glutamine or asparagine deamidation in vitri peptide 42a . ( e ) the ms / ms fragment spectrum of a c - terminal fragment of endoproteinase gluc processed vitri peptide 22a has been shown to confirm the presence of acyclic cyclotides in v. tricolor . the major b- and y - ion series have been labeled in all spectra . furthermore , peptide mass , cleavage protease , mass error , and mascot score of the illustrated peptides have been provided . although cyclotide sequencing in nonfractionated vitri plant extract using the automated proteomics workflow yielded several known and novel cyclotides , many peptides lacked identification by sequence , as compared to analysis by molecular weight ( table 1 ) . thus , to maximize the number of identified cyclotides by sequence we introduced an hplc - assisted fractionation approach with the aim to enrich cyclotides in a particular fraction and subsequently increase sensitivity of the proteomics workflow compared to the extract starting material . the vitri extract was subjected to preparative hplc , and nine fractions were collected ( supplementary figure s-2 ) . . v. tricolor fraction f1 ( vitri - f1 ) contained only minor amounts of cyclotide masses in the range of 25004500 da , and vitri - f2 did not show any cyclotide - related signals . instead , analysis of vitri - f3f9 yielded significantly more molecular weight signals as compared to malditof ms of nonfractionated vitri extract . according to previous studies , fractionation has been confirmed as an efficient tool for enrichment and increased identification of cyclotides . the results of the proteomics study of the vitri fractions have been summarized in table 1 . as compared to analysis of the vitri extract , overall 82 peptides have been validated by proteomics of the fractionated samples . most notable , 20 novel vitri peptides ( 1 , 2 , 3 , 4 , 8 , 9a/53 , 14 , 17 , 18a , 20 , 21 , 23 , 24/28 , 27a , 29 , 30 , 36/37 , 38 , 42a , 50 ) have been unambiguously characterized by ms / ms sequencing . in addition , vitri f , cycloviolacin o4 and o20 , chacur 1 , vaby c , e , viba 9 , 11 , 15 , 19 vibi c , g , and vigno 6 , 7 , 9 , and 10 have all been validated and identified by the proteomics approach . moreover , an additional 11 acyclic peptides have been found as compared to the analysis of the vitri extract ; these were acyclo - kalata b1 , -varv peptide b , -f , -g , -vitri e , -vodo n , -vitri peptide 8 , 9a/53 , 22a , 39 and -cycloviolacin o22 . most acyclic peptides were found alongside the cyclic species , except for vitri 8 of which only the acyclic peptide has been identified . in fact , vitri precursor 8 encoding for vitri 8 is missing the essential c - tail for successful cyclization in planta . in summary , sample enrichment by hplc fractionation is very effective to enhance the number of total cyclotides identified by about 2-fold . using proteomics cyclotide analysis is associated with challenges regardless if they are studied at the nucleic acid or proteome level . to highlight the benefits and drawbacks of each method , it may be useful to compare the results , i.e. the number of identified cyclotides , from transcriptome mining , mass deconvolution , and ms / ms sequencing ( figure 4a ) . analyzing cyclotides at the transcript level , by mass signals deconvolution or by ms / ms sequencing 108 , 127 , and 82 essentially , 54 cyclotides were identified by ms / ms sequencing as well as mass deconvolution , 31 by ms / ms as well as transcriptome analysis , and 17 were found by both mass deconvolution and transcriptome mining ; 11 peptides were identified by all three methods . after all , 28% of 108 vitri cyclotides identified by transcriptome mining were confirmed by ms / ms or molecular weight ; 50% of cyclotides were assigned from partial sequences , 2% constitute cyclotide - like peptides , and only 20% of all full - length cyclotides determined by transcriptome mining remained unverified by ms ( figure 4b ) . considering full - length precursor molecules , importantly , 12 out of 14 previously published vitri peptides have been identified by ms / ms sequence , which confirms the effectiveness of our approach , considering that we worked with plant extracts and fractions but avoided using purified cyclotides for proteomics analysis . comparative analysis of vitri cyclotides identified by transcriptome mining , mass deconvolution , and ms / ms sequencing . ( a ) an overview of vitri peptides discovered in this study is summarized by a venn diagram . the number of identified cyclotides by each approach ( transcriptome mining , mass deconvolution or ms / ms sequencing ) is indicated by the numbers in each of the three interwheeling circles . the overlapping regions represent the numbers of identified cyclotides by combination of two or all three approaches . ( b ) a pie chart illustrates coverage for confirmation of predicted vitri cyclotides by ms . all predicted 108 vitri cyclotides identified by transcriptome mining were grouped in cohorts : ( i ) confirmed by sequence or molecular weight ( 28% ) , ( ii ) partial sequences from transcriptome mining ( 50% ) , ( iii ) cyclotide - like molecules ( 2% ) and unverified full - length cyclotides ( 20% ) . clearly , analyzing cyclotides by molecular weight only is the most error - prone method for unambiguous characterization of cyclotides . nevertheless , combined with transcriptome analysis , a simple ms run may provide useful information about the diversity of cyclotides in a given plant extract . similarly , transcriptome mining by itself resulted in the identification of many novel cyclotides , but the sequences of many vitri precursors contained uncommon c- or n - terminal processing sites , such as the missing of the conserved asparagine or aspartic acid residues or complete lack of c - tail motifs . in combination with ms however , these powerful tools were able to verify some of these peptides as acyclic cyclotide homologues ( supplementary table s-1 ) . obviously ms / ms sequencing using an automated proteomics workflow appears to be the most effective approach for identifying and characterizing novel cyclotides from v. tricolor , since it is capable of providing evidence not only for the presence of a cyclotide and further yields detailed information sequence and ptms . thus , we are confident that our results may provide a proof - of - principle and motivation for continued efforts in cyclotide discovery in many plant families using a combined transcriptome and proteome analysis workflow . the presented study combined cyclotide peptide analysis at both the nucleic acid and the proteome level to obtain insights into the diversity of circular cysteine - rich peptides of v. tricolor . it provides a comprehensive view of the transcription and on the translation of plant cyclotides and to our knowledge the largest study to date in the field of cyclotide discovery and characterization . our results may provide a guideline for future cyclotide discovery projects combining transcriptome and proteome analysis . here we were able to shed light to the unexplored diversity of plant - derived cyclotides within the violaceae family , which comprises up to 31 genera and approximately 1000 species . previously 10 cyclotides have been estimated as a realistic number of peptides to be expressed per viola species . a more recent study determined that a single species may express up to 53 peptides . to date , 161 individual cyclotides are known in violaceae , and in total 922 cyclotides are published online in cybase ( www.cybase.org.au ) . our finding that v. tricolor contains at least 164 different cyclotides significantly augments the number of cyclotides present in violaceae . thus , we determined that violaceous plants may indeed exhibit a much higher cyclotide diversity than earlier anticipated and contain up to > 150 000 individual cyclotides . similarly , other plant families such as rubiaceae may contain 150 000 cyclotides or more , determined by the number of cyclotides per species and multiplied by the estimated number of species known to express cyclotides . this simple calculation does not account for any peptide that may be ubiquitously expressed in many species . in addition other plant families of cucurbitaceae , fabaceae , poaceae and solanaceae also contain cyclotides . encompassing the diversity of v. tricolor as a natural library of bioactive peptides , this commercially available medicinal herb may be a suitable starting point for future bioactivity - guided screening studies , and the presented methodology may provide a guideline for cyclotide analytics combining transcriptome and proteome analysis .

cyclotides are plant - derived mini proteins . they are genetically encoded as precursor proteins that become post - translationally modified to yield circular cystine - knotted molecules . because of this structural topology cyclotides resist enzymatic degradation in biological fluids , and hence they are considered as promising lead molecules for pharmaceutical applications . despite ongoing efforts to discover novel cyclotides and analyze their biodiversity , it is not clear how many individual peptides a single plant specimen can express . therefore , we investigated the transcriptome and cyclotide peptidome of viola tricolor . transcriptome mining enabled the characterization of cyclotide precursor architecture and processing sites important for biosynthesis of mature peptides . the cyclotide peptidome was explored by mass spectrometry and bottom - up proteomics using the extracted peptide sequences as queries for database searching . in total 164 cyclotides were discovered by nucleic acid and peptide analysis in v. tricolor . therefore , violaceous plants at a global scale may be the source to as many as 150 000 individual cyclotides . encompassing the diversity of v. tricolor as a combinatorial library of bioactive peptides , this commercially available medicinal herb may be a suitable starting point for future bioactivity - guided screening studies .

Introduction Experimental Section Results and Discussion Conclusion

their characteristic structure comprises a head - to - tail cyclized cystine - knot motif , which confers them great stability and resistance to enzymatic , thermal , and chemical degradation . hence , cyclotides offer an interesting scaffold for peptide drug development , and they have been utilized as templates for grafting of bioactive epitopes onto the stabilized framework . for example , they are ligands of the human oxytocin and vasopressin v1a receptors , both members of the g protein - coupled receptor family , and they inhibit the activity of human prolyl oligopeptidase , a serine - protease involved in the processing of neuropeptides . these properties were discovered in bioassay - guided fractionation approaches using plant - derived peptide extracts as starting materials ; needless to say , a robust and efficient analytical workflow is crucial to determine the molecular structure of each bioactive cyclotide . therefore , advances in the discovery and identification of cyclotides are of great interest to provide novel peptide candidates for drug discovery and pharmacological applications . cyclotide distribution within flowering plants has been explored extensively , and today members of the angiosperm families cucurbitaceae , fabaceae , solanaceae , poaceae , rubiaceae , and violaceae are known to express these circular peptides . within the violaceae mnage the species viola tricolor l. is well - known as a traditional medicinal plant . in particular , herbal extracts of v. tricolor have been described to treat inflammatory diseases such as inflammatory atopic dermatitis or psoriasis . in addition , cyclotides isolated from species of violaceae have been intensively investigated by mass spectrometry ( ms ) and nuclear magnetic resonance ( nmr ) spectroscopy to provide structural data and insight into their stability and chemical folding . as a consequence of the cyclic nature of cyclotides and therefore , the lack of c- and n - termini , enzymatic processing is required to open the backbone ring . given that an enzymatic digest of a peptide / protein - enriched plant extract yields thousands of peptides , the analysis of cyclotides in nonpurified preparations is extremely challenging , in particular due to the lack of appropriate nucleic acid / protein data sets available for proteomics analysis . in this study we combined transcriptome mining and ms to identify and characterize novel vitri cyclotides from v. tricolor . transcriptome analysis provided new insights about the structure of cyclotide precursors , processing sites , and mature peptide sequences . furthermore , we characterized the cyclotide peptidome by malditof / tof and lc ms / ms . systematic analysis and comparison of cyclotide identification at the nucleic acid level with application of different proteomics techniques provided the first comprehensive picture about cyclotide diversity and their post - translational modifications ( ptm ) in a single plant species . the outcome of this work may serve as a starting point for a routine high - throughput proteomics identification of cyclotides and other ribosomally synthesized and post - translationally modified natural peptide products . fresh leaves of v. tricolor were identified by d.s . transcriptome data of v. tricolor were accessed via the 1kp - project ( www.onekp.com ) . the v. tricolor data were accessed via tblastn using a set of published cyclotide precursor sequences as queries ( www.cybase.org.au ) . any insoluble material was removed by centrifugation at 12000 g for 10 min , and the supernatant was used for further analysis . samples were mixed with a saturated matrix solution of -cyano - hydroxy cinnamic acid dissolved in acn / ddh2o / tfa 50/50/0.1% ( v / v / v ) at a ratio of 1:6 , and 0.5 l of this mixture was transferred onto the target plate to allow air - drying in the darkness . sample desalting and preconcentration were performed with a trap column , and peptides were separated with a pepmap acclaim capillary column ( thermo fisher scientific ; 150 0.3 mm , 2 m 100 ) using a dionex ultimate 3000 uplc system . peptide sequencing and automated identification by subsequent database search were performed by ms / ms experiments on the qqtof mass spectrometer using a bottom - up shotgun proteomics approach . the available cyclotide sequence database era was updated and customized with the most recent data set of circular peptide sequences ( www.cybase.org.au ) and from recent cyclotide discovery studies to yield in total 376 individual peptide sequences , including 55 novel precursors from the vitri transcriptome analysis of this study . identified proteins with a probability score > 50 of two independent analyses of at least one digest ( as described in the section sample preparation for mass spectrometry ) were considered to be valid ; this threshold score has been found by manual ms / ms validation to yield acceptable peptide assignment . with an estimated 50 000 cyclotides present in rubiaceae alone , the current rate and performance of standard techniques of cyclotide discovery are clearly behind expectations , and it needs further optimization . hence , not surprisingly , the number of novel cyclotides reported by peptide sequencing in single studies to date is 18 and 24 , respectively , and the overall number of cyclotides deposited in cybase from > 20 years of research is currently at 922 . in the era of high - throughput nucleic acid sequencing , new tools have become available to examine cyclotide diversity on a genome / transcriptome level . therefore , we aimed to discover as many cyclotides as possible expressed in a single plant species and to examine their molecular diversity by a combination of sequence analysis at the nucleic acid level and ms studies at the peptide level . in addition , we performed a state - of - the - art bottom - up proteomics approach using plant extracts as well as hplc fractionated samples to identify the sequences and ptms of many novel cyclotides present in v. tricolor and to establish a robust workflow for cyclotide analysis . to date , only two precursor sequences have been reported for v. tricolor , and these were identified by pcr - based cdna sequencing . therefore , it is of interest to gain insight into the diversity of cyclotide precursors . v. tricolor transcriptome data were available via the 1kp - project ( www.onekp.com ) and identified by tblastn searches using known cyclotide precursor sequences as queries ( www.cybase.org.au ) . this approach led to the identification of 98 unique precursor proteins encoding 108 peptide sequences ( supplementary table s-1 and supplementary data s-1 ) . these findings nearly double the number of precursors compared to a very recent report suggesting that a single viola species contains 53 cyclotide precursor sequences . in fact we identified eight precursor proteins with two cyclotide domains ( vitri precursor 18 , 22 , 27 , 42 , 72 , 85 , 91 , and 94 ) and one precursor , vitri precursor 9 , encoding for three cyclotide domains ( figure 1a ) . analysis of precursor architecture as well as putative processing sites provides further insight into cyclotide biosynthesis in v. tricolor ( figure 1b , supplementary data s-2 ) and may also help to distinguish cyclotides from other cysteine - rich plant peptides . the majority of identified precursor proteins , namely , 54 precursors contain an asparagine ( or aspartic acid ) at the c - terminus of the cyclotide domain followed by a highly conserved sl , or rarely si , gl or ai in the positions p1/p2 . surprisingly , 10 precursors differ significantly in these common processing sites ; vitri precursors 15 , 16 , 17 , 19 , 27a , 35 , 40 , 66 , 76 , and 79 lack the common asparagine ( aspartic acid ) in their cyclotide domain vital for cyclization ( supplementary data s-1 ) , and another seven peptides completely lack a c - terminal tail sequence altogether ( vitri precursors 8 , 25 , 32 , 33 , 46 , 51 and 52 ) . in fact , it was possible to confirm occurrence of acyclic cyclotide homologues in v. tricolor ( see section ms / ms peptide sequencing of cyclotides ) . the biosynthesis of cyclotides is still not fully understood ; however research has addressed the role of processing enzymes and the involvement of single residues at the processing sites within the peptide precursors ( supplementary text s-1 ) . ( a ) a generic cyclotide precursor is ribosomally synthesized and contains an er signal , followed by a n - pro - region , n - terminal repeats ( ntr ) , the cyclotide domain , and a c - terminal tail region . novel vitri precursors discovered by transcriptome mining contain one , two , or three cyclotide domains . these novel vitri precursor sequences encode up to 108 putative cyclotides of v. tricolor , and many of them exhibit novel or very rare features ( supplementary table s-1 ) . these new and rare sequence properties underline the structural plasticity of cyclotides , and they may be of future interest for studies utilizing cyclotides in peptide engineering and drug development . although transcriptome data analysis revealed 108 peptide sequences , only a single known v. tricolor cyclotide ( vitri d ) has been identified at the transcript level . moreover , the reported vitri precursor tricyclon precursor a could not be verified by transcriptome mining . most importantly , the major limitation of in silico sequence mining is the erroneous prediction of processing sites by homology , and hence certain cyclotides may be assigned alternatively ( see footnotes supplementary table s-1 ) . having established a comprehensive view of the expression of cyclotide peptides at the nucleic acid level , we were interested in cyclotides present at the protein level . to elucidate the cyclotide proteome of v. tricolor ms was used to collect evidence of the expression of cyclotides in v. tricolor . most notably , the lc ms system takes advantage of its high resolution power by chromatographic separation and hence in total higher capacity in the scan mode . to gain deeper insight into cyclotide diversity of v. tricolor , in particular with regard to molecular structure and ptms , we utilized ms - based peptide fragmentation and database searching to identify cyclotides by an automated proteomics workflow . for this purpose , the existing era database was updated to contain all vitri precursors or mature peptide sequences that were discovered by transcriptome mining ( supplementary table s-1 and supplementary data s-1 ) . the lack of a c- or n - terminus requires enzymatic processing for opening the cyclotide backbone , and linearization facilitates sufficient fragmentation using collision induced dissociation since intact cyclotides exhibit poor fragmentation . identification of a unique full or partial sequence in at least one of the digested samples with a mascot score > 50 was considered valid , and each cyclotide hit was further supported by peptide identification using malditof or lc 38 cyclotides were determined , 26 with full and 12 with partial sequence coverage ( table 1 ) . using this proteomics workflow , it was possible to confirm the expression of vitri cyclotides varv peptide a , d , e , f , h , vitri a , d , e , cycloviolacin o2 and tricyclon a. five cyclotides ( vitri 18b , 22a , 39 , 42a , and 94b ) were detected from the novel transcriptome data set , and tricyclon b as well as oak6 cyclotide 1 were verified at protein level for the first time . further , this methodology yielded identification of the cyclotides co13 , co22 , and co28 , kalata b1 , varv peptide b , c and g , vigno 4 and 5 , viba 30 and 32 , vodo m , vodo n. identified in trypsin digest . it is noteworthy that some peptides differ only by one pivotal residue , for instance , vitri e ( n29 ) and cycloviolacin o28 ( d29 ) . judging from the ms / ms data , both cyclotides were found side - by - side in v. tricolor , but nevertheless it was necessary to manually reanalyze the automated assignments . analysis of endoproteinase gluc digested samples revealed evidence for the presence of acyclic peptides in v. tricolor , namely , acyclo - kalata s , -tricyclon a , -vigno 5 -viba 30 , -varv peptide c , d , and e and -viba 30 . since cyclized peptides emerge stabilized during collision - induced dissociation , only linear peptide species yielded ms / ms spectra with sufficient fragmentation for database searching . ( e ) the ms / ms fragment spectrum of a c - terminal fragment of endoproteinase gluc processed vitri peptide 22a has been shown to confirm the presence of acyclic cyclotides in v. tricolor . although cyclotide sequencing in nonfractionated vitri plant extract using the automated proteomics workflow yielded several known and novel cyclotides , many peptides lacked identification by sequence , as compared to analysis by molecular weight ( table 1 ) . the vitri extract was subjected to preparative hplc , and nine fractions were collected ( supplementary figure s-2 ) . v. tricolor fraction f1 ( vitri - f1 ) contained only minor amounts of cyclotide masses in the range of 25004500 da , and vitri - f2 did not show any cyclotide - related signals . using proteomics cyclotide analysis is associated with challenges regardless if they are studied at the nucleic acid or proteome level . to highlight the benefits and drawbacks of each method , it may be useful to compare the results , i.e. the number of identified cyclotides , from transcriptome mining , mass deconvolution , and ms / ms sequencing ( figure 4a ) . analyzing cyclotides at the transcript level , by mass signals deconvolution or by ms / ms sequencing 108 , 127 , and 82 essentially , 54 cyclotides were identified by ms / ms sequencing as well as mass deconvolution , 31 by ms / ms as well as transcriptome analysis , and 17 were found by both mass deconvolution and transcriptome mining ; 11 peptides were identified by all three methods . after all , 28% of 108 vitri cyclotides identified by transcriptome mining were confirmed by ms / ms or molecular weight ; 50% of cyclotides were assigned from partial sequences , 2% constitute cyclotide - like peptides , and only 20% of all full - length cyclotides determined by transcriptome mining remained unverified by ms ( figure 4b ) . comparative analysis of vitri cyclotides identified by transcriptome mining , mass deconvolution , and ms / ms sequencing . all predicted 108 vitri cyclotides identified by transcriptome mining were grouped in cohorts : ( i ) confirmed by sequence or molecular weight ( 28% ) , ( ii ) partial sequences from transcriptome mining ( 50% ) , ( iii ) cyclotide - like molecules ( 2% ) and unverified full - length cyclotides ( 20% ) . nevertheless , combined with transcriptome analysis , a simple ms run may provide useful information about the diversity of cyclotides in a given plant extract . similarly , transcriptome mining by itself resulted in the identification of many novel cyclotides , but the sequences of many vitri precursors contained uncommon c- or n - terminal processing sites , such as the missing of the conserved asparagine or aspartic acid residues or complete lack of c - tail motifs . obviously ms / ms sequencing using an automated proteomics workflow appears to be the most effective approach for identifying and characterizing novel cyclotides from v. tricolor , since it is capable of providing evidence not only for the presence of a cyclotide and further yields detailed information sequence and ptms . thus , we are confident that our results may provide a proof - of - principle and motivation for continued efforts in cyclotide discovery in many plant families using a combined transcriptome and proteome analysis workflow . the presented study combined cyclotide peptide analysis at both the nucleic acid and the proteome level to obtain insights into the diversity of circular cysteine - rich peptides of v. tricolor . it provides a comprehensive view of the transcription and on the translation of plant cyclotides and to our knowledge the largest study to date in the field of cyclotide discovery and characterization . our results may provide a guideline for future cyclotide discovery projects combining transcriptome and proteome analysis . here we were able to shed light to the unexplored diversity of plant - derived cyclotides within the violaceae family , which comprises up to 31 genera and approximately 1000 species . to date , 161 individual cyclotides are known in violaceae , and in total 922 cyclotides are published online in cybase ( www.cybase.org.au ) . our finding that v. tricolor contains at least 164 different cyclotides significantly augments the number of cyclotides present in violaceae . thus , we determined that violaceous plants may indeed exhibit a much higher cyclotide diversity than earlier anticipated and contain up to > 150 000 individual cyclotides . encompassing the diversity of v. tricolor as a natural library of bioactive peptides , this commercially available medicinal herb may be a suitable starting point for future bioactivity - guided screening studies , and the presented methodology may provide a guideline for cyclotide analytics combining transcriptome and proteome analysis .

it is accepted that the adult human being consists of approximately ten times as many microbial as mammalian cells and that about 1.25 kg of microbes is carried by the average adult . the complex indigenous microbial flora plays a role in protecting man against pathogenic organisms , contributes to our energy and vitamin supply and is pivotal in the development of our immune system . it is because of this relationship that microbiologists have for many years endeavoured to establish appropriate methods of study to elucidate this symbiosis . rumney and rowland ( 1992 ) reviewed the then available models and listed the numerous methodological challenges to studying the ecology and metabolism of the colonic flora of man and concluded that there appears to be no single ideal approach . they identified problems that included the difficulty of working with a strictly anaerobic population ; problems in sampling colon contents ; the fact that often used faecal flora may not be representative of colonic flora , variable effects of diet within human subjects , the inability to carry out toxicology studies for ethical reasons and the fact that the flora becomes disturbed when removed from the biotic and abiotic constraints of the human gut . the use of a human floraassociated ( hfa ) rodent model , whereby human flora is implanted into gnotobiotic rodents , which are subsequently maintained in a facility that protects them from being exposed to exogenous flora , was an attempt to circumvent some of these challenges . this approach allows for a level of dietary , environmental and genetic control by maintaining the microbial flora in an in vivo environment similar to that of the human gastrointestinal tract . the hfa rodent model has and continues to be used to study the relationship between the human gut flora and health and diseases such as stomach and lower bowel cancer and inflammatory bowel disease , as well as in a whole range of toxicology studies . there appear , however , to have been few studies considering the relevance of in vivo hfa rodent models that have considered the assumptions that lie behind their use . it is tacitly assumed that the hfa rodent model does indeed mimic the flora of the human gastrointestinal tract and that the animal model will therefore be predictive of the situation in man . this paper reviews the literature with regard to examining direct and indirectly measured differences between the bacterial flora of hfa rodents , conventional rodents and man to establish the microbiological basis of the use of hfa rodents as a surrogate for studying the effects on the human gastrointestinal flora . it is pertinent to make clear at the outset that there are fundamental differences between the gastrointestinal tract of rodents and man apart from the questions of bacterial flora , such differences are not addressed in this paper . this review concludes that the established microbial flora in the hfa rodent model is different to that of donor human faecal flora , and this clearly raises the question as to whether this matters , after all a model is a model and as such models can be useful even should they fail to be a true representation of , in this case , the gastrointestinal tract . what matters is that there is a proper understanding of the limitations of the model as we attempt to unravel the significance of the components of the gastrointestinal flora in health and disease the role of the indigenous microbiota has been eloquently reviewed by tannock ( 2008 ) , and two examples will be described that highlight why it is important to understand the limitations of the hfa rodent model . the first is taken directly from tannock ( 2008 ) who made the point that molecular studies of the relationship between bowel bacteria and the mouse suggest that microbes impact importantly on murine physiology . bacteroides thetaiotaomicron in the bowel , in relatively shortterm gnotobiotic mouse experiments , influenced fucosylation of the enterocyte extracellular matrix , and upregulated or downregulated murine genes whose products are associated with nutrition , epithelial integrity and angiogenesis ( hooper et al . , 1999 ; 2001 ; stappenbeck et al . , 2002 ) . while this impacts on bowel mucosa , tannock ( 2008 ) makes the point that there is also a systemic effect on the deposition of fat in relation to the bowel microbiota of mice . the latter work revealed that murine physiology is actually attuned to the downregulation of fat storage after weaning , but that the bowel microbiota negates the mouse regulatory mechanism by reducing the production of fastinginduced adipose factor ( fiaf ) in the intestinal mucosa that leads to a corresponding increase in the systemic activity of lipoprotein lipase ( lpl ) . fiaf is an inhibitor of lpl , the latter influencing the uptake of triglycerides by adipocytes ( backhed et al . , 2004 ) . tannock ( 2008 ) makes the point that it is unknown whether this phenomenon is applicable to humans . the issue here is that we do not yet understand which are the critical components of the gastrointestinal flora that are implicated in such interactions , and as such it is important that we are aware of the limitations of the models used . the second example relates to the role of bifidobacteria ; they are considered to be a significant bacterial population within the human gastrointestinal tract and play a pivotal role in health by maintaining a wellbalanced intestinal microbiota ( reuter , 2001 ; vaughan et al . , 2005 ) . ( 2008 ) described the beneficial effects attributed to bifidobacteria to include the establishment of a healthy microbiota in infants , the prevention and treatment of diarrhoea , the alleviation of constipation and the symptoms of lactose intolerance , the enhancement of immune functions , cholesterol reduction and the suppression of tumorigenesis , among others ( ouwehand et al . this genus is certainly of commercial relevance as many of the microbial components of commercial probiotics belong to the bifidobacterial group ( yeung et al . the reviewed data suggests that this important group of organisms is not always implanted in the hfa rodent model . silvi and colleagues ( 1999 ) in a study investigating the modification of gut flora by resistant starch stated that , the study was performed in human floraassociated rats , which provide greater relevance to man than using conventional flora rats . human floraassociated rats , obtained by colonizing germfree rats with human faecal bacteria , develop and maintain a flora with bacteriological and metabolic characteristics similar to that of the mature human colonic microflora . the study was performed in human floraassociated rats , which provide greater relevance to man than using conventional flora rats . human floraassociated rats , obtained by colonizing germfree rats with human faecal bacteria , develop and maintain a flora with bacteriological and metabolic characteristics similar to that of the mature human colonic microflora . the authors cited the work of mallett and colleagues ( 1987 ) , hirayama and colleagues ( 1995 ) and rumney and rowland ( 1992 ) as support for this farreaching statement and one that has been used many times as justification for the hfa model . mallett and colleagues ( 1987 ) compared gastrointestinal enzyme activities ( glucosidase , glucuronidase , nitrate reductase and nitroreductase ) in conventional and hfa rats and human faeces . they concluded that conventional and hfa rats exhibited comparable enzyme activities that were similar to those found in human faeces with the exception of nitrate reductase , which exhibited negligible activity in conventional rats . the conventional rat flora did , however , respond differently to hfa rats and to human faeces when diet was supplemented with pectin in that nitrate reductase activity was stimulated . evidence for the similarity of flora between the hfa rodent model and man was thus based not on direct bacteriological data but on similarity in enzyme activities of three of the four tested enzymes . as a prelude to the cited study of hirayama and colleagues ( 1995 ) , it is important to acknowledge that hirayama and colleagues ( 1991 ) demonstrated that most of the major components of human faecal bacteria could be transferred into hfa mice , although this was not true for the important bifidobacterium group , which were not established in the model . indeed , the authors suggested that this could be a model to study the implications on human health of this important group of bacteria . this initial study was followed by a comprehensive study comparing the composition and metabolism of faecal microbiota among human floraassociated mice inoculated with faeces from six different human donors ( hirayama et al . , 1995 ) . it was this latter study that was cited by silvi and colleagues ( 1999 ) . it is probably the most comprehensive of all studies with regard to the detailed bacteriology that was carried out and is worth considering in some detail . the bacterial composition of the microbiota , bacterial reductive and hydrolytic enzyme activities , concentrations of short chain fatty acids ( scfas ) and putrefactive metabolites in faeces of germfree mice inoculated with faecal suspensions of six different volunteers were examined . the study confirmed that human faecal bacteria could be transferred into the intestine of hfa mice , although there were differences between the human donor inoculum and the established flora . in hfa mice , the numbers of eubacterium spp . , anaerobic cocci and enterobacteriaceae were significantly higher than those in conventional mice but similar to those in man . perhaps of greatest significance is that bifidobacteria were eliminated from three out of the six hfa mouse groups . despite these differences , which are clearly detailed in the results section of the paper , the discussion merely states that , faecal bacteria could be transferred into the intestine of hfa mice with minor modification , except for a decrease or elimination of bifidobacteria in one half of the hfa mouse groups. the abstract to the paper simply says , the composition of major faecal bacteria of hfa mice was similar to that of inoculated human faeces , although bifidobacteria were eliminated from some hfa mouse groups. it is clear that unless the paper is read in some detail , the reader could easily draw the wrong conclusions . the authors make the point that the reasons for the differences between donor flora and established flora are not clear . regardless of inoculated human faeces , the numbers of bacteroides spp . , clostridium spp . , anaerobic cocci and streptococcus spp . in all hfa mice were higher than those in the inocula . hirayama and colleagues ( 1995 ) suggest that the bacterial balance in the intestine of the hfa mice might be controlled by physiological conditions of the mouse intestine , rather than by the balance of the microbes in the human faeces . this will , as they point out , reduce the variability in bacterial composition among hfa mouse groups when compared with what happens in human faecal samples . the activities of glucosidase and glucuronidase of hfa mice were similar to those in man but the activities of nitrate reductase and nitroreductase in hfa mice were different from those in both man and conventional mice . additionally , the concentrations of putrefactive products in the faeces of hfa mice were largely different from those in human faeces but similar to those found in conventional mice . the composition of faecal scfas in hfa mice was more similar to that in humans than that in conventional mice , but the acid concentrations were significantly lower than those found in man . bacterial metabolism in the intestine of hfa mice thus reflected that of human faeces with respect to some metabolic activities but not others . the authors suggest two hypotheses by which to explain the results : dominant bacterial species of each bacterial group in hfa mice might change from those present in inoculated human faeces ; andbacterial metabolism might change because of an altered intestinal environment . dominant bacterial species of each bacterial group in hfa mice might change from those present in inoculated human faeces ; and bacterial metabolism might change because of an altered intestinal environment . further comment was made that the variations of composition and metabolic activities of the faecal microbiota among hfa mice groups were not particularly obvious , and perhaps more importantly the individual variations among inoculated human faeces were not reflected in hfa mouse groups and that some of the characteristics of each human donor faeces were hardly reflected in the hfa mice . while this paper is widely cited as justification for the use of hfa rodents as models for study of the gastrointestinal tract flora in man , there appears to have been no close scrutiny of the data nor even the cautionary final sentence in the abstract : these findings indicate that hfa mice provide a stable and valuable tool for studying the ecosystem and metabolism of the human faecal microbiota , but they have some limitations as a model . these findings indicate that hfa mice provide a stable and valuable tool for studying the ecosystem and metabolism of the human faecal microbiota , but they have some limitations as a model . the final supportive reference mentioned above was from a review carried out by rumney and rowland ( 1992 ) . within the review they cited the work of hazenberg and colleagues ( 1981 ) , who through a detailed bacteriology study showed that the gross bacterial composition of the hfa mouse was similar to that from man and distinct from that of a conventional mouse . these workers further demonstrated that the caecum of the hfa mouse was much reduced in size to a level that was equivalent on a body weight basis with that of man . the differences between the hfa mouse flora and that of the conventional mouse were easily demonstrated in that they reported a replacement of the human flora by murine flora within 2 weeks if the mice were not retained in their respective isolators . this is a rather important point in that it suggests that the human flora is not really stabilized in the mice , and that it could be more readily perturbed by antibiotic exposure than would be the case in the human gastrointestinal tract . ducluzeau and colleagues ( 1984 ) confirmed the work of hazenberg and colleagues ( 1981 ) , and according to rumney and rowland ( 1992 ) this was also confirmed by pecquet and colleagues ( 1986 ) . close reading of the pecquet and colleagues ( 1986 ) study , however , reveals that while they showed similarity in flora between hfa mice and human volunteers this was only with respect to enterobacteriaceae and total anaerobes , no other groups were tested nor was there any comparison with the flora from conventional animals . another error in the rumney and rowland ( 1992 ) review occurs in their citation of the previously discussed paper from mallett and colleagues ( 1987 ) . the review concludes from the mallett and colleagues ( 1987 ) paper : when the hfa animals were compared with their conventional flora counterparts all four microbial enzymes studied showed significant differences in activity . when the hfa animals were compared with their conventional flora counterparts all four microbial enzymes studied showed significant differences in activity . examination of the mallett and colleagues ( 1987 ) paper as described previously shows this not to be the case . the rumney and rowland ( 1992 ) review also presented some previously unpublished data showing that the metabolism of 2amino3methyl3himidazo[4,5f ] quinoline occurred faster in bacterial suspensions isolated from rat and mouse faeces than from human faeces yet the rate of reaction from hfa rat faeces was similar to that from human faeces . ward and colleagues ( 1990 ) in a similar study investigated the effect of different types of dietary fat on the formation of nnitrosoproline in germfree , conventional and hfa rats . the results in hfa rats and humans were similar and contrasted with those in conventional rats . what is clear is that the evidence supporting the view that hfa rat studies are more relevant to the situation in man than studies with conventional rats ( silvi et al . , the studies of hirayama and colleagues ( 1991 ; 1995 ) are the only exhaustive supporting studies , which have carried out a thorough bacteriological analysis . there are a number of studies that provide indirect support for hfa rodent models being predictive of the situation in man with regard to gi tract metabolism . rowland and tanaka ( 1993 ) described the feeding of transgalactosylated oligosaccharides ( tos ) to hfa rats resulting in an increase in total anaerobe , bifidobacterium and lactobacillus spp . these results were consistent with those published by ito and colleagues ( 1990 ) , who fed tos to human volunteers and found significant increases in bifidobacterium spp . and lactobacillus spp . mallett and colleagues ( 1989 ) examined the influence of incubation ph on the activity of rat and human gut flora enzymes ( glucosidase , glucuronidase and nitrate reductase ) . all three enzymes were influenced by ph , as exemplified by glucosidase activity that diminished as ph increased . in other instances the rat and human flora showed distinct profiles with nitrate reductase activity undetectable in human faeces below ph 6.6 , whereas the rat caecal flora displayed optimal reduction of nitrate around neutrality . the authors considered that the diverging profile for the two sources of intestinal material may reflect differences in the occurrence of glucuronidasepositive organisms present in the rat or human gut , as different bacterial species possess widely varying levels of this enzyme ( cole et al . , 1985 ) . apart from the study of hirayama and colleagues ( 1991 ; 1995 ) there appear to be few such comprehensive studies considering comparative bacteriological analysis of the faecal microbiota of conventional , hfa rodents and donor human faeces . there are , however , other studies in the literature that provide pointers as to differences between the faecal microbiota of conventional rodents , hfa rodents and man . it has become possible to analyse the complex concentrated anaerobic bacterial fraction of the gut microbial flora by a technique known as micromorphometry . this technique utilizes sophisticated digital image processing that enables detection and quantitative as well as qualitative analysis of bacterial objects in faeces , based on their morphological appearances . analysis is determined by the distribution of bacterial species present in faeces without dilution of the sample . using this technique , veenendaal and colleagues ( 1996 ) showed that the micromorphological patterns of faecal anaerobes from hfa mice were different to those from exgermfree mice associated with mousespecific pathogenfree flora . in particular , the authors studied germfree mice associated with either related rodent spf microflora ( spfmf ) or unrelated human microflora ( hummf ) . the micromorphological pattern of the bacteria from faeces of hummfassociated mice was significantly different from spfmfassociated mice . there was interestingly gross morphological variability between individual hummf mice but not between the individual spfmf mice . no differences were found between the donor human flora and the donor flora from the spf mice . while not citing any supporting data the authors , in the introduction , made the statement that , the highly concentrated anaerobic fraction in rodents is known to be micromorphologically different compared with those in humans , e.g. stool specimens of mice contain large numbers of fusiform shaped bacteria whereas small rod shaped and coccoid bacteria are predominant in human faeces. the findings from their reported study appear to conflict somewhat with this statement . koopman and colleagues ( 1989 ) and veenendaal and colleagues ( 1988 ) have similarly described differences in faecal flora between different rodent species . as far back as 1980 raibaud and colleagues ( 1980 ) carried out a series of fascinating experiments in which bacteria from the digestive tract of man and various animals were implanted into gnotobiotic mice . in the first experiment fourteen microbial strains isolated from conventional rats were inoculated into axenic rats and mice receiving identical diets . the populations of these organisms , which became established in the faeces of the gnotobiotic adult recipient rats and mice , were quite similar apart from one strain of clostridium sp . , which established in rats but not the mice . the complex flora from a conventional chicken was then successfully implanted in adult gnotobiotic mice such that it exhibited an intact barrier effect against salmonella typhimurium . finally raibaud and colleagues ( 1980 ) inoculated faeces from a human donor into adult gnotobiotic recipient mice . colonization was achieved although members of the genera bifidobacterium , lactobacillus and clostridium were not able to establish despite being present in the donor inoculum . nevertheless the colonizing flora did still exert an effective barrier against a toxigenic strain of clostridium difficile , although this barrier effect spontaneously disappeared several weeks later . garcalafuente and colleagues ( 2001 ) working with pathogenfree spraguedawley rats have clearly shown that the colonic barrier function is modulated by the composition of the commensal gi tract flora . colonization of the colon by escherichia coli , klebsiella pneumoniae and streptococcus viridans was seen to compromise colonic barrier function whereas colonization with lactobacillus brevis reduced colonic permeability and thus improved barrier function . other experimental studies have shown that certain strains of lactobacillus may prevent or reverse intestinal permeability disorders . prolonged cow 's milk challenge in suckling rats increases gut permeability to intact protein and oral bacteriotherapy with lactobacillus casei strain gg counteracts the increase in intestinal permeability ( isolauri et al . , 1993 ) . what is clear from these studies is that the commensal bacteria can influence the colonic barrier function in normal nonpathological conditions and that the interaction between the colonic flora and the gut wall can also be important . this last mentioned point is particularly important with regard to speciesspecific attachment mechanisms whereby many lactobacillus spp the reason for highlighting these findings is that failure of one bacterial group to colonize a model may prejudice the results . our current state of knowledge does not allow us to be sure of the importance of the respective bacterial groupings . are an important group within the gastrointestinal tract ecosystem yet they are a group that consistently fail to become established in the hfa model . the epithelial structure of mammalian intestinal mucosa reflects the absorptive and secretory activity of its various segments . the apical surface membrane of intestinal epithelial cells has a welldefined layer of glycoproteins and glycolipids , the glycocalyx , which is involved in key cellular processes , such as adhesion , antigenic recognition , differentiation and transport . in an elegant study investigating the influence of diet and gut microflora on lectin binding patterns of intestinal mucins in rats sharma and schumacher ( 1995 ) described an overall reduction in sialic acidlinked dgalactose residues in conventional compared with germfree rats and a loss of crypttosurface gradient of fucosyl expression in the large intestine of hfa rats . these differences were considered to be due to differing strains of glycosidases in the two different floras . in a further study , sharma and colleagues ( 1995 ) reported that while the effects of the human flora on the gut structure and mucus composition of hfa rats were in many ways similar to conventional rats , there were differences notably in the composition of the surface and goblet mucus and the length of the large intestinal crypt cells . while the authors acknowledge that these results may reflect real differences in response to the two types of flora they also consider it possible that when a human flora is inoculated in the germfree animal , its establishment and subsequent interaction with the intestinal tract may induce changes not seen with an indigenous flora where colonization is not subject to experimental manipulation . tach and colleagues ( 2000 ) investigated the effect of carrageenans as potential tumourinducing agents in conventional and hfa rats . at high dose levels in conventional rats aberrant crypt foci were detected whereas in contrast no effects were observed in hfa rats . these findings supported the hypothesis that it is the gut microflora of the host animal that is involved in the toxic effects of carrageenans in the rat colon . andrieux and colleagues ( 1993 ) investigated inulin fermentation in germfree rats associated with a human intestinal flora from methane and nonmethane producers . the donor human flora in both cases retained their fermentative properties when inoculated into germfree rats fed control diet . bacterial floras from human methane producers only gave rise to methane production , whereas hydrogen production was similar in both methane and nonmethaneproducing rats . caecal scfa concentrations were lower in methaneproducing rats than in nonmethane producers as was found in human faeces , but the lactic acid concentration was higher as compared with that in nonmethaneproducing rats . hirayama ( 1999 ) concluded that exgermfree animals harbouring intestinal microbiota originating in other animal species provide a stable and valuable tool for studying the ecosystem and metabolism of human faecal microbiota , but they have some limitations as a model . hirayama believes that further studies concerning the composition of intestinal bacteria of hfa mice at the species level ( nb bifidobacterium spp . ) and differences in intestinal physiological conditions among human , domestic animals and mice are needed . he further highlighted the findings of rumney and colleagues ( 1993 ) , who reported that the metabolic activities of intestinal bacteria of hfa rats appeared to be dependent on diet and that the results obtained with hfa rats were most relevant to humans when the animals were fed a human diet . hirayama ( 1999 ) clearly stated that the development of a special diet for hfa mice is required to establish a better model to study the metabolism of human intestinal microbiota . hentges and colleagues ( 1995 ) has similarly shown that diet is important when interpreting results in that gnotobiotic mice associated with human infant intestinal microorganisms were more resistant to colonization with s. typhimurium when consuming human milk than when consuming bovine milk , cows ' milkbased formula or formula modifications . a summary of the conclusions from the pivotal papers in this area is presented in table 1 . the epithelial structure of mammalian intestinal mucosa reflects the absorptive and secretory activity of its various segments . the apical surface membrane of intestinal epithelial cells has a welldefined layer of glycoproteins and glycolipids , the glycocalyx , which is involved in key cellular processes , such as adhesion , antigenic recognition , differentiation and transport . in an elegant study investigating the influence of diet and gut microflora on lectin binding patterns of intestinal mucins in rats sharma and schumacher ( 1995 ) described an overall reduction in sialic acidlinked dgalactose residues in conventional compared with germfree rats and a loss of crypttosurface gradient of fucosyl expression in the large intestine of hfa rats . these differences were considered to be due to differing strains of glycosidases in the two different floras . in a further study , sharma and colleagues ( 1995 ) reported that while the effects of the human flora on the gut structure and mucus composition of hfa rats were in many ways similar to conventional rats , there were differences notably in the composition of the surface and goblet mucus and the length of the large intestinal crypt cells . while the authors acknowledge that these results may reflect real differences in response to the two types of flora they also consider it possible that when a human flora is inoculated in the germfree animal , its establishment and subsequent interaction with the intestinal tract may induce changes not seen with an indigenous flora where colonization is not subject to experimental manipulation . tach and colleagues ( 2000 ) investigated the effect of carrageenans as potential tumourinducing agents in conventional and hfa rats . at high dose levels in conventional rats aberrant crypt foci were detected whereas in contrast no effects were observed in hfa rats . these findings supported the hypothesis that it is the gut microflora of the host animal that is involved in the toxic effects of carrageenans in the rat colon . andrieux and colleagues ( 1993 ) investigated inulin fermentation in germfree rats associated with a human intestinal flora from methane and nonmethane producers . the donor human flora in both cases retained their fermentative properties when inoculated into germfree rats fed control diet . bacterial floras from human methane producers only gave rise to methane production , whereas hydrogen production was similar in both methane and nonmethaneproducing rats . caecal scfa concentrations were lower in methaneproducing rats than in nonmethane producers as was found in human faeces , but the lactic acid concentration was higher as compared with that in nonmethaneproducing rats . hirayama ( 1999 ) concluded that exgermfree animals harbouring intestinal microbiota originating in other animal species provide a stable and valuable tool for studying the ecosystem and metabolism of human faecal microbiota , but they have some limitations as a model . hirayama believes that further studies concerning the composition of intestinal bacteria of hfa mice at the species level ( nb bifidobacterium spp . ) and differences in intestinal physiological conditions among human , domestic animals and mice are needed . he further highlighted the findings of rumney and colleagues ( 1993 ) , who reported that the metabolic activities of intestinal bacteria of hfa rats appeared to be dependent on diet and that the results obtained with hfa rats were most relevant to humans when the animals were fed a human diet . hirayama ( 1999 ) clearly stated that the development of a special diet for hfa mice is required to establish a better model to study the metabolism of human intestinal microbiota . hentges and colleagues ( 1995 ) has similarly shown that diet is important when interpreting results in that gnotobiotic mice associated with human infant intestinal microorganisms were more resistant to colonization with s. typhimurium when consuming human milk than when consuming bovine milk , cows ' milkbased formula or formula modifications . a summary of the conclusions from the pivotal papers in this area is presented in table 1 . in the absence of hard microbiological data one wonders why so many workers simply cite the work of the pioneers of these models claiming the utility without describing the limitations ; is this merely because they simply read abstracts and not the details of published papers or more likely because they have access to a model yet themselves have made no attempts to validate the test system ? perringuyomard and colleagues ( 2001 ) used the hfa model to evaluate residual and therapeutic doses of tetracycline and apply this to impacts upon the human gastrointestinal flora . they based this on the fact that the model , may have high relevance in determining the effects of low levels of antibiotics on human microflora and in this context cited studies of corpet ( 1980 ; 1987 ) , yet they did state that the model needed validation but did not appear to make any attempts to validate nor critically evaluate the model but rather reported data that they claimed related to human safety of antimicrobial residues . indeed , they did not report how the bacterial counts related the donor counts but rather presented data showing the stability of the bacterial populations from 4 days post implantation . where they did present comparative data for enzyme activities and volatile fatty acid production there were clear differences between the data from human and hfa samples , yet they concluded that the model was acceptable for studying doserelated effects of tetracycline on human intestinal flora . the same group perringuyomard and colleagues ( 2005 ) again used the model to address residue and therapeutic doses of ciprofloxacin on human gastrointestinal flora and , despite showing significant differences between bacteriological counts of the implanted and human flora , suggested that the model was appropriate for regulatory decision making . the adequacy of a human faecal microbiota associated mouse as a model for studying the activities of human intestinal microorganisms was examined . during a 6 month period , several predominant aerobic and anaerobic components of the human faecal bacteria persisted at stable numbers in the intestinal tracts of mice . however , bacillus species and both aerobic and anaerobic lactobacillus species disappeared within 7 days after association . an inverse relationship existed between the presence of shortchain fatty acids and nonfatty organic acids in the caeca of the associated mice . the relative concentrations of the two acid groups changed over a 21 days period , suggesting an alteration in the pattern of metabolism by the bacteria during the course of study . the total amount of organic acid produced by the microorganisms in the caeca of the associated mice was approximately 25% of the published value for humans , suggesting that the human microbiota retained only a portion of its metabolic activities in the mouse host . when challenged orogastrically with s. typhimurium , associated mice were as resistant to colonization as conventional mice , but germfree mice were very susceptible . furthermore , kibe and colleagues ( 2005 ) while using hfa pointed out the limitations of the model . they showed shifts in dominant species of microbiota in hfa mice after administration of human intestinal microbiota by using 16s rrna gene sequence and terminal restriction fragment length polymorphism analyses . they concluded that the intestinal microbiota of hfa mice and their offspring reflected the composition of individual human intestinal bacteria with some modifications and that the intestinal microbiota of hfa mice represents a limited sample of bacteria from the human source and are selected by unknown interactions between the host and bacteria . they made the point that there is a need to further the establishment of a suitable model for study and to clarify the details of the interaction between the host and the associated bacteria . indeed , the use of molecular techniques has greatly aided our understanding of the human gastrointestinal flora . licht and colleagues ( 2007 ) showed that while the hfa rat model may have some utility there are indeed differences between the stabilized implanted bacterial flora and that of the donor . in particular , they found that the rat gut environment specifically selected for ruminococcus spp . originating from the human faecal sample , speculating that it was the high content of cellulose fibres in the standard rat feed that provided a selective advantage for these cellulosedegrading species . they speculated if hfa rats were fed with a diet more closely resembling a human diet , the dominating strains in the rat gut may be even more similar to those of the human gut , and that for example the ruminococcus spp . the results raise questions about the adequacy of the human faecal microorganisms associated rodent as a definitive model to study the ecology of the human intestinal tract . the disappearances of selected species and failure of others to colonize may influence the outcomes of the studies . moreover , the greatly reduced metabolic activity of the human faecal microbiota in the mouse may diminish the significance of the results of metabolic studies when extrapolated to the human situation . however , the model might have utility in studies on colonization resistance against various enteric pathogens . while there have been few studies that have carried out sufficiently detailed direct comparisons of the microbial flora of conventional rodents , hfa rodents and donor human faeces , there are a number of indirect studies that have compared the predictive nature of the hfa rodent and conventional rodent model with that of man . these suggest , on balance , that the hfa rodent model is more likely to be predictive of what occurs in man than will the conventional rodent model . there are of course limitations to the hfa rodent model , which have been highlighted by a number of workers most notably the failure of bifidobacterium spp . whatever stance is taken workers in this area must be mindful of the views of rogers and fox ( 2004 ) , who considered the role of rodent models in the study of infectious gastrointestinal and liver cancer and stated that no meaningful conclusions can be drawn from studies in rodents without knowledge of how the species , strain and gender of the animal may affect experimental outcomes . they argued that results may be highly divergent when the same protocol is applied to different mouse populations , and care must be taken when extrapolating findings from a single inbred mouse to human disease . they made the point that little attention is often given to animal husbandry and the environment when designing and interpreting rodent models of infectious gastrointestinal and liver cancer . of importance to this paper was their view that animal stress associated with overcrowding , inadequate sanitation and variations in temperature , humidity and light cycles may predispose otherwise resistant animals to adverse disease outcomes and in the case of the enterohepatic system , differences in study outcomes may be attributed to endogenous gut microflora . microbial population differences may be pronounced even between different rooms and cages within the same facility . as we move forward in our understanding of the gastrointestinal ecosystem , it will be crucial to embrace a systems biology approach involving not only microbiologists but scientists from other disciplines . as tannock ( 2008 ) recently explained , the technological approaches to achieve these goals are at the fingertips of microbiologists : metagenomics to access and assess community genetics and metabolomics to analyze functional attributes of the the indigenous microbiota in concert with that of the host. clearly , one model will not suffice yet data from models such as the hfa rodent will no doubt be important as we seek to unravel the mysteries of the gastrointestinal tract flora , the implications of that data can only be fully understood if we are aware of the limitations of the model .

summarythere is little direct literature detailing exhaustive bacteriological studies comparing human donor faecal flora , human floraassociated ( hfa ) mouse models and conventional rodent faecal flora . while there is a premise that the implanted donor faecal flora from humans is established in the rodent model the evidence is incomplete and indeed for groups such as bifidobacterium spp . it is lacking . the reviewed bacteriology studies are generally lacking in detail with the exception of one study from which the data have mostly been overlooked when cited by other workers . while there are studies that suggest that the hfa rodent model is more relevant to man than studies with conventional rodents , the hypothesis remains to be proven . this review concludes that the established microbial flora in the hfa rodent model is different to that of donor human faecal flora , and this clearly raises the question as to whether this matters , after all a model is a model and as such models can be useful even should they fail to be a true representation of , in this case , the gastrointestinal tract . what matters is that there is a proper understanding of the limitations of the model as we attempt to unravel the significance of the components of the gastrointestinal flora in health and disease ; examples of why such an analysis is important are provided with regard to obesity and nutritional studies . the data do unsurprisingly suggest that diet is an extremely influential variable when interpreting hfa and conventional rodent data . the microbiology data from direct bacteriology and indirect enzyme studies show that the established microbial flora in the hfa rodent model is different to that of donor human faecal flora . the significance of this conclusion remains to be established .

Introduction Basis for the HFA model Differences between germfree and conventional rodents Studies comparing metabolism in HFA rodents and man No questions? Conclusions

it is accepted that the adult human being consists of approximately ten times as many microbial as mammalian cells and that about 1.25 kg of microbes is carried by the average adult . rumney and rowland ( 1992 ) reviewed the then available models and listed the numerous methodological challenges to studying the ecology and metabolism of the colonic flora of man and concluded that there appears to be no single ideal approach . they identified problems that included the difficulty of working with a strictly anaerobic population ; problems in sampling colon contents ; the fact that often used faecal flora may not be representative of colonic flora , variable effects of diet within human subjects , the inability to carry out toxicology studies for ethical reasons and the fact that the flora becomes disturbed when removed from the biotic and abiotic constraints of the human gut . the use of a human floraassociated ( hfa ) rodent model , whereby human flora is implanted into gnotobiotic rodents , which are subsequently maintained in a facility that protects them from being exposed to exogenous flora , was an attempt to circumvent some of these challenges . this approach allows for a level of dietary , environmental and genetic control by maintaining the microbial flora in an in vivo environment similar to that of the human gastrointestinal tract . the hfa rodent model has and continues to be used to study the relationship between the human gut flora and health and diseases such as stomach and lower bowel cancer and inflammatory bowel disease , as well as in a whole range of toxicology studies . it is tacitly assumed that the hfa rodent model does indeed mimic the flora of the human gastrointestinal tract and that the animal model will therefore be predictive of the situation in man . this paper reviews the literature with regard to examining direct and indirectly measured differences between the bacterial flora of hfa rodents , conventional rodents and man to establish the microbiological basis of the use of hfa rodents as a surrogate for studying the effects on the human gastrointestinal flora . it is pertinent to make clear at the outset that there are fundamental differences between the gastrointestinal tract of rodents and man apart from the questions of bacterial flora , such differences are not addressed in this paper . this review concludes that the established microbial flora in the hfa rodent model is different to that of donor human faecal flora , and this clearly raises the question as to whether this matters , after all a model is a model and as such models can be useful even should they fail to be a true representation of , in this case , the gastrointestinal tract . what matters is that there is a proper understanding of the limitations of the model as we attempt to unravel the significance of the components of the gastrointestinal flora in health and disease the role of the indigenous microbiota has been eloquently reviewed by tannock ( 2008 ) , and two examples will be described that highlight why it is important to understand the limitations of the hfa rodent model . bacteroides thetaiotaomicron in the bowel , in relatively shortterm gnotobiotic mouse experiments , influenced fucosylation of the enterocyte extracellular matrix , and upregulated or downregulated murine genes whose products are associated with nutrition , epithelial integrity and angiogenesis ( hooper et al . fiaf is an inhibitor of lpl , the latter influencing the uptake of triglycerides by adipocytes ( backhed et al . the issue here is that we do not yet understand which are the critical components of the gastrointestinal flora that are implicated in such interactions , and as such it is important that we are aware of the limitations of the models used . the second example relates to the role of bifidobacteria ; they are considered to be a significant bacterial population within the human gastrointestinal tract and play a pivotal role in health by maintaining a wellbalanced intestinal microbiota ( reuter , 2001 ; vaughan et al . this genus is certainly of commercial relevance as many of the microbial components of commercial probiotics belong to the bifidobacterial group ( yeung et al . the reviewed data suggests that this important group of organisms is not always implanted in the hfa rodent model . silvi and colleagues ( 1999 ) in a study investigating the modification of gut flora by resistant starch stated that , the study was performed in human floraassociated rats , which provide greater relevance to man than using conventional flora rats . human floraassociated rats , obtained by colonizing germfree rats with human faecal bacteria , develop and maintain a flora with bacteriological and metabolic characteristics similar to that of the mature human colonic microflora . the study was performed in human floraassociated rats , which provide greater relevance to man than using conventional flora rats . human floraassociated rats , obtained by colonizing germfree rats with human faecal bacteria , develop and maintain a flora with bacteriological and metabolic characteristics similar to that of the mature human colonic microflora . they concluded that conventional and hfa rats exhibited comparable enzyme activities that were similar to those found in human faeces with the exception of nitrate reductase , which exhibited negligible activity in conventional rats . evidence for the similarity of flora between the hfa rodent model and man was thus based not on direct bacteriological data but on similarity in enzyme activities of three of the four tested enzymes . as a prelude to the cited study of hirayama and colleagues ( 1995 ) , it is important to acknowledge that hirayama and colleagues ( 1991 ) demonstrated that most of the major components of human faecal bacteria could be transferred into hfa mice , although this was not true for the important bifidobacterium group , which were not established in the model . indeed , the authors suggested that this could be a model to study the implications on human health of this important group of bacteria . it is probably the most comprehensive of all studies with regard to the detailed bacteriology that was carried out and is worth considering in some detail . the study confirmed that human faecal bacteria could be transferred into the intestine of hfa mice , although there were differences between the human donor inoculum and the established flora . perhaps of greatest significance is that bifidobacteria were eliminated from three out of the six hfa mouse groups . despite these differences , which are clearly detailed in the results section of the paper , the discussion merely states that , faecal bacteria could be transferred into the intestine of hfa mice with minor modification , except for a decrease or elimination of bifidobacteria in one half of the hfa mouse groups. the abstract to the paper simply says , the composition of major faecal bacteria of hfa mice was similar to that of inoculated human faeces , although bifidobacteria were eliminated from some hfa mouse groups. it is clear that unless the paper is read in some detail , the reader could easily draw the wrong conclusions . hirayama and colleagues ( 1995 ) suggest that the bacterial balance in the intestine of the hfa mice might be controlled by physiological conditions of the mouse intestine , rather than by the balance of the microbes in the human faeces . additionally , the concentrations of putrefactive products in the faeces of hfa mice were largely different from those in human faeces but similar to those found in conventional mice . further comment was made that the variations of composition and metabolic activities of the faecal microbiota among hfa mice groups were not particularly obvious , and perhaps more importantly the individual variations among inoculated human faeces were not reflected in hfa mouse groups and that some of the characteristics of each human donor faeces were hardly reflected in the hfa mice . while this paper is widely cited as justification for the use of hfa rodents as models for study of the gastrointestinal tract flora in man , there appears to have been no close scrutiny of the data nor even the cautionary final sentence in the abstract : these findings indicate that hfa mice provide a stable and valuable tool for studying the ecosystem and metabolism of the human faecal microbiota , but they have some limitations as a model . these findings indicate that hfa mice provide a stable and valuable tool for studying the ecosystem and metabolism of the human faecal microbiota , but they have some limitations as a model . within the review they cited the work of hazenberg and colleagues ( 1981 ) , who through a detailed bacteriology study showed that the gross bacterial composition of the hfa mouse was similar to that from man and distinct from that of a conventional mouse . these workers further demonstrated that the caecum of the hfa mouse was much reduced in size to a level that was equivalent on a body weight basis with that of man . the differences between the hfa mouse flora and that of the conventional mouse were easily demonstrated in that they reported a replacement of the human flora by murine flora within 2 weeks if the mice were not retained in their respective isolators . this is a rather important point in that it suggests that the human flora is not really stabilized in the mice , and that it could be more readily perturbed by antibiotic exposure than would be the case in the human gastrointestinal tract . close reading of the pecquet and colleagues ( 1986 ) study , however , reveals that while they showed similarity in flora between hfa mice and human volunteers this was only with respect to enterobacteriaceae and total anaerobes , no other groups were tested nor was there any comparison with the flora from conventional animals . another error in the rumney and rowland ( 1992 ) review occurs in their citation of the previously discussed paper from mallett and colleagues ( 1987 ) . examination of the mallett and colleagues ( 1987 ) paper as described previously shows this not to be the case . what is clear is that the evidence supporting the view that hfa rat studies are more relevant to the situation in man than studies with conventional rats ( silvi et al . there are a number of studies that provide indirect support for hfa rodent models being predictive of the situation in man with regard to gi tract metabolism . the authors considered that the diverging profile for the two sources of intestinal material may reflect differences in the occurrence of glucuronidasepositive organisms present in the rat or human gut , as different bacterial species possess widely varying levels of this enzyme ( cole et al . apart from the study of hirayama and colleagues ( 1991 ; 1995 ) there appear to be few such comprehensive studies considering comparative bacteriological analysis of the faecal microbiota of conventional , hfa rodents and donor human faeces . there are , however , other studies in the literature that provide pointers as to differences between the faecal microbiota of conventional rodents , hfa rodents and man . using this technique , veenendaal and colleagues ( 1996 ) showed that the micromorphological patterns of faecal anaerobes from hfa mice were different to those from exgermfree mice associated with mousespecific pathogenfree flora . while not citing any supporting data the authors , in the introduction , made the statement that , the highly concentrated anaerobic fraction in rodents is known to be micromorphologically different compared with those in humans , e.g. the populations of these organisms , which became established in the faeces of the gnotobiotic adult recipient rats and mice , were quite similar apart from one strain of clostridium sp . colonization was achieved although members of the genera bifidobacterium , lactobacillus and clostridium were not able to establish despite being present in the donor inoculum . garcalafuente and colleagues ( 2001 ) working with pathogenfree spraguedawley rats have clearly shown that the colonic barrier function is modulated by the composition of the commensal gi tract flora . this last mentioned point is particularly important with regard to speciesspecific attachment mechanisms whereby many lactobacillus spp the reason for highlighting these findings is that failure of one bacterial group to colonize a model may prejudice the results . are an important group within the gastrointestinal tract ecosystem yet they are a group that consistently fail to become established in the hfa model . these differences were considered to be due to differing strains of glycosidases in the two different floras . in a further study , sharma and colleagues ( 1995 ) reported that while the effects of the human flora on the gut structure and mucus composition of hfa rats were in many ways similar to conventional rats , there were differences notably in the composition of the surface and goblet mucus and the length of the large intestinal crypt cells . these findings supported the hypothesis that it is the gut microflora of the host animal that is involved in the toxic effects of carrageenans in the rat colon . the donor human flora in both cases retained their fermentative properties when inoculated into germfree rats fed control diet . hirayama ( 1999 ) concluded that exgermfree animals harbouring intestinal microbiota originating in other animal species provide a stable and valuable tool for studying the ecosystem and metabolism of human faecal microbiota , but they have some limitations as a model . he further highlighted the findings of rumney and colleagues ( 1993 ) , who reported that the metabolic activities of intestinal bacteria of hfa rats appeared to be dependent on diet and that the results obtained with hfa rats were most relevant to humans when the animals were fed a human diet . hentges and colleagues ( 1995 ) has similarly shown that diet is important when interpreting results in that gnotobiotic mice associated with human infant intestinal microorganisms were more resistant to colonization with s. typhimurium when consuming human milk than when consuming bovine milk , cows ' milkbased formula or formula modifications . a summary of the conclusions from the pivotal papers in this area is presented in table 1 . while the authors acknowledge that these results may reflect real differences in response to the two types of flora they also consider it possible that when a human flora is inoculated in the germfree animal , its establishment and subsequent interaction with the intestinal tract may induce changes not seen with an indigenous flora where colonization is not subject to experimental manipulation . these findings supported the hypothesis that it is the gut microflora of the host animal that is involved in the toxic effects of carrageenans in the rat colon . he further highlighted the findings of rumney and colleagues ( 1993 ) , who reported that the metabolic activities of intestinal bacteria of hfa rats appeared to be dependent on diet and that the results obtained with hfa rats were most relevant to humans when the animals were fed a human diet . hentges and colleagues ( 1995 ) has similarly shown that diet is important when interpreting results in that gnotobiotic mice associated with human infant intestinal microorganisms were more resistant to colonization with s. typhimurium when consuming human milk than when consuming bovine milk , cows ' milkbased formula or formula modifications . in the absence of hard microbiological data one wonders why so many workers simply cite the work of the pioneers of these models claiming the utility without describing the limitations ; is this merely because they simply read abstracts and not the details of published papers or more likely because they have access to a model yet themselves have made no attempts to validate the test system ? they based this on the fact that the model , may have high relevance in determining the effects of low levels of antibiotics on human microflora and in this context cited studies of corpet ( 1980 ; 1987 ) , yet they did state that the model needed validation but did not appear to make any attempts to validate nor critically evaluate the model but rather reported data that they claimed related to human safety of antimicrobial residues . where they did present comparative data for enzyme activities and volatile fatty acid production there were clear differences between the data from human and hfa samples , yet they concluded that the model was acceptable for studying doserelated effects of tetracycline on human intestinal flora . the same group perringuyomard and colleagues ( 2005 ) again used the model to address residue and therapeutic doses of ciprofloxacin on human gastrointestinal flora and , despite showing significant differences between bacteriological counts of the implanted and human flora , suggested that the model was appropriate for regulatory decision making . the adequacy of a human faecal microbiota associated mouse as a model for studying the activities of human intestinal microorganisms was examined . during a 6 month period , several predominant aerobic and anaerobic components of the human faecal bacteria persisted at stable numbers in the intestinal tracts of mice . an inverse relationship existed between the presence of shortchain fatty acids and nonfatty organic acids in the caeca of the associated mice . the relative concentrations of the two acid groups changed over a 21 days period , suggesting an alteration in the pattern of metabolism by the bacteria during the course of study . the total amount of organic acid produced by the microorganisms in the caeca of the associated mice was approximately 25% of the published value for humans , suggesting that the human microbiota retained only a portion of its metabolic activities in the mouse host . furthermore , kibe and colleagues ( 2005 ) while using hfa pointed out the limitations of the model . they made the point that there is a need to further the establishment of a suitable model for study and to clarify the details of the interaction between the host and the associated bacteria . indeed , the use of molecular techniques has greatly aided our understanding of the human gastrointestinal flora . licht and colleagues ( 2007 ) showed that while the hfa rat model may have some utility there are indeed differences between the stabilized implanted bacterial flora and that of the donor . originating from the human faecal sample , speculating that it was the high content of cellulose fibres in the standard rat feed that provided a selective advantage for these cellulosedegrading species . they speculated if hfa rats were fed with a diet more closely resembling a human diet , the dominating strains in the rat gut may be even more similar to those of the human gut , and that for example the ruminococcus spp . moreover , the greatly reduced metabolic activity of the human faecal microbiota in the mouse may diminish the significance of the results of metabolic studies when extrapolated to the human situation . while there have been few studies that have carried out sufficiently detailed direct comparisons of the microbial flora of conventional rodents , hfa rodents and donor human faeces , there are a number of indirect studies that have compared the predictive nature of the hfa rodent and conventional rodent model with that of man . these suggest , on balance , that the hfa rodent model is more likely to be predictive of what occurs in man than will the conventional rodent model . there are of course limitations to the hfa rodent model , which have been highlighted by a number of workers most notably the failure of bifidobacterium spp . whatever stance is taken workers in this area must be mindful of the views of rogers and fox ( 2004 ) , who considered the role of rodent models in the study of infectious gastrointestinal and liver cancer and stated that no meaningful conclusions can be drawn from studies in rodents without knowledge of how the species , strain and gender of the animal may affect experimental outcomes . of importance to this paper was their view that animal stress associated with overcrowding , inadequate sanitation and variations in temperature , humidity and light cycles may predispose otherwise resistant animals to adverse disease outcomes and in the case of the enterohepatic system , differences in study outcomes may be attributed to endogenous gut microflora . as we move forward in our understanding of the gastrointestinal ecosystem , it will be crucial to embrace a systems biology approach involving not only microbiologists but scientists from other disciplines . as tannock ( 2008 ) recently explained , the technological approaches to achieve these goals are at the fingertips of microbiologists : metagenomics to access and assess community genetics and metabolomics to analyze functional attributes of the the indigenous microbiota in concert with that of the host. clearly , one model will not suffice yet data from models such as the hfa rodent will no doubt be important as we seek to unravel the mysteries of the gastrointestinal tract flora , the implications of that data can only be fully understood if we are aware of the limitations of the model .

alzheimer s disease ( ad ) is the most common neurologic condition affecting the elderly in the us . the cost associated with ad is currently high and is expected to grow over the next 40 years . total payments in 2012 for health care and long - term care services for people with ad and other dementias are expected to be $ 200 billion ( not including the contributions of unpaid caregivers).1 the cost of care for ad in the us is expected to rise above $ 1 trillion by 2050.2 pharmacologic strategies for the treatment of ad have focused on modulating disease - associated neurotransmitter alterations.3 two classes of pharmacologic agents approved for treatment of ad are cholinesterase inhibitors and an n - methyl - d - aspartate receptor antagonist.35 these agents are considered symptomatic treatments based on their ability to slow the clinical progression of symptoms across cognitive , behavioral , and functional domains . they do not provide detectable and sustained improvements in cognitive function above baseline.35 new therapies are needed . the brains of patients with ad display progressive , region - specific declines in the cerebral metabolic rate of glucose,6 and these changes are apparent early in the course of ad before there is demonstrable cell loss and plaque deposition.7 under most conditions , cerebral metabolism is fueled almost exclusively by glucose . however , under certain conditions , such as neonatal development and extended fasting , the brain will derive much of its energy from ketone bodies.8,9 ketone bodies are comprised of beta - hydroxybutyrate , acetoacetate , and acetone . they are normally derived from fat stores and offer an alternative fuel for brain metabolism . therefore , if cerebral glucose metabolism is compromised in the ad patient , perhaps ketone bodies can substitute for glucose and improve cognitive performance.9,10 ketone bodies are an efficient fuel for cells,11,12 and results from preclinical studies have suggested that induced ketosis may be beneficial in ad.13,14 in addition , studies have suggested that ketone bodies may be useful in a variety of neurologic disorders.15 a previous study has demonstrated that administration of 20 g of caprylic triglyceride ( ct ) increases blood levels of beta - hydroxybutyrate ( the major ketone body in humans ) 2 hours post dose to a mean value of 0.39 mm , and this therapy has been shown to be beneficial in patients with mild - to - moderate ad.10,16 ct is composed of a glycerol backbone and three caprylic ( c8:0 ) fatty acid chains , well known for their ketogenic properties.17 the efficacy and safety of a medical food containing ct in subjects with mild - to - moderate ad has been demonstrated in a multicenter , randomized , double - blind , placebo - controlled study that included 152 outpatients with probable mild - to - moderate ad.10 it is important to continue evaluations of interventions in routine clinical practice . this approach has been taken with pharmacotherapies for ad,1820 and ct should be evaluated in a similar manner . the primary objective of this study was to assess patient outcomes of ct used alone or in combination with ongoing pharmacotherapy in mild - to - moderate ad in routine clinical practice . due to the small number of patients enrolled in the study , patient outcomes were evaluated by chart review carried out at 11 practices in which ct was prescribed in patients with mild - to - moderate ad . investigators at each site were instructed to review all charts for patients who were taking or had taken ct and determine their eligibility for the study . the criteria for inclusion were male or female outpatients aged 50 years with a diagnosis of probable ad of mild - to - moderate severity ; mini - mental state examination ( mmse ) score between 14 and 24 inclusive at the initiation of ct ( if available ) , or diagnosis of mild - to - moderate ad ; and receipt of ct for 6 months . potential patients were excluded if they had an mmse score < 14 at the time when ct was initiated or comorbid disease known to adversely impact cognitive function ( eg , parkinson s disease , history of cerebral infarcts , dementia with lewy bodies , vascular dementia ) . the sample size was based on estimates of numbers of patients who were available for inclusion as reported by participating physicians , and not based on any statistical considerations . after an independent evaluation of potential patients , only 55 were found to be eligible for participation . the trial was carried out with institutional review board approval . patients and caregivers were required to provide written informed consent prior to inclusion of their information in the study . a caregiver was defined to be at least 21 years of age ( this age was believed to be appropriate for a caregiver who would provide a patient assessment as part of the study ) , had been the primary caregiver since prior to the initiation of ct , provided at least 4 hours of supervision or direct assistance per day for the patient , and was able to perform assessments . the first was the physician s overall assessment of patient status at the most recent evaluation on ct . the second was the change in mmse score from initiation of ct to the most recent mmse assessment on ct . first , status at initiation of ct therapy , scored as check boxes of : mild ad or moderate ad . second , status at most recent evaluation , scored as check boxes of : mild ad , moderate ad , severe ad , very severe ad . third , change from beginning of ct to present , scored as check boxes of : marked improvement , moderate improvement , improvement , stable , worsening , moderate worsening , or marked worsening . secondary measures included changes from baseline in patient s living situation ; medication changes for the management of ad and for psychiatric symptoms related to ad ; changes in patient s memory and ability to carry out instrumental activities of daily living as assessed by caregivers ; and adverse events . caregiver questionnaires were also employed to determine ct dosing frequency ( number of days per week ) ; to estimate routine dose level ( full dose or less than a full dose is generally taken ) ; and adherence to ct ( if available ) . caregivers were also asked to assess the patient s memory since initiation of ct , based on a modified version of the memory complaints questionnaire developed by memory assessment clinics ; and ability to carry out activities of daily living adapted from lawton and brody.21 scores from the physician s overall assessment of change from baseline after initiation of ct were summarized with descriptive statistics . the primary analysis of the mmse was a paired t - test of baseline score and last available score while on ct . changes from baseline in caregiver assessments of patients ability to use the telephone , prepare food , and dress were evaluated with mann all statistical tests were two - sided and were carried out at the 5% level of significance . results for all demographic and secondary study endpoints were tabulated and appropriate descriptive statistics were calculated . pearson correlation between physician s overall assessment of change and caregiver assessment was performed with r 2.15.0 ( www.r-project.org ) . patient outcomes were evaluated by chart review carried out at 11 practices in which ct was prescribed in patients with mild - to - moderate ad . investigators at each site were instructed to review all charts for patients who were taking or had taken ct and determine their eligibility for the study . the criteria for inclusion were male or female outpatients aged 50 years with a diagnosis of probable ad of mild - to - moderate severity ; mini - mental state examination ( mmse ) score between 14 and 24 inclusive at the initiation of ct ( if available ) , or diagnosis of mild - to - moderate ad ; and receipt of ct for 6 months . potential patients were excluded if they had an mmse score < 14 at the time when ct was initiated or comorbid disease known to adversely impact cognitive function ( eg , parkinson s disease , history of cerebral infarcts , dementia with lewy bodies , vascular dementia ) . the sample size was based on estimates of numbers of patients who were available for inclusion as reported by participating physicians , and not based on any statistical considerations . after an independent evaluation of potential patients , only 55 were found to be eligible for participation . the trial was carried out with institutional review board approval . patients and caregivers were required to provide written informed consent prior to inclusion of their information in the study . a caregiver was defined to be at least 21 years of age ( this age was believed to be appropriate for a caregiver who would provide a patient assessment as part of the study ) , had been the primary caregiver since prior to the initiation of ct , provided at least 4 hours of supervision or direct assistance per day for the patient , and was able to perform assessments . the first was the physician s overall assessment of patient status at the most recent evaluation on ct . the second was the change in mmse score from initiation of ct to the most recent mmse assessment on ct . first , status at initiation of ct therapy , scored as check boxes of : mild ad or moderate ad . second , status at most recent evaluation , scored as check boxes of : mild ad , moderate ad , severe ad , very severe ad . third , change from beginning of ct to present , scored as check boxes of : marked improvement , moderate improvement , improvement , stable , worsening , moderate worsening , or marked worsening . secondary measures included changes from baseline in patient s living situation ; medication changes for the management of ad and for psychiatric symptoms related to ad ; changes in patient s memory and ability to carry out instrumental activities of daily living as assessed by caregivers ; and adverse events . caregiver questionnaires were also employed to determine ct dosing frequency ( number of days per week ) ; to estimate routine dose level ( full dose or less than a full dose is generally taken ) ; and adherence to ct ( if available ) . caregivers were also asked to assess the patient s memory since initiation of ct , based on a modified version of the memory complaints questionnaire developed by memory assessment clinics ; and ability to carry out activities of daily living adapted from lawton and brody.21 scores from the physician s overall assessment of change from baseline after initiation of ct were summarized with descriptive statistics . the primary analysis of the mmse was a paired t - test of baseline score and last available score while on ct . changes from baseline in caregiver assessments of patients ability to use the telephone , prepare food , and dress were evaluated with mann all statistical tests were two - sided and were carried out at the 5% level of significance . results for all demographic and secondary study endpoints were tabulated and appropriate descriptive statistics were calculated . pearson correlation between physician s overall assessment of change and caregiver assessment was performed with r 2.15.0 ( www.r-project.org ) . a total of 437 records were reviewed at 11 study sites , and those of 55 patients who met the inclusion and exclusion criteria and provided informed written consent were included in the analysis . the most common reasons for exclusion were mmse scores out of the designated inclusion range , taking ct for < 6 months , no formal diagnosis of ad , and comorbidities that may have affected cognitive function . a minimum of 28 hours of caregiving per week was set as a criterion for inclusion in the study protocol . five caregivers did not meet this minimum , and were not included in the analysis . the demographic and clinical characteristics of the patients whose records were analyzed are summarized in table 1 . nearly all patients ( 96.4% ) were receiving medications indicated for the treatment of ad ; and 54.6% were receiving agents indicated for other psychiatric symptoms ( most often depression ) . at baseline , 88.7% of patients were living with caregivers ( relatives ) and 11.3% were residing in assisted living facilities . patients who had received ct , regardless of the dose , for 6 months or longer were eligible for the study . for the 55 patients whose records were reviewed , the mean duration of ct was 18.8 9.2 months . chart review indicated that most patients ( 89.1% ) were titrated to a full 20 g daily ct dose . physician s overall assessment of changes from baseline in patient status ( figure 1a ) were marked improvement for 5.5% , moderate improvement for 5.5% , improvement for 24.1% , stable for 44.4% , worsening for 14.8% , moderate worsening for 3.7% , and marked worsening for 1.8% . the mean baseline score for these patients was 20.6 3.0 and that for the most recent measurement on ct was 20.1 5.6 ( difference 0.48 , 95% confidence interval 1.05 to 2.01 , t = 0.6470 , p = 0.5233 ) . at baseline , 43.6% of patients were rated by their physicians as having mild ad and 56.4% were considered to have moderate ad . of the 55 participants , 80% ( n = 44 ) did not change in severity of disease , while 20% ( n = 11 ) were diagnosed with a more advanced disease state ( figure 1b ) . no patients whose records were reviewed had changes in their living situation during ct therapy . there were also few changes in medications patients were taking for ad ; 87.3% of patients had no changes ; 7.3% had switches or augmentation in pharmacotherapy , and 5.4% had addition of a medical food that included phosphatidylserine enriched with docosahexaenoic acid . pharmacotherapy for psychiatric / behavioral symptoms also remained stable for most patients ( 70.9% ) . there were 25 patients taking no medications for psychiatric illness at baseline and six of these ( 10.9% of all patients , 24.0% of those not taking psychiatric medications at baseline ) had agents added to therapy , most often antidepressants or atypical antipsychotics . in addition , 33.0% of patients receiving medications at baseline had switches and/or additions to their therapy . these changes most often included switching and/or augmentation of antidepressant treatment and addition of an anxiolytic . caregivers responses indicated that addition of ct to therapy resulted in some degree of improvement or stability in remembering the name of a person to whom they were just introduced in 80.9% ( 17/21 ) of patients , recalling telephone numbers or zip codes that were used on a daily or weekly basis in 80.9% ( 17/21 ) , and in finding commonly used objects in 85.7% ( 18/21 ) , as shown in figure 2 . overall assessments of memory also indicated that the majority of patients ( 75.0% ) ( 15/20 ) were stable or improved over the follow - up period ( figure 2 ) . assessment of ability to use the telephone , prepare food , and dressing indicated no significant changes from baseline ( mann whitney u - tests , p = 0.696 , p = 0.904 , and p = 0.596 , respectively ) . however , a significant correlation was found between the physician s overall assessment of change and caregiver surveys ( r = 0.5423 , confidence interval 0.80 to 0.12 , p = 0.016 , figure 3 ) . of the 55 patients whose charts were reviewed , 41 ( 74.5% ) had no adverse events . the remaining 14 patients had a total of 30 adverse events , eight of which were considered to be related to ct . there was one severe adverse event , a brain tumor that was not related to ct . a total of eight patients ( 14.5% ) discontinued ct and four of these ( 7.3% ) discontinued due to adverse events . a total of 437 records were reviewed at 11 study sites , and those of 55 patients who met the inclusion and exclusion criteria and provided informed written consent were included in the analysis . the most common reasons for exclusion were mmse scores out of the designated inclusion range , taking ct for < 6 months , no formal diagnosis of ad , and comorbidities that may have affected cognitive function . a minimum of 28 hours of caregiving per week was set as a criterion for inclusion in the study protocol . five caregivers did not meet this minimum , and were not included in the analysis . the demographic and clinical characteristics of the patients whose records were analyzed are summarized in table 1 . nearly all patients ( 96.4% ) were receiving medications indicated for the treatment of ad ; and 54.6% were receiving agents indicated for other psychiatric symptoms ( most often depression ) . at baseline , 88.7% of patients were living with caregivers ( relatives ) and 11.3% were residing in assisted living facilities . patients who had received ct , regardless of the dose , for 6 months or longer were eligible for the study . for the 55 patients whose records were reviewed , the mean duration of ct was 18.8 9.2 months . chart review indicated that most patients ( 89.1% ) were titrated to a full 20 g daily ct dose . physician s overall assessment of changes from baseline in patient status ( figure 1a ) were marked improvement for 5.5% , moderate improvement for 5.5% , improvement for 24.1% , stable for 44.4% , worsening for 14.8% , moderate worsening for 3.7% , and marked worsening for 1.8% . the mean baseline score for these patients was 20.6 3.0 and that for the most recent measurement on ct was 20.1 5.6 ( difference 0.48 , 95% confidence interval 1.05 to 2.01 , t = 0.6470 , p = 0.5233 ) . at baseline , 43.6% of patients were rated by their physicians as having mild ad and 56.4% were considered to have moderate ad . of the 55 participants , 80% ( n = 44 ) did not change in severity of disease , while 20% ( n = 11 ) were diagnosed with a more advanced disease state ( figure 1b ) . no patients whose records were reviewed had changes in their living situation during ct therapy . there were also few changes in medications patients were taking for ad ; 87.3% of patients had no changes ; 7.3% had switches or augmentation in pharmacotherapy , and 5.4% had addition of a medical food that included phosphatidylserine enriched with docosahexaenoic acid . pharmacotherapy for psychiatric / behavioral symptoms also remained stable for most patients ( 70.9% ) . there were 25 patients taking no medications for psychiatric illness at baseline and six of these ( 10.9% of all patients , 24.0% of those not taking psychiatric medications at baseline ) had agents added to therapy , most often antidepressants or atypical antipsychotics . in addition , 33.0% of patients receiving medications at baseline had switches and/or additions to their therapy . these changes most often included switching and/or augmentation of antidepressant treatment and addition of an anxiolytic . physician s overall assessment of changes from baseline in patient status ( figure 1a ) were marked improvement for 5.5% , moderate improvement for 5.5% , improvement for 24.1% , stable for 44.4% , worsening for 14.8% , moderate worsening for 3.7% , and marked worsening for 1.8% . the mean baseline score for these patients was 20.6 3.0 and that for the most recent measurement on ct was 20.1 5.6 ( difference 0.48 , 95% confidence interval 1.05 to 2.01 , t = 0.6470 , p = 0.5233 ) . at baseline , 43.6% of patients were rated by their physicians as having mild ad and 56.4% were considered to have moderate ad . of the 55 participants , 80% ( n = 44 ) did not change in severity of disease , while 20% ( n = 11 ) were diagnosed with a more advanced disease state ( figure 1b ) . no patients whose records were reviewed had changes in their living situation during ct therapy . there were also few changes in medications patients were taking for ad ; 87.3% of patients had no changes ; 7.3% had switches or augmentation in pharmacotherapy , and 5.4% had addition of a medical food that included phosphatidylserine enriched with docosahexaenoic acid . pharmacotherapy for psychiatric / behavioral symptoms also remained stable for most patients ( 70.9% ) . there were 25 patients taking no medications for psychiatric illness at baseline and six of these ( 10.9% of all patients , 24.0% of those not taking psychiatric medications at baseline ) had agents added to therapy , most often antidepressants or atypical antipsychotics . in addition , 33.0% of patients receiving medications at baseline had switches and/or additions to their therapy . these changes most often included switching and/or augmentation of antidepressant treatment and addition of an anxiolytic . caregivers responses indicated that addition of ct to therapy resulted in some degree of improvement or stability in remembering the name of a person to whom they were just introduced in 80.9% ( 17/21 ) of patients , recalling telephone numbers or zip codes that were used on a daily or weekly basis in 80.9% ( 17/21 ) , and in finding commonly used objects in 85.7% ( 18/21 ) , as shown in figure 2 . overall assessments of memory also indicated that the majority of patients ( 75.0% ) ( 15/20 ) were stable or improved over the follow - up period ( figure 2 ) . assessment of ability to use the telephone , prepare food , and dressing indicated no significant changes from baseline ( mann whitney u - tests , p = 0.696 , p = 0.904 , and p = 0.596 , respectively ) . however , a significant correlation was found between the physician s overall assessment of change and caregiver surveys ( r = 0.5423 , confidence interval 0.80 to 0.12 , p = 0.016 , figure 3 ) . of the 55 patients whose charts were reviewed , 41 ( 74.5% ) had no adverse events . the remaining 14 patients had a total of 30 adverse events , eight of which were considered to be related to ct . there was one severe adverse event , a brain tumor that was not related to ct . a total of eight patients ( 14.5% ) discontinued ct and four of these ( 7.3% ) discontinued due to adverse events . results from this chart review suggest that ct helped to stabilize or improve the clinical status and cognitive function in patients receiving therapy for a mean of approximately 19 months . the physician s overall assessment of change from baseline indicated that 79.5% of patients either showed improvement or remained stable , while 20.3% worsened . results for the 27 patients for whom mmse scores were available at baseline and on therapy indicate that , on average , patients remained stable with no significant change over a mean of 15 months of follow - up . no patients had changes in their living situation during ct therapy , and most patients remained stable with respect to medications for ad and those for psychiatric / behavioral conditions . caregiver assessments also indicated stability or improvement in cognitive function and no significant changes in the ability to carry out activities of daily living . the physician s overall assessment indicated that ~80% of patients who had ct added to their ongoing pharmacotherapy were stable or improved . a large number of different global severity / status scales have been employed in assessing effects of treatment in patients with ad . that most similar to the measure employed in this study is the clinical global impressions of change , which provides minimal instructions to the rater.22 results from global assessments of the type carried out in this study have also been shown to be well correlated with quantitative measures of cognitive function / dementia.23 the mmse is the most widely used brief mental status screening instrument,24,25 and has been used extensively for evaluation of patients with ad in both clinical trials and follow - up of patients being managed in routine clinical practice.2629 in order to evaluate the impact of ct on mild - to - moderate ad in routine practice , comparison with subjects on standard therapy without ct was examined by literature review . this approach permitted comparison of the present results with those from much larger numbers of patients receiving pharmacotherapy for ad . a summary of results from clinical trials and epidemiologic studies published prior to 2005 indicated a decline in mmse scores of 0.64.4 points per year in different studies.30 more recently , a study of 686 patients with mild - to - moderate ad with 90% receiving ad - specific medication indicated an annual decline in mmse scores of 2.4 points per year.31 in comparison , the decline in mmse scores over 15 months in the present study was 0.48 and not statistically significant . it should be noted that there are limitations to the use of the mmse for assessment of cognitive function , particularly when comparisons are made across studies . the rate of change for mmse has been shown to be strongly influenced by the baseline score.32,33 other variables , including patient age , duration of ad , and education , have been linked to rate of decline for mmse scores , but these relationships appear to be weaker and more variable than those for baseline mmse score.3440 thus , comparison of results from the present chart review and those from other studies should be undertaken with caution . caregiver assessments also indicate that memory and ability to carry out activities of daily living remained stable or improved during therapy with ct . in an assessment of overall memory , 40% of the caregivers functional impairments in ad place the greatest burden on both caregivers and the economy;41 and results from several studies have suggested that neuropsychologic test performance is predictive of activities of daily living performance in patients with ad.42,43 results from observational studies and clinical trials indicate that deterioration in the ability to carry out activities of daily living can be detected among patients with ad over follow - up periods comparable with that in the present study.44,45 changes in the requirement for psychiatric medications were used as a surrogate measure for effects of ct on neuropsychiatric symptoms . these symptoms are a source of caregiver burden and are associated with increased probability of institutionalization.46 treatment aimed at improving cognition in patients with ad has also been shown to decrease the severity of neuropsychiatric symptoms.47 results from the record review indicate that medications for psychiatric illness remained stable for most patients during the period of ct . results from the chart review indicated the adverse event most often associated with ct was gastrointestinal upset and that this event led to discontinuation in 7.3% of patients . most patients ( 89.1% ) were titrated to a full 20 g daily ct dose and maintained at this dose . while the presumed mechanism of action of ct is the induction of ketosis and subsequent provision of ketone bodies as an alternative fuel for neurons , ketone bodies have been shown to have other neuroprotective effects that may be beneficial , not only in ad but also in other neurologic conditions.15 however , it is important to note that there have been reports of detrimental effects of caloric restriction and ketogenic diets on neurologic function.48,49 however , induction of mild ketosis by ct does not require restriction of macronutrients , and therefore may not lead to the detrimental effects attributed to ketogenic diets . one limitation of this study was that the apolipoprotein e4 ( apoe4 ) carriage status was evaluated and recorded in the medical records of only seven patients ( two were apoe4-positive ) . prior clinical results for ct indicated significant efficacy in apoe4-negative but not apoe4-positive patients.10,16 in conclusion , results from this small chart review study indicate that addition of ct to pharmacotherapy for ad was associated with stable disease or improvement for most patients over a follow - up period of 18.8 months . addition of ct was also associated with stabilization or improvements in the ability to carry out activities of daily living for most patients and warrants further investigation in a larger study .

backgroundthe purpose of this study was to evaluate the effects of caprylic triglyceride ( ct ) in patients with mild - to - moderate alzheimer s disease ( ad ) in routine clinical practice via review of medical records and caregiver questionnaires.methodsparticipants were outpatients aged 50 years with a diagnosis of probable mild - to - moderate ad who had received ct for 6 months . the primary outcome was change from baseline in the patient s condition as rated by the treating physician using a physician s overall assessment.resultsa total of 55 patients were included . the physician s overall assessment indicated that ~80% of patients who had ct added to ongoing pharmacotherapy were stable or improved . mini - mental state examination scores also remained stable over 15 months of therapy ( 20.6 3.0 at baseline and 20.1 5.6 at follow - up , p = 0.5233 , n = 27 ) . caregiver assessments indicated that most patients were stable or improved with respect to memory and ability to carry out activities of daily living . the most frequent adverse events with ct involved the gastrointestinal system.conclusionresults from this chart review indicate that addition of ct to pharmacotherapy was associated with stable disease or improvement over a follow - up period of 18.8 months .

Introduction Materials and methods Study design Assessments Data analysis Results Records reviewed CT dosing Patient outcomes Primary measures Secondary measures Results from caregiver surveys Correlation between physician and caregiver assessments Safety outcomes Discussion

alzheimer s disease ( ad ) is the most common neurologic condition affecting the elderly in the us . total payments in 2012 for health care and long - term care services for people with ad and other dementias are expected to be $ 200 billion ( not including the contributions of unpaid caregivers).1 the cost of care for ad in the us is expected to rise above $ 1 trillion by 2050.2 pharmacologic strategies for the treatment of ad have focused on modulating disease - associated neurotransmitter alterations.3 two classes of pharmacologic agents approved for treatment of ad are cholinesterase inhibitors and an n - methyl - d - aspartate receptor antagonist.35 these agents are considered symptomatic treatments based on their ability to slow the clinical progression of symptoms across cognitive , behavioral , and functional domains . the brains of patients with ad display progressive , region - specific declines in the cerebral metabolic rate of glucose,6 and these changes are apparent early in the course of ad before there is demonstrable cell loss and plaque deposition.7 under most conditions , cerebral metabolism is fueled almost exclusively by glucose . therefore , if cerebral glucose metabolism is compromised in the ad patient , perhaps ketone bodies can substitute for glucose and improve cognitive performance.9,10 ketone bodies are an efficient fuel for cells,11,12 and results from preclinical studies have suggested that induced ketosis may be beneficial in ad.13,14 in addition , studies have suggested that ketone bodies may be useful in a variety of neurologic disorders.15 a previous study has demonstrated that administration of 20 g of caprylic triglyceride ( ct ) increases blood levels of beta - hydroxybutyrate ( the major ketone body in humans ) 2 hours post dose to a mean value of 0.39 mm , and this therapy has been shown to be beneficial in patients with mild - to - moderate ad.10,16 ct is composed of a glycerol backbone and three caprylic ( c8:0 ) fatty acid chains , well known for their ketogenic properties.17 the efficacy and safety of a medical food containing ct in subjects with mild - to - moderate ad has been demonstrated in a multicenter , randomized , double - blind , placebo - controlled study that included 152 outpatients with probable mild - to - moderate ad.10 it is important to continue evaluations of interventions in routine clinical practice . the primary objective of this study was to assess patient outcomes of ct used alone or in combination with ongoing pharmacotherapy in mild - to - moderate ad in routine clinical practice . due to the small number of patients enrolled in the study , patient outcomes were evaluated by chart review carried out at 11 practices in which ct was prescribed in patients with mild - to - moderate ad . the criteria for inclusion were male or female outpatients aged 50 years with a diagnosis of probable ad of mild - to - moderate severity ; mini - mental state examination ( mmse ) score between 14 and 24 inclusive at the initiation of ct ( if available ) , or diagnosis of mild - to - moderate ad ; and receipt of ct for 6 months . a caregiver was defined to be at least 21 years of age ( this age was believed to be appropriate for a caregiver who would provide a patient assessment as part of the study ) , had been the primary caregiver since prior to the initiation of ct , provided at least 4 hours of supervision or direct assistance per day for the patient , and was able to perform assessments . the first was the physician s overall assessment of patient status at the most recent evaluation on ct . the second was the change in mmse score from initiation of ct to the most recent mmse assessment on ct . third , change from beginning of ct to present , scored as check boxes of : marked improvement , moderate improvement , improvement , stable , worsening , moderate worsening , or marked worsening . secondary measures included changes from baseline in patient s living situation ; medication changes for the management of ad and for psychiatric symptoms related to ad ; changes in patient s memory and ability to carry out instrumental activities of daily living as assessed by caregivers ; and adverse events . caregivers were also asked to assess the patient s memory since initiation of ct , based on a modified version of the memory complaints questionnaire developed by memory assessment clinics ; and ability to carry out activities of daily living adapted from lawton and brody.21 scores from the physician s overall assessment of change from baseline after initiation of ct were summarized with descriptive statistics . changes from baseline in caregiver assessments of patients ability to use the telephone , prepare food , and dress were evaluated with mann all statistical tests were two - sided and were carried out at the 5% level of significance . pearson correlation between physician s overall assessment of change and caregiver assessment was performed with r 2.15.0 ( www.r-project.org ) . patient outcomes were evaluated by chart review carried out at 11 practices in which ct was prescribed in patients with mild - to - moderate ad . the criteria for inclusion were male or female outpatients aged 50 years with a diagnosis of probable ad of mild - to - moderate severity ; mini - mental state examination ( mmse ) score between 14 and 24 inclusive at the initiation of ct ( if available ) , or diagnosis of mild - to - moderate ad ; and receipt of ct for 6 months . a caregiver was defined to be at least 21 years of age ( this age was believed to be appropriate for a caregiver who would provide a patient assessment as part of the study ) , had been the primary caregiver since prior to the initiation of ct , provided at least 4 hours of supervision or direct assistance per day for the patient , and was able to perform assessments . the first was the physician s overall assessment of patient status at the most recent evaluation on ct . the second was the change in mmse score from initiation of ct to the most recent mmse assessment on ct . third , change from beginning of ct to present , scored as check boxes of : marked improvement , moderate improvement , improvement , stable , worsening , moderate worsening , or marked worsening . secondary measures included changes from baseline in patient s living situation ; medication changes for the management of ad and for psychiatric symptoms related to ad ; changes in patient s memory and ability to carry out instrumental activities of daily living as assessed by caregivers ; and adverse events . caregivers were also asked to assess the patient s memory since initiation of ct , based on a modified version of the memory complaints questionnaire developed by memory assessment clinics ; and ability to carry out activities of daily living adapted from lawton and brody.21 scores from the physician s overall assessment of change from baseline after initiation of ct were summarized with descriptive statistics . changes from baseline in caregiver assessments of patients ability to use the telephone , prepare food , and dress were evaluated with mann all statistical tests were two - sided and were carried out at the 5% level of significance . pearson correlation between physician s overall assessment of change and caregiver assessment was performed with r 2.15.0 ( www.r-project.org ) . a total of 437 records were reviewed at 11 study sites , and those of 55 patients who met the inclusion and exclusion criteria and provided informed written consent were included in the analysis . the most common reasons for exclusion were mmse scores out of the designated inclusion range , taking ct for < 6 months , no formal diagnosis of ad , and comorbidities that may have affected cognitive function . at baseline , 88.7% of patients were living with caregivers ( relatives ) and 11.3% were residing in assisted living facilities . patients who had received ct , regardless of the dose , for 6 months or longer were eligible for the study . for the 55 patients whose records were reviewed , the mean duration of ct was 18.8 9.2 months . chart review indicated that most patients ( 89.1% ) were titrated to a full 20 g daily ct dose . physician s overall assessment of changes from baseline in patient status ( figure 1a ) were marked improvement for 5.5% , moderate improvement for 5.5% , improvement for 24.1% , stable for 44.4% , worsening for 14.8% , moderate worsening for 3.7% , and marked worsening for 1.8% . the mean baseline score for these patients was 20.6 3.0 and that for the most recent measurement on ct was 20.1 5.6 ( difference 0.48 , 95% confidence interval 1.05 to 2.01 , t = 0.6470 , p = 0.5233 ) . at baseline , 43.6% of patients were rated by their physicians as having mild ad and 56.4% were considered to have moderate ad . there were also few changes in medications patients were taking for ad ; 87.3% of patients had no changes ; 7.3% had switches or augmentation in pharmacotherapy , and 5.4% had addition of a medical food that included phosphatidylserine enriched with docosahexaenoic acid . pharmacotherapy for psychiatric / behavioral symptoms also remained stable for most patients ( 70.9% ) . there were 25 patients taking no medications for psychiatric illness at baseline and six of these ( 10.9% of all patients , 24.0% of those not taking psychiatric medications at baseline ) had agents added to therapy , most often antidepressants or atypical antipsychotics . caregivers responses indicated that addition of ct to therapy resulted in some degree of improvement or stability in remembering the name of a person to whom they were just introduced in 80.9% ( 17/21 ) of patients , recalling telephone numbers or zip codes that were used on a daily or weekly basis in 80.9% ( 17/21 ) , and in finding commonly used objects in 85.7% ( 18/21 ) , as shown in figure 2 . overall assessments of memory also indicated that the majority of patients ( 75.0% ) ( 15/20 ) were stable or improved over the follow - up period ( figure 2 ) . assessment of ability to use the telephone , prepare food , and dressing indicated no significant changes from baseline ( mann whitney u - tests , p = 0.696 , p = 0.904 , and p = 0.596 , respectively ) . however , a significant correlation was found between the physician s overall assessment of change and caregiver surveys ( r = 0.5423 , confidence interval 0.80 to 0.12 , p = 0.016 , figure 3 ) . the remaining 14 patients had a total of 30 adverse events , eight of which were considered to be related to ct . a total of 437 records were reviewed at 11 study sites , and those of 55 patients who met the inclusion and exclusion criteria and provided informed written consent were included in the analysis . the most common reasons for exclusion were mmse scores out of the designated inclusion range , taking ct for < 6 months , no formal diagnosis of ad , and comorbidities that may have affected cognitive function . at baseline , 88.7% of patients were living with caregivers ( relatives ) and 11.3% were residing in assisted living facilities . patients who had received ct , regardless of the dose , for 6 months or longer were eligible for the study . for the 55 patients whose records were reviewed , the mean duration of ct was 18.8 9.2 months . chart review indicated that most patients ( 89.1% ) were titrated to a full 20 g daily ct dose . physician s overall assessment of changes from baseline in patient status ( figure 1a ) were marked improvement for 5.5% , moderate improvement for 5.5% , improvement for 24.1% , stable for 44.4% , worsening for 14.8% , moderate worsening for 3.7% , and marked worsening for 1.8% . the mean baseline score for these patients was 20.6 3.0 and that for the most recent measurement on ct was 20.1 5.6 ( difference 0.48 , 95% confidence interval 1.05 to 2.01 , t = 0.6470 , p = 0.5233 ) . at baseline , 43.6% of patients were rated by their physicians as having mild ad and 56.4% were considered to have moderate ad . there were also few changes in medications patients were taking for ad ; 87.3% of patients had no changes ; 7.3% had switches or augmentation in pharmacotherapy , and 5.4% had addition of a medical food that included phosphatidylserine enriched with docosahexaenoic acid . pharmacotherapy for psychiatric / behavioral symptoms also remained stable for most patients ( 70.9% ) . there were 25 patients taking no medications for psychiatric illness at baseline and six of these ( 10.9% of all patients , 24.0% of those not taking psychiatric medications at baseline ) had agents added to therapy , most often antidepressants or atypical antipsychotics . physician s overall assessment of changes from baseline in patient status ( figure 1a ) were marked improvement for 5.5% , moderate improvement for 5.5% , improvement for 24.1% , stable for 44.4% , worsening for 14.8% , moderate worsening for 3.7% , and marked worsening for 1.8% . the mean baseline score for these patients was 20.6 3.0 and that for the most recent measurement on ct was 20.1 5.6 ( difference 0.48 , 95% confidence interval 1.05 to 2.01 , t = 0.6470 , p = 0.5233 ) . at baseline , 43.6% of patients were rated by their physicians as having mild ad and 56.4% were considered to have moderate ad . there were also few changes in medications patients were taking for ad ; 87.3% of patients had no changes ; 7.3% had switches or augmentation in pharmacotherapy , and 5.4% had addition of a medical food that included phosphatidylserine enriched with docosahexaenoic acid . pharmacotherapy for psychiatric / behavioral symptoms also remained stable for most patients ( 70.9% ) . there were 25 patients taking no medications for psychiatric illness at baseline and six of these ( 10.9% of all patients , 24.0% of those not taking psychiatric medications at baseline ) had agents added to therapy , most often antidepressants or atypical antipsychotics . caregivers responses indicated that addition of ct to therapy resulted in some degree of improvement or stability in remembering the name of a person to whom they were just introduced in 80.9% ( 17/21 ) of patients , recalling telephone numbers or zip codes that were used on a daily or weekly basis in 80.9% ( 17/21 ) , and in finding commonly used objects in 85.7% ( 18/21 ) , as shown in figure 2 . overall assessments of memory also indicated that the majority of patients ( 75.0% ) ( 15/20 ) were stable or improved over the follow - up period ( figure 2 ) . assessment of ability to use the telephone , prepare food , and dressing indicated no significant changes from baseline ( mann whitney u - tests , p = 0.696 , p = 0.904 , and p = 0.596 , respectively ) . however , a significant correlation was found between the physician s overall assessment of change and caregiver surveys ( r = 0.5423 , confidence interval 0.80 to 0.12 , p = 0.016 , figure 3 ) . results from this chart review suggest that ct helped to stabilize or improve the clinical status and cognitive function in patients receiving therapy for a mean of approximately 19 months . the physician s overall assessment of change from baseline indicated that 79.5% of patients either showed improvement or remained stable , while 20.3% worsened . results for the 27 patients for whom mmse scores were available at baseline and on therapy indicate that , on average , patients remained stable with no significant change over a mean of 15 months of follow - up . no patients had changes in their living situation during ct therapy , and most patients remained stable with respect to medications for ad and those for psychiatric / behavioral conditions . caregiver assessments also indicated stability or improvement in cognitive function and no significant changes in the ability to carry out activities of daily living . the physician s overall assessment indicated that ~80% of patients who had ct added to their ongoing pharmacotherapy were stable or improved . a large number of different global severity / status scales have been employed in assessing effects of treatment in patients with ad . that most similar to the measure employed in this study is the clinical global impressions of change , which provides minimal instructions to the rater.22 results from global assessments of the type carried out in this study have also been shown to be well correlated with quantitative measures of cognitive function / dementia.23 the mmse is the most widely used brief mental status screening instrument,24,25 and has been used extensively for evaluation of patients with ad in both clinical trials and follow - up of patients being managed in routine clinical practice.2629 in order to evaluate the impact of ct on mild - to - moderate ad in routine practice , comparison with subjects on standard therapy without ct was examined by literature review . a summary of results from clinical trials and epidemiologic studies published prior to 2005 indicated a decline in mmse scores of 0.64.4 points per year in different studies.30 more recently , a study of 686 patients with mild - to - moderate ad with 90% receiving ad - specific medication indicated an annual decline in mmse scores of 2.4 points per year.31 in comparison , the decline in mmse scores over 15 months in the present study was 0.48 and not statistically significant . caregiver assessments also indicate that memory and ability to carry out activities of daily living remained stable or improved during therapy with ct . in an assessment of overall memory , 40% of the caregivers functional impairments in ad place the greatest burden on both caregivers and the economy;41 and results from several studies have suggested that neuropsychologic test performance is predictive of activities of daily living performance in patients with ad.42,43 results from observational studies and clinical trials indicate that deterioration in the ability to carry out activities of daily living can be detected among patients with ad over follow - up periods comparable with that in the present study.44,45 changes in the requirement for psychiatric medications were used as a surrogate measure for effects of ct on neuropsychiatric symptoms . these symptoms are a source of caregiver burden and are associated with increased probability of institutionalization.46 treatment aimed at improving cognition in patients with ad has also been shown to decrease the severity of neuropsychiatric symptoms.47 results from the record review indicate that medications for psychiatric illness remained stable for most patients during the period of ct . results from the chart review indicated the adverse event most often associated with ct was gastrointestinal upset and that this event led to discontinuation in 7.3% of patients . one limitation of this study was that the apolipoprotein e4 ( apoe4 ) carriage status was evaluated and recorded in the medical records of only seven patients ( two were apoe4-positive ) . prior clinical results for ct indicated significant efficacy in apoe4-negative but not apoe4-positive patients.10,16 in conclusion , results from this small chart review study indicate that addition of ct to pharmacotherapy for ad was associated with stable disease or improvement for most patients over a follow - up period of 18.8 months . addition of ct was also associated with stabilization or improvements in the ability to carry out activities of daily living for most patients and warrants further investigation in a larger study .

a total of 81 participants , including 52 patients with des ( 21 males and 31 females ; mean age , 52.37 10.18 years ; range , 32 to 75 years ) and 29 age- and sex - matched healthy individuals as controls ( 17 males and 12 females ; mean age , 50.69 10.37 years ; range , 24 to 68 years ) , were enrolled in this study . des was diagnosed by a physician based on the results of a patient questionnaire and clinical examinations according to the international dry eye workshop report . in the 52 patients with des not associated with sjgren 's syndrome , coexisting lid margin disease , or altered tear distribution and clearance , des severity was categorized into 1 of 4 levels , according to the delphi panel classification ( des grade 1 , n = 10 ; grade 2 , n = 10 ; grade 3 , n = 22 ; grade 4 , n = 10 ) . then , each individual underwent a complete examination of the ocular surface in the following order : tear break - up time ( tbut ) , corneal staining with f luorescein , conjunctival staining with lissamine green , and schirmer 's test without anesthesia . corneal staining was evaluated using the national eye institute method , and conjunctival staining was evaluated using the oxford scheme . the exclusion criteria included an age less than 18 years , previous usage of ophthalmic medications except artificial tears , a history of surgical intervention , chemical injury , a corneal pathology such as a corneal infection , complaints of ocular pain or discomfort due to any recent history of ocular disease besides des , use of contact lenses in the previous 6 months , connective tissue disease ( other than rheumatoid arthritis ) , autoimmune disease , diabetes mellitus , and parkinson 's disease . all control individuals had no abnormalities in their ocular surface examinations , i.e. , a schirmer test i value greater than 10 mm/5 min , a tbut value greater than 10 seconds , no complaint of ocular discomfort , and no usage of ocular medications . the research followed the tenets of the declaration of helsinki and informed consent was obtained from the participants after explanation of the nature and possible consequences of the study . the research was approved by an institutional review board ( no . c2012136 - 831 ) . samples of pure , undiluted tear fluid were collected from all 81 participants in 50-l glass capillaries placed with one end at the lateral canthus in contact with the tear film of the right eye . each tear sample was placed in a 1.5-ml eppendorf tube and immediately stored at -70 until further examination . to keep tear stimulation at a low level , contact with the conjunctival epithelium no additional solution that could induce irritation and stimulate tear fluids was used during tear collection . from each participant , approximately 10 l of unstimulated tears were collected by a single physician ( sww ) . the total tear protein concentration was evaluated with a nano - drop spectrophotometer ( nd-1000 ; nano - drop technologies , wilmington , de , usa ) . as the concentrations of ang and lf in an individual 's tear fluids used for elisa may vary , we normalized the concentration of each protein to the total tear protein concentration for both groups . we used 3 l of tear sample from each normal individual and des patient , and diluted this aliquot to a volume of 1 ml with buffer . tear protein profiling of all participants was performed with an immunodot blot assay following the manufacturer 's instructions . tear proteins such as basic fibroblast growth factor ( bfgf ) , vascular endothelial growth factor , transforming growth factor ( tgf)-2 , tumor necrosis factor- , interleukin ( il)-10 , il-17 , angiostatin , stromal cell - derived factor-1 , fas ligand , and ang were investigated with a human cytokine antibody array kit ( raybiotech , atlanta , ga , usa ) . collected tear samples were centrifuged to eliminate any cell debris and were applied to a membrane . the tear samples were applied to each membrane for 2 hours at room temperature , after which the non - specific areas of the membranes were blocked for 30 minutes . all membranes were washed three times for 5 minutes with wash buffer 1 , and then washed twice for 5 minutes with wash buffer 2 . tear proteins were detected by diluted biotin - conjugated antibodies added to each membrane for 2 hours of incubation . the membranes were visualized using a chemidoc xrs ( bio - rad laboratories , berkeley , ca , usa ) . the density of each blot was measured with a personal molecular imager fx ( bio - rad laboratories ) supported by imaging analysis software ( quantity one , bio - rad laboratories ) . each tear sample was diluted to a total volume of 300 l with buffer . to confirm the results of the immunodot blot assay , the amounts of ang and lf in tears from normal controls and des patients were compared quantitatively by elisa using a human lf detection kit ( bethyl laboratories , montgomery , tx , usa ) and a human ang elisa detection kit ( abcam , cambridge , england ) following the manufacturers ' instructions . in brief , standards or samples were added to 96-well plates and incubated for 1 hour at 4. the plates were washed four times , and a biotin antibody for ang or lf detection was added to each well . the plates were covered , incubated for 1 hour at room temperature , and then washed four times . a streptavidin solution for ang or an horseradish peroxidase solution the plates were covered and incubated at room temperature for 45 minutes with ang or 30 minutes with lf . after the washing step was repeated , the 3,3',5,5'-tetramethylbenzidine liquid substrate solution was added to each well . the plates were incubated for 30 minutes at room temperature in the dark , and the stop solution was added to each well . the optical density was measured at 450 nm on a microplate photometer spectramax 340pc using softmax pro 5.4.1 ( mds inc . , 16.0 ( spss inc . , chicago , il , usa ) . for all tests , the significance level was set at p < 0.05 . based on the normality of the data distribution , comparative analyses among individual samples of proteins were performed using the mann - whitney u - test , student 's t - test , and the chi - square test . correlations between the protein concentrations and dry eye severity were assessed using spearman 's correlation test . a total of 81 participants , including 52 patients with des ( 21 males and 31 females ; mean age , 52.37 10.18 years ; range , 32 to 75 years ) and 29 age- and sex - matched healthy individuals as controls ( 17 males and 12 females ; mean age , 50.69 10.37 years ; range , 24 to 68 years ) , were enrolled in this study . des was diagnosed by a physician based on the results of a patient questionnaire and clinical examinations according to the international dry eye workshop report . in the 52 patients with des not associated with sjgren 's syndrome , coexisting lid margin disease , or altered tear distribution and clearance , des severity was categorized into 1 of 4 levels , according to the delphi panel classification ( des grade 1 , n = 10 ; grade 2 , n = 10 ; grade 3 , n = 22 ; grade 4 , n = 10 ) . then , each individual underwent a complete examination of the ocular surface in the following order : tear break - up time ( tbut ) , corneal staining with f luorescein , conjunctival staining with lissamine green , and schirmer 's test without anesthesia . corneal staining was evaluated using the national eye institute method , and conjunctival staining was evaluated using the oxford scheme . the exclusion criteria included an age less than 18 years , previous usage of ophthalmic medications except artificial tears , a history of surgical intervention , chemical injury , a corneal pathology such as a corneal infection , complaints of ocular pain or discomfort due to any recent history of ocular disease besides des , use of contact lenses in the previous 6 months , connective tissue disease ( other than rheumatoid arthritis ) , autoimmune disease , diabetes mellitus , and parkinson 's disease . all control individuals had no abnormalities in their ocular surface examinations , i.e. , a schirmer test i value greater than 10 mm/5 min , a tbut value greater than 10 seconds , no complaint of ocular discomfort , and no usage of ocular medications . the research followed the tenets of the declaration of helsinki and informed consent was obtained from the participants after explanation of the nature and possible consequences of the study . the research was approved by an institutional review board ( no . c2012136 - 831 ) . samples of pure , undiluted tear fluid were collected from all 81 participants in 50-l glass capillaries placed with one end at the lateral canthus in contact with the tear film of the right eye . each tear sample was placed in a 1.5-ml eppendorf tube and immediately stored at -70 until further examination . to keep tear stimulation at a low level , contact with the conjunctival epithelium no additional solution that could induce irritation and stimulate tear fluids was used during tear collection . from each participant , approximately 10 l of unstimulated tears were collected by a single physician ( sww ) . the total tear protein concentration was evaluated with a nano - drop spectrophotometer ( nd-1000 ; nano - drop technologies , wilmington , de , usa ) . as the concentrations of ang and lf in an individual 's tear fluids used for elisa may vary , we normalized the concentration of each protein to the total tear protein concentration for both groups . we used 3 l of tear sample from each normal individual and des patient , and diluted this aliquot to a volume of 1 ml with buffer . tear protein profiling of all participants was performed with an immunodot blot assay following the manufacturer 's instructions . tear proteins such as basic fibroblast growth factor ( bfgf ) , vascular endothelial growth factor , transforming growth factor ( tgf)-2 , tumor necrosis factor- , interleukin ( il)-10 , il-17 , angiostatin , stromal cell - derived factor-1 , fas ligand , and ang were investigated with a human cytokine antibody array kit ( raybiotech , atlanta , ga , usa ) . collected tear samples were centrifuged to eliminate any cell debris and were applied to a membrane . the tear samples were applied to each membrane for 2 hours at room temperature , after which the non - specific areas of the membranes were blocked for 30 minutes . all membranes were washed three times for 5 minutes with wash buffer 1 , and then washed twice for 5 minutes with wash buffer 2 . tear proteins were detected by diluted biotin - conjugated antibodies added to each membrane for 2 hours of incubation . the membranes were visualized using a chemidoc xrs ( bio - rad laboratories , berkeley , ca , usa ) . the density of each blot was measured with a personal molecular imager fx ( bio - rad laboratories ) supported by imaging analysis software ( quantity one , bio - rad laboratories ) . was diluted to a total volume of 300 l with buffer . to confirm the results of the immunodot blot assay , the amounts of ang and lf in tears from normal controls and des patients were compared quantitatively by elisa using a human lf detection kit ( bethyl laboratories , montgomery , tx , usa ) and a human ang elisa detection kit ( abcam , cambridge , england ) following the manufacturers ' instructions . in brief , standards or samples were added to 96-well plates and incubated for 1 hour at 4. the plates were washed four times , and a biotin antibody for ang or lf detection was added to each well . the plates were covered , incubated for 1 hour at room temperature , and then washed four times . a streptavidin solution for ang or an horseradish peroxidase solution was added to each well . the plates were covered and incubated at room temperature for 45 minutes with ang or 30 minutes with lf . after the washing step was repeated , the 3,3',5,5'-tetramethylbenzidine liquid substrate solution was added to each well . the plates were incubated for 30 minutes at room temperature in the dark , and the stop solution was added to each well . the optical density was measured at 450 nm on a microplate photometer spectramax 340pc using softmax pro 5.4.1 ( mds inc . , all data were analyzed with statistical software spss ver . 16.0 ( spss inc . , based on the normality of the data distribution , comparative analyses among individual samples of proteins were performed using the mann - whitney u - test , student 's t - test , and the chi - square test . correlations between the protein concentrations and dry eye severity were assessed using spearman 's correlation test . demographic data from the patients and healthy controls , including age , sex , and clinical test results , are summarized in table 1 . the ang areas in des grades 3 and 4 patients were significantly lower than those of normal controls and des patients with lower grades . however , there were no significant differences between normal controls and des patients with lower grades . the ang blot densities were 508.15 60.80 in normal controls , 511.39 63.34 in des grade 1 patients , 460.40 35.72 in des grade 2 patients , 49.05 23.57 in des grade 3 patients , and 38.70 11.71 in des grade 4 patients . the areas of bfgf , tgf-2 , and il-10 were significantly greater than those of normal controls only in grade 4 des patients , but these proteins did not correlate significantly with dry eye severity . il-17 , angiostatin , stromal cell - derived factor-1 , fas ligand , and vascular endothelial growth factor did not differ significantly among participants , regardless of des grade . 1a and 1b and 2a-2e summarize the results of tear protein profiling from the immunodot blot assay . the mean concentrations of ang in the tears of normal controls ( n = 29 ) and des grade 1 ( n = 10 ) , and 2 ( n = 10 ) patients were 206.65 100.23 the concentrations of ang in the tears of des grade 3 ( n = 22 ) and 4 ( n = 10 ) patients were 71.03 44.67 pg / ml and 41.16 33.83 the mean concentrations of lf in the tears of normal controls and des grade 1 , and 2 patients were 0.41 0.19 mg / ml , 0.27 0.11 mg / ml , and 0.27 0.19 mg / ml , respectively . the concentrations of lf in the tears of des grade 3 and 4 patients were 0.10 0.09 mg / ml and 0.06 0.04 mg / ml , respectively . ang and lf concentrations in des patients were lower than those of normal controls and decreased significantly with increasing des severity , except in grades 1 and 2 . the group - to - group differences in lf and ang were statistically significant ( p < 0.05 ) , except for the differences between des severity grades 1 and 2 . table 2 summarizes the mean concentrations of ang and lf in control and des tears . the ratios of ang and lf to total tear proteins were measured to supplement the concentration results in normal controls and des patients . the percentage concentration of ang / total tear protein was 11.48 5.90 ( 10)% in the normal control group , 6.14 2.93 ( 10)% in des grade 1 , 5.74 2.12 ( 10)% in des grade 2 , 3.78 2.27 ( 10)% in des grade 3 , and 2.15 1.62 ( 10)% in des grade 4 patients . the percentage concentration of lf / total tear protein was 21.69 7.58% in the normal control group , 13.65 6.70% in des grade 1 , 13.36 7.74% in des grade 2 , 5.54 4.68% in des grade 3 , and 3.05 2.31% in des grade 4 patients . the group - to - group differences in the percentage concentrations of lf and ang were statistically significant ( p < 0.05 ) , except for those between des severity grades 1 and 2 . fig . 4 shows the significant correlation between the percentage concentrations of ang and lf in des patients [ lf ( % ) = 1.0 ang ( % ) 10 + 4.7828 ] . demographic data from the patients and healthy controls , including age , sex , and clinical test results , are summarized in table 1 . the ang areas in des grades 3 and 4 patients were significantly lower than those of normal controls and des patients with lower grades . however , there were no significant differences between normal controls and des patients with lower grades . the ang blot densities were 508.15 60.80 in normal controls , 511.39 63.34 in des grade 1 patients , 460.40 35.72 in des grade 2 patients , 49.05 23.57 in des grade 3 patients , and 38.70 11.71 in des grade 4 patients . the areas of bfgf , tgf-2 , and il-10 were significantly greater than those of normal controls only in grade 4 des patients , but these proteins did not correlate significantly with dry eye severity . il-17 , angiostatin , stromal cell - derived factor-1 , fas ligand , and vascular endothelial growth factor did not differ significantly among participants , regardless of des grade . 1a and 1b and 2a-2e summarize the results of tear protein profiling from the immunodot blot assay . the mean concentrations of ang in the tears of normal controls ( n = 29 ) and des grade 1 ( n = 10 ) , and 2 ( n = 10 ) patients were 206.65 100.23 pg / ml , 123.43 52.54 pg / ml , and 109.85 38.76 pg / ml , respectively . the concentrations of ang in the tears of des grade 3 ( n = 22 ) and 4 ( n = 10 ) patients were 71.03 44.67 pg / ml and 41.16 33.83 the mean concentrations of lf in the tears of normal controls and des grade 1 , and 2 patients were 0.41 0.19 mg / ml , 0.27 0.11 mg / ml , and 0.27 0.19 mg / ml , respectively . the concentrations of lf in the tears of des grade 3 and 4 patients were 0.10 0.09 mg / ml and 0.06 0.04 mg / ml , respectively . ang and lf concentrations in des patients were lower than those of normal controls and decreased significantly with increasing des severity , except in grades 1 and 2 . the group - to - group differences in lf and ang were statistically significant ( p < 0.05 ) , except for the differences between des severity grades 1 and 2 . table 2 summarizes the mean concentrations of ang and lf in control and des tears . the ratios of ang and lf to total tear proteins were measured to supplement the concentration results in normal controls and des patients . the percentage concentration of ang / total tear protein was 11.48 5.90 ( 10)% in the normal control group , 6.14 2.93 ( 10)% in des grade 1 , 5.74 2.12 ( 10)% in des grade 2 , 3.78 2.27 ( 10)% in des grade 3 , and 2.15 1.62 ( 10)% in des grade 4 patients . the percentage concentration of lf / total tear protein was 21.69 7.58% in the normal control group , 13.65 6.70% in des grade 1 , 13.36 7.74% in des grade 2 , 5.54 4.68% in des grade 3 , and 3.05 2.31% in des grade 4 patients . the group - to - group differences in the percentage concentrations of lf and ang were statistically significant ( p < 0.05 ) , except for those between des severity grades 1 and 2 . fig . 4 shows the significant correlation between the percentage concentrations of ang and lf in des patients [ lf ( % ) = 1.0 ang ( % ) 10 + 4.7828 ] . several diagnostic tests for des , such as fluorescein staining , tbut measurement , schirmer 's test , rose bengal staining , impression cytology , and tear clearance rate measurement , have been widely used . however , these commonly used diagnostic tests are reported to be poorly associated with patients ' symptoms , and no definite standards for measuring the therapeutic response to treatment in patients with des have been established . recently , several studies have evaluated and graded des via proteomic analysis ; a proteomic approach is becoming more common as a novel diagnostic tool for des . tear protein levels have been compared between evaporative dry eye patients and controls . in a previous study , lf and lipophilin a - c were lower in evaporative dry eye patients than in controls . on the other hand , as des is understood to be a multifactorial disorder and inflammation of the ocular surface is considered to be closely related with des , alterations in tear protein concentrations in dry eye patients may reflect the inflammatory status of the eye . we hypothesized that the tear protein levels of des patients would differ from those of normal controls , and evaluated the correlation between the des severity and the concentrations of certain proteins . in the present study , concentrations of bfgf , tgf-2 , and il-10 were significantly greater than those of normal controls only in grade 4 des patients , but these proteins did not correlate significantly with dry eye severity . as tgf-2 and il-10 are reported to be anti - inflammatory proteins secreted by cd4 + regulatory t cells upon inflammation of the ocular surface , and bfgf is reported to potentiate inflammatory mediator - induced leukocyte recruitment in chronic inflammation , we concluded that the results of our immunodot blot assay were similar to those of the previous studies . however , the concentrations of bfgf , tgf-2 , tumor necrosis factor- , and il-10 were not significantly correlated with des severity . in contrast , ang was linearly associated with des severity , except between grades 1 and 2 . we performed a further study of ang and lf by elisa to confirm that the ang levels in participants ' tears were inversely proportional to the disease severity grade in des patients , as lf has been reported to indicate reflect the severity of ocular surface damage due to des . in the present study , therefore , we concluded that both ang and lf are specific proteins that are significantly correlated with des severity , except between grades 1 and 2 . this study has valuable implications for the practical use of proteomic analysis in diagnosing des severity . although there have been few studies regarding ang in the field of ophthalmology , various biological functions have been disclosed . we can infer the characteristics and functions of ang from studies that have investigated the effects of lf and the association between lf and des . lf is a glycoprotein present in human breast milk and is secreted into tears by the lacrimal and meibomian glands . the tear lf level has been reported to be an indicator of lacrimal secretory function . lf has several functions , including anti - inflammatory effects that reduce tumor necrosis factor- and t - cell proliferation , increase il- 10 levels , and actions that promote cell growth and dna synthesis . previous studies also have reported that tear lf levels correlated with the severity of conjunctivocorneal epithelial lesions in des patients . as des is a multifactorial disease of the tears and the ocular surface , including an inflammatory process that alters the tear protein composition , it is worth considering lf as a therapeutic agent that may reduce des severity . indeed , dogru et al . reported that oral lf supplements improved tear stability and the state of the ocular surface epithelium , confirming the above - mentioned hypothesis . possible interactions between ang and lf in the immune and inflammatory systems have been reported previously , and we found a significant positive correlation between ang and lf in the present study . although the roles of ang in inflammatory cascades are not clear , we could hypothesize that ang and lf interact with each other in des progression . we also may suppose that the gradual decrease in ang levels with the increasing des severity reflects the progression of the inflammatory response , inferring from a previous study regarding the action of ang as a defensive agent in pro - inflammatory conditions . together , these findings suggest that ang could be used as a diagnostic tool for grading des and its associated inflammatory symptoms . in addition , as ang is a natural protein present in the body , it is expected that an exogenous ang supplement may improve des symptoms , much like supplements of lf and albumin do . it is necessary to investigate the possible actions of ang in the eye and the safety of its biological use through in vitro and in vivo studies of therapeutic exogenous supplements . in general , ang has been reported to induce the synthesis of ribosomal rna and neovascularization . furthermore , there have been several reports regarding the action of ang in the posterior segments of the eye . reported that ang may participate in pathologic angiogenesis in eyes with choroidal neovascularization . in contrast , marek et al . postulated that ang is not associated with the pathogenesis of diabetic retinopathy , contrary to vascular endothelial growth factor , which is the main mediator responsible for pathological neovascularization in diabetic retinopathy . as the action of ang is still a matter of debate , further studies are needed regarding the action of ang in various regions of the eye . first , ang levels did not differ significantly between the normal controls and des grade 1 and 2 patients in the immunodot blot assay . these results do not support the conclusion of the present study that ang could be used for the diagnosis and grading of des . on the other hand , unlike the results of the immunodot blot assay , the elisa results indicated that the concentration of ang was negatively correlated with dry eye severity , and the group - to - group difference in ang was statistically significant , except between des grades 1 and 2 . therefore , we believe that the present study is important and will be valuable because it is the first to discuss the correlation among ang , lf , and des severity . second , we analyzed the participants ' clinical test results and the tear fluids collected from their right eyes , because it was uncertain whether both eyes would have the same des grade . we recognize that this study is limited in evaluating the true state of dry eye severity , as only one eye was studied , and there may be variability in dry eye conditions between both eyes . however , we thought that this methodology was appropriate , because the goal of this study was to evaluate the correlation between ang levels and dry eye severity by analyzing tear fluids and the diagnostic test results . thirdly , although we tried to categorize the des patients based on completely consistent test results using the delphi classification , there were some discrepancies among the results of the tbut , schirmer 's test , and corneal staining . in such cases , we placed priority on the corneal staining , as it is more reliable than both tbut and schirmer 's test . in conclusion , ang and lf concentrations were significantly lower in des patients than in normal controls , and were significantly correlated with des severity , except between grades 1 and 2 . also , ang may be expected to act as a therapeutic agent in des patients , inferring from the results of previous and recent studies of lf . further research should be carried out to evaluate the safety of ang for clinical use .

purposeto investigate the properties of angiogenin ( ang ) as a potential tool for the diagnosis and grading of dry eye syndrome ( des ) by analyzing tear protein profiles.methodstear samples were collected with capillary tubes from 52 des patients and 29 normal individuals as controls . tear protein profiles were analyzed with an immunodot blot assay as a screening test . to confirm that the tear ang levels were in inverse proportion to the disease severity grade , the ang and lactoferrin ( lf ) tear contents of normal controls and des patients were compared in an enzyme - linked immunosorbent assay.resultsin the immunodot blot assay , the ang area was lower in patients with grades 3 and 4 des than in normal controls . the areas of basic fibroblast growth factor , transforming growth factor 2 , and interleukin 10 were significantly greater than those of normal controls only in grade 4 des patients , but these proteins were not linearly correlated with dry eye severity . upon enzyme - linked immunosorbent assay analysis , the mean concentrations of ang and lf decreased significantly as dry eye severity increased , except between grades 1 and 2 . in addition , the ratios of ang and lf to total tear proteins were correlated significantly with des severity.conclusionsang level was significantly lower in des patients than in normal controls , and was significantly correlated with the worsening severity of des , except between grades 1 and 2 , as was lf . therefore , ang may be a useful measure of des severity through proteomic analysis .

Materials and Methods Participants Tear collection Immunodot blot assay Enzyme-linked immunosorbent assay Statistical analysis Results Demographics of the participants Immunodot blot assay Enzyme-linked immunosorbent assay Discussion

a total of 81 participants , including 52 patients with des ( 21 males and 31 females ; mean age , 52.37 10.18 years ; range , 32 to 75 years ) and 29 age- and sex - matched healthy individuals as controls ( 17 males and 12 females ; mean age , 50.69 10.37 years ; range , 24 to 68 years ) , were enrolled in this study . des was diagnosed by a physician based on the results of a patient questionnaire and clinical examinations according to the international dry eye workshop report . in the 52 patients with des not associated with sjgren 's syndrome , coexisting lid margin disease , or altered tear distribution and clearance , des severity was categorized into 1 of 4 levels , according to the delphi panel classification ( des grade 1 , n = 10 ; grade 2 , n = 10 ; grade 3 , n = 22 ; grade 4 , n = 10 ) . the exclusion criteria included an age less than 18 years , previous usage of ophthalmic medications except artificial tears , a history of surgical intervention , chemical injury , a corneal pathology such as a corneal infection , complaints of ocular pain or discomfort due to any recent history of ocular disease besides des , use of contact lenses in the previous 6 months , connective tissue disease ( other than rheumatoid arthritis ) , autoimmune disease , diabetes mellitus , and parkinson 's disease . samples of pure , undiluted tear fluid were collected from all 81 participants in 50-l glass capillaries placed with one end at the lateral canthus in contact with the tear film of the right eye . as the concentrations of ang and lf in an individual 's tear fluids used for elisa may vary , we normalized the concentration of each protein to the total tear protein concentration for both groups . we used 3 l of tear sample from each normal individual and des patient , and diluted this aliquot to a volume of 1 ml with buffer . tear protein profiling of all participants was performed with an immunodot blot assay following the manufacturer 's instructions . tear proteins such as basic fibroblast growth factor ( bfgf ) , vascular endothelial growth factor , transforming growth factor ( tgf)-2 , tumor necrosis factor- , interleukin ( il)-10 , il-17 , angiostatin , stromal cell - derived factor-1 , fas ligand , and ang were investigated with a human cytokine antibody array kit ( raybiotech , atlanta , ga , usa ) . the tear samples were applied to each membrane for 2 hours at room temperature , after which the non - specific areas of the membranes were blocked for 30 minutes . to confirm the results of the immunodot blot assay , the amounts of ang and lf in tears from normal controls and des patients were compared quantitatively by elisa using a human lf detection kit ( bethyl laboratories , montgomery , tx , usa ) and a human ang elisa detection kit ( abcam , cambridge , england ) following the manufacturers ' instructions . a total of 81 participants , including 52 patients with des ( 21 males and 31 females ; mean age , 52.37 10.18 years ; range , 32 to 75 years ) and 29 age- and sex - matched healthy individuals as controls ( 17 males and 12 females ; mean age , 50.69 10.37 years ; range , 24 to 68 years ) , were enrolled in this study . des was diagnosed by a physician based on the results of a patient questionnaire and clinical examinations according to the international dry eye workshop report . in the 52 patients with des not associated with sjgren 's syndrome , coexisting lid margin disease , or altered tear distribution and clearance , des severity was categorized into 1 of 4 levels , according to the delphi panel classification ( des grade 1 , n = 10 ; grade 2 , n = 10 ; grade 3 , n = 22 ; grade 4 , n = 10 ) . the exclusion criteria included an age less than 18 years , previous usage of ophthalmic medications except artificial tears , a history of surgical intervention , chemical injury , a corneal pathology such as a corneal infection , complaints of ocular pain or discomfort due to any recent history of ocular disease besides des , use of contact lenses in the previous 6 months , connective tissue disease ( other than rheumatoid arthritis ) , autoimmune disease , diabetes mellitus , and parkinson 's disease . samples of pure , undiluted tear fluid were collected from all 81 participants in 50-l glass capillaries placed with one end at the lateral canthus in contact with the tear film of the right eye . the total tear protein concentration was evaluated with a nano - drop spectrophotometer ( nd-1000 ; nano - drop technologies , wilmington , de , usa ) . as the concentrations of ang and lf in an individual 's tear fluids used for elisa may vary , we normalized the concentration of each protein to the total tear protein concentration for both groups . tear protein profiling of all participants was performed with an immunodot blot assay following the manufacturer 's instructions . tear proteins such as basic fibroblast growth factor ( bfgf ) , vascular endothelial growth factor , transforming growth factor ( tgf)-2 , tumor necrosis factor- , interleukin ( il)-10 , il-17 , angiostatin , stromal cell - derived factor-1 , fas ligand , and ang were investigated with a human cytokine antibody array kit ( raybiotech , atlanta , ga , usa ) . the tear samples were applied to each membrane for 2 hours at room temperature , after which the non - specific areas of the membranes were blocked for 30 minutes . tear proteins were detected by diluted biotin - conjugated antibodies added to each membrane for 2 hours of incubation . to confirm the results of the immunodot blot assay , the amounts of ang and lf in tears from normal controls and des patients were compared quantitatively by elisa using a human lf detection kit ( bethyl laboratories , montgomery , tx , usa ) and a human ang elisa detection kit ( abcam , cambridge , england ) following the manufacturers ' instructions . , all data were analyzed with statistical software spss ver . correlations between the protein concentrations and dry eye severity were assessed using spearman 's correlation test . demographic data from the patients and healthy controls , including age , sex , and clinical test results , are summarized in table 1 . the ang areas in des grades 3 and 4 patients were significantly lower than those of normal controls and des patients with lower grades . however , there were no significant differences between normal controls and des patients with lower grades . the ang blot densities were 508.15 60.80 in normal controls , 511.39 63.34 in des grade 1 patients , 460.40 35.72 in des grade 2 patients , 49.05 23.57 in des grade 3 patients , and 38.70 11.71 in des grade 4 patients . the areas of bfgf , tgf-2 , and il-10 were significantly greater than those of normal controls only in grade 4 des patients , but these proteins did not correlate significantly with dry eye severity . il-17 , angiostatin , stromal cell - derived factor-1 , fas ligand , and vascular endothelial growth factor did not differ significantly among participants , regardless of des grade . 1a and 1b and 2a-2e summarize the results of tear protein profiling from the immunodot blot assay . the mean concentrations of ang in the tears of normal controls ( n = 29 ) and des grade 1 ( n = 10 ) , and 2 ( n = 10 ) patients were 206.65 100.23 the concentrations of ang in the tears of des grade 3 ( n = 22 ) and 4 ( n = 10 ) patients were 71.03 44.67 pg / ml and 41.16 33.83 the mean concentrations of lf in the tears of normal controls and des grade 1 , and 2 patients were 0.41 0.19 mg / ml , 0.27 0.11 mg / ml , and 0.27 0.19 mg / ml , respectively . the concentrations of lf in the tears of des grade 3 and 4 patients were 0.10 0.09 mg / ml and 0.06 0.04 mg / ml , respectively . ang and lf concentrations in des patients were lower than those of normal controls and decreased significantly with increasing des severity , except in grades 1 and 2 . the group - to - group differences in lf and ang were statistically significant ( p < 0.05 ) , except for the differences between des severity grades 1 and 2 . table 2 summarizes the mean concentrations of ang and lf in control and des tears . the ratios of ang and lf to total tear proteins were measured to supplement the concentration results in normal controls and des patients . the percentage concentration of ang / total tear protein was 11.48 5.90 ( 10)% in the normal control group , 6.14 2.93 ( 10)% in des grade 1 , 5.74 2.12 ( 10)% in des grade 2 , 3.78 2.27 ( 10)% in des grade 3 , and 2.15 1.62 ( 10)% in des grade 4 patients . the percentage concentration of lf / total tear protein was 21.69 7.58% in the normal control group , 13.65 6.70% in des grade 1 , 13.36 7.74% in des grade 2 , 5.54 4.68% in des grade 3 , and 3.05 2.31% in des grade 4 patients . the group - to - group differences in the percentage concentrations of lf and ang were statistically significant ( p < 0.05 ) , except for those between des severity grades 1 and 2 . 4 shows the significant correlation between the percentage concentrations of ang and lf in des patients [ lf ( % ) = 1.0 ang ( % ) 10 + 4.7828 ] . demographic data from the patients and healthy controls , including age , sex , and clinical test results , are summarized in table 1 . the ang areas in des grades 3 and 4 patients were significantly lower than those of normal controls and des patients with lower grades . however , there were no significant differences between normal controls and des patients with lower grades . the ang blot densities were 508.15 60.80 in normal controls , 511.39 63.34 in des grade 1 patients , 460.40 35.72 in des grade 2 patients , 49.05 23.57 in des grade 3 patients , and 38.70 11.71 in des grade 4 patients . the areas of bfgf , tgf-2 , and il-10 were significantly greater than those of normal controls only in grade 4 des patients , but these proteins did not correlate significantly with dry eye severity . il-17 , angiostatin , stromal cell - derived factor-1 , fas ligand , and vascular endothelial growth factor did not differ significantly among participants , regardless of des grade . 1a and 1b and 2a-2e summarize the results of tear protein profiling from the immunodot blot assay . the mean concentrations of ang in the tears of normal controls ( n = 29 ) and des grade 1 ( n = 10 ) , and 2 ( n = 10 ) patients were 206.65 100.23 pg / ml , 123.43 52.54 pg / ml , and 109.85 38.76 pg / ml , respectively . the concentrations of ang in the tears of des grade 3 ( n = 22 ) and 4 ( n = 10 ) patients were 71.03 44.67 pg / ml and 41.16 33.83 the mean concentrations of lf in the tears of normal controls and des grade 1 , and 2 patients were 0.41 0.19 mg / ml , 0.27 0.11 mg / ml , and 0.27 0.19 mg / ml , respectively . the concentrations of lf in the tears of des grade 3 and 4 patients were 0.10 0.09 mg / ml and 0.06 0.04 mg / ml , respectively . ang and lf concentrations in des patients were lower than those of normal controls and decreased significantly with increasing des severity , except in grades 1 and 2 . the group - to - group differences in lf and ang were statistically significant ( p < 0.05 ) , except for the differences between des severity grades 1 and 2 . table 2 summarizes the mean concentrations of ang and lf in control and des tears . the ratios of ang and lf to total tear proteins were measured to supplement the concentration results in normal controls and des patients . the percentage concentration of ang / total tear protein was 11.48 5.90 ( 10)% in the normal control group , 6.14 2.93 ( 10)% in des grade 1 , 5.74 2.12 ( 10)% in des grade 2 , 3.78 2.27 ( 10)% in des grade 3 , and 2.15 1.62 ( 10)% in des grade 4 patients . the percentage concentration of lf / total tear protein was 21.69 7.58% in the normal control group , 13.65 6.70% in des grade 1 , 13.36 7.74% in des grade 2 , 5.54 4.68% in des grade 3 , and 3.05 2.31% in des grade 4 patients . the group - to - group differences in the percentage concentrations of lf and ang were statistically significant ( p < 0.05 ) , except for those between des severity grades 1 and 2 . 4 shows the significant correlation between the percentage concentrations of ang and lf in des patients [ lf ( % ) = 1.0 ang ( % ) 10 + 4.7828 ] . however , these commonly used diagnostic tests are reported to be poorly associated with patients ' symptoms , and no definite standards for measuring the therapeutic response to treatment in patients with des have been established . recently , several studies have evaluated and graded des via proteomic analysis ; a proteomic approach is becoming more common as a novel diagnostic tool for des . tear protein levels have been compared between evaporative dry eye patients and controls . in a previous study , lf and lipophilin a - c were lower in evaporative dry eye patients than in controls . on the other hand , as des is understood to be a multifactorial disorder and inflammation of the ocular surface is considered to be closely related with des , alterations in tear protein concentrations in dry eye patients may reflect the inflammatory status of the eye . we hypothesized that the tear protein levels of des patients would differ from those of normal controls , and evaluated the correlation between the des severity and the concentrations of certain proteins . in the present study , concentrations of bfgf , tgf-2 , and il-10 were significantly greater than those of normal controls only in grade 4 des patients , but these proteins did not correlate significantly with dry eye severity . as tgf-2 and il-10 are reported to be anti - inflammatory proteins secreted by cd4 + regulatory t cells upon inflammation of the ocular surface , and bfgf is reported to potentiate inflammatory mediator - induced leukocyte recruitment in chronic inflammation , we concluded that the results of our immunodot blot assay were similar to those of the previous studies . however , the concentrations of bfgf , tgf-2 , tumor necrosis factor- , and il-10 were not significantly correlated with des severity . in contrast , ang was linearly associated with des severity , except between grades 1 and 2 . we performed a further study of ang and lf by elisa to confirm that the ang levels in participants ' tears were inversely proportional to the disease severity grade in des patients , as lf has been reported to indicate reflect the severity of ocular surface damage due to des . in the present study , therefore , we concluded that both ang and lf are specific proteins that are significantly correlated with des severity , except between grades 1 and 2 . this study has valuable implications for the practical use of proteomic analysis in diagnosing des severity . we can infer the characteristics and functions of ang from studies that have investigated the effects of lf and the association between lf and des . previous studies also have reported that tear lf levels correlated with the severity of conjunctivocorneal epithelial lesions in des patients . as des is a multifactorial disease of the tears and the ocular surface , including an inflammatory process that alters the tear protein composition , it is worth considering lf as a therapeutic agent that may reduce des severity . possible interactions between ang and lf in the immune and inflammatory systems have been reported previously , and we found a significant positive correlation between ang and lf in the present study . although the roles of ang in inflammatory cascades are not clear , we could hypothesize that ang and lf interact with each other in des progression . we also may suppose that the gradual decrease in ang levels with the increasing des severity reflects the progression of the inflammatory response , inferring from a previous study regarding the action of ang as a defensive agent in pro - inflammatory conditions . in addition , as ang is a natural protein present in the body , it is expected that an exogenous ang supplement may improve des symptoms , much like supplements of lf and albumin do . it is necessary to investigate the possible actions of ang in the eye and the safety of its biological use through in vitro and in vivo studies of therapeutic exogenous supplements . postulated that ang is not associated with the pathogenesis of diabetic retinopathy , contrary to vascular endothelial growth factor , which is the main mediator responsible for pathological neovascularization in diabetic retinopathy . first , ang levels did not differ significantly between the normal controls and des grade 1 and 2 patients in the immunodot blot assay . these results do not support the conclusion of the present study that ang could be used for the diagnosis and grading of des . on the other hand , unlike the results of the immunodot blot assay , the elisa results indicated that the concentration of ang was negatively correlated with dry eye severity , and the group - to - group difference in ang was statistically significant , except between des grades 1 and 2 . therefore , we believe that the present study is important and will be valuable because it is the first to discuss the correlation among ang , lf , and des severity . we recognize that this study is limited in evaluating the true state of dry eye severity , as only one eye was studied , and there may be variability in dry eye conditions between both eyes . however , we thought that this methodology was appropriate , because the goal of this study was to evaluate the correlation between ang levels and dry eye severity by analyzing tear fluids and the diagnostic test results . thirdly , although we tried to categorize the des patients based on completely consistent test results using the delphi classification , there were some discrepancies among the results of the tbut , schirmer 's test , and corneal staining . in conclusion , ang and lf concentrations were significantly lower in des patients than in normal controls , and were significantly correlated with des severity , except between grades 1 and 2 . also , ang may be expected to act as a therapeutic agent in des patients , inferring from the results of previous and recent studies of lf .

our discussion will not address the massive scale of unmet medical need to be found in populations suffering from neurological and psychiatric illness . nor will we analyze the lack of substantive change in treatment options for these patients . suffice it to say that , in spite of the billions of dollars spent searching for better treatments for neurological and psychiatric disorders , and the myriad advances made in basic neuroscience , when it comes to real - world therapeutic drug options , the situation has been one of near stasis . our antidepressants do not differ significantly from those that were available 20 years ago ; the same can be said of our antipsychotic options , used primarily for schizophrenia and bipolar disorder ; and for the modest cognitive enhancers marketed for alzheimer s . the one exception an area where significant advances have occurred in terms of efficacy and ease - of - use is in the treatment of relapsing - remitting multiple sclerosis ( rrms ) . the beta - interferons gave rise to iv natalizumab , then the oral s1p targeting compounds , and the oral fumarate - based drugs . it is the one disorder where we are now able to actually slow the progression of the disease , rather than simply masking or suppressing symptoms , and the repertoire comprises a range of choices with a spectrum of risk - reward profiles from which patients and their physicians can choose . it is also the area where the regeneration of what has been lost , in terms of neural circuits and functioning , is now being first attempted . advances for rrms represent the way we hope neurotherapeutics for other disorders will evolve over the next two decades . first , the great psychiatric drug classes that emerged during the 1950s and 1960s were largely the product of serendipity ; the clinical observation of therapeutic effects that led to post - hoc hypotheses explaining how these drugs might work they failed to provide a road map for their successors ; many scientists embarked on research journeys launched by assumptions that turned out to be incorrect and guided by processes that misinformed . there was a false sense of confidence based on the commercial success of new drug classes that had become popular due to a better side - effect profile , rather than improved efficacy , like the ssris and second - generation antipsychotics . the pharmaceutical industry became , for lack of a better word , lazy when it came to internal r&d ; imitation was frequently more prized than discovery , as many companies tried to piggyback on the success of earlier drugs through tweaking rather than innovating . to the degree to which innovation was permitted and funded , there was a tendency towards premature closure , choosing new mechanisms for full development without adequately auditioning the range of alternatives . the single best example of this is alzheimer s , where the bulk of research funding and testing over the past 20 years has relied upon an amyloid hypothesis that , even now , has yet to prove itself to be valid . because the brain is often described as the most complex structure in the known universe , neuroscience is fundamentally more challenging and less advanced than other areas of medicine . one key problem has been and continues to be that the tools with which neuroscience r&d is carried out have been inadequate to the task , and generally less effective in their application than those available to other therapeutic endeavors . this has yielded an inevitable string of failures . in the world of animal models , the theoretical underpinnings have mostly been dubious , and the issues numerous . the system used for classifying disorders is based on categories that date back a century or more . what s more , the pathophysiological roots of most disorders are unknown ; targets for intervention have been generally based on theories derived from animal models of ambiguous relevance , and are located behind the blood - brain - barrier , making it difficult to get drug candidates where they were needed . it has been for the most part impossible to be sure if target engagement has been achieved . and the endpoints by which clinical status and progress are measured in human testing still tend to be ambiguous and subjective , particularly in psychiatry . from a pragmatist s point - of - view , the question might better boil down to : why would anyone invest in this area ? unmet medical need , but the existence of such needs , great as they may be , does not in itself form a bridge to the treatments being developed for them . to a large extent , we have been flying blind , without much in the way of instrument - assistance . the definition of success has been binary , determined by whether the flight landed , or ended in a crash . the wreckage of many highly - touted programs litters the runways of the biopharma industry , and has come to dominate the perception of the neurotherapeutics area in the eyes of many ( albeit not all ) investors and pharma companies as being too risky . confidence in biopharma s neuro - skillset was gradually ground down to a nub via a drawn - out series of high - profile failures , which led investors to question whether neuroscience had any idea what it was doing . the full list of failures is too long and disheartening to review in detail , but clinical landmarks that played an important role in squandering industry credibility include : myotrophin and als ( 1997)substance p and depression ( 1999)free radicals and stroke ( 2006)bapineuzumab and alzheimer s ( 2012)pomaglumetad methionil and schizophrenia ( 2013 ) myotrophin and als ( 1997 ) substance p and depression ( 1999 ) free radicals and stroke ( 2006 ) bapineuzumab and alzheimer s ( 2012 ) pomaglumetad methionil and schizophrenia ( 2013 ) the net effect of these failures over a period of 15 years was to flag neurology and psychiatry as too hard, leading to the nearly complete departure of glaxosmithkline from neuroscience , and significant contractions of neuroscience programming at sanofi , merck , lilly , and others . even as the success rate of neuroscience r&d plummeted , the first world was approaching a macro - economic near - death experience . the fiscal crisis of 20082010 led to a dramatic retraction of capital investment , and investors became highly skittish when it came to investment risk . this made it extremely difficult for privately held biotech companies to go public , and threw a major obstacle into the cycle by which capital enters and exits the biopharma system . while travelers tend to ignore the safety feature demonstrations provided by a flight attendant before each take - off , assuming that exits available for emergencies will not be needed , such is not the case for venture capital and institutional investors , for whom the location and timing of an exit are core components of their investment model . in biopharma , such exits take the form of ipos , wherein public money comes into a company , replacing much of the private investment that sustained the company to that point , and provides continued liquidity via stock sales . the simultaneous withdrawal of big pharma as a potential acquirer , along with this closing of the ipo window , meant one thing above all for anyone contemplating investment in the neurotherapeutics area : once invested in a small company , there was no near - term exit at hand . even worse , with no promise of new investors , the initial investors were increasingly confronted by the choice of either doubling down on a high - risk venture , or letting it wither and die . venture capitalists , who operate in pre - defined life - cycles , typically less than ten years , could not credibly assure investors that they would be able to retrieve their investment , hopefully with profits attached , in that promised timeframe . this , in turn , reduced the inflow of resources to these venture capitalists , impairing their ability to sustain old investments , let alone make new ones , and thus produced a vicious cycle of the first order . to illustrate , consider the casualty rate amongst 81 private cognitive neuroscience companies that ni research tracked , beginning in 2003 : of those companies , only 19.7 percent provided an exit for their investors ( 12.3 percent via acquisition , 7.4 percent via ipo ) , and 61 percent went out of business entirely , representing a complete loss for their investors . the remaining 19 percent have continued in private operation , many of them barely alive , a herd of neurotech zombies . they are far more likely to end up in the failure than the success column . vc entrances and exits : ipos 200415 this chart illustrates the correlation between the availability of ipo exits and the availability of investment for new and young cns companies , which represents the entrance of money into the small company cns arena . over the ten years from 2004 through 2013 , there were a total of just 21 ipos by private cns companies . as the number and total raised by these ipos began to accelerate , there was an even more dramatic flow of investment into the cns sector , far exceeding the amount that exited . reinforcing this transformation was the less dramatic , but still vitally important return of some large and midsize pharma companies to neuroscience , while partnering external research . indeed , many large companies have essentially outsourced much of their neuroscience research to smaller companies . resourcing neurotherapeutics 200415 this chart shows the past decade s transformation of resources overall for the small cns company world , from both investors and pharma partners . the spectacular escalation of resource availability , particularly from investors , began towards the end of 2013 , and accelerated over the next two years . what accounted for this turnaround ? to some degree it was due to the economic recovery overall , particularly in the us , yielding capital that was looking for a home . in spite of the legacy of failure and frustration , there were factors that began to make the neurotherapeutics area palatable , even desirable , to investors . there is the belief , perhaps more akin to faith , that the genomics advances that allowed the development of precision medicines in oncology may yet prove useful in neuroscience , the greatest untapped market of all . beyond these nascent genomics advances were the maturation of neuroimaging ( e.g. amyloid plaque , tau ) , csf biomarkers , changes in nomenclature ( the national institute of mental health research domain criteria [ rdoc ] initiative ) , and technologies allowing the tracking of trial and treatment compliance . in aggregate , these constitute the harbinger of an era in neuroscience wherein guesswork is being replaced with something more substantive . we are better equipped to track target engagement at least for some targets and rrms is now seen as the leading edge , rather than the sole outlier . induced pluripotent stem cell ( ipsc ) models , modified via gene editing , offer a more face - valid screen for early drug development than the animal models upon which the sector has too long relied . remote biosensors and big data analytics offer the prospect of being able to sift huge datasets for meaningful relationships , defining pathways where new and more impactful targets may exist , and the concept of brain and brain pathology as based on networks of circuits can be reified and tested via techniques like optogenetics . parsing this list , in a highly oversimplified fashion , it can be argued that there were three events or dynamics that set the stage for this transformation : personalized oncology , hepatitis c treatment , and neuroimaging . personalized oncology : the human genome was sequenced in 2000 , but it has taken a long time for the genomics revolution to make a tangible difference in the practice of medicine . we would argue that it is in the area of personalized medicine in the treatment of cancer that the highest profile gain from genomics has been realized : rather than relying upon trial - and - error ( and tradition ) in offering patients various cocktails of chemotherapeutics , oncology began to parse cancer into subcategories based on genetic factors . this altered the climate of frustration and delay that had grown around genomics , and raised the question : where else might this work ? when it comes to large - scale , heterogeneous populations of uncertain etiology , nothing exceeds the scale offered by the worlds of neurology and psychiatry , and the belief system within the investment world has begun to shift towards the anticipation that genomics will render these disorders more comprehensible and tractable.hepatitis c : a disease that formerly could only be treated palliatively turned into a disease for which magic bullets can provide a cure . the fact that these treatments can be premium - priced and yet make pharmacoeconomic sense has also hit home for the investment community . in an era where treatment pricing had turned into a war of attrition between payors , generic manufacturers , and pharma companies , and like personalized treatments for cancer , this portended a time where a successful new product could be once again expected to produce outsized profits . no area had been hit harder by the advent of generics than neuroscience as a whole , psychiatry in particular , and this provided a road map for returning some pricing control to the neuropharm world.neuroimaging : as was mentioned earlier , it was the success of disease - modifiers in the treatment of relapsing - remitting multiple sclerosis that for a very long time was the only example of substantive , rather than incremental , progress in the treatments of cns disorders . one critical element was the availability of imaging technology that could provide an objective , empirical measurement of therapeutic impact on the rate at which the disease progressed . as gadolinium - enhanced imaging was for ms , the advent of florbetapir as a means of empirically quantifying amyloid plaque in alzheimer s has served as a beacon of hope that biomarkers would begin to take neurotherapeutics out of its familiar morass of squishy , subjective endpoints . the fact that amyloid plaque s utility as a biomarker has yet to be fully established has been less important than its role as the poster child for a new era of objective measures in neuroscience . other imaging markers ( e.g. , tau ) and a plethora of blood and csf biomarkers have emerged , albeit yet - to - be - proven in their ability to focus and accelerate cns drug development . personalized oncology : the human genome was sequenced in 2000 , but it has taken a long time for the genomics revolution to make a tangible difference in the practice of medicine . we would argue that it is in the area of personalized medicine in the treatment of cancer that the highest profile gain from genomics has been realized : rather than relying upon trial - and - error ( and tradition ) in offering patients various cocktails of chemotherapeutics , oncology began to parse cancer into subcategories based on genetic factors . this altered the climate of frustration and delay that had grown around genomics , and raised the question : where else might this work ? when it comes to large - scale , heterogeneous populations of uncertain etiology , nothing exceeds the scale offered by the worlds of neurology and psychiatry , and the belief system within the investment world has begun to shift towards the anticipation that genomics will render these disorders more comprehensible and tractable . hepatitis c : a disease that formerly could only be treated palliatively turned into a disease for which magic bullets can provide a cure . the fact that these treatments can be premium - priced and yet make pharmacoeconomic sense has also hit home for the investment community . in an era where treatment pricing had turned into a war of attrition between payors , generic manufacturers , and pharma companies , and like personalized treatments for cancer , this portended a time where a successful new product could be once again expected to produce outsized profits . no area had been hit harder by the advent of generics than neuroscience as a whole , psychiatry in particular , and this provided a road map for returning some pricing control to the neuropharm world . neuroimaging : as was mentioned earlier , it was the success of disease - modifiers in the treatment of relapsing - remitting multiple sclerosis that for a very long time was the only example of substantive , rather than incremental , progress in the treatments of cns disorders . what made rrms different ? one critical element was the availability of imaging technology that could provide an objective , empirical measurement of therapeutic impact on the rate at which the disease progressed . as gadolinium - enhanced imaging was for ms , the advent of florbetapir as a means of empirically quantifying amyloid plaque in alzheimer s has served as a beacon of hope that biomarkers would begin to take neurotherapeutics out of its familiar morass of squishy , subjective endpoints . the fact that amyloid plaque s utility as a biomarker has yet to be fully established has been less important than its role as the poster child for a new era of objective measures in neuroscience . other imaging markers ( e.g. , tau ) and a plethora of blood and csf biomarkers have emerged , albeit yet - to - be - proven in their ability to focus and accelerate cns drug development . while the process of testing candidate drugs in clinical trials remains a high - risk , high - anxiety endeavor for neuroscience , new tools offer greater assurance that participants in drug trials are real patients , not professional patients, and patients , who are not ill , or who do not take the medication being evaluated , simply are not a valid template for testing the efficacy of such a drug . we will never know how many clinical trials have been ruined by noncompliant patients ; one must wonder how many potentially useful drugs had the signal of their therapeutic impact obscured by a flawed clinical testing process . clinical trial professionals have come to realize , sometimes at odds with the companies sponsoring the trials , that in this context , speed kills . fake patients ; and are beginning to explore technologies ( like a chip on a pill ) that allow accurate monitoring of what a patient takes , and when . and in an environment where generic drugs have become king , the pharma industry and its investors have finally come to recognize that redundancy no longer is remunerative , and that they will have to grapple with the risk , along with the potential reward of novel mechanisms for intervention . as confidence in the tools has grown , so too has the willingness of both pharma partners and investors to bet on higher - innovation , higher - risk programs , because making significant inroads on the huge , unmet needs of neurology and psychiatry will require new and disruptive technologies . neurotherapeutics is , of course , not a unitary construct , and it is informative to consider where resources are flowing more specifically . the breakdown over the past decade , parsing the area into four categories ( neurodegenerative disease - modifiers , neurology - symptomatics , psychiatry , pain ) yields the following in terms of funding therapeutic subareas from 2009 to 2015 : after several years where no neuroscience area was favored in terms of funding , there was a dramatic surge in 2014 , with the most spectacular rise in funding for symptomatic treatments for neurological disorders , including levodopa - induced dyskinesia , and cognition or psychosis associated with neurodegeneration . this is a relatively lower risk area compared to programs aimed at slowing or stopping the course of a neurodegenerative disease , which received the least funding of the four in 2014 . but in 2015 , while there was continued , slowly growing interest in symptomatic treatments , investors finally became willing to fund disease - modifiers , reflecting the belief that improvements in neuroscience tools would mean that these high - risk programs stood a better chance of success . it should be noted that the willingness to put venture capital into higher - risk programs has been anything but across - the - board , but there are some vcs who have the long - term perspective and neuroscience background that allows them to be more risk - tolerant . thus , in 201415 , the roster of vcs leading investment rounds in highly innovative research included fidelity biosciences ( denali therapeutics , yumanity therapeutics , forum pharmaceuticals ) ; third rock ( voyager therapeutics ) ; atlas ventures ( lysosomal therapeutics and rodin therapeutics ) and clarus ventures ( annexon ) . the fact that the denali series a round of $ 217 million was by far the largest such round ever completed by a cns company provided a signal to more reticent vcs that smart money is starting to find its way into neurodegeneration research and that they should consider participating , even if not leading . another important development has been heightened activity from pharma companies investing through venture arms , giving them the benefit of the investment itself and insight into ongoing research activities , without having to buy in completely . in the other therapeutic subdomains , pain enjoyed steadily increasing investment , partly due to the growing visibility of opioid abuse , enhancing the potential prospects for novel analgesia alternatives . finally , psychiatry , long out of favor , rebounded somewhat in 2014 , the most apparent trigger being the rising profile of the rapid - acting - antidepressant class , epitomized by ketamine , which for the first time in 20 years seemed to offer the potential of a genuinely differentiated new antidepressant option . but the investment in psychiatry plateaued in 2015 , its flat growth leaving it well behind the other three areas in garnering investment dollars . overall , the past decade or two have constituted a humbling process to which the neurotherapeutics sector has had to submit , where scientists have had to accept the limitations of their knowledge base and research tools , finally going back to the drawing board . this has led to the growing salience of genomics , biomarker analyses , brain imaging , and sophisticated behavioral assessment technologies , providing an entirely revised approach to drug target delineation and validation . to be clear , the confluence of these technologies has yet to come to fruition . outside of ms , no new drugs have been identified , refined , and proven via the new generation of techniques . but there is a renewed emphasis upon empirical , scientific inquiry and validation , which has given the investment community hope that the brain will not continue to be a black box whose workings and our impact upon them can only be guessed at . beyond imaging and biomarkers , there is an array of new tools that portend an era of greater productivity for neurotherapeutics , which inspired at least through the end of 2015a resurgence of optimism and an influx of resources . whether that will continue will depend on macro - economic forces completely beyond the control and influence of the biopharma industry , and the degree to which clinical successes begin to provide tangible proof that this is in fact a new era for neuroscience .

editor s note : compared to the money dedicated to cancer and cardiology , funding for neuroscience research has lagged behind for decades . but things are starting to change . from the white house s brain initiative to the ice bucket challenge for als to some recent sizeable gifts to universities , money for brain research appears to be on the rise . but , as our author explains , research and development funding from private and corporate lenders for cognitive neuroscience an area that he has spent years tracking is also vital to the quality of life for millions of people .

Serendipity and Stasis A Crisis of Faith No Exit: The Existential Angst of the Venture Capitalist Other Factors Carrying Weight Playing Favorites: Where the Therapeutic Areas Rank No Place for Vertigo: The Oscillations Continue

our discussion will not address the massive scale of unmet medical need to be found in populations suffering from neurological and psychiatric illness . nor will we analyze the lack of substantive change in treatment options for these patients . suffice it to say that , in spite of the billions of dollars spent searching for better treatments for neurological and psychiatric disorders , and the myriad advances made in basic neuroscience , when it comes to real - world therapeutic drug options , the situation has been one of near stasis . our antidepressants do not differ significantly from those that were available 20 years ago ; the same can be said of our antipsychotic options , used primarily for schizophrenia and bipolar disorder ; and for the modest cognitive enhancers marketed for alzheimer s . the one exception an area where significant advances have occurred in terms of efficacy and ease - of - use is in the treatment of relapsing - remitting multiple sclerosis ( rrms ) . the beta - interferons gave rise to iv natalizumab , then the oral s1p targeting compounds , and the oral fumarate - based drugs . it is the one disorder where we are now able to actually slow the progression of the disease , rather than simply masking or suppressing symptoms , and the repertoire comprises a range of choices with a spectrum of risk - reward profiles from which patients and their physicians can choose . it is also the area where the regeneration of what has been lost , in terms of neural circuits and functioning , is now being first attempted . first , the great psychiatric drug classes that emerged during the 1950s and 1960s were largely the product of serendipity ; the clinical observation of therapeutic effects that led to post - hoc hypotheses explaining how these drugs might work they failed to provide a road map for their successors ; many scientists embarked on research journeys launched by assumptions that turned out to be incorrect and guided by processes that misinformed . there was a false sense of confidence based on the commercial success of new drug classes that had become popular due to a better side - effect profile , rather than improved efficacy , like the ssris and second - generation antipsychotics . the pharmaceutical industry became , for lack of a better word , lazy when it came to internal r&d ; imitation was frequently more prized than discovery , as many companies tried to piggyback on the success of earlier drugs through tweaking rather than innovating . to the degree to which innovation was permitted and funded , there was a tendency towards premature closure , choosing new mechanisms for full development without adequately auditioning the range of alternatives . the single best example of this is alzheimer s , where the bulk of research funding and testing over the past 20 years has relied upon an amyloid hypothesis that , even now , has yet to prove itself to be valid . because the brain is often described as the most complex structure in the known universe , neuroscience is fundamentally more challenging and less advanced than other areas of medicine . one key problem has been and continues to be that the tools with which neuroscience r&d is carried out have been inadequate to the task , and generally less effective in their application than those available to other therapeutic endeavors . in the world of animal models , the theoretical underpinnings have mostly been dubious , and the issues numerous . the system used for classifying disorders is based on categories that date back a century or more . what s more , the pathophysiological roots of most disorders are unknown ; targets for intervention have been generally based on theories derived from animal models of ambiguous relevance , and are located behind the blood - brain - barrier , making it difficult to get drug candidates where they were needed . it has been for the most part impossible to be sure if target engagement has been achieved . and the endpoints by which clinical status and progress are measured in human testing still tend to be ambiguous and subjective , particularly in psychiatry . from a pragmatist s point - of - view , the question might better boil down to : why would anyone invest in this area ? unmet medical need , but the existence of such needs , great as they may be , does not in itself form a bridge to the treatments being developed for them . to a large extent , we have been flying blind , without much in the way of instrument - assistance . the definition of success has been binary , determined by whether the flight landed , or ended in a crash . the wreckage of many highly - touted programs litters the runways of the biopharma industry , and has come to dominate the perception of the neurotherapeutics area in the eyes of many ( albeit not all ) investors and pharma companies as being too risky . confidence in biopharma s neuro - skillset was gradually ground down to a nub via a drawn - out series of high - profile failures , which led investors to question whether neuroscience had any idea what it was doing . the full list of failures is too long and disheartening to review in detail , but clinical landmarks that played an important role in squandering industry credibility include : myotrophin and als ( 1997)substance p and depression ( 1999)free radicals and stroke ( 2006)bapineuzumab and alzheimer s ( 2012)pomaglumetad methionil and schizophrenia ( 2013 ) myotrophin and als ( 1997 ) substance p and depression ( 1999 ) free radicals and stroke ( 2006 ) bapineuzumab and alzheimer s ( 2012 ) pomaglumetad methionil and schizophrenia ( 2013 ) the net effect of these failures over a period of 15 years was to flag neurology and psychiatry as too hard, leading to the nearly complete departure of glaxosmithkline from neuroscience , and significant contractions of neuroscience programming at sanofi , merck , lilly , and others . even as the success rate of neuroscience r&d plummeted , the first world was approaching a macro - economic near - death experience . the fiscal crisis of 20082010 led to a dramatic retraction of capital investment , and investors became highly skittish when it came to investment risk . in biopharma , such exits take the form of ipos , wherein public money comes into a company , replacing much of the private investment that sustained the company to that point , and provides continued liquidity via stock sales . the simultaneous withdrawal of big pharma as a potential acquirer , along with this closing of the ipo window , meant one thing above all for anyone contemplating investment in the neurotherapeutics area : once invested in a small company , there was no near - term exit at hand . even worse , with no promise of new investors , the initial investors were increasingly confronted by the choice of either doubling down on a high - risk venture , or letting it wither and die . venture capitalists , who operate in pre - defined life - cycles , typically less than ten years , could not credibly assure investors that they would be able to retrieve their investment , hopefully with profits attached , in that promised timeframe . to illustrate , consider the casualty rate amongst 81 private cognitive neuroscience companies that ni research tracked , beginning in 2003 : of those companies , only 19.7 percent provided an exit for their investors ( 12.3 percent via acquisition , 7.4 percent via ipo ) , and 61 percent went out of business entirely , representing a complete loss for their investors . the remaining 19 percent have continued in private operation , many of them barely alive , a herd of neurotech zombies . they are far more likely to end up in the failure than the success column . vc entrances and exits : ipos 200415 this chart illustrates the correlation between the availability of ipo exits and the availability of investment for new and young cns companies , which represents the entrance of money into the small company cns arena . over the ten years from 2004 through 2013 , there were a total of just 21 ipos by private cns companies . reinforcing this transformation was the less dramatic , but still vitally important return of some large and midsize pharma companies to neuroscience , while partnering external research . indeed , many large companies have essentially outsourced much of their neuroscience research to smaller companies . resourcing neurotherapeutics 200415 this chart shows the past decade s transformation of resources overall for the small cns company world , from both investors and pharma partners . the spectacular escalation of resource availability , particularly from investors , began towards the end of 2013 , and accelerated over the next two years . what accounted for this turnaround ? to some degree it was due to the economic recovery overall , particularly in the us , yielding capital that was looking for a home . in spite of the legacy of failure and frustration , there were factors that began to make the neurotherapeutics area palatable , even desirable , to investors . beyond these nascent genomics advances were the maturation of neuroimaging ( e.g. amyloid plaque , tau ) , csf biomarkers , changes in nomenclature ( the national institute of mental health research domain criteria [ rdoc ] initiative ) , and technologies allowing the tracking of trial and treatment compliance . we are better equipped to track target engagement at least for some targets and rrms is now seen as the leading edge , rather than the sole outlier . induced pluripotent stem cell ( ipsc ) models , modified via gene editing , offer a more face - valid screen for early drug development than the animal models upon which the sector has too long relied . remote biosensors and big data analytics offer the prospect of being able to sift huge datasets for meaningful relationships , defining pathways where new and more impactful targets may exist , and the concept of brain and brain pathology as based on networks of circuits can be reified and tested via techniques like optogenetics . personalized oncology : the human genome was sequenced in 2000 , but it has taken a long time for the genomics revolution to make a tangible difference in the practice of medicine . we would argue that it is in the area of personalized medicine in the treatment of cancer that the highest profile gain from genomics has been realized : rather than relying upon trial - and - error ( and tradition ) in offering patients various cocktails of chemotherapeutics , oncology began to parse cancer into subcategories based on genetic factors . when it comes to large - scale , heterogeneous populations of uncertain etiology , nothing exceeds the scale offered by the worlds of neurology and psychiatry , and the belief system within the investment world has begun to shift towards the anticipation that genomics will render these disorders more comprehensible and tractable.hepatitis c : a disease that formerly could only be treated palliatively turned into a disease for which magic bullets can provide a cure . no area had been hit harder by the advent of generics than neuroscience as a whole , psychiatry in particular , and this provided a road map for returning some pricing control to the neuropharm world.neuroimaging : as was mentioned earlier , it was the success of disease - modifiers in the treatment of relapsing - remitting multiple sclerosis that for a very long time was the only example of substantive , rather than incremental , progress in the treatments of cns disorders . one critical element was the availability of imaging technology that could provide an objective , empirical measurement of therapeutic impact on the rate at which the disease progressed . the fact that amyloid plaque s utility as a biomarker has yet to be fully established has been less important than its role as the poster child for a new era of objective measures in neuroscience . , tau ) and a plethora of blood and csf biomarkers have emerged , albeit yet - to - be - proven in their ability to focus and accelerate cns drug development . personalized oncology : the human genome was sequenced in 2000 , but it has taken a long time for the genomics revolution to make a tangible difference in the practice of medicine . we would argue that it is in the area of personalized medicine in the treatment of cancer that the highest profile gain from genomics has been realized : rather than relying upon trial - and - error ( and tradition ) in offering patients various cocktails of chemotherapeutics , oncology began to parse cancer into subcategories based on genetic factors . this altered the climate of frustration and delay that had grown around genomics , and raised the question : where else might this work ? when it comes to large - scale , heterogeneous populations of uncertain etiology , nothing exceeds the scale offered by the worlds of neurology and psychiatry , and the belief system within the investment world has begun to shift towards the anticipation that genomics will render these disorders more comprehensible and tractable . hepatitis c : a disease that formerly could only be treated palliatively turned into a disease for which magic bullets can provide a cure . the fact that these treatments can be premium - priced and yet make pharmacoeconomic sense has also hit home for the investment community . in an era where treatment pricing had turned into a war of attrition between payors , generic manufacturers , and pharma companies , and like personalized treatments for cancer , this portended a time where a successful new product could be once again expected to produce outsized profits . no area had been hit harder by the advent of generics than neuroscience as a whole , psychiatry in particular , and this provided a road map for returning some pricing control to the neuropharm world . neuroimaging : as was mentioned earlier , it was the success of disease - modifiers in the treatment of relapsing - remitting multiple sclerosis that for a very long time was the only example of substantive , rather than incremental , progress in the treatments of cns disorders . what made rrms different ? one critical element was the availability of imaging technology that could provide an objective , empirical measurement of therapeutic impact on the rate at which the disease progressed . as gadolinium - enhanced imaging was for ms , the advent of florbetapir as a means of empirically quantifying amyloid plaque in alzheimer s has served as a beacon of hope that biomarkers would begin to take neurotherapeutics out of its familiar morass of squishy , subjective endpoints . the fact that amyloid plaque s utility as a biomarker has yet to be fully established has been less important than its role as the poster child for a new era of objective measures in neuroscience . other imaging markers ( e.g. , tau ) and a plethora of blood and csf biomarkers have emerged , albeit yet - to - be - proven in their ability to focus and accelerate cns drug development . while the process of testing candidate drugs in clinical trials remains a high - risk , high - anxiety endeavor for neuroscience , new tools offer greater assurance that participants in drug trials are real patients , not professional patients, and patients , who are not ill , or who do not take the medication being evaluated , simply are not a valid template for testing the efficacy of such a drug . we will never know how many clinical trials have been ruined by noncompliant patients ; one must wonder how many potentially useful drugs had the signal of their therapeutic impact obscured by a flawed clinical testing process . clinical trial professionals have come to realize , sometimes at odds with the companies sponsoring the trials , that in this context , speed kills . fake patients ; and are beginning to explore technologies ( like a chip on a pill ) that allow accurate monitoring of what a patient takes , and when . and in an environment where generic drugs have become king , the pharma industry and its investors have finally come to recognize that redundancy no longer is remunerative , and that they will have to grapple with the risk , along with the potential reward of novel mechanisms for intervention . as confidence in the tools has grown , so too has the willingness of both pharma partners and investors to bet on higher - innovation , higher - risk programs , because making significant inroads on the huge , unmet needs of neurology and psychiatry will require new and disruptive technologies . neurotherapeutics is , of course , not a unitary construct , and it is informative to consider where resources are flowing more specifically . the breakdown over the past decade , parsing the area into four categories ( neurodegenerative disease - modifiers , neurology - symptomatics , psychiatry , pain ) yields the following in terms of funding therapeutic subareas from 2009 to 2015 : after several years where no neuroscience area was favored in terms of funding , there was a dramatic surge in 2014 , with the most spectacular rise in funding for symptomatic treatments for neurological disorders , including levodopa - induced dyskinesia , and cognition or psychosis associated with neurodegeneration . this is a relatively lower risk area compared to programs aimed at slowing or stopping the course of a neurodegenerative disease , which received the least funding of the four in 2014 . but in 2015 , while there was continued , slowly growing interest in symptomatic treatments , investors finally became willing to fund disease - modifiers , reflecting the belief that improvements in neuroscience tools would mean that these high - risk programs stood a better chance of success . it should be noted that the willingness to put venture capital into higher - risk programs has been anything but across - the - board , but there are some vcs who have the long - term perspective and neuroscience background that allows them to be more risk - tolerant . thus , in 201415 , the roster of vcs leading investment rounds in highly innovative research included fidelity biosciences ( denali therapeutics , yumanity therapeutics , forum pharmaceuticals ) ; third rock ( voyager therapeutics ) ; atlas ventures ( lysosomal therapeutics and rodin therapeutics ) and clarus ventures ( annexon ) . the fact that the denali series a round of $ 217 million was by far the largest such round ever completed by a cns company provided a signal to more reticent vcs that smart money is starting to find its way into neurodegeneration research and that they should consider participating , even if not leading . another important development has been heightened activity from pharma companies investing through venture arms , giving them the benefit of the investment itself and insight into ongoing research activities , without having to buy in completely . in the other therapeutic subdomains , pain enjoyed steadily increasing investment , partly due to the growing visibility of opioid abuse , enhancing the potential prospects for novel analgesia alternatives . finally , psychiatry , long out of favor , rebounded somewhat in 2014 , the most apparent trigger being the rising profile of the rapid - acting - antidepressant class , epitomized by ketamine , which for the first time in 20 years seemed to offer the potential of a genuinely differentiated new antidepressant option . but the investment in psychiatry plateaued in 2015 , its flat growth leaving it well behind the other three areas in garnering investment dollars . overall , the past decade or two have constituted a humbling process to which the neurotherapeutics sector has had to submit , where scientists have had to accept the limitations of their knowledge base and research tools , finally going back to the drawing board . this has led to the growing salience of genomics , biomarker analyses , brain imaging , and sophisticated behavioral assessment technologies , providing an entirely revised approach to drug target delineation and validation . to be clear , the confluence of these technologies has yet to come to fruition . but there is a renewed emphasis upon empirical , scientific inquiry and validation , which has given the investment community hope that the brain will not continue to be a black box whose workings and our impact upon them can only be guessed at . beyond imaging and biomarkers , there is an array of new tools that portend an era of greater productivity for neurotherapeutics , which inspired at least through the end of 2015a resurgence of optimism and an influx of resources . whether that will continue will depend on macro - economic forces completely beyond the control and influence of the biopharma industry , and the degree to which clinical successes begin to provide tangible proof that this is in fact a new era for neuroscience .

owing largely to early marriage , adolescent women in india become sexually active at an early age and face tremendous social and familial pressure for child bearing soon after marriage , a situation that leads to multiple reproductive health problems . many of them lack knowledge about proper use of family planning methods and safe abortions . the improper use of contraception , intrauterine devices ( iud ) insertion in the presence of infections , female sterilization in unsterile condition , and unsafe abortion also increases the risks of rtis[2 - 4 ] . silent epidemic and are among the leading public health problems significantly contributing to gynecological morbidity and maternal mortality in india and other developing countries . moreover , in india , women with self- reported symptoms of reproductive morbidity do not seek treatment due to existing taboos and inhibitions regarding sexual and reproductive health . they hesitate to discuss their reproductive health problems especially , due to shame and embarrassment[6 - 7 ] . untreated infections are among the underlying causes of pelvic inflammatory diseases ( pid ) , ectopic pregnancy , infertility , cervical cancer , fetal loss , health problems of new born , and increased risk of hiv transmission . in addition to health consequences , women experience social consequences in terms of emotional distress related to gynecological morbidity . as most of these illnesses progresses to a chronic state and remain with women for the rest of their lives , the importance of early detection and management becomes much more evident . the prevalence of rti infections is low in india ; however , the number of adolescent affected by infections is a real concern due to the large adolescent population . india has the largest proportion of adolescents ( more than 243 million ) in the world , accounting for almost 20 per cent of the country s population . around 340 million incidences of curable stis are diagnosed each year , out of which 151 million are from south and southeast asia . furthermore , two third of all stis occur among young and adolescent people who are in their early twenties . until now , very few studies have focused on reproductive tract infections ( rti ) or sexually ransmitted diseases ( stis ) among adolescent women in developing countries such as india . a study conducted in southern indian state of tamil nadu on young married women aged 16 - 22 years in a rural community reported a very high level of morbidity . the study showed that more than half of the women were suffering from at least one or more rtis . similarly , very few attempts have been made to study the health seeking behavior for reproductive morbidity of adolescent women in india[1 , 13 - 14 ] . one of the studies explained the familial influence in treatment seeking behavior of married adolescents in maharashtra , a western state of india . however , most of the studies are micro - level data collected from districts or regions . therefore , the present study focuses on knowledge and prevalence of reproductive tract infection among married adolescent women using a national level large scale data with a special attention on different categories of infection and treatment seeking behavior . more importantly , this study attempts to group the symptoms by factor analysis and to examine the factors associated with symptomatic clusters . the study uses data from third round of the india district level household survey ( dlhs ) , which provides district level information about reproductive and health care of women aged 15 - 49 . the survey used multistage stratified systematic sampling design and 50 census villages in rural areas and wards from urban areas were selected from each district based on probability proportional size ( pps ) sampling . in this paper , ever married women aged 15 - 19 consisting of 39 , 164 are considered . although this age group does not cover the complete gamut of adolescents , this age - group is more suitable for examining the prevalence of rti and knowledge as most of the adolescents get married and starts active sexual life at this age . factor analysis was used to create an index using all the 11 symptoms of rti / sti reported in the survey . factor analysis is generally used for the determination of a small number of factors based on a particular number of inter - related quantitative variables . cross tabulation were used to explore the prevalence of rtis symptoms and treatment seeking behavior among women by different socio - demographic , medical and behavioral characteristics . in this study , we tried to find statistical association between different type of symptoms of rti / sti among adolescents married women in india and reduced the numbers of factors using principal component analysis method of factor analysis . simple linear regression was used to know the relation between different types of infections and socio - demographic medical and behavioral characteristics . the explanatory variables are : religion , caste , educational levels , type of house , age of women , age at marriage , abortion , children ever born , contraceptive users , aware of rti / sti . in order to estimate the net effect of each variable on the probability of seeking treatment or consultation , binary logistic regression model was used . this study used district level household survey dataset which is conducted by international institute for population sciences ( iips ) and funded by ministry of health and family welfare ( mohfw ) , government of india ( goi ) . this study used district level household survey dataset which is conducted by international institute for population sciences ( iips ) and funded by ministry of health and family welfare ( mohfw ) , government of india ( goi ) . this study used district level household survey dataset which is conducted by international institute for population sciences ( iips ) and funded by ministry of health and family welfare ( mohfw ) , government of india ( goi ) . in the present study , we found that awareness among married adolescent women is very low with only one - fourth ( 25% ) of them are aware of any symptoms of rti / sti . the awareness among older women is better than the adolescent women ( table 1 ) . unsafe sex with persons having many partners ( 60% ) is the most perceived mode of transmission among women in india . knowledge of other modes of transmission like unsafe delivery , unsafe abortion , unsafe iud insertion and unsafe sex with homosexuals is low among adolescent women than their adult counterparts . the awareness of any rtis/stis symptoms is low among married adolescent women in rural areas ( 24% ) than in the urban areas . however , only 31 percent of the urban adolescent women in india are aware of any rtis/stis symptoms . less percentage of younger , illiterate and women from poorest wealth quintiles are aware of rti / sti as compared to older , higher educated and women from richest wealth quintile respectively ( table not shown ) . knowledge of different mode of transmission of rti / sti of ever married women by their age groups , india , 2007 - 08 percentage of adolescent married women reporting different symptoms of rtis / stis and the duration of the illness , india , 2007 - 08 excluding abnormal discharge . linear regression showing beta values infection in and around the vulva and other infection in upper tract problems among women 15 - 19 years the of sexual intercourse related problem is not given as none of the covariates are significant in the model . among the adolescent women , around 15 percent reported having any symptoms of rtis / stis and 11 percent reported having abnormal vaginal discharge in the last three months of the survey . low backache ( 8 % ) , pain during sexual intercourse ( 5 % ) , pain in the lower abdomen not related to menstruation ( 5% ) , itching or irritation over the vulva ( 4% ) , pain on urination or defecation ( 2.5 % ) and boils / ulcers / warts around the vulva ( 1.6% ) are the major problems reported by married adolescents . the result shows that more than half of the adolescent women have at least one reproductive infections for more than three months . the proportion of other morbidities varied from 46 to 60 percent . low backache , and swelling in the groin is most neglected by more than 60 percent of women reporting the symptoms exist for more than 3 months . some of the infections may exist due to other type of infections or may simultaneously exist together . in order to understand the association between different symptoms , we used factor analysis using all eleven symptoms of rtis / stis reported by adolescent women . using principal component analysis method the factors are a ) infections in and around the vulva i.e. boils / ulcers / warts around the vulva , painful blister like lesions in and around the vagina , itching or irritation over the vulva and swelling in the groin . all the symptoms in the first factor are in around the vulva and mostly lower track infections . b ) other reproductive infection related to the upper tract of the vagina like pain in the lower abdomen not related to menses , pain during urination and defecation and low backache . abnormal vaginal discharge which is a lower tract infection is also found to be associated with the second factor c ) sexual intercourse related problem of pain and spotting among women . we used linear regression to understand different socioeconomic and demographic factors affecting these three variables which are in continuous form . dummy variables were created from categorical socioeconomic variables and age was used as a continuous variable . the result of linear regression shows that following variables have a significant and positive association with lower tract infections , use of modern contraception ( =0.36 ) , abortion ( =0.4 ) , poorest ( =0.6 ) and middle ( =0.19 ) quintiles of wealth index ; and north - east ( =0 . 51 ) , west ( =0.25 ) , and central ( =0.13 ) regions. other infections related to the upper tract shows positive and significant association with age ( =0 . 04 ) , education , region , the use of modern contraception , abortion , wealth index and regions . the value for use of modern contraception and abortion is 0.18 and 0.67 respectively . while caste status shows a negative association with other infections related to upper tract . although premarital sexual relationship is not as prevalent in india as in other countries , early induction into marriage compels women into early and long sexual lives with their husbands at a young age when they are not accustomed to the use of family planning practices and other sexual health education . in this study , we found that treatment seeking among adolescent women is poor . only 62 percent of the adolescent women discuss the infections with their husbands / partner whereas only one out of four of them go for treatment . comparing different age groups , the treatment seeking behavior is much higher among older women ( 25 + years ) as compared to the adolescents . this may be due to the fact that older women enjoy better status in households than younger and newly married women . more than three - fifths of the women preferred private hospitals / clinics as compared to only a little more than one - fourth going to government hospitals for treatment . this may be attributed to privacy , better quality of care , as well as lack of special treatment division for rtis in government hospitals . more than half the poorest and poorer section of the control prefers to private hospitals or clinics . this shows that going to private hospitals might well be a compulsion rather than choice . in order to understand the adjusted effect of different socioeconomic and demographic determinants , a binary logistic regression was performed taking care sought the result shows that adolescent age , education , religion , caste , wealth index categories , and awareness about rtis / stis are significant determinants of her care seeking behavior ( table 4 ) . age , religion , residence , education and wealth are positively associated with treatment seeking of rtis / stis of women . the older adolescent women ( age 19 years ) are two times more likely to seek treatment than the younger adolescent ( age 15 years ) . muslims ( or=1.45 , p<0.05 ) and other ( or=1.29 , p<0.1 ) religious adolescent women are more likely to seek treatment than the hindu adolescents . husband s education also shows significant association and women of higher educated husbands ( or=1.18 , p<0.1 ) have higher odds of seeking care than women of illiterate husbands . the adolescent women with secondary education ( or=1.28 , p<0.05 ) and women in richest quintile of the households ( or=1.39 , p<0.05 ) have a higher chance of seeking treatment than women with no education and women belonging to the poorest households . in comparison to adolescent from the north , the southern adolescent women have a higher odd ( or=1.25 , p<0.05 ) and north - eastern adolescent have a lower odd ( or=0.54 , p<0.1 ) of seeking treatment . those who are not aware ( or=0.72 , p<0.05 ) of any rtis / stis are less likely to seek treatment than those married adolescent aware about the infections . logistic regression showing odds ratio the treatment seeking behavior among married adolescents in india note : education of women and their husbands , children ever born , and caste are used as control variable in the regression model . in the present study , we found that awareness among married adolescent women is very low with only one - fourth ( 25% ) of them are aware of any symptoms of rti / sti . the awareness among older women is better than the adolescent women ( table 1 ) . unsafe sex with persons having many partners ( 60% ) is the most perceived mode of transmission among women in india . knowledge of other modes of transmission like unsafe delivery , unsafe abortion , unsafe iud insertion and unsafe sex with homosexuals is low among adolescent women than their adult counterparts . the awareness of any rtis/stis symptoms is low among married adolescent women in rural areas ( 24% ) than in the urban areas . however , only 31 percent of the urban adolescent women in india are aware of any rtis/stis symptoms . less percentage of younger , illiterate and women from poorest wealth quintiles are aware of rti / sti as compared to older , higher educated and women from richest wealth quintile respectively ( table not shown ) . knowledge of different mode of transmission of rti / sti of ever married women by their age groups , india , 2007 - 08 percentage of adolescent married women reporting different symptoms of rtis / stis and the duration of the illness , india , 2007 - 08 excluding abnormal discharge . linear regression showing beta values infection in and around the vulva and other infection in upper tract problems among women 15 - 19 years the of sexual intercourse related problem is not given as none of the covariates are significant in the model . among the adolescent women , around 15 percent reported having any symptoms of rtis / stis and 11 percent reported having abnormal vaginal discharge in the last three months of the survey . low backache ( 8 % ) , pain during sexual intercourse ( 5 % ) , pain in the lower abdomen not related to menstruation ( 5% ) , itching or irritation over the vulva ( 4% ) , pain on urination or defecation ( 2.5 % ) and boils / ulcers / warts around the vulva ( 1.6% ) are the major problems reported by married adolescents . the result shows that more than half of the adolescent women have at least one reproductive infections for more than three months . the proportion of other morbidities varied from 46 to 60 percent . low backache , and swelling in the groin is most neglected by more than 60 percent of women reporting the symptoms exist for more than 3 months . some of the infections may exist due to other type of infections or may simultaneously exist together . in order to understand the association between different symptoms , we used factor analysis using all eleven symptoms of rtis / stis reported by adolescent women . using principal component analysis method the factors are a ) infections in and around the vulva i.e. boils / ulcers / warts around the vulva , painful blister like lesions in and around the vagina , itching or irritation over the vulva and swelling in the groin . all the symptoms in the first factor are in around the vulva and mostly lower track infections . b ) other reproductive infection related to the upper tract of the vagina like pain in the lower abdomen not related to menses , pain during urination and defecation and low backache . abnormal vaginal discharge which is a lower tract infection is also found to be associated with the second factor c ) sexual intercourse related problem of pain and spotting among women . we used linear regression to understand different socioeconomic and demographic factors affecting these three variables which are in continuous form . dummy variables were created from categorical socioeconomic variables and age was used as a continuous variable . the result of linear regression shows that following variables have a significant and positive association with lower tract infections , use of modern contraception ( =0.36 ) , abortion ( =0.4 ) , poorest ( =0.6 ) and middle ( =0.19 ) quintiles of wealth index ; and north - east ( =0 . 51 ) , west ( =0.25 ) , and central ( =0.13 ) regions. other infections related to the upper tract shows positive and significant association with age ( =0 . 04 ) , education , region , the use of modern contraception , abortion , wealth index and regions . the value for use of modern contraception and abortion is 0.18 and 0.67 respectively . while caste status shows a negative association with other infections related to upper tract . although premarital sexual relationship is not as prevalent in india as in other countries , early induction into marriage compels women into early and long sexual lives with their husbands at a young age when they are not accustomed to the use of family planning practices and other sexual health education . in this study only 62 percent of the adolescent women discuss the infections with their husbands / partner whereas only one out of four of them go for treatment . comparing different age groups , the treatment seeking behavior is much higher among older women ( 25 + years ) as compared to the adolescents . this may be due to the fact that older women enjoy better status in households than younger and newly married women . more than three - fifths of the women preferred private hospitals / clinics as compared to only a little more than one - fourth going to government hospitals for treatment . this may be attributed to privacy , better quality of care , as well as lack of special treatment division for rtis in government hospitals . more than half the poorest and poorer section of the control prefers to private hospitals or clinics . this shows that going to private hospitals might well be a compulsion rather than choice . in order to understand the adjusted effect of different socioeconomic and demographic determinants , a binary logistic regression was performed taking care sought the result shows that adolescent age , education , religion , caste , wealth index categories , and awareness about rtis / stis are significant determinants of her care seeking behavior ( table 4 ) . age , religion , residence , education and wealth are positively associated with treatment seeking of rtis / stis of women . the older adolescent women ( age 19 years ) are two times more likely to seek treatment than the younger adolescent ( age 15 years ) . muslims ( or=1.45 , p<0.05 ) and other ( or=1.29 , p<0.1 ) religious adolescent women are more likely to seek treatment than the hindu adolescents . husband s education also shows significant association and women of higher educated husbands ( or=1.18 , p<0.1 ) have higher odds of seeking care than women of illiterate husbands . the adolescent women with secondary education ( or=1.28 , p<0.05 ) and women in richest quintile of the households ( or=1.39 , p<0.05 ) have a higher chance of seeking treatment than women with no education and women belonging to the poorest households . in comparison to adolescent from the north , the southern adolescent women have a higher odd ( or=1.25 , p<0.05 ) and north - eastern adolescent have a lower odd ( or=0.54 , p<0.1 ) of seeking treatment . those who are not aware ( or=0.72 , p<0.05 ) of any rtis / stis are less likely to seek treatment than those married adolescent aware about the infections . logistic regression showing odds ratio the treatment seeking behavior among married adolescents in india note : education of women and their husbands , children ever born , and caste are used as control variable in the regression model . in this study , we highlight the state of knowledge , prevalence and treatment seeking behavior among married adolescents in india . awareness of rtis / stis among married is very low in india and level of knowledge of rti is even lower than in other developing countries including many african countries[16 - 17 ] . only one - fourth of the adolescent married women of are aware about any mode of transmission considered in the study and this is little higher among the older women . due to low education and low age at marriage adolescent women are mostly not accustomed to rtis . many studies in south - east asia had also reported low understanding of rti / stis among women . for instance , a study in rural bangladesh reported that only 12% of the study population had the basic understanding of rtis . the study explained this great disparity in the proportion of the various populations who were aware of rtis could be explained from the rural nature of india and bangladesh where most of the respondents were illiterates , when compared to the urban settings of nigeria and kenya where the literacy level is higher . however , we found that only 31 percent of the urban adolescent women in india are aware of any rtis/stis symptoms . earlier studies on south asia found the prevalence of reproductive varies in between 22 - 92%[3,6,19 - 21 ] . however , most of these studies included all three types of reproductive morbidities including gynecological morbidities . the factor analysis shows the concentration of diseases in three clusters - infection in around the vulva , other reproductive infection and abnormal discharge ; and intercourse - related problems . multivariate results show that the use of modern contraception and abortion were significantly associated with both infection in around the vulva and other reproductive infection and abnormal discharge . the treatment seeking for any rti / sti infections is found to be low in our study . three - fifths of women discuss rtis with their husbands / partner but only a little more than one - fourth of them prefers going to seek treatment . this finding is consistent with some of the earlier studies in india which accentuates that the country s health programs which has been special importance to adolescents and youths has failed to reach them[13 , 22 - 23 ] . our multivariate results shows younger adolescents , rural and poor women are less likely to seek treatment than older urban and rich adolescent women respectively . with respect to geography , women from northern region and north - eastern women have less probability to seek treatment than their southern counterparts . those who are not aware of rti / sti symptoms are less likely to seek treatment . thus , here is need to focus on rural and poor adolescents with a special focus on strengthening their knowledge about rti / sti infections . the result of this study shows that awareness of life threatening reproductive infections still remains low despite several government efforts in reproductive and child health ( rch ) programs and the current national rural health mission ( nrhm ) . the awareness remains dismal among adolescent women who have very low knowledge of these infections . not many of the women are aware that rtis / stis can be transmitted through unsafe delivery , unsafe abortion or iud insertion in the presence of infections . the study found although the prevalence is not very high many of the women ignore these infections for long duration . furthermore , the number of unqualified and illegal private medical practitioners practicing allopathic medicine and doing abortions has remained the priority among people in the rural areas and small towns of india . people willingly pay for their services rather than availing themselves of free services at the government health facilities .

background : india is home to the highest number of adolescents in the world . adolescents in india suffer from lack of knowledge and empowerment to make informed sexual and reproductive health decisions . this paper analyses the prevalence of reproductive tract infections and sexually transmitted infections ( rti / sti ) and treatment seeking behavior among married adolescent women in india aged 15 - 19 years.methods:data from the district level household survey ( dlhs , 2007 - 08 ) of india were used . the prevalence of rtis symptoms and treatment seeking behavior among women by different socio - demographic characteristics was analyzed . factor analysis was utilized to create an index using information about 11 symptoms of rti / sti collected in the survey . linear and binary logistic regressions were used to know the association between infections and treatment seeking behavior with socio - demographic factors.results:about 15 percent of adolescent women reported having any symptoms of rti / sti . the main symptoms reported were low backache , pain in the lower abdomen , pain during intercourse and itching or irritation around the vulvar region . factor analysis showed the concentration of diseases in three clusters - infection in around the vulva , other reproductive infection and abnormal discharge ; and intercourse related problems . major predictors of both symptoms of reproductive infections and treatment seeking behavior from multivariate analysis are age , education , wealth , region and awareness about rti / sti.conclusions and public health implications : knowledge and treatment seeking behavior is poor among adolescent women in india . there is need for programmatic and policy emphasis on increasing knowledge and awareness through family life education including in educational curriculum at school level .

Introduction Methods None Ethical Statement Results Awareness of Reproductive Tract Infections among Adolescent Women Prevalence and Duration of Symptoms Factor Analysis Determinants of RTIs/STIs Treatment seeking behavior of RTIs/STIs among married adolescent women in India Factors Affecting Treatment Seeking Behaviour of Adolescents Discussion Conclusion

owing largely to early marriage , adolescent women in india become sexually active at an early age and face tremendous social and familial pressure for child bearing soon after marriage , a situation that leads to multiple reproductive health problems . the improper use of contraception , intrauterine devices ( iud ) insertion in the presence of infections , female sterilization in unsterile condition , and unsafe abortion also increases the risks of rtis[2 - 4 ] . silent epidemic and are among the leading public health problems significantly contributing to gynecological morbidity and maternal mortality in india and other developing countries . moreover , in india , women with self- reported symptoms of reproductive morbidity do not seek treatment due to existing taboos and inhibitions regarding sexual and reproductive health . the prevalence of rti infections is low in india ; however , the number of adolescent affected by infections is a real concern due to the large adolescent population . india has the largest proportion of adolescents ( more than 243 million ) in the world , accounting for almost 20 per cent of the country s population . until now , very few studies have focused on reproductive tract infections ( rti ) or sexually ransmitted diseases ( stis ) among adolescent women in developing countries such as india . similarly , very few attempts have been made to study the health seeking behavior for reproductive morbidity of adolescent women in india[1 , 13 - 14 ] . one of the studies explained the familial influence in treatment seeking behavior of married adolescents in maharashtra , a western state of india . therefore , the present study focuses on knowledge and prevalence of reproductive tract infection among married adolescent women using a national level large scale data with a special attention on different categories of infection and treatment seeking behavior . more importantly , this study attempts to group the symptoms by factor analysis and to examine the factors associated with symptomatic clusters . the study uses data from third round of the india district level household survey ( dlhs ) , which provides district level information about reproductive and health care of women aged 15 - 49 . the survey used multistage stratified systematic sampling design and 50 census villages in rural areas and wards from urban areas were selected from each district based on probability proportional size ( pps ) sampling . in this paper , ever married women aged 15 - 19 consisting of 39 , 164 are considered . although this age group does not cover the complete gamut of adolescents , this age - group is more suitable for examining the prevalence of rti and knowledge as most of the adolescents get married and starts active sexual life at this age . factor analysis was used to create an index using all the 11 symptoms of rti / sti reported in the survey . factor analysis is generally used for the determination of a small number of factors based on a particular number of inter - related quantitative variables . cross tabulation were used to explore the prevalence of rtis symptoms and treatment seeking behavior among women by different socio - demographic , medical and behavioral characteristics . in this study , we tried to find statistical association between different type of symptoms of rti / sti among adolescents married women in india and reduced the numbers of factors using principal component analysis method of factor analysis . simple linear regression was used to know the relation between different types of infections and socio - demographic medical and behavioral characteristics . the explanatory variables are : religion , caste , educational levels , type of house , age of women , age at marriage , abortion , children ever born , contraceptive users , aware of rti / sti . this study used district level household survey dataset which is conducted by international institute for population sciences ( iips ) and funded by ministry of health and family welfare ( mohfw ) , government of india ( goi ) . this study used district level household survey dataset which is conducted by international institute for population sciences ( iips ) and funded by ministry of health and family welfare ( mohfw ) , government of india ( goi ) . this study used district level household survey dataset which is conducted by international institute for population sciences ( iips ) and funded by ministry of health and family welfare ( mohfw ) , government of india ( goi ) . in the present study , we found that awareness among married adolescent women is very low with only one - fourth ( 25% ) of them are aware of any symptoms of rti / sti . the awareness among older women is better than the adolescent women ( table 1 ) . unsafe sex with persons having many partners ( 60% ) is the most perceived mode of transmission among women in india . knowledge of other modes of transmission like unsafe delivery , unsafe abortion , unsafe iud insertion and unsafe sex with homosexuals is low among adolescent women than their adult counterparts . the awareness of any rtis/stis symptoms is low among married adolescent women in rural areas ( 24% ) than in the urban areas . however , only 31 percent of the urban adolescent women in india are aware of any rtis/stis symptoms . less percentage of younger , illiterate and women from poorest wealth quintiles are aware of rti / sti as compared to older , higher educated and women from richest wealth quintile respectively ( table not shown ) . knowledge of different mode of transmission of rti / sti of ever married women by their age groups , india , 2007 - 08 percentage of adolescent married women reporting different symptoms of rtis / stis and the duration of the illness , india , 2007 - 08 excluding abnormal discharge . linear regression showing beta values infection in and around the vulva and other infection in upper tract problems among women 15 - 19 years the of sexual intercourse related problem is not given as none of the covariates are significant in the model . among the adolescent women , around 15 percent reported having any symptoms of rtis / stis and 11 percent reported having abnormal vaginal discharge in the last three months of the survey . low backache ( 8 % ) , pain during sexual intercourse ( 5 % ) , pain in the lower abdomen not related to menstruation ( 5% ) , itching or irritation over the vulva ( 4% ) , pain on urination or defecation ( 2.5 % ) and boils / ulcers / warts around the vulva ( 1.6% ) are the major problems reported by married adolescents . the result shows that more than half of the adolescent women have at least one reproductive infections for more than three months . low backache , and swelling in the groin is most neglected by more than 60 percent of women reporting the symptoms exist for more than 3 months . in order to understand the association between different symptoms , we used factor analysis using all eleven symptoms of rtis / stis reported by adolescent women . using principal component analysis method the factors are a ) infections in and around the vulva i.e. boils / ulcers / warts around the vulva , painful blister like lesions in and around the vagina , itching or irritation over the vulva and swelling in the groin . all the symptoms in the first factor are in around the vulva and mostly lower track infections . b ) other reproductive infection related to the upper tract of the vagina like pain in the lower abdomen not related to menses , pain during urination and defecation and low backache . abnormal vaginal discharge which is a lower tract infection is also found to be associated with the second factor c ) sexual intercourse related problem of pain and spotting among women . we used linear regression to understand different socioeconomic and demographic factors affecting these three variables which are in continuous form . the result of linear regression shows that following variables have a significant and positive association with lower tract infections , use of modern contraception ( =0.36 ) , abortion ( =0.4 ) , poorest ( =0.6 ) and middle ( =0.19 ) quintiles of wealth index ; and north - east ( =0 . other infections related to the upper tract shows positive and significant association with age ( =0 . 04 ) , education , region , the use of modern contraception , abortion , wealth index and regions . although premarital sexual relationship is not as prevalent in india as in other countries , early induction into marriage compels women into early and long sexual lives with their husbands at a young age when they are not accustomed to the use of family planning practices and other sexual health education . in this study , we found that treatment seeking among adolescent women is poor . only 62 percent of the adolescent women discuss the infections with their husbands / partner whereas only one out of four of them go for treatment . comparing different age groups , the treatment seeking behavior is much higher among older women ( 25 + years ) as compared to the adolescents . this may be due to the fact that older women enjoy better status in households than younger and newly married women . this may be attributed to privacy , better quality of care , as well as lack of special treatment division for rtis in government hospitals . in order to understand the adjusted effect of different socioeconomic and demographic determinants , a binary logistic regression was performed taking care sought the result shows that adolescent age , education , religion , caste , wealth index categories , and awareness about rtis / stis are significant determinants of her care seeking behavior ( table 4 ) . age , religion , residence , education and wealth are positively associated with treatment seeking of rtis / stis of women . the older adolescent women ( age 19 years ) are two times more likely to seek treatment than the younger adolescent ( age 15 years ) . muslims ( or=1.45 , p<0.05 ) and other ( or=1.29 , p<0.1 ) religious adolescent women are more likely to seek treatment than the hindu adolescents . the adolescent women with secondary education ( or=1.28 , p<0.05 ) and women in richest quintile of the households ( or=1.39 , p<0.05 ) have a higher chance of seeking treatment than women with no education and women belonging to the poorest households . in comparison to adolescent from the north , the southern adolescent women have a higher odd ( or=1.25 , p<0.05 ) and north - eastern adolescent have a lower odd ( or=0.54 , p<0.1 ) of seeking treatment . those who are not aware ( or=0.72 , p<0.05 ) of any rtis / stis are less likely to seek treatment than those married adolescent aware about the infections . logistic regression showing odds ratio the treatment seeking behavior among married adolescents in india note : education of women and their husbands , children ever born , and caste are used as control variable in the regression model . in the present study , we found that awareness among married adolescent women is very low with only one - fourth ( 25% ) of them are aware of any symptoms of rti / sti . the awareness among older women is better than the adolescent women ( table 1 ) . unsafe sex with persons having many partners ( 60% ) is the most perceived mode of transmission among women in india . knowledge of other modes of transmission like unsafe delivery , unsafe abortion , unsafe iud insertion and unsafe sex with homosexuals is low among adolescent women than their adult counterparts . the awareness of any rtis/stis symptoms is low among married adolescent women in rural areas ( 24% ) than in the urban areas . however , only 31 percent of the urban adolescent women in india are aware of any rtis/stis symptoms . less percentage of younger , illiterate and women from poorest wealth quintiles are aware of rti / sti as compared to older , higher educated and women from richest wealth quintile respectively ( table not shown ) . knowledge of different mode of transmission of rti / sti of ever married women by their age groups , india , 2007 - 08 percentage of adolescent married women reporting different symptoms of rtis / stis and the duration of the illness , india , 2007 - 08 excluding abnormal discharge . linear regression showing beta values infection in and around the vulva and other infection in upper tract problems among women 15 - 19 years the of sexual intercourse related problem is not given as none of the covariates are significant in the model . among the adolescent women , around 15 percent reported having any symptoms of rtis / stis and 11 percent reported having abnormal vaginal discharge in the last three months of the survey . low backache ( 8 % ) , pain during sexual intercourse ( 5 % ) , pain in the lower abdomen not related to menstruation ( 5% ) , itching or irritation over the vulva ( 4% ) , pain on urination or defecation ( 2.5 % ) and boils / ulcers / warts around the vulva ( 1.6% ) are the major problems reported by married adolescents . the result shows that more than half of the adolescent women have at least one reproductive infections for more than three months . low backache , and swelling in the groin is most neglected by more than 60 percent of women reporting the symptoms exist for more than 3 months . in order to understand the association between different symptoms , we used factor analysis using all eleven symptoms of rtis / stis reported by adolescent women . using principal component analysis method the factors are a ) infections in and around the vulva i.e. boils / ulcers / warts around the vulva , painful blister like lesions in and around the vagina , itching or irritation over the vulva and swelling in the groin . all the symptoms in the first factor are in around the vulva and mostly lower track infections . b ) other reproductive infection related to the upper tract of the vagina like pain in the lower abdomen not related to menses , pain during urination and defecation and low backache . abnormal vaginal discharge which is a lower tract infection is also found to be associated with the second factor c ) sexual intercourse related problem of pain and spotting among women . the result of linear regression shows that following variables have a significant and positive association with lower tract infections , use of modern contraception ( =0.36 ) , abortion ( =0.4 ) , poorest ( =0.6 ) and middle ( =0.19 ) quintiles of wealth index ; and north - east ( =0 . other infections related to the upper tract shows positive and significant association with age ( =0 . 04 ) , education , region , the use of modern contraception , abortion , wealth index and regions . although premarital sexual relationship is not as prevalent in india as in other countries , early induction into marriage compels women into early and long sexual lives with their husbands at a young age when they are not accustomed to the use of family planning practices and other sexual health education . in this study only 62 percent of the adolescent women discuss the infections with their husbands / partner whereas only one out of four of them go for treatment . comparing different age groups , the treatment seeking behavior is much higher among older women ( 25 + years ) as compared to the adolescents . this may be due to the fact that older women enjoy better status in households than younger and newly married women . this may be attributed to privacy , better quality of care , as well as lack of special treatment division for rtis in government hospitals . in order to understand the adjusted effect of different socioeconomic and demographic determinants , a binary logistic regression was performed taking care sought the result shows that adolescent age , education , religion , caste , wealth index categories , and awareness about rtis / stis are significant determinants of her care seeking behavior ( table 4 ) . age , religion , residence , education and wealth are positively associated with treatment seeking of rtis / stis of women . the older adolescent women ( age 19 years ) are two times more likely to seek treatment than the younger adolescent ( age 15 years ) . muslims ( or=1.45 , p<0.05 ) and other ( or=1.29 , p<0.1 ) religious adolescent women are more likely to seek treatment than the hindu adolescents . the adolescent women with secondary education ( or=1.28 , p<0.05 ) and women in richest quintile of the households ( or=1.39 , p<0.05 ) have a higher chance of seeking treatment than women with no education and women belonging to the poorest households . in comparison to adolescent from the north , the southern adolescent women have a higher odd ( or=1.25 , p<0.05 ) and north - eastern adolescent have a lower odd ( or=0.54 , p<0.1 ) of seeking treatment . those who are not aware ( or=0.72 , p<0.05 ) of any rtis / stis are less likely to seek treatment than those married adolescent aware about the infections . logistic regression showing odds ratio the treatment seeking behavior among married adolescents in india note : education of women and their husbands , children ever born , and caste are used as control variable in the regression model . in this study , we highlight the state of knowledge , prevalence and treatment seeking behavior among married adolescents in india . awareness of rtis / stis among married is very low in india and level of knowledge of rti is even lower than in other developing countries including many african countries[16 - 17 ] . only one - fourth of the adolescent married women of are aware about any mode of transmission considered in the study and this is little higher among the older women . due to low education and low age at marriage adolescent women are mostly not accustomed to rtis . many studies in south - east asia had also reported low understanding of rti / stis among women . for instance , a study in rural bangladesh reported that only 12% of the study population had the basic understanding of rtis . the study explained this great disparity in the proportion of the various populations who were aware of rtis could be explained from the rural nature of india and bangladesh where most of the respondents were illiterates , when compared to the urban settings of nigeria and kenya where the literacy level is higher . however , we found that only 31 percent of the urban adolescent women in india are aware of any rtis/stis symptoms . earlier studies on south asia found the prevalence of reproductive varies in between 22 - 92%[3,6,19 - 21 ] . the factor analysis shows the concentration of diseases in three clusters - infection in around the vulva , other reproductive infection and abnormal discharge ; and intercourse - related problems . multivariate results show that the use of modern contraception and abortion were significantly associated with both infection in around the vulva and other reproductive infection and abnormal discharge . the treatment seeking for any rti / sti infections is found to be low in our study . this finding is consistent with some of the earlier studies in india which accentuates that the country s health programs which has been special importance to adolescents and youths has failed to reach them[13 , 22 - 23 ] . our multivariate results shows younger adolescents , rural and poor women are less likely to seek treatment than older urban and rich adolescent women respectively . with respect to geography , women from northern region and north - eastern women have less probability to seek treatment than their southern counterparts . those who are not aware of rti / sti symptoms are less likely to seek treatment . thus , here is need to focus on rural and poor adolescents with a special focus on strengthening their knowledge about rti / sti infections . the result of this study shows that awareness of life threatening reproductive infections still remains low despite several government efforts in reproductive and child health ( rch ) programs and the current national rural health mission ( nrhm ) . the awareness remains dismal among adolescent women who have very low knowledge of these infections . not many of the women are aware that rtis / stis can be transmitted through unsafe delivery , unsafe abortion or iud insertion in the presence of infections . the study found although the prevalence is not very high many of the women ignore these infections for long duration . furthermore , the number of unqualified and illegal private medical practitioners practicing allopathic medicine and doing abortions has remained the priority among people in the rural areas and small towns of india .

the intestinal epithelium permits the traffic of nutrients from the lumen to the blood , while at the same time restricting the passage of potentially harmful microorganisms and toxins.9 , 10 two main characteristics are key in establishing and maintaining the epithelial barrier . first , a capacity for controlled cell renewal , which is achieved through a tightly regulated balance between epithelial cell proliferation and apoptosis , and , second , the presence of effective intercellular junctions . intestinal epithelial cells are replaced every 45 days , an impressive feat considering that the adult intestinal epithelium has a surface area of approximately 300 m. the control of epithelial cell renewal is based on a continuous balance between proliferation and apoptosis - dependent cell death.11 , 13 , 14 epithelial cells are generated from stem cell progenitors in the intestinal crypts and migrate from the crypt to the lumen - facing mucosal surface . the process of intestinal cell proliferation and migration is regulated through complex signaling events in which the wnt pathway is a key player.11 , 15 an equally balanced rate of epithelial cell death also is essential to maintain controlled renewal of the epithelial barrier . alterations in the balance between proliferation and apoptosis are known to be involved in barrier dysfunction , which leads to disease . for example , in the context of ibd , the primed inflammatory environment can lead to the generation of pro - apoptotic signals and , consequently , to the activation of apoptotic pathways in epithelial cells.16 , 17 , 18 ultimately , this compromises the capacity of the epithelium to maintain barrier integrity . this is in contrast to intestinal epithelial cancers in which epithelial proliferation exceeds the rate of apoptosis and promotes the development of intestinal tumors . effective sealing of the intercellular space is a second factor necessary to fulfill the provision of a continuous intestinal epithelial barrier . this sealing property is defined primarily by the tight junctions , adherens junctions , and desmosomes . adherens junctions and desmosomes are responsible for the maintenance of the proximity between cells through intercellular molecular connections , whereas tight junctions are responsible for sealing the paracellular space . tight junctions consist of complexes formed through the interaction of proteins including occludin , zonula occludens ( zo ) , and claudins ( of which multiple isoforms exist).21 , 22 although occludin and claudins are transmembrane proteins , zo acts as an anchoring component that binds transmembrane tight junction proteins.23 , 24 in addition to these constituent proteins , regulatory enzymes can play an important role in controlling the permeability of the tight junction . an example of this is myosin light - chain kinase , which regulates tight junction permeability.26 , 27 , 28 thus , in addition to transcellular transport , the paracellular space defines an alternative transport route . therefore , the dynamic regulation of the tight junction is critical for the maintenance of effective barrier function while allowing the absorption of nutrients and other macromolecules . importantly , inflammatory mediators such as tumor necrosis factor- ( tnf- ) or interleukin ( il)13 are capable of causing alterations in the structure of the tight junction ( tj ) complexes and changes in permeability.29 , 30 as discussed later , such alterations represent another mechanism of induction of barrier dysfunction - related intestinal disease . the intestinal epithelium is in direct contact with a large number of microorganisms that are part of the normal intestinal microbiota.31 , 32 at the same time , the epithelium separates the microbiota from the intestinal immune system that is resident in the lamina propria ( figure 1 ) . therefore , the intestinal epithelium is a highly regulated and interactive surface that communicates signals between the mucosal immune system and the gut microbiota.31 , 33 , 34 the number of bacterial cells that reside in the intestinal tract exceeds the number of human cells in the body.7 , 35 this rich intestinal microenvironment has important interactions with the epithelial cells that form the intestinal barrier . intestinal epithelial cells express pattern recognition receptors such as toll - like receptors ( tlrs ) . furthermore , there is evidence that supports a role for the microbiome in both digestive and immune functions . for example , the gut microbiome is implicated in the absorption of fats.38 , 39 moreover , components of the microbiome also are required for the degradation of complex polysaccharides that can not be absorbed into simpler carbohydrates that can be absorbed.40 , 41 furthermore , the gut microbiome is known to regulate mucins derived from intestinal goblet cells . secreted mucins form a mucous layer that covers the epithelial surface ( figure 1 ) . this mucous layer serves as a defense system against physical and chemical injury and aids the clearance of pathogenic microorganisms.43 , 44 components of the microbiome are involved in both mucus production as well as degradation.45 , 46 , 47 , 48 , 49 this implicates a mechanism of cross - talk between the intestinal epithelium and microbiota that contributes to both digestion and barrier function . beyond barrier formation , the intestinal epithelium actively regulates the immune response . in fact , as explained earlier , intestinal epithelial cells express multiple tlrs , including tlr-4 and tlr-3 , and are responsive to microbial stimuli.50 , 51 , 52 these receptors are common immune receptors responsible for the recognition of bacterial components such as lipopolysaccharide . in response to such stimuli , human intestinal epithelial cells are capable of producing a variety of inflammatory cytokines including il8 , c - x - c motif chemokine ligand 2 , or c - c motif chemokine ligand 20 . these mediators actively recruit different components of the immune system and participate in the activation of inflammatory responses . a final mechanism whereby the intestinal epithelium plays a role in the maintenance of homeostasis is through the production of mediators such as trefoil factors ( tffs ) and defensins.55 , 56 , 57 , 58 for example , in a study comparing the expression of tff isoforms between healthy and intestinal biopsy specimens from patients with ibd , the messenger rna levels of the tffs analyzed were found to be correlated to the pathologic state . in specimens from patients with crohn s disease ( cd ) in remission , tffs were up - regulated , showing an inverse correlation between tff expression and inflammation . moreover , local delivery of recombinant tffs ameliorates dextran sodium sulfate ( dss)-induced colitis , providing further evidence for the beneficial role of these peptides in intestinal physiology . in line with these results , podolsky et al showed that the deleterious effects of a mutation of tlr-2 were caused by altered production of tff3 . defensins are antimicrobial peptides , which in the gut are produced mainly by paneth cells . these peptides participate in the response against infectious microorganisms and also are involved in the control of the intestinal microbiota.57 , 58 salzman et al showed that transgenic expression of human defensin-5 in mice makes them resistant to salmonella typhimurium infection . in addition , the expression of these defensive peptides is altered in ibd.61 , 62 in summary , the intestinal epithelium has critical interactions with both the luminal microenvironment and the immune system , as well as active roles in the immune response . these functions highlight the importance of an effective epithelial barrier in normal intestinal physiology and health . aerobic organisms have developed systems for the absorption and utilization of molecular oxygen for metabolic purposes . the success of this metabolic approach has rendered most aerobic metazoans fully dependent on a constant supply of molecular oxygen for survival . the human body is working constantly to maintain an adequate oxygen supply from its absorption in the lungs , its distribution via blood vessels , and its transport and consumption at the cellular level . the intestinal epithelium is in a continuous state of physiologic hypoxia as a result of 2 main events.64 , 65 first , it is juxtaposed between the largely anoxic intestinal lumen and the well - perfused lamina propria ( figure 1 ) . second , the oxygen pressure fluctuates in the lamina propria depending on blood volume in the gut . during fasting , blood flow to the intestine is relatively low and , as a result , so is the oxygen tension . however , when food is ingested , there is an increase in intestinal blood flow with the purpose of facilitating the absorption of nutrients . thus , under physiologic conditions , the intestinal epithelium often is subjected to states of transient oxygen deprivation . this ability of the intestinal epithelial cells to tolerate transient periods of hypoxia in physiologic conditions has led to the concept of physiologic hypoxia . given the critical dependence of mammalian cells for oxygen , the development of adaptive mechanisms to hypoxia have been key to our survival . as discussed in the previous section , the intestinal epithelium is exposed constantly to low concentrations of oxygen , and thus represents a paradigm environment in which adaptation to hypoxia is key . at the cellular level , our ability to adapt to hypoxia depends on the activation of the hypoxia - inducible factor ( hif ) signaling pathway.68 , 69 hif is a ubiquitously expressed family of heterodimeric transcription factors formed by the binding of hif- and hif- subunits . the mechanisms underpinning the regulation of hif-1 and hif-2 are well characterized and recently were reviewed extensively.69 , 70 , 71 , 72 , 73 , 74 hif-1 is expressed constitutively and is present in the nucleus , whereas hif- subunits are expressed constitutively in the cytoplasm . under hypoxic conditions , the formation of functional hif transcription factors in the nucleus triggers a reprogramming of gene expression that controls cell fate , activates alternative mechanisms of energy generation , or enhances oxygen absorption among many other functions.74 , 75 , 76 , 77 , 78 thus , hif responses are critical in the control of cell survival , metabolism , and other functions under low oxygenation . a group of 3 prolyl hydroxylases ( phd ) , phd-1 , 2 , and 3 , and an asparaginyl hydroxylase known as factor inhibiting hif ( fih ) , provide an efficient mechanism by which to control hif - dependent responses.79 , 80 hif- subunits are synthesized constitutively at high levels in all cells . under normoxic conditions , hif- subunits are hydroxylated on 2 prolyl residues ( pro402 and pro564 for hif-1 and pro405 and pro531 for hif-2 ) within their oxygen - dependent degradation domain by phd1 - 3.81 , 82 , 83 , 84 prolyl hydroxylation makes hif- a target for the e3 ubiquitin ligase von hippel - lindau protein that ubiquitinates hif- , marking it for proteosomal degradation . transactivation domain ( asn803 in hif-1 ) , blocking its binding to the cofactor creb - binding protein / p300 and preventing hif - mediated transcriptional activation.79 , 85 , 86 , 87 in hypoxia , hydroxylases no longer can hydroxylate hif- subunits ( because of the lack of availability of molecular oxygen as a co - factor ) . in this situation , hif- is stabilized and translocates to the nucleus where it binds hif-1 and forms transcriptionally active complexes . ibd is a chronic intestinal inflammatory disorder that comprises cd and ulcerative colitis ( uc ) . furthermore , ibd progression is associated with the development of a number of common complications . current therapies for ibd are focused primarily on controlling inflammation using drugs such as mesalamine and/or corticosteroids.91 , 92 however , these therapeutics often are insufficient to maintain patients in remission . in these cases , immunomodulator therapies such as azathioprine or methotrexate more recently developed approaches are focused on the use of antibodies targeting cytokines that are known to play critical roles in the development of the pathology . among those , tnf- is one of the most important drivers of inflammation in ibd , and thus therapies such as infliximab that specifically target tnf- are common in maintenance therapy.91 , 93 , 94 unwanted side effects of current ibd treatments are common . for example , the use of azathioprine is related to a higher risk of malignancy.92 , 95 anti - tnf therapy can lead to immune - compromised states and is related to a higher risk of infectious disease as well as a higher risk of malignancy . moreover , complications such as fibrosis still can appear and represent major indication for surgical interventions owing to the lack of pharmacologic options . this emphasizes the need for new therapeutic approaches that can prolong remission and control the development of complications . ibd is the result of a combination of environmental factors leading to recurrent inflammatory insults , which , in genetically predisposed patients , leads to the development of chronic inflammation.91 , 92 , 96 the development of chronic unresolved inflammation has been linked to intestinal epithelial barrier dysfunction including both disruption of the physical barrier as well as loss of tolerance against components of the microbiota.1 , 97 first , the loss of the physical barrier function increases permeability , facilitating contact between luminal antigens and the mucosal immune cells ( figure 2 ) . second , the intestinal epithelial cells actively participate in the recognition of pathogens and recruitment of other immune cells and the initiation of inflammation ( figure 2 ) . at the cellular level , the loss of epithelial barrier function seen in ibd can be caused by either enhanced apoptosis98 , 99 , 100 or alterations in the expression and assembly of the tj proteins ( or a combination of both).101 , 102 this is supported , for example , by evidence linking tnf- to epithelial cell apoptosis and tj regulation.99 , 101 , 102 , 103 studies have shown that anti - tnf therapies reduce intestinal epithelial cell apoptosis in models of colitis and cd patients.98 , 104 , 105 tnf- down - regulates expression and alters localization of zo-1 and promotes occludin endocytosis , actively regulating the architecture of the tj.101 , 102 tnf - mediated effects on intestinal epithelial tj occur via the activation of myosin light - chain kinase signaling.29 , 106 , 107 thus , alterations in the regulation of epithelial barrier function appear to be determinants in the development of chronic inflammation in the intestinal tract . tissue oxygen levels are reduced in different pathologic foci including tumors and inflammatory environments such as ibd.64 , 65 , 66 , 108 , 109 , 110 in the case of ibd , inflamed areas of the colon experience an increase in activity and subsequently oxygen consumption as a result of the increased presence of activated immune cells , including neutrophils , as well as the activation of reparative processes ( figure 2).65 , 111 , 112 furthermore , the epithelial surface , which is in direct contact with the profoundly hypoxic intestinal lumen , is damaged in ibd and becomes leaky , allowing infiltration of luminal contents into the mucosa and lamina propria ( figure 2 ) . the presence of profound hypoxia on the epithelial surface was shown by karhausen et al using 2-(2-nitro-1h - imidazol-1yl)-n-(2,2,3,3,-pentafluoropropyl ) acetamide - based measurement of tissue oxygenation in murine colitis . in this study , the presence of hypoxia was restricted to the epithelial surface in healthy colons but became penetrant in diseased colons.65 , 112 another factor that accounts for the poor tissue oxygenation is the vasculopathy that affects inflamed tissues and reduces blood perfusion and , as a result , compromises oxygen supply.66 , 112 therefore , hypoxia is a common feature of inflammatory bowel disease . in the following section , we discuss evidence showing the involvement of hif and oxygen - sensing hydroxylases in epithelial barriers the knowledge that hypoxia is common in disease environments led to the interest in unravelling the consequences of low oxygenation in different pathologies . in the context of ibd , a key observation was the increased presence of hif in the colon from both murine colitis specimens and ibd patients.112 , 113 these observations led to the investigation of the effects of pharmacologic and genetic manipulation of the hypoxia - inducible factor pathway to analyze its involvement in disease . initial studies have shown that 2 hydroxylase inhibitors , dimethyloxalylglycine ( dmog ) and fg-4497 , reduce inflammation in different models of murine ibd ( dss- and trinitrobenzene sulfonic acid induced colitis , respectively).115 , 116 subsequently , multiple studies have confirmed the anti - inflammatory actions of these drugs . by using phd - deficient mice for the 3 main phd isoforms ( phd13 ) this provided evidence that the protective effects of hydroxylase inhibitors may be mediated via protection of the intestinal barrier . hindryckx et al subsequently showed hif - dependent reduction of intestinal epithelial cell apoptosis in dmog - treated mice in a model of tnf - induced colitis . moreover , a study investigating the expression of phd13 in uc and cd biopsy specimens showed an up - regulation of phd1 in ibd . altogether these data suggest that the hydroxylase inhibition mediated protection against intestinal inflammation relies at least in part on barrier - protective effects . in line with this hypothesis , other barrier - protective mechanisms regard , hif has been linked to the production of trefoil factors , mucins,120 , 121 -defensins , and the regulation of mucosal immune responses.121 , 122 through the regulation of these factors hif provides a barrier - protective mechanism in intestinal disease . however , in some studies , indirect negative effects of hif in colitis also have been reported . knock - out of intestinal von hippel - lindau tumor suppressor protein , the ubiquitin ligase responsible for hif degradation , led to increased severity of colitis indirectly correlating hif to increased inflammation . however , the protective effects of pharmacologic hydroxylase inhibition now has been shown to be reproducible in multiple models of colitis . another potential mechanism whereby hydroxylase inhibitors may be implicated in barrier protection is through the regulation of the tight junction . in 2 different reports , claudin-1 expression and occludin stabilization were found to be regulated by hif and phd3 , respectively.124 , 125 interestingly , in 2 studies using an in vitro model of neural vasculature and an in vivo model of brain ischemic stroke , hypoxia and pharmacologic hydroxylase inhibition were found to modulate permeability via regulation of the tight junction.126 , 127 in addition , a hif-2mediated increase in caveolin 1 has been reported to increase intestinal permeability via down - regulation of occludin and disruption of tight junction . further research is required to fully elucidate the role of hydroxylases in the regulation of the tight junction in the intestinal epithelium . beyond their fundamental role in the regulation of hif , hif hydroxylases have been shown to have important functions in the regulation of other processes . for example , there is a close relationship between hif , hif hydroxylases , and the nuclear factor-b ( nf-b ) pathway . initial evidence for this comes from the observation that inflammatory cytokines such as il1 , lipopolysaccharide , and tnf- up - regulate hif-1 at both rna and protein levels.129 , 130 , 131 indeed , these and other studies also have shown the involvement of different subunits of the nf-b pathway in the regulation of hif-1 expression and stabilization,131 , 132 as well as in the expression of hif - dependent genes.132 , 133 similar nf-b dependent regulatory mechanisms have been described for the expression of hif-1 , giving further insights into the nf-b mediated hif regulation . cummins et al reported phd1 silencing to up - regulate the nf-b response , and fitzpatrick et al showed a phd1-dependent regulation of nf-b mediated apoptosis . moreover , knock - down of phd1 and fih in combination regulates il1-mediated inflammatory gene expression via reduced nf-b activity . in addition to this phd1-dependent regulation , a number of studies have described an important role for phd3 in the regulation of nf-b signaling.139 , 140 , 141 thus , there is now significant evidence supporting the involvement of phds in the regulation of nf-b dependent inflammatory pathways . one of these complications is a deficiency in iron , causing low levels of hemoglobin and leading to anemia . the causes of anemia in ibd could be linked to intestinal bleeding or malabsorption , which turns ibd into a debilitating disorder . as a regulator of hypoxic responses , hif is a strong inducer of the expression of erythropoietin , which stimulates erythrocyte proliferation , contributing to increased oxygen transport in the blood.144 , 145 , 146 , 147 this erythropoietin - inducing property now is being investigated in clinical trials for the treatment of anemia.146 , 148 this could represent a further beneficial effect of hydroxylase inhibition in anemic ibd patients . another common complication in ibd is intestinal fibrosis , which is more common in cd patients although still is significant in uc.149 , 150 fibrosis is the consequence of the overactivation of wound healing responses leading to the deposition of excessive extracellular matrix and the formation of scar tissue ( figure 2).111 , 150 intestinal fibrosis is the main cause of surgical intervention in cd patients owing to the lack of pharmacologic tools to prevent it . the role of hypoxia and hydroxylases in wound healing and fibrosis has been investigated in many fibrotic pathologies and has generated variable results.152 , 153 , 154 , 155 , 156 however , little is known about the effects of hydroxylase inhibitors in intestinal fibrosis . in recently published work , we described a role of hydroxylase inhibition as a negative regulator of transforming growth factor-1induced intestinal fibrosis . these effects were hif independent and appear to be caused by hydroxylase - dependent modulation of the noncanonical transforming growth factor given the relevance of the extracellular regulated kinase pathway in fibrotic pathology , these observations may represent a first step toward investigating hydroxylase inhibition as antifibrotic therapy in ibd.158 , 159 , 160 some side effects of hydroxylase inhibitors can be expected and are related mainly to the ability of these drugs to activate the hif pathway . as discussed in the previous section , hif activation can mediate erythropoietin production and lead to increased erythropoiesis . although this effect can be exploited in anemic patients , the up - regulation of erythropoietin a potential solution to this side effect would be the formulation of hydroxylase inhibitors in targeted release forms . we recently described that the use of a colonic release form can diminish the systemic exposure of dmog and allows dose reduction without affecting its intestinal protective effects . other potential source of negative effects come from the observation that hif is activated in tumors and may contribute to tumor survival and growth.162 , 163 , 164 , 165 hif induces vascular endothelial growth factor in different cancer models.166 , 167 , 168 , 169 this effect is related to the hif - driven response that enhances oxygen supply , in this case via promotion of angiogenesis . vascular endothelial growth factor angiogenic properties are a contributing factor to tumor growth and could represent a negative side effect in hydroxylase inhibition therapy . furthermore , other cancer - related processes such as metastasis or cancer stem cell differentiation also have been related to hypoxia and hif , representing other potential side effects.171 , 172 this single layer of epithelial cells is involved in both nutrition and defense , playing fundamental roles in the transport of nutrients , the formation of the external mucosal environment , the tolerance toward microbiota components , and the control of inflammatory responses . epithelial disruption is one of the key drivers of inflammatory bowel diseases and restoring the epithelial permeability is seen as one of the main challenges in the development of novel therapies . hypoxia is another central feature of ibd as a result of the excessive oxygen consumption combined with the reduced supply that is typical of chronic inflamed environments . pharmacologic targeting of oxygen - sensing hydroxylases represents a promising therapeutic approach in multiple models of ibd . this protective effect likely is related to both hif - dependent and hif - independent mechanisms that contribute to barrier function ( figure 3 ) . given the reported beneficial effects of the inhibition of single subunits of the oxygen - sensing hydroxylase family , the possibility of developing specific drugs represents a promise toward the improvement of this potential new therapeutic approach . therefore , future research should focus on a more detailed characterization of the actions of single oxygen - sensing hydroxylases in epithelial function and on the development and test of specific inhibitors .

human health is dependent on the ability of the body to extract nutrients , fluids , and oxygen from the external environment while at the same time maintaining a state of internal sterility . therefore , the cell layers that cover the surface areas of the body such as the lung , skin , and gastrointestinal mucosa provide vital semipermeable barriers that allow the transport of essential nutrients , fluid , and waste products , while at the same time keeping the internal compartments free of microbial organisms . these epithelial surfaces are highly specialized and differ in their anatomic structure depending on their location to provide appropriate and effective site - specific barrier function . given this important role , it is not surprising that significant disease often is associated with alterations in epithelial barrier function . examples of such diseases include inflammatory bowel disease , chronic obstructive pulmonary disease , and atopic dermatitis . these chronic inflammatory disorders often are characterized by diminished tissue oxygen levels ( hypoxia ) . hypoxia triggers an adaptive transcriptional response governed by hypoxia - inducible factors ( hifs ) , which are repressed by a family of oxygen - sensing hif hydroxylases . here , we review recent evidence suggesting that pharmacologic hydroxylase inhibition may be of therapeutic benefit in inflammatory bowel disease through the promotion of intestinal epithelial barrier function through both hif - dependent and hif - independent mechanisms .

The Intestinal Epithelial Barrier Oxygen Tension and the IntestinalEpithelium Inflammatory Bowel Disease: The Involvement of Barrier Dysfunction Hydroxylase Inhibitors: A New Therapeutic Approach in IBD Conclusions

the intestinal epithelium permits the traffic of nutrients from the lumen to the blood , while at the same time restricting the passage of potentially harmful microorganisms and toxins.9 , 10 two main characteristics are key in establishing and maintaining the epithelial barrier . first , a capacity for controlled cell renewal , which is achieved through a tightly regulated balance between epithelial cell proliferation and apoptosis , and , second , the presence of effective intercellular junctions . intestinal epithelial cells are replaced every 45 days , an impressive feat considering that the adult intestinal epithelium has a surface area of approximately 300 m. the control of epithelial cell renewal is based on a continuous balance between proliferation and apoptosis - dependent cell death.11 , 13 , 14 epithelial cells are generated from stem cell progenitors in the intestinal crypts and migrate from the crypt to the lumen - facing mucosal surface . the process of intestinal cell proliferation and migration is regulated through complex signaling events in which the wnt pathway is a key player.11 , 15 an equally balanced rate of epithelial cell death also is essential to maintain controlled renewal of the epithelial barrier . alterations in the balance between proliferation and apoptosis are known to be involved in barrier dysfunction , which leads to disease . for example , in the context of ibd , the primed inflammatory environment can lead to the generation of pro - apoptotic signals and , consequently , to the activation of apoptotic pathways in epithelial cells.16 , 17 , 18 ultimately , this compromises the capacity of the epithelium to maintain barrier integrity . this is in contrast to intestinal epithelial cancers in which epithelial proliferation exceeds the rate of apoptosis and promotes the development of intestinal tumors . effective sealing of the intercellular space is a second factor necessary to fulfill the provision of a continuous intestinal epithelial barrier . this sealing property is defined primarily by the tight junctions , adherens junctions , and desmosomes . adherens junctions and desmosomes are responsible for the maintenance of the proximity between cells through intercellular molecular connections , whereas tight junctions are responsible for sealing the paracellular space . tight junctions consist of complexes formed through the interaction of proteins including occludin , zonula occludens ( zo ) , and claudins ( of which multiple isoforms exist).21 , 22 although occludin and claudins are transmembrane proteins , zo acts as an anchoring component that binds transmembrane tight junction proteins.23 , 24 in addition to these constituent proteins , regulatory enzymes can play an important role in controlling the permeability of the tight junction . an example of this is myosin light - chain kinase , which regulates tight junction permeability.26 , 27 , 28 thus , in addition to transcellular transport , the paracellular space defines an alternative transport route . therefore , the dynamic regulation of the tight junction is critical for the maintenance of effective barrier function while allowing the absorption of nutrients and other macromolecules . importantly , inflammatory mediators such as tumor necrosis factor- ( tnf- ) or interleukin ( il)13 are capable of causing alterations in the structure of the tight junction ( tj ) complexes and changes in permeability.29 , 30 as discussed later , such alterations represent another mechanism of induction of barrier dysfunction - related intestinal disease . the intestinal epithelium is in direct contact with a large number of microorganisms that are part of the normal intestinal microbiota.31 , 32 at the same time , the epithelium separates the microbiota from the intestinal immune system that is resident in the lamina propria ( figure 1 ) . therefore , the intestinal epithelium is a highly regulated and interactive surface that communicates signals between the mucosal immune system and the gut microbiota.31 , 33 , 34 the number of bacterial cells that reside in the intestinal tract exceeds the number of human cells in the body.7 , 35 this rich intestinal microenvironment has important interactions with the epithelial cells that form the intestinal barrier . intestinal epithelial cells express pattern recognition receptors such as toll - like receptors ( tlrs ) . for example , the gut microbiome is implicated in the absorption of fats.38 , 39 moreover , components of the microbiome also are required for the degradation of complex polysaccharides that can not be absorbed into simpler carbohydrates that can be absorbed.40 , 41 furthermore , the gut microbiome is known to regulate mucins derived from intestinal goblet cells . this mucous layer serves as a defense system against physical and chemical injury and aids the clearance of pathogenic microorganisms.43 , 44 components of the microbiome are involved in both mucus production as well as degradation.45 , 46 , 47 , 48 , 49 this implicates a mechanism of cross - talk between the intestinal epithelium and microbiota that contributes to both digestion and barrier function . in fact , as explained earlier , intestinal epithelial cells express multiple tlrs , including tlr-4 and tlr-3 , and are responsive to microbial stimuli.50 , 51 , 52 these receptors are common immune receptors responsible for the recognition of bacterial components such as lipopolysaccharide . a final mechanism whereby the intestinal epithelium plays a role in the maintenance of homeostasis is through the production of mediators such as trefoil factors ( tffs ) and defensins.55 , 56 , 57 , 58 for example , in a study comparing the expression of tff isoforms between healthy and intestinal biopsy specimens from patients with ibd , the messenger rna levels of the tffs analyzed were found to be correlated to the pathologic state . the human body is working constantly to maintain an adequate oxygen supply from its absorption in the lungs , its distribution via blood vessels , and its transport and consumption at the cellular level . the intestinal epithelium is in a continuous state of physiologic hypoxia as a result of 2 main events.64 , 65 first , it is juxtaposed between the largely anoxic intestinal lumen and the well - perfused lamina propria ( figure 1 ) . second , the oxygen pressure fluctuates in the lamina propria depending on blood volume in the gut . thus , under physiologic conditions , the intestinal epithelium often is subjected to states of transient oxygen deprivation . this ability of the intestinal epithelial cells to tolerate transient periods of hypoxia in physiologic conditions has led to the concept of physiologic hypoxia . as discussed in the previous section , the intestinal epithelium is exposed constantly to low concentrations of oxygen , and thus represents a paradigm environment in which adaptation to hypoxia is key . at the cellular level , our ability to adapt to hypoxia depends on the activation of the hypoxia - inducible factor ( hif ) signaling pathway.68 , 69 hif is a ubiquitously expressed family of heterodimeric transcription factors formed by the binding of hif- and hif- subunits . under hypoxic conditions , the formation of functional hif transcription factors in the nucleus triggers a reprogramming of gene expression that controls cell fate , activates alternative mechanisms of energy generation , or enhances oxygen absorption among many other functions.74 , 75 , 76 , 77 , 78 thus , hif responses are critical in the control of cell survival , metabolism , and other functions under low oxygenation . a group of 3 prolyl hydroxylases ( phd ) , phd-1 , 2 , and 3 , and an asparaginyl hydroxylase known as factor inhibiting hif ( fih ) , provide an efficient mechanism by which to control hif - dependent responses.79 , 80 hif- subunits are synthesized constitutively at high levels in all cells . under normoxic conditions , hif- subunits are hydroxylated on 2 prolyl residues ( pro402 and pro564 for hif-1 and pro405 and pro531 for hif-2 ) within their oxygen - dependent degradation domain by phd1 - 3.81 , 82 , 83 , 84 prolyl hydroxylation makes hif- a target for the e3 ubiquitin ligase von hippel - lindau protein that ubiquitinates hif- , marking it for proteosomal degradation . transactivation domain ( asn803 in hif-1 ) , blocking its binding to the cofactor creb - binding protein / p300 and preventing hif - mediated transcriptional activation.79 , 85 , 86 , 87 in hypoxia , hydroxylases no longer can hydroxylate hif- subunits ( because of the lack of availability of molecular oxygen as a co - factor ) . furthermore , ibd progression is associated with the development of a number of common complications . current therapies for ibd are focused primarily on controlling inflammation using drugs such as mesalamine and/or corticosteroids.91 , 92 however , these therapeutics often are insufficient to maintain patients in remission . in these cases , immunomodulator therapies such as azathioprine or methotrexate more recently developed approaches are focused on the use of antibodies targeting cytokines that are known to play critical roles in the development of the pathology . among those , tnf- is one of the most important drivers of inflammation in ibd , and thus therapies such as infliximab that specifically target tnf- are common in maintenance therapy.91 , 93 , 94 unwanted side effects of current ibd treatments are common . for example , the use of azathioprine is related to a higher risk of malignancy.92 , 95 anti - tnf therapy can lead to immune - compromised states and is related to a higher risk of infectious disease as well as a higher risk of malignancy . ibd is the result of a combination of environmental factors leading to recurrent inflammatory insults , which , in genetically predisposed patients , leads to the development of chronic inflammation.91 , 92 , 96 the development of chronic unresolved inflammation has been linked to intestinal epithelial barrier dysfunction including both disruption of the physical barrier as well as loss of tolerance against components of the microbiota.1 , 97 first , the loss of the physical barrier function increases permeability , facilitating contact between luminal antigens and the mucosal immune cells ( figure 2 ) . second , the intestinal epithelial cells actively participate in the recognition of pathogens and recruitment of other immune cells and the initiation of inflammation ( figure 2 ) . at the cellular level , the loss of epithelial barrier function seen in ibd can be caused by either enhanced apoptosis98 , 99 , 100 or alterations in the expression and assembly of the tj proteins ( or a combination of both).101 , 102 this is supported , for example , by evidence linking tnf- to epithelial cell apoptosis and tj regulation.99 , 101 , 102 , 103 studies have shown that anti - tnf therapies reduce intestinal epithelial cell apoptosis in models of colitis and cd patients.98 , 104 , 105 tnf- down - regulates expression and alters localization of zo-1 and promotes occludin endocytosis , actively regulating the architecture of the tj.101 , 102 tnf - mediated effects on intestinal epithelial tj occur via the activation of myosin light - chain kinase signaling.29 , 106 , 107 thus , alterations in the regulation of epithelial barrier function appear to be determinants in the development of chronic inflammation in the intestinal tract . tissue oxygen levels are reduced in different pathologic foci including tumors and inflammatory environments such as ibd.64 , 65 , 66 , 108 , 109 , 110 in the case of ibd , inflamed areas of the colon experience an increase in activity and subsequently oxygen consumption as a result of the increased presence of activated immune cells , including neutrophils , as well as the activation of reparative processes ( figure 2).65 , 111 , 112 furthermore , the epithelial surface , which is in direct contact with the profoundly hypoxic intestinal lumen , is damaged in ibd and becomes leaky , allowing infiltration of luminal contents into the mucosa and lamina propria ( figure 2 ) . the presence of profound hypoxia on the epithelial surface was shown by karhausen et al using 2-(2-nitro-1h - imidazol-1yl)-n-(2,2,3,3,-pentafluoropropyl ) acetamide - based measurement of tissue oxygenation in murine colitis . in this study , the presence of hypoxia was restricted to the epithelial surface in healthy colons but became penetrant in diseased colons.65 , 112 another factor that accounts for the poor tissue oxygenation is the vasculopathy that affects inflamed tissues and reduces blood perfusion and , as a result , compromises oxygen supply.66 , 112 therefore , hypoxia is a common feature of inflammatory bowel disease . in the following section , we discuss evidence showing the involvement of hif and oxygen - sensing hydroxylases in epithelial barriers the knowledge that hypoxia is common in disease environments led to the interest in unravelling the consequences of low oxygenation in different pathologies . in the context of ibd , a key observation was the increased presence of hif in the colon from both murine colitis specimens and ibd patients.112 , 113 these observations led to the investigation of the effects of pharmacologic and genetic manipulation of the hypoxia - inducible factor pathway to analyze its involvement in disease . by using phd - deficient mice for the 3 main phd isoforms ( phd13 ) this provided evidence that the protective effects of hydroxylase inhibitors may be mediated via protection of the intestinal barrier . hindryckx et al subsequently showed hif - dependent reduction of intestinal epithelial cell apoptosis in dmog - treated mice in a model of tnf - induced colitis . altogether these data suggest that the hydroxylase inhibition mediated protection against intestinal inflammation relies at least in part on barrier - protective effects . in line with this hypothesis , other barrier - protective mechanisms regard , hif has been linked to the production of trefoil factors , mucins,120 , 121 -defensins , and the regulation of mucosal immune responses.121 , 122 through the regulation of these factors hif provides a barrier - protective mechanism in intestinal disease . knock - out of intestinal von hippel - lindau tumor suppressor protein , the ubiquitin ligase responsible for hif degradation , led to increased severity of colitis indirectly correlating hif to increased inflammation . however , the protective effects of pharmacologic hydroxylase inhibition now has been shown to be reproducible in multiple models of colitis . another potential mechanism whereby hydroxylase inhibitors may be implicated in barrier protection is through the regulation of the tight junction . in 2 different reports , claudin-1 expression and occludin stabilization were found to be regulated by hif and phd3 , respectively.124 , 125 interestingly , in 2 studies using an in vitro model of neural vasculature and an in vivo model of brain ischemic stroke , hypoxia and pharmacologic hydroxylase inhibition were found to modulate permeability via regulation of the tight junction.126 , 127 in addition , a hif-2mediated increase in caveolin 1 has been reported to increase intestinal permeability via down - regulation of occludin and disruption of tight junction . for example , there is a close relationship between hif , hif hydroxylases , and the nuclear factor-b ( nf-b ) pathway . initial evidence for this comes from the observation that inflammatory cytokines such as il1 , lipopolysaccharide , and tnf- up - regulate hif-1 at both rna and protein levels.129 , 130 , 131 indeed , these and other studies also have shown the involvement of different subunits of the nf-b pathway in the regulation of hif-1 expression and stabilization,131 , 132 as well as in the expression of hif - dependent genes.132 , 133 similar nf-b dependent regulatory mechanisms have been described for the expression of hif-1 , giving further insights into the nf-b mediated hif regulation . in addition to this phd1-dependent regulation , a number of studies have described an important role for phd3 in the regulation of nf-b signaling.139 , 140 , 141 thus , there is now significant evidence supporting the involvement of phds in the regulation of nf-b dependent inflammatory pathways . as a regulator of hypoxic responses , hif is a strong inducer of the expression of erythropoietin , which stimulates erythrocyte proliferation , contributing to increased oxygen transport in the blood.144 , 145 , 146 , 147 this erythropoietin - inducing property now is being investigated in clinical trials for the treatment of anemia.146 , 148 this could represent a further beneficial effect of hydroxylase inhibition in anemic ibd patients . another common complication in ibd is intestinal fibrosis , which is more common in cd patients although still is significant in uc.149 , 150 fibrosis is the consequence of the overactivation of wound healing responses leading to the deposition of excessive extracellular matrix and the formation of scar tissue ( figure 2).111 , 150 intestinal fibrosis is the main cause of surgical intervention in cd patients owing to the lack of pharmacologic tools to prevent it . in recently published work , we described a role of hydroxylase inhibition as a negative regulator of transforming growth factor-1induced intestinal fibrosis . these effects were hif independent and appear to be caused by hydroxylase - dependent modulation of the noncanonical transforming growth factor given the relevance of the extracellular regulated kinase pathway in fibrotic pathology , these observations may represent a first step toward investigating hydroxylase inhibition as antifibrotic therapy in ibd.158 , 159 , 160 some side effects of hydroxylase inhibitors can be expected and are related mainly to the ability of these drugs to activate the hif pathway . other potential source of negative effects come from the observation that hif is activated in tumors and may contribute to tumor survival and growth.162 , 163 , 164 , 165 hif induces vascular endothelial growth factor in different cancer models.166 , 167 , 168 , 169 this effect is related to the hif - driven response that enhances oxygen supply , in this case via promotion of angiogenesis . furthermore , other cancer - related processes such as metastasis or cancer stem cell differentiation also have been related to hypoxia and hif , representing other potential side effects.171 , 172 this single layer of epithelial cells is involved in both nutrition and defense , playing fundamental roles in the transport of nutrients , the formation of the external mucosal environment , the tolerance toward microbiota components , and the control of inflammatory responses . epithelial disruption is one of the key drivers of inflammatory bowel diseases and restoring the epithelial permeability is seen as one of the main challenges in the development of novel therapies . pharmacologic targeting of oxygen - sensing hydroxylases represents a promising therapeutic approach in multiple models of ibd . this protective effect likely is related to both hif - dependent and hif - independent mechanisms that contribute to barrier function ( figure 3 ) . given the reported beneficial effects of the inhibition of single subunits of the oxygen - sensing hydroxylase family , the possibility of developing specific drugs represents a promise toward the improvement of this potential new therapeutic approach . therefore , future research should focus on a more detailed characterization of the actions of single oxygen - sensing hydroxylases in epithelial function and on the development and test of specific inhibitors .

the latrunculins constitute a family of macrolide natural products isolated from the taxologically unique marine sponges cacospongia mycofijiensis and negombata magnifica ( figure 1 ) that have been widely studied given of both their reversible inhibition of actin polymerization and their cytotoxicity against several human cancer cell lines . latrunculin a ( 2 ) in particular has served as an important molecular probe used to explore the biological implications of actin depolymerization , and as such , it has been the target of several successful synthetic ventures . crews and co - workers reported the isolation , characterization , and initial biological assessment of a series of new , naturally occurring latrunculin analogues . one compound , ( + ) -18-epi - latrunculol a ( 1 ) , exhibited selective solid tumor cytotoxicity when tested against hct-116 ( 5.5 m ) and mda - mb-435 ( > 50 m ) , but unlike the other members of the latrunculin family , 1 was devoid of the ability to inhibit actin polymerization . on the other hand , the parent compounds ( + ) -latrunculin a ( 2 ) and b ( 3 ) demonstrate nonselective cytotoxicity profiles , thus limiting their use as chemotherapeutics . given our longstanding interest in the latrunculins and the intriguing biological profile of the epimeric latrunculin congener ( + ) -18-epi - latrunculol a ( 1 ) , we undertook the development of a scalable total synthesis of ( + ) -1 to confirm both the assigned structure and absolute stereochemistry and to provide sufficient quantities for additional biological evaluation . described herein is a full account of the evolution of the total synthesis of ( + ) -18-epi - latrunculol a ( 1 ) , which led not only to ( + ) -1 but also to the preparation of ca . 50 mg of the penultimate synthetic precursor for use in for future analogue development . while at the outset of this synthetic venture the total synthesis of ( + ) -18-epi - latrunculol a ( 1 ) had not been reported , effective total syntheses of the parent latrunculins a ( 2 ) and b ( 3 ) had been published by white , frstner , and our laboratory . not surprisingly , our synthetic strategy for ( + ) -18-epi - latrunculol a ( 1 ) was envisioned to exploit the lessons learned in our earlier syntheses , with important modifications to ensure efficient asymmetric access to the natural product ( scheme 1 ) . specifically , we envisioned a late - stage mitsunobu macrolactonization and either a wittig olefination or , given our earlier difficulties with the wittig union for latrunculin a , a nucleophilic addition protocol to construct the 16-membered macrolactone . the requisite southern hemisphere coupling partner 4 was in turn envisioned to be readily accessible from cyclic ketal 5 via a strategy level acid - mediated cyclization / equilibration of -hydroxy enone 6 . the latter would be constructed through the aegis of a functional - group - compatible cross - metathesis reaction , which was anticipated to circumvent both protecting group manipulation and oxidation , as required in all previous latrunculin syntheses . the requisite cross - metathesis partners 7 and 9 in turn would arise from known aldehyde ( )-8 and d - cysteine , respectively . in the synthetic direction , d - cysteine was converted to thiazolidinone ( + ) -10 upon treatment with phenyl chloroformate followed by chemoselective n - protection with p - methoxybenzyl chloride , which provided ( + ) -10 in a 65% yield for the two steps ( scheme 2 ) . acid ( + ) -10 was then converted to the weinreb amide , which upon treatment with freshly prepared vinylmagnesium bromide furnished enone ( + ) -9 in 55% yield for the two steps . quenching of the addition reaction with aqueous hcl was critical to obtain high yields of ( + ) -9 . when saturated aqueous ammonium chloride was employed , a mixture of enone ( + ) -9 and byproduct ( + ) -11 was obtained , presumably via addition of the liberated hydroxylamine to the electrophilic enone . the structure of ( + ) -11 was confirmed by single - crystal x - ray analysis . with gram quantities of enone ( + ) -9 in hand , we turned to the construction of the remaining cross - metathesis partner ( scheme 3 ) . alcohol ( )-13 was readily prepared utilizing a previously reported three - step sequence beginning with commercially available 5-hexenoic acid ( 12 ) . alcohol ( )-13 was then protected as the tbs ether , and subsequent ozonolysis of the terminal olefin provided known aldehyde ( )-8 . an asymmetric brown allylation followed to deliver the desired homoallylic alcohol , which was contaminated with small and variable amounts of the epimeric alcohol . without separation , treatment of this mixture with 3 equiv of enone ( + ) -9 and the hoveyda grubbs second - generation catalyst to achieve cross - metathesis provided pure cyclization precursor ( + ) -6 in 70% yield from ( )-8 after standard flash column chromatography with 72% recovery of unreacted enone ( + ) -9 . the key acid - mediated cyclization was next achieved after extensive experimentation by subjecting -hydroxy enone ( + ) -6 to a 1:1.3 ( v / v ) mixture of 6 n hcl and thf to furnish lactol ( + ) -14 ( scheme 4 ) . upon standing , lactol ( + ) -14 slowly epimerized at room temperature ; accordingly , the conversion to methyl ketal ( + ) -5 was conducted immediately following the cyclization by treatment of lactol ( + ) -14 with acidic methanol to provide ( + ) -5 as a single diastereomer in 43% yield over two steps . multiple byproducts and a small amount of the minor -diastereomer accounted for the remaining mass balance of the acid - mediated cyclization of ( + ) -6 . the mechanism proposed for the observed acid - mediated cyclization is depicted in scheme 4 . we reason that reversible formation of unsaturated oxonium intermediate 15 accounts for enrichment in the mixture of the diastereomeric secondary alcohols , thus providing the -diastereomer ( + ) -16 as the major product . wittig olefination was next envisaged to unite the northern and southern hemispheres of ( + ) -18-epi - latrunculol a ( 1 ) . however , we were cognizant of the challenges that a wittig union had provided in the original synthesis of ( + ) -latrunculin a ( 2 ) . as indicated in our synthetic analysis , ketal ( + ) -5 also held the promise of further elaboration to explore alternative coupling protocols ( scheme 5 ) . proceeding with the wittig olefination tactic , wittig reagent ( + ) -17 was prepared in three steps and 55% yield from ( + ) -5 ( scheme 6 ) . with ( + ) -17 in hand , we explored the olefination protocol with benzaldehyde as a model coupling partner , employing nahmds to achieve deprotonation . full conversion to a major product was observed , but to our surprise the product did not incorporate benzaldehyde ; instead , phosphine oxide ( + ) -21 was identified as the major product . importantly , the reaction had been conducted under strictly anhydrous and deoxygenated conditions . nonetheless , all attempts afforded ( + ) -21 as the major product , and no olefination was observed . undeterred , we turned to the alternative coupling protocols that could be accessed quickly from ketal ( + ) -5 ( scheme 5 ) . vinyl iodide 18 and/or alkyne 19 , for example , would permit the union of the derived vinyl or alkynyl organometallic reagents to a northern hemisphere in the form of an aldehyde . toward this end , vinyl iodide ( + ) -18 and terminal alkyne ( + ) -19 were each constructed in three steps from ketal ( + ) -5 ( scheme 7 ) . first , chemoselective oxidation of the primary alcohol provided the corresponding aldehyde . subsequent stork zhao olefination was followed by tbs protection of the secondary hydroxyl , which led to cis - vinyl iodide ( + ) -18 in 32% yield over the three steps . again , the secondary tetrahydropyran hydroxyl group was protected as a tbs ether to provide alkyne ( + ) -19 in 56% yield from ketal ( + ) -5 . the requisite northern - hemisphere aldehyde ( + ) -24 was next prepared in six steps from known alkynyl diol ( )-22 ( scheme 8) . the synthesis began with the two - step chemoselective protection of ( )-22 , which was followed by deprotonation of the alkynyl proton with n - buli and , in turn , addition of the resulting lithium acetylide to methyl chloroformate to deliver alkynoate ( )-23 . conjugate addition of me2culi then provided the z - trisubstituted enoate , which was exposed to acetic acid - buffered tbaf to remove the primary tbs group . interestingly , employing unbuffered tbaf to remove the tbs group led to complete isomerization of the trisubstituted enoate . doering conditions then provided the requisite northern - hemisphere aldehyde ( + ) -24 in a yield of 35% over the six steps . on the basis of the transition state outlined in scheme 9a , we reasoned that the chelation - controlled addition of a vinyl nucleophile , prepared via metalation of vinyl iodide ( + ) -18 , to aldehyde ( + ) -24 would lead to the desired syn stereochemistry . somewhat surprisingly , however , addition of various metalated species derived from ( + ) -18 to aldehyde ( + ) -24 resulted only in protodemetalation and aldehyde decomposition ( scheme 9b ) . presumably the decomposition of ( + ) -24 is due to facile deprotonation of the acidic - and/or -protons of the enoate ( shown in red in scheme 9 ) upon treatment with the strongly basic nucleophiles . aware of the pronounced base sensitivity of aldehyde ( + ) -24 , we explored a carreira alkynylation employing alkyne ( + ) -19 . the mild nature of the alkynylzinc nucleophile employed in this reaction was anticipated to circumvent decomposition . pleasingly , after brief optimization of the carreira alkynylation , the desired union of the northern and southern hemispheres was achieved to funish ( + ) -25 in 95% yield , impressively as a single diastereomer ( scheme 10 ) . of considerable importance , prolonged drying of the zn(otf)2 proved to be critical for the success of this union.the stereochemistry of the newly formed propargylic alcohol was confirmed via conversion to acetonide ( + ) -26 , with the latter structure assigned by 2d nmr analysis . with construction of the carbon skeleton of ( + ) -18-epi - latrunculol a ( 1 ) now achieved , we turned to the requisite alkyne semireduction . no reduction was observed when ( + ) -25 was subjected to either the lindlar or p-2 nickel boride catalyst under a hydrogen atmosphere . to determine whether the steric environment of the internal alkyne was precluding the metal coordination necessary for hydrogenation , we turned to molecular mechanics calculations employing the mm2 force field . on the basis of these calculations as well as examination of a physical model of ( + ) -25 , we discovered that in spite of the linear geometry of the alkyne , intermediate ( + ) -25 could in fact attain the requisite orientation for the key mitsunobu macrolactonization . with this scenario in mind as well as with the expectation that semireduction of an alkyne in a 16-membered macrolactone might be enhanced by ring strain , we proceeded with the synthesis of the envisioned alkyne - containing seco acid . propargyl alcohol ( + ) -25 was protected as the sem ether to maintain rotational freedom of the northern hemisphere ( scheme 11 ) . removal of the tbs ether was achieved by employing acetic acid - buffered tbaf . hydrolysis of the methyl ester followed by warming of the ester to 50 c with naoh in ethanol provided the alkyne - containing seco acid ( + ) -27 in 85% yield from ( + ) -25 . the anticipated mitsunobu macrolactonization was then attempted by addition of triphenylphosphine and diisopropyl azodicarboxylate ( diad ) to seco acid ( + ) -27 ( scheme 12 ) . although no reaction was observed in thf , toluene provided full conversion to macrolactone 28 and the reduced diad byproduct ( diad - h2 ) , which unfortunately proved to be inseparable via standard silica gel chromatography . treatment of the mixture with ceric ammonium nitrate ( can ) in aqueous acetonitrile pleasingly removed the pmb group to furnish deprotected macrolactone ( + ) -29 in 36% yield from ( + ) -27 , which could be readily purified via flash chromatography . a similar mitsunobu macrolactonization employed in the original smith synthesis of ( + ) -latrunculin a ( 2 ) , although yielding a similarly 16-membered macrolactone in a 31% yield , proved to be completely unworkable in this case because of the incompatible conditions required for removal of the pmb protecting group . frstner and co - workers observed similar decompositions of their late - stage macrolactones when attempting to remove the robust pmb protecting group and likewise required a protecting group interchange before completing their total synthesis of ( + ) -latrunculin a ( 2 ) . presumably the macrolactone in ( + ) -29 is more stable toward the strong oxidizing conditions required to remove the pmb group because ( + ) -29 lacks the conjugated diene moiety present in the advanced ( + ) -latrunculin a ( 2 ) intermediates . we next undertook global deprotection to remove both sem protecting groups with concomitant hydrolysis of the ketal ( scheme 13 ) . extensive experimentation culminated in the use of aqueous acetic acid with catalytic camphorsulfonic acid ( csa ) at 50 c to provide the penultimate alkyne ( + ) -30 . although the global deprotection was achieved , removal of the sem groups required extended reaction times ( > 12 h ) at elevated temperature ( 50 c ) , which resulted in varying degrees of decomposition as well as inconsistent yields of ( + ) -30 ranging from ca . while moderate in yield , the global deprotection provided sufficient alkyne ( + ) -30 to evaluate the final semireduction ( scheme 14 ) . again , semireduction employing either the lindlar or p-2 nickel boride catalyst under a hydrogen atmosphere did not proceed . other reduction conditions , including wilkinson s catalyst , homogeneous palladium - catalyzed transfer hydrogenation , adams catalyst , and a two - step hydroboration / protodeborylation sequence , also proved ineffective ; no reaction , over - reduction , and/or decomposition of starting material resulted . ultimately we discovered that a catalytic quantity of palladium on carbon , without a poisoning agent , delivered the semireduction product , albeit with incomplete consumption of alkyne ( + ) -30 . fortunately , the use of 1.2 equiv of pd / c ( 10 wt % ) did eventually provide full conversion , but only a 29% isolated yield of ( + ) -18-epi - latrunculol a ( 1 ) was obtained . the spectral data of synthetic ( + ) -18-epi - latrunculol a ( 1 ) , including the h nmr ( 500 mhz ) , hrms parent ion identification , and chiroptic properties , proved identical in all respects to those reported for natural ( + ) -18-epi - latrunculol a ( 1 ) . importantly , the observation of identical chiroptic properties for synthetic ( + ) -1 permitted the assignment of the absolute stereochemistry of ( + ) -18-epi - latrunculol a ( 1 ) . the c nmr spectrum , however , proved problematic . when taken in acetone - d6 as reported by crews , the carbon resonances , while identical in chemical shift to those reported for natural ( + ) -18-epi - latrunculol a ( 1 ) , appeared doubled in several cases , a hallmark of course of a diastereomeric mixture ( figure 2a ) . on the other hand , when the c nmr spectrum of synthetic ( + ) -18-epi - latrunculol a ( 1 ) was taken in cdcl3 instead of acetone - d6 , the spectrum revealed the correct number of chemical shifts required for ( + ) -1 . tracing the problem back to acetone - d6 , we observed both h2o and dho peaks in the h nmr spectrum and thus speculated that a deuterium equilibrium exchange had occurred to account for the mixture observed in the c nmr spectrum . taking extreme care to introduce the acetone - d6 under a strictly nitrogen atmosphere greatly reduced the amount of h2o and dho , and although the doubled peaks were still observable , they were considerably reduced ( figure 2a ) . importantly , upon addition of d2o to the nmr sample in acetone - d6 , the doubled carbon resonances were converted to a single set of resonances for deuterated 18-epi - latrunculol a ( 1 ) ( figure 2b ; see the supporting information for the nmr spectra of deuterated 18-epi - latrunculol a ) . c nmr spectra ( 6080 ppm ) of synthetic ( + ) -18-epi - latrunculol a ( 1 ) in ( a ) acetone - d6 and ( b ) acetone - d6 with added d2o . with conclusive evidence that the first total synthesis , structural confirmation , and absolute configuration assignment of ( + ) -18-epi - latrunculol a ( 1 ) had been achieved , we returned to the optimization of the global deprotection and final semireduction to facilitate a preparatively useful synthesis of the natural product . toward this end , we elected to explore an acetonide group to protect the vicinal diol , as such a group exchange would result in the same overall step count as in the bis - sem sequence and , importantly , the acetonide was anticipated to be more acid - labile . treatment of ( + ) -25 with acidic methanol as described earlier ( scheme 10 ) removed both the sem and tbs protecting groups while maintaining the mixed methyl ketal . the vicinal diol was in turn protected chemoselectively as the acetonide to provide ( + ) -26 in 77% yield over the two steps . upon hydrolysis of the methyl ester , the mitsunobu macrolactonization and subsequent pmb removal proceeded in a yield comparable to that in the sem - protected sequence of intermediates to furnish macrolactone ( + ) -31 in 35% yield from the methyl ester ( scheme 15 ) . we were then particularly pleased to find that global deprotection delivered the penultimate alkyne ( + ) -30 in 86% yield after only 2 h. the low yield of the final semireduction was reasoned to be a consequence of the excess adsorbing carbon solid support ( scheme 14 ) . pleasingly , a change to barium carbonate as a less adsorbent solid support provided the semireduction of ( + ) -30 in nearly quantitative yield , although a stoichiometric quantity of palladium was still required ( scheme 15 ) . synthetic ( + ) -18-epi - latrunculol a ( 1 ) was thus available upon semihydrogenation in 86% overall yield for the final two steps , a marked improvement from the previous protecting group strategy . we have reported here the total synthesis , structural validation , and assignment of the relative and absolute stereochemistry of ( + ) -18-epi - latrunculol a ( 1 ) , exploiting a longest linear sequence of 20 steps from commercially available 5-hexenoic acid . key steps in the successful route include a functional - group - compatible cross - metathesis reaction that avoids protection and oxidation steps required in all previous latrunculin synthetic ventures , an acid - mediated cyclization / equilibration sequence , an effective carreira alkynylation , and a late - stage mitsunobu macrolactonization . in addition , judicious selection of diol protection and successful optimization of the alkyne semireduction now permits access to synthetic ( + ) -18-epi - latrunculol a ( 1 ) . biological evaluation of the natural product and synthetic intermediates and further development of the tandem cyclization / equilibration of trans--hydroxy enones are currently underway and will be reported in due course . reactions were carried out in flame - dried or oven - dried glassware under a nitrogen atmosphere unless noted otherwise . anhydrous diethyl ether ( et2o ) , tetrahydrofuran ( thf ) , dichloromethane ( ch2cl2 ) , and toluene were obtained from a solvent purification system . all of the commercially available reagents were used without purification unless otherwise noted . triethylamine , diisopropylethylamine , and pyridine were freshly distilled from calcium hydride under a nitrogen atmosphere . reactions were magnetically stirred unless stated otherwise and monitored by thin - layer chromatography ( tlc ) with 0.25 mm silacycle precoated silica gel plates . silica gel chromatography was performed utilizing acs - grade solvents and silica gel from either silacycle or sorbent technologies . h nmr spectra ( 500 mhz field strength ) and c nmr spectra ( 125 mhz field strength ) were obtained on a 500 mhz spectrometer or a cryomagnet ( 500 mhz/52 mm ) with a 5 mm dual cryoprobe . chemical shifts are reported relative to chloroform ( 7.27 ) or acetone ( 2.05 ) for h nmr spectra and chloroform ( 77.23 ) or acetone ( 206.68 , 29.92 ) for c spectra . high - resolution mass spectrometry ( hrms ) was performed on an lc - tof mass spectrometer . to a solution of d - cysteine hydrochloride hydrate ( 25 g , 142.34 mmol ) in an aqueous sodium hydroxide solution ( 28.47 g of naoh , 140 ml of h2o ) cooled to 0 c was added phenyl chloroformate ( 39 ml , 313.15 mmol ) in toluene ( 60 ml ) dropwise via an addition funnel . after the addition was complete , the reaction mixture was allowed to warm to room temperature , where it was stirred overnight and then quenched with toluene ( 60 ml ) and h2o ( 60 ml ) . the aqueous layer was acidified by dropwise addition of 1 n hcl to ph < 1 , and the solution was extracted with etoac ( 3 50 ml ) . the combined organic layers were washed with brine , dried with na2so4 , filtered , and concentrated in vacuo to provide a white solid that was used without further purification . to a solution of the previously obtained white solid in h2o ( 14 ml ) , dmso ( 48 ml ) , and naoh ( 11.1 g , 278 mmol ) cooled to 0 c was added p - methoxybenzyl chloride ( 25 ml , 184.4 mmol ) dropwise . after the addition was complete , the ice water bath was removed and the reaction mixture was stirred at room temperature for 14 h. the reaction mixture became cloudy with a white precipitate . the reaction mixture was partitioned between diethyl ether ( 50 ml ) and 0.5 n naoh(aq ) ( 50 ml ) . the aqueous layer was separated and washed with diethyl ether ( 2 50 ml ) . the aqueous layer was acidified to ph < 1 by dropwise addition of 6 n hcl to the stirring basic aqueous solution . the cloudy white mixture was extracted with etoac ( 3 75 ml ) and concentrated in vacuo to yield ( + ) -10 ( 16.0 g , 59.86 mmol , 65% over two steps ) as a brown oil . [ ]d21 + 53.2 ( c 0.36 , chcl3 ) ; ir ( neat , cm ) 2934 , 1740 , 1612 , 1514 , 1444 , 1396 , 1248 ; h nmr ( 500 mhz , cdcl3 ) 7.20 ( d , j = 8.3 hz , 2h ) , 6.89 ( d , j = 8.3 hz , 2h ) , 5.15 ( d , j = 14.5 hz , 1h ) , 4.20 ( dd , j = 2.8 , 8.3 hz , 1h ) , 4.02 ( d , j = 15.7 hz , 1h ) , 3.81 ( s , 3h ) , 3.54 ( dd , j = 9.3 , 11.7 hz , 1h ) , 3.42 ( dd , j = 2.8 , 12.3 hz , 1h ) ; c nmr ( 125 mhz , cdcl3 ) 174.5 , 172.0 , 159.7 , 130.1 , 127.5 , 114.6 , 59.0 , 55.6 , 47.6 , 29.2 ; hrms ( esi - tof ) m / z ( m h ) calcd for c12h12no4s 266.0487 , found 266.0475 . to a solution of acid ( + ) -10 ( 16 g , 59.86 mmol ) in ch2cl2 ( 200 ml ) was added i - pr2net ( 31.4 ml , 179.58 mmol ) n , o - dimethylhydroxylamine hydrochloride ( 9.93 g , 179.58 mmol ) , and then tbtu ( 28.8 g , 89.79 mmol ) portionwise . the reaction mixture became cloudy with a white precipitate and was stirred overnight . the reaction mixture was quenched with 1.2 n hcl ( 100 ml ) , and the biphasic mixture was extracted with ch2cl2 ( 4 100 ml ) . the combined organic layers were washed with 0.5 n naoh ( 100 ml ) and then concentrated in vacuo . the crude mixture was purified via column chromatography on sio2 ( 60% etoac / hexanes ) to provide weinreb amide ( + ) -s1 ( for structures s1s11 see the supporting information ) ( 15.35 g , 49.46 mmol , 83% ) as a brown oil . [ ]d21 + 76.1 ( c 1 , chcl3 ) ; ir ( neat , cm ) 2935 , 1678 , 1513 , 1444 , 1393 , 1303 ; h nmr ( 500 mhz , cdcl3 ) 7.16 ( d , j = 8.5 hz , 2h ) , 6.87 ( d , j = 8.5 hz , 2h ) , 5.15 ( d , j = 15.3 hz , 1h ) , 4.40 ( dd , j = 5.7 , 8.1 hz , 1h ) , 3.85 ( d , j = 14.9 hz , 1h ) , 3.80 ( s , 3h ) , 3.47 ( dd , j = 8.9 , 11.3 hz , 1h ) , 3.38 ( s , 3h ) , 3.21 ( s , 3h ) , 3.16 ( dd , j = 4.8 , 11.3 hz , 1h ) ; c nmr ( 125 mhz , cdcl3 ) 172.5 , 169.4 , 159.6 , 130.3 , 127.9 , 114.4 , 61.5 , 57.7 , 55.5 , 47.2 , 32.8 , 28.3 ; hrms ( esi - tof ) m / z [ m + na ] calcd for c14h18n2nao4s 333.0885 , found 333.0887 . to a solution of weinreb amide ( + ) -s1 ( 316 mg , 1.018 mmol ) in thf ( 3 ml ) cooled to 0 c was added dropwise a solution of freshly prepared vinylmagnesium bromide in thf ( 2.1 m , 2.5 ml ) . the reaction mixture was stirred for 15 min before it was slowly poured into stirring 2 n hcl ( 10 ml ) at room temperature and extracted with ch2cl2 ( 3 15 ml ) . the combined organic layers were washed with a saturated aqueous solution of nahco3 , dried over na2so4 , decanted , and concentrated in vacuo . the crude mixture was purified via column chromatography on sio2 ( 60% etoac / hexanes ) to provide ( + ) -9 ( 181 mg , 0.653 mmol , 64% ) as a yellow oil . [ ]d21 + 69.4 ( c 0.75 , chcl3 ) ; ir ( neat , cm ) 1672 , 1612 , 1513 , 1248 , 1175 ; h nmr ( 500 mhz , cdcl3 ) 7.12 ( d , j = 8.9 hz , 2h ) , 6.85 ( d , j = 8.5 hz , 2h ) , 6.49 ( dd , j = 11.1 , 17.4 hz , 1h ) , 6.36 ( dd , j = 1.0 , 17.0 hz , 1h ) , 5.90 ( dd , j = 1.0 , 10.5 hz , 1h ) , 5.07 ( d , j = 14.7 hz , 1h ) , 4.34 ( dd , j = 4.6 , 9.3 hz , 1h ) , 3.83 ( d , j = 15.1 hz , 1h ) , 3.80 ( s , 3h ) , 3.51 ( dd , j = 9.7 , 11.9 hz , 1h ) , 3.14 ( dd , j = 4.6 , 11.5 hz , 1h ) ; c nmr ( 125 mhz , cdcl3 ) 195.4 , 172.0 , 159.7 , 131.7 , 131.4 , 130.2 , 127.4 , 114.4 , 63.7 , 55.4 , 47.4 , 27.9 ; hrms ( esi - tof ) m / z [ m + na ] calcd for c14h15nnao3s 300.0670 , found 300.0684 . orange crystalline solid , melting point 8789 c ; [ ]d21 + 40.9 ( c 1.0 , chcl3 ) ; ir ( neat , cm ) 2935 , 1724 , 1674 , 1611 , 1513 ; h nmr ( 500 mhz , cdcl3 ) 7.13 ( d , j = 8.5 hz , 2h ) , 6.86 ( d , j = 8.5 hz , 2h ) , 5.06 ( d , j = 13.9 hz , 1h ) , 4.19 ( dd , j = 5.2 , 9.3 hz , 1h ) , 3.86 ( d , j = 13.3 hz , 1h ) , 3.80 ( s , 3h ) , 3.51 ( dd , j = 10.3 , 11.3 hz , 1h ) , 3.43 ( s , 3h ) , 3.18 ( dd , j = 4.0 , 12.3 hz , 1h ) , 2.972.83 ( m , 2h ) , 2.66 ( t , j = 6.3 hz , 2h ) , 2.57 ( s , 3h ) ; c nmr ( 125 mhz , cdcl3 ) 205.4 , 171.9 , 159.7 , 130.1 , 127.6 , 114.5 , 65.2 , 60.0 , 55.5 , 54.8 , 47.5 , 45.0 , 36.9 , 27.9 ; hrms ( esi - tof ) m / z ( m + h ) calcd for c16h23n2o4s 339.1379 , found 339.1374 . to a solution of alcohol ( )-13 ( 1 g , 8.76 mmol ) in ch2cl2 ( 30 ml ) at rt was added imidazole ( 775 mg , 11.39 mmol ) followed by tbscl ( 1.39 g , 9.2 mmol ) . after 30 min , 0.5 m hcl ( 20 ml ) and ch2cl2 ( 20 ml ) were added . the aqueous layer was separated and extracted with ch2cl2 ( 2 20 ml ) . the combined organic layers were washed with a saturated aqueous solution of nahco3 ( 10 ml ) and brine ( 10 ml ) sequentially and then dried over na2so4 , decanted , and concentrated in vacuo . the crude mixture was purified via column chromatography on sio2 ( 5% et2o / hexanes ) to provide ( )-s2 ( 1.804 g , 7.89 mmol , 90% ) as a clear oil . h nmr ( 500 mhz , cdcl3 ) 5.915.75 ( m , 1h ) , 5.02 ( dd , j = 17.1 , 1.7 hz , 1h ) , 4.95 ( app d , j = 10 hz , 1h ) , 3.46 ( dd , j = 5.9 , 9.5 hz , 1h ) , 3.39 ( dd , j = 6.7 , 9.5 hz , 1h ) , 2.202.08 ( m , 1h ) , 2.071.95 ( m , 1h ) , 1.701.58 ( m , 1h ) , 1.571.46 ( m , 1h ) , 1.231.11 ( m , 1h ) , 0.91 ( s , 9h ) , 0.89 ( d , j = 6.7 hz , 3h ) , 0.05 ( s , 6h ) ; c nmr ( 125 mhz , cdcl3 ) 139.5 , 114.3 , 68.5 , 35.5 , 32.6 , 31.5 , 26.2 , 18.6 , 16.8 , 5.1 . ozone was bubbled through a solution of olefin ( )-s2 ( 3.98 g , 17.42 mmol ) in ch2cl2 ( 60 ml ) at 78 c until the reaction mixture appeared blue ( 3 h ) . a stream of nitrogen was then bubbled through the reaction mixture until the reaction mixture was again clear and no blue color remained . triphenylphosphine ( 4.71 g , 17.94 mmol ) was then added in one portion at 78 c , and after the addition the reaction mixture was allowed to warm to rt and stirred overnight . et3n - buffered silica ( stirred with 2 ml of et3n and 100 ml of hexanes ) was added , and the mixture was concentrated in vacuo . the crude mixture , adsorbed onto et3n - buffered silica , was purified via column chromatography on sio2 ( 100% hexanes to 5% et2o / hexanes ) to provide ( )-8 ( 3.15 g , 13.67 mmol , 79% ) as a clear oil . the spectral data matched those previously reported.h nmr ( 500 mhz , cdcl3 ) 9.829.72 ( m , 1h ) , 3.43 ( s , 2h ) , 2.532.38 ( m , 2h ) , 1.831.73 ( m , 1h ) , 1.681.57 ( m , 1h ) , 1.501.39 ( m , 1h ) , 0.980.81 ( m , 12h ) , 0.04 ( s , 6h ) ; c nmr ( 125 mhz , cdcl3 ) 203.0 , 68.0 , 41.9 , 35.5 , 26.1 , 25.7 , 18.5 , 16.7 , 5.2 , 5.2 . to a solution of ( )-b - methoxydiisopinocampheylborane ( 3.72 g , 11.8 mmol ) in et2o ( 29 ml ) at 0 c was added a 1 m solution of allylmagnesium bromide in et2o ( 11.8 ml , 11.8 mmol ) . after the addition was complete , the ice bath was removed and the reaction mixture was stirred for 1 h at rt . the mixture was cooled to 78 c , and a solution of aldehyde ( )-8 ( 2.58 g , 11.2 mmol ) in et2o ( 10 ml ) was added dropwise down the side of the flask ; additional et2o ( 5 ml ) was used to wash any residual aldehyde . the reaction mixture was stirred at 78 c for 3 h and then allowed to slowly warm to rt overnight , after which naoh ( 3 n , 8 ml ) and h2o2 ( 30% w / w , 3 ml ) were added and the mixture was refluxed for 2 h. after cooling , the mixture was extracted with et2o ( 2 100 ml ) , and the combined organic layers were dried over na2so4 , decanted , and concentrated in vacuo . the crude mixture was purified via column chromatography on sio2 ( 10% etoac / hexanes ) to provide the allylic alcohol ( 3.10 g ) contaminated with a minor amount of the epimeric alcohol , which was used in next reaction without further purification . to a portion of the allylic alcohol mixture ( 197 mg ) in dce ( 4 ml ) was added ( + ) -9 ( 600 mg , 2.16 mmol , 3 equiv ) , and the mixture was sparged with n2 for 20 min . hoveyda grubbs second - generation catalyst ( 45 mg , 0.072 mmol , 10 mol % ) was then added at rt , after which n2 sparging was resumed and the reaction mixture was heated to 50 c . . 50 mg ) was added , and the reaction mixture was stirred for 1 h. silica ( ca . 1 g ) was then added , and the solvent was removed in vacuo . the crude mixture was purified via column chromatography on sio2 ( 10% etoac / ch2cl2 ) to provide ( + ) -6 [ 262 mg , 0.502 mmol , 70% from aldehyde ( )-8 ] and recovered ( + ) -9 ( 287 mg , 1.04 mmol , 72% recovery ) . ( + ) -6 : [ ]d21 + 66.7 ( c 0.93 , chcl3 ) ; ir ( neat ) 3459 , 2929 , 2856 , 1682 , 1514 , 1250 ; h nmr ( 500 mhz , cdcl3 ) 7.12 ( d , j = 8.7 hz , 2h ) , 7.03 ( dt , j = 15.5 , 6.9 hz , 1h ) , 6.84 ( d , j = 7.9 hz , 2h ) , 6.27 ( d , j = 15.9 hz , 1h ) , 5.04 ( d , j = 15.7 hz , 1h ) , 4.30 ( dd , j = 4.6 , 9.3 hz , 1h ) , 3.84 ( d , j = 15.9 hz , 1h ) , 3.79 ( s , 3h ) , 3.763.70 ( m , 1h ) , 3.49 ( dd , j = 9.3 , 11.7 hz , 1h ) , 3.43 ( d , j = 5.9 hz , 2h ) , 3.14 ( dd , j = 4.8 , 10.1 hz , 1h ) , 2.472.39 ( m , 1h ) , 2.382.28 ( m , 1h ) , 1.901.81 ( m , 1h ) , 1.661.59 ( m , 1h ) , 1.591.52 ( m , 2h ) , 1.511.39 ( m , 1h ) , 1.181.09 ( m , 1h ) , 0.910.86 ( m , 12h ) , 0.05 ( s , 6h ) ; c nmr ( 125 mhz , cdcl3 ) 195.0 , 172.2 , 159.6 , 148.1 , 130.2 , 127.6 , 127.0 , 114.4 , 71.1 , 68.3 , 64.1 , 55.5 , 47.5 , 40.6 , 35.9 , 35.1 , 29.3 , 28.2 , 26.1 , 18.5 , 17.0 , 5.2 , 5.2 ; hrms ( esi - tof ) m / z ( m + h ) calcd for c27h44no5ssi 522.2709 , found 522.2688 . to a solution of enone ( + ) -6 ( 25 mg , 0.048 mmol ) in thf ( 0.28 ml ) was added 6 n hcl ( 0.21 ml ) dropwise at 20 c . after the reaction mixture was stirred for 19 h , a saturated aqueous solution of nahco3 ( 5 ml ) was added , followed by extraction with ch2cl2 ( 3 10 ml ) . the combined organic layers were dried over na2so4 , decanted , and concentrated in vacuo . the crude mixture was purified via column chromatography on sio2 ( 100% etoac ) to provide lactol ( + ) -16 ( 16 mg ) as a yellow oil with minor impurities that was used in the following reaction without further purification . [ ]d21 + 30.0 ( c 1.0 , chcl3 ) ; ir ( neat ) 3370 , 2934 , 1650 , 1513 , 1444 , 1400 ; h nmr ( 500 mhz , cdcl3 ) 7.2 ( d , j = 8.2 hz , 1h ) , 6.87 ( d , j = 8.5 hz , 1h ) , 5.16 ( d , j = 14.9 hz , 1h ) , 4.45 ( d , j = 14.3 hz , 1h ) , 4.194.11 ( m , 1h ) , 3.933.85 ( m , 1h ) , 3.81 ( s , 3h ) , 3.64 ( dd , j = 5.2 , 7.7 hz , 1h ) , 3.45 ( d , j = 5.7 hz , 2h ) , 3.293.23 ( m , 2h ) , 2.15 ( dd , j = 4.1 , 12.0 hz , 1h ) , 2.0 ( d , j = 12 hz , 1h ) , 1.89 ( bs , 2h ) , 1.661.53 ( m , 4h ) , 1.351.11 ( m , 3h ) , 0.92 ( d , j = 6.5 hz , 3h ) ; c nmr ( 125 mhz , cdcl3 ) 174.3 , 159.4 , 129.8 , 128.8 , 114.4 , 100.5 , 70.0 , 68.1 , 64.7 , 64.5 , 55.5 , 48.4 , 40.5 , 38.9 , 35.9 , 33.1 , 29.1 , 26.7 , 16.7 ; hrms ( esi - tof ) m / z ( m + na ) calcd for c21h31nnao6s 448.1770 , found 448.1782 . to a solution of lactol ( + ) -16 ( 16 mg ) with minor impurities in meoh ( 0.4 ml ) was added camphorsulfonic acid ( 1 mg , 0.004 mmol ) , and the reaction mixture was stirred overnight at rt . the reaction was quenched with a saturated aqueous solution of nahco3 ( 5 ml ) , which was followed by extraction with ch2cl2 ( 3 10 ml ) . the combined organic layers were dried over na2so4 , decanted , and concentrated in vacuo . the crude mixture was purified via column chromatography on sio2 ( 80% etoac / hexanes ) to provide ( + ) -5 [ 9 mg , 0.021 mmol , 43% from enone ( + ) -6 ] as a clear oil . [ ]d21 + 46.1 ( c 0.95 , chcl3 ) ; ir ( neat ) 3411 , 2933 , 1655 , 1513 , 1452 , 1396 , 1248 ; h nmr ( 500 mhz , cdcl3 ) 7.15 ( d , j = 9.1 hz , 2h ) , 6.88 ( d , j = 8.5 hz , 2h ) , 5.24 ( d , j = 15.1 hz , 1h ) , 4.22 ( d , j = 14.9 hz , 1h ) , 4.134.03 ( m , 1h ) , 3.95 ( dd , j = 4.8 , 10.3 hz , 1h ) , 3.81 ( s , 3h ) , 3.563.49 ( m , 1h ) , 3.46 ( t , j = 5.7 hz , 1h ) , 3.413.26 ( m , 2h ) , 3.02 ( s , 3h ) , 2.11 ( dd , j = 4.6 , 11.9 hz , 1h ) , 1.99 ( d , j = 11.7 hz , 1h ) , 1.731.43 ( m , 8h ) , 1.291.16 ( m , 1h ) , 1.151.07 ( m , 1h ) , 0.91 ( d , j = 6.5 hz , 3h ) ; c nmr ( 125 mhz , cdcl3 ) 173.4 , 159.2 , 128.8 , 128.6 , 114.3 , 102.6 , 70.5 , 68.1 , 64.8 , 57.0 , 55.4 , 47.9 , 46.9 , 40.2 , 37.8 , 35.8 , 33.1 , 28.9 , 26.4 , 16.6 ; hrms ( esi - tof ) m / z ( m + na ) calcd for c22h33nnao6s 462.1926 , found 462.1912 . to a solution of methyl ketal ( + ) -5 ( 165 mg , 0.375 mmol ) in ch2cl2 ( 4 ml ) were added imidazole ( 153 mg , 2.25 mmol ) , triphenyl phosphine ( 296 mg , 1.13 mmol ) , and finally iodine ( 248 mg , 0.975 mmol ) , and the reaction mixture was stirred overnight at rt . a 1:1 mixture of a 10% aqueous solution of na2s2o3 ( 5 ml ) and a saturated aqueous solution of sodium bicarbonate ( 5 ml ) was added to quench the reaction mixture . to the resulting biphasic solution was added additional ch2cl2 ( 5 ml ) , and the organic layer was removed . the aqueous layer was extracted with ch2cl2 ( 2 5 ml ) , and the combined organic layers were dried over na2so4 , decanted , and concentrated in vacuo . the crude mixture was purified via column chromatography on sio2 ( 40% etoac / hexanes ) to provide the primary iodide ( 172 mg , 0.313 mmol , 84% ) as a clear oil that was used directly in the next reaction . to a solution of the prepared primary iodide ( 170 mg , 0.31 mmol ) in ch2cl2 ( 3 ml ) was added imidazole ( 53 mg , 0.78 mmol ) , dmap ( 19 mg , 0.16 mmol ) , and then tbscl ( 71 mg , 0.47 mmol ) portionwise . after the reaction mixture was stirred overnight , a saturated aqueous solution of sodium bicarbonate ( 10 ml ) was added to quench the reaction mixture . to the resulting biphasic solution was added additional ch2cl2 ( 15 ml ) , and the organic layer was removed . the aqueous layer was extracted with ch2cl2 ( 2 15 ml ) , and the combined organic layers were dried over na2so4 , decanted , and concentrated in vacuo . the crude mixture was purified via column chromatography on sio2 ( 10% etoac / hexanes ) to provide the tbs - ether ( 175 mg , 0.264 mmol , 84% ) as a clear oil that was used directly in the next reaction . to a solution of the tbs - ether ( 108 mg , 0.163 mmol ) in acetonitrile ( 2 ml ) were added triphenylphosphine ( 640 mg , 2.44 mmol ) and i - pr2net ( 0.2 ml , 1.14 mmol ) , and the reaction mixture was heated to 55 c for 48 h and then concentrated in vacuo to afford an orange oil that was purified via filtration through a short pad of sio2 . washing with etoac to remove residual triphenylphosphine and triphenylphosphine oxide followed by washing with 5% meoh in ch2cl2 provided wittig reagent ( + ) -17 as an orange foam ( 118 mg , 0.13 mmol , 78% ) . [ ]d21 + 24.6 ( c 1.0 , chcl3 ) ; ir ( neat , cm ) 2927 , 1667 , 1512 , 1438 ; h nmr ( 500 mhz , cdcl3 ) 7.907.82 ( m , 4h ) , 7.717.61 ( m , 8h ) , 7.55 ( t , j = 7.7 hz , 1h ) , 7.507.44 ( m , 2h ) , 7.16 ( d , j = 8.6 hz , 2h ) , 6.91 ( d , j = 8.4 hz , 2h ) , 5.15 ( d , j = 16.1 hz , 1h ) , 4.19 ( d , j = 16.1 hz , 1h ) , 4.023.86 ( m , 2h ) , 3.81 ( s , 3h ) , 3.763.63 ( m , 2h ) , 3.40 ( t , j = 9.6 hz , 1h ) , 3.273.18 ( m , 2h ) , 2.84 ( s , 3h ) , 1.86 ( dd , j = 4.5 , 13.5 hz , 1h ) , 1.861.78 ( m , 1h ) , 1.771.65 ( m , 4h ) , 1.581.47 ( m , 3h ) , 1.251.18 ( m , 1h ) , 1.04 ( d , j = 6.4 hz , 3h ) , 0.87 , ( s , 9h ) , 0.06 ( s , 6h ) ; c nmr ( 125 mhz , cdcl3 ) 173.5 , 159.3 , 135.2 , 133.9 ( d , j = 9.6 hz ) , 132.3 ( d , j = 11.8 hz ) , 132.1 , 130.6 ( d , j = 11.8 hz ) , 129.0 , 128.8 ( d , j = 3.2 hz ) , 128.6 , 119.5 , 118.8 , 114.4 , 102.6 , 69.8 , 65.2 , 57.0 , 55.6 , 48.3 , 46.9 , 37.8 , 33.7 ( d , j = 10.2 hz ) , 33.3 , 30.3 , 29.9 , 29.8 ( d , j = 3.3 hz ) , 26.5 , 26.1 , 21.0 ( d , j = 8.7 hz ) , 18.2 , 4.3 , 4.4 ; hrms ( esi - tof ) m / z ( m ) calcd for c46h61no5pssi 798.3777 , found 798.3763 . to a solution of wittig reagent ( + ) -17 ( 5 mg , 5 mol ) in thf ( 0.1 ml ) at 0 c was slowly added nahmds ( 1 m , 50 l ) , which turned the reaction mixture red . after the reaction mixture was stirred at 0 c for 10 min , benzaldehyde ( 10 l , 10 mg , 0.01 mmol ) was added . after 5 min of stirring , the reaction mixture was quenched with a saturated aqueous solution of ammonium chloride ( 5 ml ) . to the resulting biphasic solution was added additional ch2cl2 ( 5 ml ) , and the organic layer was removed . the aqueous layer was extracted with ch2cl2 ( 2 5 ml ) , and the combined organic layers were dried over na2so4 , decanted , and concentrated in vacuo . the crude mixture was purified via column chromatography on sio2 ( 40% etoac / hexanes ) to provide ( + ) -21 with an unidentified contaminant . the mixture was then further purified via preparative tlc ( 250 m , 60% etoac / hexanes ) to provide ( + ) -21 ( 1.6 mg , 2.2 mol , 44% ) as a clear film . [ ]d21 + 33.0 ( c 0.11 , chcl3 ) ; ir ( neat , cm ) 2926 , 2854 , 1673 , 1514 , 1459 , 1386 ; h nmr ( 500 mhz , cdcl3 ) 7.767.69 ( m , 4h ) , 7.527.36 ( m , 6h ) , 7.15 ( d , j = 8.5 hz , 2h ) , 6.89 ( d , j = 8.5 hz , 2h ) , 5.19 ( d , j = 15.6 hz , 1h ) , 4.21 ( d , j = 15.6 hz , 1h ) , 3.96 ( sept , j = 5.9 hz , 1h ) , 3.83 ( dd , j = 4.4 , 9.3 hz , 1h ) , 3.81 ( s , 3h ) , 3.373.20 ( m , 3h ) , 2.88 ( s , 3h ) , 2.312.22 ( m , 1h ) , 2.122.11 ( m , 1h ) , 2.081.96 ( m , 1h ) , 1.87 ( dd , j = 4.7 , 13.1 hz ) , 1.72 ( app dt , j = 2.2 , 12.8 hz , 1h ) , 1.55 ( dd , j = 10.4 , 13.1 hz , 2h ) , 1.471.33 ( m , 2h ) , 1.15 ( q , j = 12.7 hz , 2h ) , 0.98 ( d , j = 6.7 hz , 3h ) , 0.88 ( s , 9h ) , 0.06 ( s , 6h ) ; c nmr ( 125 mhz , cdcl3 ) 173.6 , 159.0 , 131.6 , 131.6 , 130.7 ( d , j = 10.4 hz ) , 130.6 ( d , j = 9.5 hz ) , 128.8 , 128.7 ( d , j = 5.3 hz ) , 128.6 ( d , j = 5.6 hz ) , 128.5 , 114.1 , 102.5 , 69.9 , 65.1 , 57.0 , 55.3 , 47.7 , 46.7 , 40.8 , 37.6 , 37.1 , 36.5 , 34.0 , 32.7 , 29.7 , 28.1 ( d , j = 3.4 hz ) , 26.3 , 25.9 , 21.5 ( d , j = 8.3 hz ) , 18.1 , 4.5 , 4.6 . hrms ( esi - tof ) m / z ( m + h ) calcd for c40h57no6pssi 738.3414 , found 738.3427 . to a solution of ketal ( + ) -5 ( 400 mg , 0.91 mmol ) in ch2cl2 ( 9 ml ) cooled to 0 c was added tempo ( 21 mg , 0.137 mmol ) followed by ( diacetoxyiodo)benzene ( 264 mg , 0.819 mmol ) portionwise . after 12 h , the reaction mixture was partitioned between ch2cl2 ( 10 ml ) and a saturated aqueous solution of na2s2o3 ( 10 ml ) . the combined organic layers were dried over na2so4 , decanted , and concentrated in vacuo . the crude mixture was purified via column chromatography on sio2 ( 60% etoac / hexanes ) to provide hydroxy aldehyde ( + ) -s3 ( 286 mg , 0.654 mmol , 72% ) as a clear oil . [ ]d21 + 40.9 ( c 0.39 , chcl3 ) ; ir ( neat ) 3438 , 2929 , 2845 , 1721 , 1669 , 1612 , 1513 , 1456 , 1395 ; h nmr ( 500 mhz , cdcl3 ) 9.59 ( d , j = 1.6 hz , 1h ) , 7.14 ( d , j = 8.5 hz , 2h ) , 6.87 ( d , j = 8.5 hz , 2h ) , 5.22 ( d , j = 15.3 hz , 1h ) , 4.21 ( d , j = 14.9 hz , 1h ) , 4.07 ( sept , j = 4.2 hz , 1h ) , 3.96 ( dd , j = 3.2 , 9.3 hz , 1h ) , 3.80 ( s , 3h ) , 3.583.48 ( m , 1h ) , 3.433.27 ( m , 2h ) , 3.00 ( s , 3h ) , 2.32 ( q , j = 7.1 hz , 1h ) , 2.10 ( dd , j = 5.2 , 12.5 hz , 1h ) , 1.97 ( d , j = 12.7 hz , 2h ) , 1.931.77 ( m , 1h ) , 1.65 ( t , j = 11.1 hz , 1h ) , 1.53 ( q , j = 7.5 hz , 2h ) , 1.401.30 ( m , 1h ) , 1.291.15 ( m , 2h ) , 1.10 ( d , j = 7.1 hz , 3h ) , 0.910.75 ( m , 1h ) ; c nmr ( 125 mhz , cdcl3 ) 204.8 , 173.5 , 159.2 , 128.7 , 128.6 , 114.3 , 102.7 , 70.1 , 64.5 , 56.9 , 55.4 , 47.8 , 46.8 , 46.3 , 40.1 , 37.7 , 33.0 , 26.4 , 26.3 , 13.6 ; hrms ( esi - tof ) m / z ( m + h ) calcd for c22h32no6s 438.1950 , found 438.1938 . to a solution of iph3pch2i ( 1.26 g , 2.38 mmol ) in thf ( 24 ml ) was added nahmds ( 1 m , 1.9 ml ) at rt , and the reaction mixture was stirred for ca . hmpa ( 0.66 ml , 3.81 mmol ) was added , and the reaction mixture was further cooled to 78 c , after which hydroxy aldehyde ( + ) -s3 ( 104 mg , 0.24 mmol ) in thf ( ca . 1 after 1 h of stirring at 78 c , the reaction mixture was quenched by the addition of a saturated aqueous solution of ammonium chloride ( 5 ml ) . the biphasic mixture was extracted with diethyl ether ( 3 5 ml ) , and the combined organic layers were dried over na2so4 , decanted , and concentrated in vacuo . the crude mixture was purified via column chromatography on sio2 ( 4550% etoac / hexanes ) to provide the hydroxyvinyl iodide ( 60 mg , 0.107 mmol , 45% ) as a brown foam that was used directly in the next reaction . to a solution of the hydroxy vinyl iodide ( 60 mg , 0.11 mmol ) in ch2cl2 ( 0.4 ml ) were added imidazole ( 15 mg , 0.214 mmol ) , dmap ( 7 mg , 0.054 mmol ) , and finally tbscl ( 24 mg , 0.16 mmol ) , and the resulting mixture was stirred at rt overnight . the reaction was incomplete after 14 h , so again imidazole ( 15 mg , 0.214 mmol ) , dmap ( 7 mg , 0.054 mmol ) , and finally tbscl ( 24 mg , 0.16 mmol ) were added . after 12 h , a saturated aqueous solution of sodium bicarbonate ( ca . 5 ml ) was added to quench the reaction . the biphasic mixture was extracted with ch2cl2 ( 3 5 ml ) , and the combined organic layers were dried over na2so4 , decanted , and concentrated in vacuo . the crude mixture was purified via column chromatography on sio2 ( 30% etoac / hexanes ) to provide vinyl iodide ( + ) -18 ( 74 mg , 0.11 mmol , quant . ) as an oil . [ ]d21 + 15.4 ( c 0.14 , chcl3 ) ; ir ( neat , cm ) 2927 , 2856 , 1676 , 1513 , 1457 , 1389 ; h nmr ( 500 mhz , cdcl3 ) 7.16 ( d , j = 8.7 hz , 2h ) , 6.87 ( d , j = 8.7 hz , 2h ) , 6.15 ( d , 7.1 hz , 1h ) , 5.88 ( dd , j = 7.4 , 9.0 hz , 1h ) , 5.20 ( d , j = 15.3 hz , 1h ) , 4.26 ( d , j = 15.4 hz , 1h ) , 4.02 ( sept , j = 5.0 hz , 1h ) , 3.95 ( dd , j = 4.0 , 9.3 hz , 1h ) , 3.81 ( s , 3h ) , 3.563.50 ( m , 1h ) , 3.393.29 ( m , 2h ) , 3.04 ( s , 3h ) , 2.50 ( quin , j = 6.0 hz , 1h ) , 1.92 ( dd , j = 1.4 , 4.9 hz , 1h ) , 1.81 ( dt , j = 2.3 , 12.4 hz , 1h ) , 1.59 ( dd , j = 10.5 , 13.0 hz , 1h ) , 1.521.36 ( m , 5h ) , 1.24 ( d , j = 12.2 hz , 2h ) , 0.98 ( d , j = 7.1 hz , 3h ) , 0.89 ( s , 9h ) , 0.076 ( s , 6h ) ; c nmr ( 125 mhz , cdcl3 ) 173.8 , 159.2 , 146.5 , 129.0 , 128.8 , 114.4 , 102.8 , 81.2 , 70.0 , 65.4 , 57.5 , 55.5 , 48.0 , 47.1 , 41.0 , 39.4 , 38.1 , 33.2 , 31.9 , 29.9 , 26.5 , 26.1 , 19.6 , 18.3 , 4.3 , 4.4 ; hrms ( esi - tof ) m / z ( m + na ) calcd for c29h46innao5ssi 698.1808 , found 698.1774 . to a 1.0 m solution of t - buok in thf ( 4.6 ml , 4.6 mmol ) cooled to 78 c was added seyferth gilbert reagent ( 789 mg , 5.26 mmol ) in thf ( 7 ml ) down the side of the reaction vessel . the reagent was washed with thf ( 3 ml ) , and the reaction mixture turned yellow - orange but remained transparent . after 25 min , aldehyde ( + ) -s3 ( 1.15 g , 2.63 mmol ) in thf ( 10 ml ) was added to the solution dropwise and then washed with additional thf ( 5 ml ) . the yellow - orange reaction mixture was quenched with a saturated aqueous solution of nahco3 ( 15 ml ) and ch2cl2 ( 20 ml ) at 78 c . the aqueous layer was separated and extracted with ch2cl2 ( 2 30 ml ) . the combined organic layers were dried over na2so4 , decanted , and concentrated in vacuo . the crude mixture was purified via column chromatography on sio2 ( 50% etoac / hexanes ) to provide alkyne ( + ) -s4 ( 852 mg , 1.97 mmol , 75% ) as a white foam . [ ]d21 + 52.8 ( c 0.23 , chcl3 ) ; ir ( neat ) 3416 , 2931 , 1670 , 1513 ; h nmr ( 500 mhz , cdcl3 ) 7.14 ( d , j = 8.5 hz , 2h ) , 6.87 ( d , j = 8.5 hz , 2h ) , 5.22 ( d , j = 16.1 hz , 1h ) , 4.22 ( d , j = 15.5 hz , 1h ) , 4.07 ( sept , j = 4.8 hz , 1h ) , 3.95 ( dd , j = 4.2 , 7.9 hz , 1h ) , 3.80 ( s , 3h ) , 3.583.47 ( m , 1h ) , 3.433.28 ( m , 2h ) , 3.01 ( s , 3h ) , 2.482.32 ( m , 1h ) , 2.10 ( dd , j = 4.2 , 12.7 hz , 1h ) , 2.04 ( d , j = 2.6 hz , 1h ) , 1.98 ( d , j = 14.9 hz , 1h ) , 1.811.68 ( m , 1h ) , 1.671.54 ( m , 3h ) , 1.491.35 ( m , 1h ) , 1.301.20 ( m , 1h ) , 1.17 ( d , j = 6.9 hz , 3h ) ; c nmr ( 125 mhz , cdcl3 ) 173.6 , 159.2 , 128.8 , 128.7 , 114.4 , 102.7 , 88.6 , 70.0 , 68.9 , 64.7 , 57.1 , 55.5 , 47.9 , 46.9 , 40.3 , 37.9 , 33.4 , 32.5 , 26.4 , 25.9 , 21.1 ; hrms ( esi - tof ) m / z ( m + h ) calcd for c23h32no5s 434.2001 , found 434.2007 . to a solution of alkyne ( + ) -s4 ( 270 mg , 0.623 mmol ) in ch2cl2 ( 2.1 ml ) was added 2,6-lutidine ( 0.14 ml , 1.246 mmol ) . the reaction mixture was cooled to 0 c , and tbsotf ( 0.17 ml , 0.747 mmol ) was added dropwise . after 1 h , the reaction mixture was diluted with ch2cl2 ( 5 ml ) , and 0.5 n hcl ( 5 ml ) was added , the aqueous layer was separated and extracted with ch2cl2 ( 3 10 ml ) , and the combined organic layers were dried over na2so4 , decanted , and concentrated in vacuo . the crude mixture was purified via column chromatography on sio2 ( 30% etoac / hexanes ) to provide ( + ) -19 ( 341 mg , 0.623 mmol , near quant . ) as white crystals . [ ]d21 + 16.7 ( c 0.69 , chcl3 ) ; mp 7984 c ; ir ( neat ) 3307 , 2930 , 2856 , 1731 , 1675 , 1513 ; h nmr ( 500 mhz , cdcl3 ) 7.16 ( d , j = 8.3 hz , 2h ) , 6.87 ( d , j = 8.9 hz , 2h ) , 5.20 ( d , j = 15.3 hz , 1h ) , 4.26 ( d , j = 15.9 hz , 1h ) , 4.083.97 ( m , 1h ) , 3.94 ( dd , j = 4.6 , 9.7 hz , 1h ) , 3.853.76 ( m , 3h ) , 3.563.46 ( m , 1h ) , 3.34 ( s , 2h ) , 2.472.36 ( m , 1h ) , 2.04 ( d , j = 2.2 hz , 1h ) , 1.92 ( dd , j = 4.4 , 12.9 hz , 1h ) , 1.871.80 ( m , 1h ) , 1.761.67 ( m , 1h ) , 1.621.55 ( m , 5h ) , 1.461.37 ( m , 1h ) , 1.311.21 ( m , 2h ) , 1.18 ( d , j = 6.9 hz , 3h ) , 0.89 ( s , 9h ) , 0.08 ( s , 6h ) ; c nmr ( 125 mhz , cdcl3 ) 173.7 , 159.2 , 128.9 , 128.7 , 114.3 , 102.7 , 88.7 , 69.7 , 68.9 , 65.4 , 57.4 , 55.5 , 47.9 , 47.0 , 40.9 , 38.0 , 33.4 , 32.5 , 26.4 , 26.0 , 25.9 , 21.1 , 18.2 ; hrms ( esi - tof ) m / z ( m + h ) calcd for c29h46no5ssi 548.2866 , found 548.2878 . to a solution of diol ( )-22 ( 0.9 g , 7.89 mmol ) in ch2cl2 ( 80 ml ) was added imidazole ( 1.61 g , 23.67 mmol ) . the resulting mixture was cooled to 0 c , and tbscl ( 1.19 g , 7.89 mmol ) was added portionwise . the ice bath was removed after 30 min , and the reaction mixture was stirred at room temperature . after 14 h , 0.5 n hcl ( 40 ml ) was added , and the aqueous layer was extracted with ch2cl2 ( 3 40 ml ) . the organic layers were combined , washed sequentially with a saturated aqueous solution of nahco3 and brine , dried over na2so4 , decanted , and concentrated in vacuo . the crude mixture was purified via column chromatography on sio2 ( 20% to 100% etoac / hexanes ) to provide ( )-s5 ( 1.58 g , 6.9 mmol , 88% ) as a brown oil . [ ]d21 1.1 ( c 0.15 , chcl3 ) ; ir ( neat , cm ) 3447 , 3313 , 2118 , 1738 , 1471 , 1256 , 1121 ; h nmr ( 500 mhz , cdcl3 ) 3.79 ( sept , j = 4.2 hz , 1h ) , 3.66 ( dd , j = 3.6 , 9.7 hz , 1h ) , 3.45 ( dd , j = 7.1 , 9.9 hz , 1h ) , 2.36 ( dt , j = 2.1 , 7.1 hz , 2h ) , 1.97 ( t , j = 2.6 hz , 1h ) , 1.721.57 ( m , 2h ) , 0.91 ( s , 9h ) , 0.08 ( s , 6h ) ; c nmr ( 125 mhz , cdcl3 ) 84.2 , 70.7 , 68.7 , 67.1 , 31.8 , 26.1 , 18.5 , 15.0 , 5.2 , 5.2 ; hrms ( esi - tof ) m / z ( m + na ) calcd for c12h24nao2si 251.1443 , found 251.1441 . to a solution of tbs ether ( )-s5 ( 556 mg , 2.43 mmol ) in ch2cl2 ( 8 ml ) were added i - pr2net ( 1.7 ml , 9.72 mmol ) and semcl ( 0.52 ml , 72.92 mmol ) dropwise . an exit needle was placed through the septum to allow the smoky atmosphere to clear . after 14 h , a saturated aqueous solution of nahco3 ( 20 ml ) was added to quench the reaction mixture , and the aqueous layer was extracted with ch2cl2 ( 3 20 ml ) . the combined organic layers were washed with 10% citric acid ( 50 ml ) , dried over na2so4 , decanted , and concentrated in vacuo . the crude mixture was filtered through a pad of sio2 to yield a yellow oil that was used without further purification . to a solution of the previously obtained yellow oil in thf ( 8 ml ) cooled to 78 c was added a solution of n - buli in thf ( 2.4 m , 1.5 ml ) dropwise . after 20 min of stirring , methyl chloroformate ( 0.33 ml , 4.13 mmol ) was added dropwise . the reaction mixture was stirred for 1 h , and the dry ice bath was removed . after 3 h of stirring , et2o ( 10 ml ) was added , followed by a saturated aqueous solution of nahco3 ( 10 ml ) . the aqueous layer was extracted with etoac ( 3 20 ml ) , and the combined organic layers were washed with brine , dried over na2so4 , decanted , and concentrated in vacuo . the crude mixture was purified via column chromatography on sio2 ( 5% ether / hexanes ) to provide ( )-23 ( 737 mg , 1.77 mmol , 73% over two steps ) as a free - flowing oil . [ ]d21 36.6 ( c 1.0 , chcl3 ) ; ir ( neat , cm ) 2953 , 2239 , 1718 , 1435 , 1253 ; h nmr ( 500 mhz , cdcl3 ) 4.78 ( d , j = 6.7 hz , 1h ) , 4.71 ( d , j = 6.5 hz , 1h ) , 3.76 ( s , 3h ) , 3.713.53 ( m , 5h ) , 2.532.44 ( m , 2h ) , 1.931.81 ( m , 1h ) , 1.801.71 ( m , 1h ) , 0.95 ( t , j = 8.3 hz , 2h ) , 0.89 ( s , 9h ) , 0.06 ( s , 6h ) , 0.02 ( s , 9h ) ; c nmr ( 125 mhz , cdcl3 ) 154.4 , 94.9 , 89.6 , 73.2 , 65.6 , 65.3 , 52.7 , 30.1 , 26.1 , 18.5 , 18.3 , 15.0 , 1.2 , 5.2 , 5.2 ; hrms ( esi - tof ) m / z ( m + na ) calcd for c20h40nao5si2 439.2312 , found 439.2296 . to a suspension of cui ( 322 mg , 1.69 mmol ) in thf ( 8 ml ) cooled to 0 c was added a solution of meli in et2o ( 0.4 m , 2.25 ml , 3.38 mmol ) dropwise , and the reaction mixture turned orange and then clear . after 45 min of stirring , the me2culi solution was cooled to 78 c , and a solution of alkynoate ( )-23 ( 587 mg , 1.41 mmol ) in thf ( 8 ml ) was added . after 2 h , ph 7 buffer ( 10 ml ) and meoh ( 2 ml ) were added , and then the reaction mixture was extracted with etoac ( 3 20 ml ) . the combined organic layers were washed with brine , dried over na2so4 , decanted , and concentrated in vacuo . the crude mixture was purified via column chromatography on sio2 ( 10% ether / hexanes ) to provide enoate ( )-s6 ( 351 mg , 0.81 mmol , 58% ) as an oil . [ ]d21 6.8 ( c 0.67 , chcl3 ) ; ir ( neat , cm ) 2953 , 2929 , 2858 , 1722 , 1649 , 1250 ; h nmr ( 500 mhz , cdcl3 ) 5.67 ( s , 1h ) , 4.82 ( d , j = 7.5 hz , 1h ) , 4.74 ( d , j = 6.7 hz , 1h ) , 3.713.59 ( m , 6h ) , 3.67 ( s , 3h ) , 2.812.63 ( m , 2h ) , 1.90 ( s , 3h ) , 1.791.70 ( m , 1h ) , 1.641.59 ( m , 1h ) , 0.960.94 ( m , 1h ) , 0.90 ( s , 9h ) , 0.05 ( br s , 6h ) , 0.02 ( s , 9h ) ; c nmr ( 125 mhz , cdcl3 ) 166.8 , 160.8 , 116.1 , 94.9 , 78.5 , 65.8 , 65.3 , 51.0 , 30.3 , 29.7 , 26.1 , 25.3 , 18.5 , 18.3 , 1.2 , 5.2 , 5.2 ; hrms ( esi - tof ) m / z ( m + na ) calcd for c21h44nao5si2 455.2625 , found 455.2638 . to a solution of enoate ( )-s6 ( 351 mg , 0.811 mmol ) in thf ( 8 ml ) was added a solution of tbaf in thf ( 1 m , 1.6 ml ) that was premixed with acoh ( 0.12 ml ) . after 14 h , the reaction was quenched with a saturated aqueous solution of nh4cl ( 10 ml ) , and the aqueous layer was extracted with etoac ( 3 20 ml ) . the combined organic layers were washed with brine , dried over na2so4 , decanted , and concentrated in vacuo . the crude mixture was purified via column chromatography on sio2 ( 20% etoac / hexanes ) to provide ( + ) -s7 ( 257 mg , 0.807 mmol , 99% ) as an oil . [ ]d21 + 41.7 ( c 0.39 , chcl3 ) ; ir ( neat , cm ) 3443 , 2951 , 2891 , 1719 , 1647 , 1436 , 1248 ; h nmr ( 500 mhz , cdcl3 ) 5.755.64 ( m , 1h ) , 4.82 ( d , j = 7.3 hz , 1h ) , 4.70 ( d , j = 6.7 hz , 1h ) , 3.843.74 ( m , 1h ) , 3.68 ( s , 3h ) , 3.643.54 ( m , 4h ) , 2.792.68 ( m , 1h ) , 2.682.55 ( m , 1h ) , 1.91 ( s , 3h ) , 1.761.58 ( m , 2h ) , 1.040.90 ( m , 2h ) , 0.03 ( s , 9h ) ; c nmr ( 125 mhz , cdcl3 ) 166.9 , 160.6 , 116.2 , 95.5 , 82.2 , 65.9 , 65.5 , 51.1 , 30.1 , 29.6 , 25.4 , 18.4 , 1.3 ; hrms ( esi - tof ) m / z ( m + h ) calcd for c15h31o5si 319.1941 , found 319.1940 . to a solution of alcohol ( + ) -s7 ( 149 mg , 0.468 mmol ) in ch2cl2 ( 5 ml ) cooled to 0 c were added i - pr2net ( 0.41 ml , 2.34 mmol ) and dmso ( 0.33 ml , 4.68 mmol ) . so3pyridine ( 223 mg , 1.4 mmol ) was then added in one portion . after 15 min , the reaction mixture was diluted with ch2cl2 ( 10 ml ) , and a saturated aqueous solution of nahco3 ( 10 ml ) was added . the aqueous layer was separated and extracted with ch2cl2 ( 2 15 ml ) . the combined organic layers were washed with brine , dried over na2so4 , decanted , and concentrated in vacuo . the crude mixture was purified via short column chromatography on sio2 ( 30% etoac / hexanes ) to provide aldehyde ( + ) -24 ( 144 mg , 0.455 mmol , 97% ) as a brown oil . [ ]d21 + 5.2 ( c 1.3 , chcl3 ) ; ir ( neat ) 2952 , 1719 , 1650 , 1437 , 1378 , 1249 , 1193 ; h nmr ( 500 mhz , cdcl3 ) 9.66 ( d , j = 1.58 hz , 1h ) , 5.70 ( s , 1h ) , 4.82 ( d , j = 7.13 hz , 1h ) , 4.75 ( d , j = 6.94 hz , 1h ) , 3.94 ( ddd , j = 7.13 , 5.15 , 1.39 hz , 1h ) , 3.713.82 ( m , 1h ) , 3.68 ( s , 3h ) , 3.583.67 ( m , 1h ) , 2.732.83 ( m , 1h ) , 2.622.72 ( m , 1h ) , 1.90 ( s , 3h ) , 1.781.89 ( m , 2h ) , 0.93 ( s , 2h ) , 0.02 ( s , 9h ) ; c nmr ( 125 mhz , cdcl3 ) 202.9 , 166.7 , 159.3 , 116.8 , 95.3 , 82.4 , 66.1 , 51.1 , 29.2 , 28.6 , 25.3 , 18.2 , 1.3 ; hrms ( esi - tof ) m / z ( m + na ) calcd for c15h28nao5si 339.1604 , found 339.1605 . to zn(otf)2 ( dried by stirring overnight at 120 c under high vacuum , 1.12 g , 3.078 mmol ) were added ( + ) -nme ( azeotroped three times with toluene , 600 mg , 3.35 mmol ) and triethylamine ( distilled prior to use , 0.46 ml , 3.35 mmol ) in toluene ( 3.5 ml ) . the reaction mixture was stirred vigorously for 3 h. alkyne ( + ) -19 ( 300 mg , 0.548 mmol ) in toluene ( 1.3 ml ) was then added , and the mixture was stirred for 3 h. aldehyde ( + ) -24 ( 95 mg , 0.300 mmol ) was then added in toluene ( 0.5 ml ) , and the reaction mixture was stirred at rt overnight . the mixture was portioned between a saturated aqueous solution of nh4cl ( 10 ml ) and etoac ( 10 ml ) , and the aqueous layer was extracted with etoac ( 3 20 ml ) . the combined organic layers were washed with brine , dried over na2so4 , decanted , and concentrated in vacuo . the crude mixture was purified via column chromatography on sio2 ( 20% etoac / hexanes ) to provide alcohol ( + ) -25 ( 247 mg , 0.287 mmol , 95% ) as a clear oil . [ ]d21 + 51.5 ( c 0.79 , chcl3 ) ; ir ( neat ) 3440 , 2951 , 1719 , 1678 , 1513 ; h nmr ( 500 mhz , cdcl3 ) 7.15 ( d , j = 8.5 hz , 2h ) , 6.87 ( d , j = 8.7 hz , 2h ) , 5.67 ( s , 1h ) , 5.19 ( d , j = 15.3 hz , 1h ) , 4.88 ( d , j = 7.1 hz , 1h ) , 4.69 ( d , j = 6.9 hz , 1h ) , 4.28 ( d , j = 6.9 hz , 1h ) , 4.24 ( d , j = 15.3 hz , 1h ) , 4.01 ( sept , j = 5.0 hz , 1h ) , 3.93 ( dd , j = 4.0 , 9.5 hz , 1h ) , 3.79 ( s , 3h ) , 3.773.69 ( m , 1h ) , 3.693.59 ( m , 4h ) , 3.533.46 ( m , 2h ) , 3.383.28 ( m , 2h ) , 2.99 ( s , 3h ) , 2.902.82 ( m , 1h ) , 2.642.55 ( m , 1h ) , 2.492.40 ( m , 1h ) , 1.951.89 ( m , 2h ) , 1.88 ( d , j = 1.0 hz , 3h ) , 1.831.76 ( m , 1h ) , 1.751.63 ( m , 2h ) , 1.631.51 ( m , 3h ) , 1.451.36 ( m , 1h ) , 1.291.20 ( m , 2h ) , 1.14 ( s , 3h ) , 0.990.92 ( m , 2h ) , 0.88 ( s , 9h ) , 0.07 ( s , 6h ) , 0.01 ( s , 9h ) ; c nmr ( 125 mhz , cdcl3 ) 173.7 , 166.7 , 160.1 , 159.2 , 128.9 , 128.7 , 116.4 , 114.3 , 102.8 , 96.3 , 90.5 , 84.7 , 79.3 , 69.8 , 66.2 , 65.6 , 65.4 , 57.3 , 55.5 , 51.0 , 47.8 , 47.0 , 41.0 , 37.9 , 33.5 , 32.5 , 30.2 , 29.5 , 26.5 , 26.1 , 26.0 , 25.1 , 21.0 , 18.3 , 18.2 , 1.3 , 4.3 , 4.4 ; hrms ( esi - tof ) m / z ( m + na ) calcd for c44h73nnao10ssi2 886.4391 , found 886.4396 . to propargyl alcohol ( + ) -25 ( 43 mg , 0.0498 mmol ) was added meohhcl ( 1.5% ) ( 0.7 ml ) followed by ch2cl2 ( 0.5 ml ) to rinse the sides of the flask . after 3.5 h of stirring at rt , a saturated aqueous solution of nahco3 ( 5 ml ) was added to quench the reaction . the resulting mixture was extracted with ch2cl2 ( 2 20 ml ) . the combined organic layers were dried over na2so4 , decanted , and concentrated in vacuo . the crude mixture was purified via column chromatography on sio2 ( 80% to 100% etoac / hexanes ) to provide ( + ) -s8 ( 27 mg , 0.0436 mmol , 88% ) as a clear oil . [ ]d21 + 58.0 ( c 1.0 , chcl3 ) ; ir ( neat ) 3423 , 2945 , 1651 , 1513 , 1442 , 1395 ; h nmr ( 500 mhz , cdcl3 ) 7.15 ( d , j = 8.5 hz , 2h ) , 6.88 ( d , j = 8.5 hz , 2h ) , 5.835.72 ( m , 1h ) , 5.23 ( d , j = 16.2 hz , 1h ) , 4.22 ( d , j = 15.5 hz , 1h ) , 4.19 ( br s , 1h ) , 4.124.05 ( m , 1h ) , 4.03 ( d , j = 3.4 hz , 1h ) , 3.95 ( dd , j = 2.6 , 8.9 hz , 1h ) , 3.80 ( s , 3h ) , 3.70 ( s , 3h ) , 3.593.50 ( m , 1h ) , 3.503.43 ( m , 1h ) , 3.423.29 ( m , 2h ) , 3.193.09 ( m , 1h ) , 3.00 ( s , 3h ) , 2.81 ( d , j = 2.8 hz , 1h ) , 2.45 ( q , j = 6.1 hz , 1h ) , 2.32 ( quin , j = 5.5 hz , 1h ) , 2.11 ( dd , j = 3.2 , 12.1 hz , 1h ) , 1.97 ( d , j = 14.3 hz , 1h ) , 1.911.88 ( m , 3h ) , 1.66 ( br s , 2h ) , 1.63 ( br s , 3h ) , 1.601.55 ( m , 2h ) , 1.471.39 ( m , 1h ) , 1.251.20 ( m , 1h ) , 1.15 ( d , j = 6.9 hz , 3h ) ; c nmr ( 125 mhz , cdcl3 ) 173.5 , 168.2 , 160.0 , 159.3 , 128.8 , 128.7 , 117.2 , 114.4 , 102.7 , 90.7 , 79.2 , 73.6 , 70.0 , 66.3 , 64.8 , 57.1 , 55.5 , 51.6 , 47.9 , 46.9 , 40.3 , 37.9 , 33.4 , 32.6 , 30.8 , 29.3 , 26.4 , 26.1 , 24.9 , 21.1 ; hrms ( esi - tof ) m / z ( m + na ) calcd for c32h45nnao9s 642.2713 , found 642.2715 . to a solution of ( + ) -s8 ( 27 mg , 0.0436 mmol ) in 2,2-dimethoxypropane ( 1 ml ) were added acetone ( 0.2 ml ) and a small crystal of p - tsohh2o . after the reaction mixture was stirred for 30 min at rt , a saturated aqueous solution of nahco3 ( 5 ml ) was added to quench the reaction . the resulting mixture was extracted with ch2cl2 ( 2 20 ml ) . the combined organic layers were dried over na2so4 , decanted , and concentrated in vacuo . the crude mixture was purified via column chromatography on sio2 ( 40% etoac / hexanes ) to provide acetonide ( + ) -26 ( 25 mg , 0.0379 mmol , 87% ) as a clear oil . [ ]d21 + 27.5 ( c 1.0 , chcl3 ) ; ir ( neat ) 3447 , 2944 , 1715 , 1673 , 1513 , 1444 , 1379 ; h nmr ( 500 mhz , cdcl3 ) 7.15 ( d , j = 8.5 hz , 2h ) , 6.88 ( d , j = 8.7 hz , 2h ) , 5.765.63 ( m , 1h ) , 5.23 ( d , j = 16.6 hz , 1h ) , 4.29 ( dd , j = 1.6 , 7.3 hz , 1h ) , 4.22 ( d , j = 15.1 hz , 1h ) , 4.114.02 ( m , 1h ) , 4.023.91 ( m , 2h ) , 3.80 ( s , 3h ) , 3.68 ( s , 3h ) , 3.583.48 ( m , 1h ) , 3.423.29 ( m , 2h ) , 3.00 ( s , 3h ) , 2.882.77 ( m , 1h ) , 2.712.60 ( m , 1h ) , 2.46 ( q , j = 6.1 hz , 1h ) , 2.10 ( dd , j = 5.0 , 12.7 hz , 1h ) , 1.97 ( s , 1h ) , 1.91 ( d , j = 1.0 hz , 3h ) , 1.811.72 ( m , 3h ) , 1.711.65 ( m , 1h ) , 1.62 ( s , 3h ) , 1.601.56 ( m , 1h ) , 1.44 ( s , 3h ) , 1.40 ( s , 3h ) , 1.251.19 ( m , 1h ) , 1.15 ( d , j = 6.9 hz , 3h ) ; c nmr ( 125 mhz , cdcl3 ) 173.5 , 166.8 , 160.0 , 159.3 , 128.9 , 128.7 , 116.4 , 114.4 , 109.8 , 102.7 , 91.2 , 81.8 , 77.9 , 70.9 , 69.9 , 64.8 , 57.1 , 55.5 , 51.1 , 47.9 , 46.9 , 40.3 , 37.8 , 33.4 , 32.5 , 31.1 , 29.8 , 27.4 , 26.6 , 26.4 , 26.1 , 25.4 , 21.0 ; hrms ( esi - tof ) m / z ( m + na ) calcd for c35h49nnao9s 682.3026 , found 682.3026 . to a solution of propargylic alcohol ( + ) -25 ( 208 mg , 0.241 mmol ) and i - prnet2 in ch2cl2 ( 0.8 ml ) was added semcl dropwise . the combined organic layers were dried over na2so4 , decanted , and concentrated in vacuo . the crude mixture was purified via column chromatography on sio2 ( 12% etoac / hexanes ) to provide bis - sem ether ( + ) -s9 ( 210 mg , 0.211 mmol , 88% ) as a clear oil . [ ]d21 + 98.1 ( c 0.3 , chcl3 ) ; ir ( neat ) 2951 , 2895 , 1719 , 1681 , 1513 , 1456 ; h nmr ( 500 mhz , cdcl3 ) 7.15 ( d , j = 8.1 hz , 2h ) , 6.87 ( d , j = 8.1 hz , 2h ) , 5.65 ( s , 1h ) , 5.19 ( d , j = 15.3 hz , 1h ) , 4.91 ( d , j = 7.1 hz , 1h ) , 4.85 ( d , j = 8.3 hz , 1h ) , 4.75 ( d , j = 7.3 hz , 1h ) , 4.66 ( d , j = 7.1 hz , 1h ) , 4.45 ( d , j = 4.8 hz , 1h ) , 4.24 ( d , j = 16.4 hz , 1h ) , 4.02 ( sept , j = 4.6 hz , 1h ) , 3.94 ( dd , j = 3.6 , 9.1 hz , 1h ) , 3.80 ( s , 3h ) , 3.66 ( s , 7h ) , 3.573.44 ( m , 2h ) , 3.393.27 ( m , 2h ) , 2.98 ( s , 3h ) , 2.862.77 ( m , 1h ) , 2.712.61 ( m , 1h ) , 2.482.39 ( m , 1h ) , 1.89 ( br s , 1h ) , 1.901.85 ( m , 3h ) , 1.861.76 ( m , 1h ) , 1.55 ( br s , 6h ) , 1.441.36 ( m , 1h ) , 1.251.20 ( m , 1h ) , 1.14 ( d , j = 6.7 hz , 3h ) , 0.970.92 ( m , 4h ) , 0.89 ( s , 9h ) , 0.07 ( s , 6h ) , 0.030.01 ( m , 18h ) ; c nmr ( 125 mhz , cdcl3 ) 173.8 , 166.6 , 160.4 , 159.2 , 128.9 , 128.7 , 116.2 , 114.4 , 102.8 , 95.6 , 92.4 , 91.3 , 79.7 , 77.2 , 69.8 , 68.4 , 65.6 , 65.5 , 65.4 , 57.2 , 55.5 , 51.0 , 47.8 , 47.0 , 41.0 , 37.9 , 33.6 , 32.7 , 29.9 , 29.7 , 26.5 , 26.2 , 26.1 , 25.2 , 21.2 , 18.3 , 1.2 , 4.2 , 4.4 ; hrms ( esi - tof ) m / z ( m + na ) calcd for c50h87nnao11ssi3 1016.5205 , found 1016.5207 . to a solution of bis - sem ether ( + ) -s9 ( 346 mg , 0.348 mmol ) in thf ( 1.5 ml ) was added a premixed solution of tbaf in thf ( 1 m , 3.5 ml , 3.5 mmol ) and acetic acid ( 52 mg , 0.87 mmol ) at room temperature . after 14 h , a saturated aqueous solution of nh4cl ( 10 ml ) was added , and the biphasic mixture was extracted with ch2cl2 ( 3 40 ml ) . the combined organic layers were dried over na2so4 , decanted , and concentrated in vacuo . the crude mixture was purified via column chromatography on sio2 ( 40% etoac / hexanes ) to provide alcohol ( + ) -s10 ( 305 mg , 0.347 mmol , quant . ) as a clear oil . [ ]d21 + 50.8 ( c 0.2 , chcl3 ) ; ir ( neat ) 3458 , 2950 , 1718 , 1675 , 1513 , 1249 ; h nmr ( 500 mhz , cdcl3 ) 7.14 ( d , j = 8.5 hz , 2h ) , 6.87 ( d , j = 8.5 hz , 2h ) , 5.66 ( s , 1h ) , 5.23 ( d , j = 16.1 hz , 1h ) , 4.90 ( d , j = 6.7 hz , 1h ) , 4.85 ( d , j = 6.7 hz , 1h ) , 4.74 ( d , j = 7.1 hz , 1h ) , 4.66 ( d , j = 6.5 hz , 1h ) , 4.46 ( d , j = 4.0 hz , 1h ) , 4.21 ( d , j = 15.5 hz , 1h ) , 4.114.02 ( m , 1h ) , 3.95 ( dd , j = 3.3 , 9.2 hz , 1h ) , 3.80 ( s , 3h ) , 3.66 ( s , 7h ) , 3.573.48 ( m , 2h ) , 3.413.29 ( m , 2h ) , 3.00 ( br s , 3h ) , 2.80 ( dt , j = 6.1 , 11.9 hz , 1h ) , 2.69 ( dt , j = 5.7 , 10.9 hz , 1h ) , 2.492.40 ( m , 1h ) , 2.10 ( dd , j = 5.0 , 12.9 hz , 1h ) , 1.96 ( d , j = 12.1 hz , 1h ) , 1.88 ( s , 3h ) , 1.841.74 ( m , 2h ) , 1.681.55 ( m , 4h ) , 1.451.37 ( m , 1h ) , 1.221.18 ( m , 1h ) , 1.14 ( d , j = 6.7 hz , 3h ) , 0.970.90 ( m , 4h ) , 0.830.78 ( m , 1h ) , 0.01 ( d , j = 2.4 hz , 18h ) ; c nmr ( 125 mhz , cdcl3 ) 173.5 , 166.8 , 160.4 , 159.3 , 128.8 , 128.7 , 116.2 , 114.4 , 102.7 , 95.6 , 92.4 , 91.3 , 79.6 , 69.9 , 68.4 , 65.6 , 65.6 , 64.7 , 57.0 , 55.5 , 51.0 , 47.8 , 40.3 , 37.8 , 33.5 , 32.6 , 29.9 , 29.7 , 26.4 , 26.1 , 25.3 , 21.2 , 18.3 , 1.2 , 1.2 ; hrms ( esi - tof ) m / z ( m + h ) calcd for c44h74no11ssi2 880.4521 , found 880.4525 . to a vigorously stirring solution of ( + ) -s10 ( 24 mg , 0.027 mmol ) in etoh ( 2.5 ml ) at room temperature was added 1 m naoh ( 1 ml ) . the reaction mixture was then stirred at 50 c for 24 h. etoh was removed in vacuo , and 1 n hcl ( 5 ml ) was added . the mixture was extracted with ch2cl2 ( 3 10 ml ) , and the combined organic layers were dried over na2so4 , decanted , and concentrated in vacuo . the crude mixture was purified by filtration through a sio2 plug with etoac to yield seco acid ( + ) -27 ( 23 mg , 0.027 mmol , 97% ) as a clear oil . [ ]d21 + 56.0 ( c 0.5 , chcl3 ) ; ir ( neat ) 3420 , 2951 , 2891 , 1679 , 1513 , 1249 ; h nmr ( 500 mhz , cdcl3 ) 7.14 ( d , j = 8.5 hz , 2h ) , 6.87 ( d , j = 8.5 hz , 2h ) , 5.68 ( s , 1h ) , 5.22 ( d , j = 16.1 hz , 1h ) , 4.90 ( d , j = 7.5 hz , 1h ) , 4.84 ( d , j = 8.7 hz , 1h ) , 4.73 ( d , j = 7.3 hz , 1h ) , 4.66 ( d , j = 7.5 hz , 1h ) , 4.45 ( d , j = 5.9 hz , 1h ) , 4.21 ( d , j = 13.5 hz , 1h ) , 4.104.01 ( m , 1h ) , 3.95 ( dd , j = 3.8 , 9.5 hz , 1h ) , 3.79 ( s , 3h ) , 3.763.59 ( m , 4h ) , 3.53 ( d , j = 6.1 hz , 2h ) , 3.423.28 ( m , 2h ) , 2.98 ( s , 3h ) , 2.842.75 ( m , 1h ) , 2.752.65 ( m , 1h ) , 2.502.40 ( m , 1h ) , 2.132.07 ( m , 1h ) , 2.001.94 ( m , 1h ) , 1.951.88 ( m , 3h ) , 1.891.80 ( m , 1h ) , 1.801.49 ( m , 5h ) , 1.451.39 ( m , 1h ) , 1.231.18 ( m , 1h ) , 1.171.11 ( m , 3h ) , 0.980.89 ( m , 4h ) , 0.01 ( d , j = 3.6 hz , 18h ) ; c nmr ( 125 mhz , cdcl3 ) 173.7 , 170.1 , 163.0 , 159.2 , 128.8 , 128.7 , 116.0 , 114.4 , 102.7 , 95.6 , 92.4 , 91.4 , 79.6 , 69.8 , 68.5 , 65.6 , 65.6 , 64.8 , 60.6 , 57.1 , 55.5 , 47.8 , 46.9 , 40.0 , 37.6 , 33.4 , 29.8 , 29.8 , 26.4 , 26.1 , 25.6 , 21.2 , 21.2 , 18.2 , 14.4 , 1.2 , 1.2 ; hrms ( esi - tof ) m / z ( m - h ) calcd for c43h71no11ssi2 864.4208 , found 864.4224 . to a solution of seco - acid ( + ) -27 ( 139 mg , 0.1605 mmol ) , and triphenylphosphine ( 210 mg , 0.8023 mmol ) in toluene ( 16 ml ) cooled to 0 c was added diad dropwise . after 14 h , sio2 was added , and the solvent was removed in vacuo . the crude reaction mixture ( adsorbed on sio2 ) was purified via column chromatography on sio2 ( 20% etoac / hexanes ) to provide a macrolactone ( 140 mg , mixture of macrolactone and diad - h2 ) as a clear oil that was used directly in the next reaction . to a solution of the macrolactone from the previous step ( 70 mg ) in mecn ( 6.4 ml ) and h2o ( 1.6 ml ) cooled to 0 c was added can ( 176 mg , 0.321 mmol ) in one portion , which turned the reaction mixture orange . the ice bath was removed after the addition , and the reaction mixture was stirred vigorously . after 1.5 h , a saturated aqueous solution of nahco3 ( 10 ml ) was added , and the aqueous solution was extracted with ch2cl2 ( 3 30 ml ) . the combined organic layers were dried over na2so4 , decanted , and concentrated in vacuo . the crude mixture was purified via column chromatography on sio2 ( 25% etoac / hexanes ) to provide lactone ( + ) -29 ( 21 mg , 0.029 mmol , 36% over two steps ) as an oil . [ ]d21 + 20.7 ( c 0.2 , chcl3 ) ; ir ( neat ) 3358 , 3193 , 2924 , 2853 , 1684 , 1463 , 1377 , 1263 ; h nmr ( 500 mhz , cdcl3 ) 5.68 ( s , 1h ) , 5.45 ( s , 1h ) , 5.10 ( br s , 1h ) , 4.91 ( t , j = 6.3 hz , 2h ) , 4.76 ( d , j = 8.5 hz , 1h ) , 4.68 ( d , j = 6.7 hz , 1h ) , 4.26 ( d , j = 8.7 hz , 1h ) , 4.154.03 ( m , 2h ) , 3.803.67 ( m , 2h ) , 3.633.52 ( m , 3h ) , 3.40 ( dd , j = 8.7 , 12.7 hz , 1h ) , 3.363.29 ( m , 1h ) , 3.28 ( s , 3h ) , 2.72 ( dt , j = 5.2 , 10.7 hz , 2h ) , 2.34 ( dt , j = 5.2 , 11.7 hz , 1h ) , 2.28 ( d , j = 15.7 hz , 1h ) , 1.961.91 ( m , 1h ) , 1.88 ( s , 2h ) , 1.821.74 ( m , 2h ) , 1.711.63 ( m , 2h ) , 1.581.48 ( m , 2h ) , 1.331.28 ( m , 1h ) , 1.18 ( d , j = 6.7 hz , 4h ) , 1.000.89 ( m , 5h ) , 0.03 ( d , j = 10.5 hz , 18h ) ; c nmr ( 125 mhz , cdcl3 ) 174.1 , 167.3 , 154.4 , 118.5 , 99.4 , 96.3 , 92.8 , 90.3 , 80.5 , 78.8 , 70.7 , 66.9 , 66.0 , 65.6 , 63.7 , 57.7 , 48.6 , 35.9 , 33.0 , 31.6 , 30.9 , 30.8 , 29.9 , 29.7 , 24.6 , 24.3 , 21.5 , 18.3 , 18.2 ; hrms ( esi - tof ) m / z ( m + na ) calcd for c35h61nnao9ssi2 750.3527 , found 750.3529 . to a solution of acetonide ( + ) -26 ( 13 mg , 0.0227 mmol ) in ethanol ( 1.7 ml ) was added an aqueous solution of naoh ( 1 m , 0.7 ml ) dropwise . the reaction mixture was stirred overnight at 50 c and then concentrated under reduced pressure to give ca . an aqueous solution of hcl ( 1 n , 5 ml ) was then added , and the resulting mixture was extracted with ch2cl2 ( 3 10 ml ) . the combined organic layers were dried over na2so4 , decanted , and concentrated in vacuo . the crude mixture was purified via sio2 plug ( etoac ) to provide the seco acid ( 13 mg , 0.0227 mmol , near quant . ) as a clear oil that was used in the next reaction without further purification . to a solution of the seco acid ( 72 mg , 0.112 mmol ) in toluene ( 11 ml ) was added ph3p ( 147 mg , 0.56 mmol ) followed by a 60% solution of dead in toluene ( 227 mg , 0.78 mmol ) dropwise at rt . after the reaction mixture the crude mixture was purified via column chromatography on sio2 ( 17.5% to 20% etoac / hexanes ) to provide a macrolactone as a mixture contaminated with reduced dead ( 90 mg ) , which was used in the next reaction without further purification . the next reaction was split into three batches . to a solution of the macrolactone mixture obtained from the previous step ( 10 mg ) in mecn ( 1.3 ml ) and h2o ( 0.3 ml ) was added can ( 35 mg , 0.064 mmol ) in one portion , which turned the reaction mixture orange . after 1 h , a saturated aqueous solution of nahco3 ( 10 ml ) was added , and the aqueous solution was extracted with ch2cl2 ( 3 5 ml ) . the combined organic layers were dried over na2so4 , decanted , and concentrated in vacuo . to a solution of the macrolactone mixture obtained from the previous step ( 34 mg ) in mecn ( 4.3 ml ) and h2o ( 1.1 ml ) was added can ( 119 mg , 0.217 mmol ) in one portion , which turned the reaction mixture orange . after 1 h 40 min , a saturated aqueous solution of nahco3 ( 10 ml ) was added , and the aqueous solution was extracted with ch2cl2 ( 3 10 ml ) . the combined organic layers were dried over na2so4 , decanted , and concentrated in vacuo . to a solution of the macrolactone mixture obtained from the previous step ( 46 mg ) in mecn ( 5.6 ml ) and h2o ( 1.4 ml ) was added can ( 161 mg , 0.293 mmol ) in one portion , which turned the reaction mixture orange . . 1 h 30 min , a saturated aqueous solution of nahco3 ( 10 ml ) was added , and the aqueous solution was extracted with ch2cl2 ( 3 10 ml ) . the combined organic layers were dried over na2so4 , decanted , and concentrated in vacuo . the crude mixtures from batches 1 , 2 , and 3 were combined and then purified via column chromatography on sio2 ( 17% etoac / hexanes ) to provide lactone ( + ) -31 ( 20 mg , 0.0394 mmol , 35% over three steps ) as a film . [ ]d21 + 105.1 ( c 0.58 , chcl3 ) ; ir ( neat ) 3273 , 2935 , 1697 , 1456 , 1378 ; h nmr ( 500 mhz , cdcl3 ) 5.66 ( s , 1h ) , 5.53 ( s , 1h ) , 5.185.14 ( m , 1h ) , 4.19 ( d , j = 9.1 hz , 1h ) , 4.15 ( t , j = 11.5 hz , 1h ) , 4.07 ( t , j = 7.9 hz , 1h ) , 3.883.78 ( m , 1h ) , 3.443.36 ( m , 1h ) , 3.363.30 ( m , 1h ) , 3.29 ( s , 3h ) , 2.822.74 ( m , 1h ) , 2.70 ( dt , j = 5.4 , 11.7 hz , 1h ) , 2.53 ( dt , j = 5.2 , 11.3 hz , 1h ) , 2.26 ( d , j = 14.5 hz , 1h ) , 1.90 ( d , j = 0.8 hz , 3h ) , 1.84 ( m , 4h ) , 1.64 ( s , 3h ) , 1.611.53 ( m , 2h ) , 1.45 ( s , 3h ) , 1.41 ( s , 3h ) , 1.19 ( d , j = 6.9 hz , 3h ) ; c nmr ( 125 mhz , cdcl3 ) 174.2 , 166.8 , 154.9 , 118.6 , 109.9 , 99.4 , 90.7 , 82.5 , 77.9 , 70.7 , 66.7 , 64.7 , 57.7 , 48.7 , 36.1 , 32.5 , 31.0 , 30.9 , 30.9 , 29.7 , 29.3 , 27.3 , 26.7 , 24.7 , 24.5 , 20.8 ; hrms ( esi - tof ) m / z ( m + na ) calcd for c26h37nnao7s 530.2188 , found 530.2184 . to a solution of macrolactone ( + ) -31 ( 7 mg , 0.0138 mmol ) in acetic acid ( 2.5 ml ) and h2o ( 1.1 ml ) was added camphorsulfonic acid ( 2 mg ) . acetic acid was removed in vacuo , and a saturated aqueous solution nahco3 ( 10 ml ) was added . the cloudy aqueous mixture was extracted with ch2cl2 ( 3 20 ml ) , and the combined organic layers were dried over na2so4 , decanted , and concentrated in vacuo . the crude mixture was purified via column chromatography on sio2 ( 75% etoac / hexanes ) to provide triol ( + ) -30 ( 5.4 mg , 0.0138 mmol , 86% ) as a white foam . [ ]d21 + 90.3 ( c 0.39 , chcl3 ) ; ir ( neat ) 3395 , 2924 , 1681 , 1279 ; h nmr ( 500 mhz , cdcl3 ) 6.02 ( s , 1h ) , 5.71 ( s , 1h ) , 5.21 ( s , 1h ) , 4.27 ( t , j = 11.6 hz , 1h ) , 4.09 ( d , j = 7.7 hz , 1h ) , 3.85 ( t , j = 7.8 hz , 1h ) , 3.48 ( t , j = 8.5 hz , 1h ) , 3.423.31 ( m , 2h ) , 2.84 ( td , j = 5.3 , 11.4 hz , 1h ) , 2.702.63 ( m , 1h ) , 2.35 ( td , j = 5.0 , 12.1 hz , 1h ) , 2.17 ( d , j = 14.6 hz , 1h ) , 1.99 ( d , j = 13.5 hz , 1h ) , 1.90 ( s , 3h ) , 1.831.48 ( m , 8h ) , 1.31.27 ( m , 1h ) , 1.25 ( bs , 2h ) , 1.17 ( d , j = 7.0 hz , 3h ) ; c nmr ( 125 mhz , cdcl3 ) 175.8 , 166.8 , 156.0 , 117.8 , 96.6 , 90.4 , 80.0 , 75.9 , 68.3 , 66.9 , 62.8 , 62.7 , 35.5 , 32.5 , 32.2 , 32.0 , 30.8 , 29.9 , 29.2 , 24.5 , 23.7 , 21.3 ; hrms ( esi - tof ) m / z ( m + na ) calcd for c22h31nnao7s 476.1719 , found 476.1711 . to a solution of triol ( + ) -30 ( 2 mg , 0.0044 mmol ) in etoac ( 0.3 ml ) was added pd on baco3 ( 20 mg ) . the reaction flask was evacuated and refilled with h2 three times and then stirred at rt under a balloon of h2 . after 4 h 30 min , a sample of the reaction mixture was filtered through celite , and lc ms analysis indicated the reaction to be complete . the reaction mixture was filtered through a pad of celite with etoac and ch2cl2 , and the solvent was removed in vacuo . residual catalyst was observed , so the crude mixture was filtered through a clean pad of celite with ch2cl2 to afford ( + ) -1 ( 2 mg , 0.0044 mmol , near quant . ) as a white foam . [ ]d21 + 21.3 ( c 0.12 , meoh ) ; ir ( neat , cm ) 3418 , 2926 , 2855 , 1681 , 1444 , 1383 , 1289 ; h nmr ( 500 mhz , cd3cocd3 ) 6.58 ( s , 1h ) , 5.63 ( t , j = 10.5 hz , 1h ) , 5.54 ( s , 1h ) , 5.15 ( bs , 1h ) , 5.05 ( t , j = 10.9 hz , 1h ) , 4.87 ( s , 1h ) , 4.394.28 ( m , 2h ) , 3.91 ( t , j = 7.8 hz , 1h ) , 3.58 ( d , j = 6.2 hz , 1h ) , 3.46 ( d , j = 7.4 hz , 1h ) , 3.43 ( dd , j = 2.4 , 8.1 hz , 2h ) , 3.403.33 ( m , 1h ) , 2.792.73 ( m , 1h ) , 2.68 ( td , j = 3.6 , 11.8 hz , 1h ) , 1.531.40 ( m , 2h ) , 1.101.02 ( m , 1h ) , 0.93 ( d , j = 6.5 hz , 3h ) ; c nmr ( 125 mhz , cd3cocd3 ) 174.1 , 166.7 , 158.8 , 136.6 , 132.4 , 118.7 , 97.3 , 76.7 , 70.2 , 68.1 , 63.9 , 62.5 , 36.8 , 35.4 , 33.2 , 32.5 , 32.2 , 31.9 , 29.7 , 29.1 , 25.6 , 23.2 ; hrms ( esi - tof ) m / z ( m + na ) calcd for c22h33nnao7s 478.1875 , found 478.1861 . see the supporting information for deuterated 18-epi - latrunculol a ( s11 ) .

the evolution of an enantioselective total synthesis of ( + ) -18-epi - latrunculol a , a congener of the marine - sponge - derived latrunculins a and b , is reported . key steps include a late - stage mitsunobu macrolactonization to construct the 16-membered macrolactone , a mild carreira alkynylation to unite the northern and southern hemispheres , a diastereoselective , acid - mediated -hydroxy enone cyclization / equilibration sequence , and a functional - group - tolerant cross - metathesis to access the enone cyclization precursor .

Introduction Results and Discussion Summary Experimental Section

one compound , ( + ) -18-epi - latrunculol a ( 1 ) , exhibited selective solid tumor cytotoxicity when tested against hct-116 ( 5.5 m ) and mda - mb-435 ( > 50 m ) , but unlike the other members of the latrunculin family , 1 was devoid of the ability to inhibit actin polymerization . on the other hand , the parent compounds ( + ) -latrunculin a ( 2 ) and b ( 3 ) demonstrate nonselective cytotoxicity profiles , thus limiting their use as chemotherapeutics . given our longstanding interest in the latrunculins and the intriguing biological profile of the epimeric latrunculin congener ( + ) -18-epi - latrunculol a ( 1 ) , we undertook the development of a scalable total synthesis of ( + ) -1 to confirm both the assigned structure and absolute stereochemistry and to provide sufficient quantities for additional biological evaluation . described herein is a full account of the evolution of the total synthesis of ( + ) -18-epi - latrunculol a ( 1 ) , which led not only to ( + ) -1 but also to the preparation of ca . while at the outset of this synthetic venture the total synthesis of ( + ) -18-epi - latrunculol a ( 1 ) had not been reported , effective total syntheses of the parent latrunculins a ( 2 ) and b ( 3 ) had been published by white , frstner , and our laboratory . not surprisingly , our synthetic strategy for ( + ) -18-epi - latrunculol a ( 1 ) was envisioned to exploit the lessons learned in our earlier syntheses , with important modifications to ensure efficient asymmetric access to the natural product ( scheme 1 ) . specifically , we envisioned a late - stage mitsunobu macrolactonization and either a wittig olefination or , given our earlier difficulties with the wittig union for latrunculin a , a nucleophilic addition protocol to construct the 16-membered macrolactone . the requisite southern hemisphere coupling partner 4 was in turn envisioned to be readily accessible from cyclic ketal 5 via a strategy level acid - mediated cyclization / equilibration of -hydroxy enone 6 . the latter would be constructed through the aegis of a functional - group - compatible cross - metathesis reaction , which was anticipated to circumvent both protecting group manipulation and oxidation , as required in all previous latrunculin syntheses . quenching of the addition reaction with aqueous hcl was critical to obtain high yields of ( + ) -9 . when saturated aqueous ammonium chloride was employed , a mixture of enone ( + ) -9 and byproduct ( + ) -11 was obtained , presumably via addition of the liberated hydroxylamine to the electrophilic enone . the structure of ( + ) -11 was confirmed by single - crystal x - ray analysis . with gram quantities of enone ( + ) -9 in hand , we turned to the construction of the remaining cross - metathesis partner ( scheme 3 ) . without separation , treatment of this mixture with 3 equiv of enone ( + ) -9 and the hoveyda grubbs second - generation catalyst to achieve cross - metathesis provided pure cyclization precursor ( + ) -6 in 70% yield from ( )-8 after standard flash column chromatography with 72% recovery of unreacted enone ( + ) -9 . the key acid - mediated cyclization was next achieved after extensive experimentation by subjecting -hydroxy enone ( + ) -6 to a 1:1.3 ( v / v ) mixture of 6 n hcl and thf to furnish lactol ( + ) -14 ( scheme 4 ) . multiple byproducts and a small amount of the minor -diastereomer accounted for the remaining mass balance of the acid - mediated cyclization of ( + ) -6 . we reason that reversible formation of unsaturated oxonium intermediate 15 accounts for enrichment in the mixture of the diastereomeric secondary alcohols , thus providing the -diastereomer ( + ) -16 as the major product . wittig olefination was next envisaged to unite the northern and southern hemispheres of ( + ) -18-epi - latrunculol a ( 1 ) . however , we were cognizant of the challenges that a wittig union had provided in the original synthesis of ( + ) -latrunculin a ( 2 ) . proceeding with the wittig olefination tactic , wittig reagent ( + ) -17 was prepared in three steps and 55% yield from ( + ) -5 ( scheme 6 ) . nonetheless , all attempts afforded ( + ) -21 as the major product , and no olefination was observed . subsequent stork zhao olefination was followed by tbs protection of the secondary hydroxyl , which led to cis - vinyl iodide ( + ) -18 in 32% yield over the three steps . on the basis of the transition state outlined in scheme 9a , we reasoned that the chelation - controlled addition of a vinyl nucleophile , prepared via metalation of vinyl iodide ( + ) -18 , to aldehyde ( + ) -24 would lead to the desired syn stereochemistry . presumably the decomposition of ( + ) -24 is due to facile deprotonation of the acidic - and/or -protons of the enoate ( shown in red in scheme 9 ) upon treatment with the strongly basic nucleophiles . aware of the pronounced base sensitivity of aldehyde ( + ) -24 , we explored a carreira alkynylation employing alkyne ( + ) -19 . pleasingly , after brief optimization of the carreira alkynylation , the desired union of the northern and southern hemispheres was achieved to funish ( + ) -25 in 95% yield , impressively as a single diastereomer ( scheme 10 ) . of considerable importance , prolonged drying of the zn(otf)2 proved to be critical for the success of this union.the stereochemistry of the newly formed propargylic alcohol was confirmed via conversion to acetonide ( + ) -26 , with the latter structure assigned by 2d nmr analysis . with construction of the carbon skeleton of ( + ) -18-epi - latrunculol a ( 1 ) now achieved , we turned to the requisite alkyne semireduction . on the basis of these calculations as well as examination of a physical model of ( + ) -25 , we discovered that in spite of the linear geometry of the alkyne , intermediate ( + ) -25 could in fact attain the requisite orientation for the key mitsunobu macrolactonization . with this scenario in mind as well as with the expectation that semireduction of an alkyne in a 16-membered macrolactone might be enhanced by ring strain , we proceeded with the synthesis of the envisioned alkyne - containing seco acid . propargyl alcohol ( + ) -25 was protected as the sem ether to maintain rotational freedom of the northern hemisphere ( scheme 11 ) . hydrolysis of the methyl ester followed by warming of the ester to 50 c with naoh in ethanol provided the alkyne - containing seco acid ( + ) -27 in 85% yield from ( + ) -25 . the anticipated mitsunobu macrolactonization was then attempted by addition of triphenylphosphine and diisopropyl azodicarboxylate ( diad ) to seco acid ( + ) -27 ( scheme 12 ) . treatment of the mixture with ceric ammonium nitrate ( can ) in aqueous acetonitrile pleasingly removed the pmb group to furnish deprotected macrolactone ( + ) -29 in 36% yield from ( + ) -27 , which could be readily purified via flash chromatography . a similar mitsunobu macrolactonization employed in the original smith synthesis of ( + ) -latrunculin a ( 2 ) , although yielding a similarly 16-membered macrolactone in a 31% yield , proved to be completely unworkable in this case because of the incompatible conditions required for removal of the pmb protecting group . frstner and co - workers observed similar decompositions of their late - stage macrolactones when attempting to remove the robust pmb protecting group and likewise required a protecting group interchange before completing their total synthesis of ( + ) -latrunculin a ( 2 ) . although the global deprotection was achieved , removal of the sem groups required extended reaction times ( > 12 h ) at elevated temperature ( 50 c ) , which resulted in varying degrees of decomposition as well as inconsistent yields of ( + ) -30 ranging from ca . other reduction conditions , including wilkinson s catalyst , homogeneous palladium - catalyzed transfer hydrogenation , adams catalyst , and a two - step hydroboration / protodeborylation sequence , also proved ineffective ; no reaction , over - reduction , and/or decomposition of starting material resulted . fortunately , the use of 1.2 equiv of pd / c ( 10 wt % ) did eventually provide full conversion , but only a 29% isolated yield of ( + ) -18-epi - latrunculol a ( 1 ) was obtained . the spectral data of synthetic ( + ) -18-epi - latrunculol a ( 1 ) , including the h nmr ( 500 mhz ) , hrms parent ion identification , and chiroptic properties , proved identical in all respects to those reported for natural ( + ) -18-epi - latrunculol a ( 1 ) . importantly , the observation of identical chiroptic properties for synthetic ( + ) -1 permitted the assignment of the absolute stereochemistry of ( + ) -18-epi - latrunculol a ( 1 ) . when taken in acetone - d6 as reported by crews , the carbon resonances , while identical in chemical shift to those reported for natural ( + ) -18-epi - latrunculol a ( 1 ) , appeared doubled in several cases , a hallmark of course of a diastereomeric mixture ( figure 2a ) . on the other hand , when the c nmr spectrum of synthetic ( + ) -18-epi - latrunculol a ( 1 ) was taken in cdcl3 instead of acetone - d6 , the spectrum revealed the correct number of chemical shifts required for ( + ) -1 . c nmr spectra ( 6080 ppm ) of synthetic ( + ) -18-epi - latrunculol a ( 1 ) in ( a ) acetone - d6 and ( b ) acetone - d6 with added d2o . with conclusive evidence that the first total synthesis , structural confirmation , and absolute configuration assignment of ( + ) -18-epi - latrunculol a ( 1 ) had been achieved , we returned to the optimization of the global deprotection and final semireduction to facilitate a preparatively useful synthesis of the natural product . treatment of ( + ) -25 with acidic methanol as described earlier ( scheme 10 ) removed both the sem and tbs protecting groups while maintaining the mixed methyl ketal . upon hydrolysis of the methyl ester , the mitsunobu macrolactonization and subsequent pmb removal proceeded in a yield comparable to that in the sem - protected sequence of intermediates to furnish macrolactone ( + ) -31 in 35% yield from the methyl ester ( scheme 15 ) . we were then particularly pleased to find that global deprotection delivered the penultimate alkyne ( + ) -30 in 86% yield after only 2 h. the low yield of the final semireduction was reasoned to be a consequence of the excess adsorbing carbon solid support ( scheme 14 ) . pleasingly , a change to barium carbonate as a less adsorbent solid support provided the semireduction of ( + ) -30 in nearly quantitative yield , although a stoichiometric quantity of palladium was still required ( scheme 15 ) . synthetic ( + ) -18-epi - latrunculol a ( 1 ) was thus available upon semihydrogenation in 86% overall yield for the final two steps , a marked improvement from the previous protecting group strategy . we have reported here the total synthesis , structural validation , and assignment of the relative and absolute stereochemistry of ( + ) -18-epi - latrunculol a ( 1 ) , exploiting a longest linear sequence of 20 steps from commercially available 5-hexenoic acid . key steps in the successful route include a functional - group - compatible cross - metathesis reaction that avoids protection and oxidation steps required in all previous latrunculin synthetic ventures , an acid - mediated cyclization / equilibration sequence , an effective carreira alkynylation , and a late - stage mitsunobu macrolactonization . in addition , judicious selection of diol protection and successful optimization of the alkyne semireduction now permits access to synthetic ( + ) -18-epi - latrunculol a ( 1 ) . biological evaluation of the natural product and synthetic intermediates and further development of the tandem cyclization / equilibration of trans--hydroxy enones are currently underway and will be reported in due course . to a solution of acid ( + ) -10 ( 16 g , 59.86 mmol ) in ch2cl2 ( 200 ml ) was added i - pr2net ( 31.4 ml , 179.58 mmol ) n , o - dimethylhydroxylamine hydrochloride ( 9.93 g , 179.58 mmol ) , and then tbtu ( 28.8 g , 89.79 mmol ) portionwise . the mixture was cooled to 78 c , and a solution of aldehyde ( )-8 ( 2.58 g , 11.2 mmol ) in et2o ( 10 ml ) was added dropwise down the side of the flask ; additional et2o ( 5 ml ) was used to wash any residual aldehyde . to a portion of the allylic alcohol mixture ( 197 mg ) in dce ( 4 ml ) was added ( + ) -9 ( 600 mg , 2.16 mmol , 3 equiv ) , and the mixture was sparged with n2 for 20 min . to a solution of lactol ( + ) -16 ( 16 mg ) with minor impurities in meoh ( 0.4 ml ) was added camphorsulfonic acid ( 1 mg , 0.004 mmol ) , and the reaction mixture was stirred overnight at rt . to a solution of methyl ketal ( + ) -5 ( 165 mg , 0.375 mmol ) in ch2cl2 ( 4 ml ) were added imidazole ( 153 mg , 2.25 mmol ) , triphenyl phosphine ( 296 mg , 1.13 mmol ) , and finally iodine ( 248 mg , 0.975 mmol ) , and the reaction mixture was stirred overnight at rt . hmpa ( 0.66 ml , 3.81 mmol ) was added , and the reaction mixture was further cooled to 78 c , after which hydroxy aldehyde ( + ) -s3 ( 104 mg , 0.24 mmol ) in thf ( ca . the crude mixture was purified via column chromatography on sio2 ( 30% etoac / hexanes ) to provide ( + ) -19 ( 341 mg , 0.623 mmol , near quant . ) to zn(otf)2 ( dried by stirring overnight at 120 c under high vacuum , 1.12 g , 3.078 mmol ) were added ( + ) -nme ( azeotroped three times with toluene , 600 mg , 3.35 mmol ) and triethylamine ( distilled prior to use , 0.46 ml , 3.35 mmol ) in toluene ( 3.5 ml ) . the reaction mixture was stirred vigorously for 3 h. alkyne ( + ) -19 ( 300 mg , 0.548 mmol ) in toluene ( 1.3 ml ) was then added , and the mixture was stirred for 3 h. aldehyde ( + ) -24 ( 95 mg , 0.300 mmol ) was then added in toluene ( 0.5 ml ) , and the reaction mixture was stirred at rt overnight . to propargyl alcohol ( + ) -25 ( 43 mg , 0.0498 mmol ) was added meohhcl ( 1.5% ) ( 0.7 ml ) followed by ch2cl2 ( 0.5 ml ) to rinse the sides of the flask . to a solution of ( + ) -s8 ( 27 mg , 0.0436 mmol ) in 2,2-dimethoxypropane ( 1 ml ) were added acetone ( 0.2 ml ) and a small crystal of p - tsohh2o . to a vigorously stirring solution of ( + ) -s10 ( 24 mg , 0.027 mmol ) in etoh ( 2.5 ml ) at room temperature was added 1 m naoh ( 1 ml ) . to a solution of seco - acid ( + ) -27 ( 139 mg , 0.1605 mmol ) , and triphenylphosphine ( 210 mg , 0.8023 mmol ) in toluene ( 16 ml ) cooled to 0 c was added diad dropwise . the crude mixtures from batches 1 , 2 , and 3 were combined and then purified via column chromatography on sio2 ( 17% etoac / hexanes ) to provide lactone ( + ) -31 ( 20 mg , 0.0394 mmol , 35% over three steps ) as a film . after 4 h 30 min , a sample of the reaction mixture was filtered through celite , and lc ms analysis indicated the reaction to be complete .

over the past two decades , it has been convincingly shown that adipose tissue is an active endocrine organ which produces a number of biologically active molecules named adipokines . more recently , the endocrine function of the skeleton and its important role in metabolic homeostasis has been revealed [ 1 , 2 ] . mainly through mouse genetic means by analysing loss - of - function models , the existence of a complex bilateral hormonal link ( crosstalk ) between bone and energy metabolism has been discovered [ 14 ] . according to the current paradigm , bone remodelling and energy metabolism are coregulated by adipocyte - derived hormones , leptin , and adiponectin , and the feedback loop between bone and energy metabolism is mediated by osteocalcin ( oc ) , an osteoblast - specific protein . the biological importance of tight connections between adipose tissue and bone remodelling is further supported by the fact that adipocytes and osteoblasts are derived from a common mesenchymal progenitor cell , leptin and adiponectin are expressed in osteoblasts [ 68 ] and oc in human adipocytes . a crosstalk between signalling pathway regulating adipocyte and osteoblast differentiation has also been recently described . results of experimental studies on reciprocal bone - energy metabolism relationships mediated by adipokines and oc are fairly consistent . however , clinical data on the association between circulating leptin and adiponectin levels and oc are controversial . previous human studies that have evaluated the relationship between leptin and oc yielded conflicting results , showing either no correlation [ 1116 ] , positive , or negative correlation [ 1821 ] . similarly , some studies reported a positive association between serum adiponectin and oc [ 2227 ] , whereas other studies were not able to demonstrate a significant and independent relationship [ 16 , 20 , 2830 ] . emerging evidence has shown that resistin , a peptide hormone classified as an adipokine , although in humans it is mainly produced by mononuclear cells and macrophages , is important in regulating insulin resistance , diabetes , inflammatory processes , immunity , and bone metabolism [ 17 , 3134 ] . however , the interrelations between resistin and oc have not been characterised . in the above - mentioned studies , such factors as serum calcium , phosphate , magnesium , 25-hydroxyvitamin d ( 25(oh)d ) , parathyroid hormone ( pth ) , renal status , and age , known to influence both bone metabolism and circulating adipokines , have rarely been measured and analysed . lack of assessment of several adipokines simultaneously , difference in study populations and the dual nature of leptin 's effect on the skeleton ( central antiosteogenic and peripheral osteogenic ) may also contribute to the inconsistency in human data . there are only a few studies evaluating leptin [ 36 , 37 ] and adiponectin in patients with hip fracture ( hf ) , but no research has been carried out showing the relationship between adipokines and oc in these patients . it remains to be determined whether the phenomenon of bidirectional adipokine - oc interaction is involved in human osteoporosis . therefore , the aim of the present study was to asses in older patients with hf the association of leptin , adiponectin , and resistin , the three most widely investigated adipokines , with oc , and to examine whether oc is a significant and independent predictor of circulating adipokine levels . we also analysed the combined effect of adipokines using their ratios , since it has been suggested that metabolic functions of adipokines , especially of leptin and adiponectin , are complementary , and the leptin / adiponectin and adiponectin / resistin ratios are better clinical indicators [ 3943 ] . a total of 294 consecutive older patients ( 60 years of age , mean age 82.1 8.0 years ) with low - trauma osteoporotic hf were included in this study . data were obtained from a prospective electronic database on all adult patients with fracture of the upper femur admitted to the orthopaedic ward of the canberra hospital ( canberra , australian capital territory , australia ) , a university - affiliated tertiary care centre . exclusion criteria were subtrochanteric and shaft fracture , age < 60 years , high trauma , and pathological hf due to primary or metastatic bone cancer , multiple myeloma , paget disease , or primary hyperparathyroidism . sociodemographic , anthropometric , clinical ( hf type , co - morbidities , complications , medication use ) and laboratory data were recorded . antecubital venous blood samples were collected after overnight fast within 48 hours of arrival at the emergency department . routine haematological and biochemical assessments were performed by standardised methods on autoanalysers at the day of collection . for assays of oc , leptin , adiponectin , and resistin serum samples were frozen in liquid nitrogen and stored at 70c , subsequently thawed and analysed in a single batch using commercially available kits . serum levels of oc were determined by electrochemiluminescence immunoassay ( elecsys 1010 ; roche diagnostics , in , usa ; analytical sensitivity 0.5 ng / ml , interassay coefficient of variation ( cv ) 2.13.1% , intraassay cv < 3% ) , leptin by enzyme - linked immunosorbent assay ( elisa ) method ( diagnostic system laboratories , webster , tx , usa ; sensitivity 0.05 ng / ml , interassay cv 3.45.5% , intraassay cv < 6% ) , total adiponectin and resistin by human elisa kits ( b - bridge international , mountain view , ca , usa ; for adiponectin sensitivity 0.5 ng / ml , intraassay cv 3.27.3% , intraassay cv < 6% ; for resistin sensitivity 0.03 ng / ml , interassay cv 4.57.2% , intraassay cv serum levels of 25(oh)d were determined by a radioimmunoassay kit ( dia sorin , stillwater , mn , usa ; sensitivity 0.7 pmol / l , interassay cv5.99.4% , intraassay cv < 11.5% ) , intact pth by solid - phase two - site chemiluminescent enzyme - linked immunometricassay on a dpc immulite 2000 analyzer ( diagnostic products , los angeles , ca , usa ; sensitivity 0.07 pmol / l , interassay cv 6.27.0% , intraassay cv < 6% ) . all analyses were performed using stata software ( version 10 ; statacorp , college station , tx , usa ) . the summary statistics are presented as the mean standard deviation for continuous variables and as the number ( percentages ) for categorical variables . continuous variables with a skewed distribution were logarithmically transformed before being used in correlation analyses . the relationships between variables were examined by pearson 's linear correlation test and multivariate logistic regression analyses . p < 0.05 ( two - sided ) was considered statistically significant . to assess the potential effect of multiple comparisons and the significance of multicollinearity phenomena in multivariate regression analyses , bonferroni 's and sidak 's corrections were used and the variance inflation factor was calculated . women were found to be slightly older than men ( 82.6 7.7 versus 80.6 8.3 years , p = 0.053 ) . the hf was of cervical type in 52% and of trochanteric in 48% of patients . the mean ( sd ) values of serum 25(oh)d and pth were 37.2 18.0 nmol / l and 6.9 5.6 pmol / l , respectively . nmol / l was found in 84.6% of females and 67.5% of males ( p < 0.008 ) and secondary hyperparathyroidism ( pth > 6.8 pmol / l ) in 39.4% and 25.3% , respectively ( p = 0.028 ) . the mean serum osteocalcin level was 17.2 15.2 ng / ml . the serum osteocalcin levels were low ( < 14 ng / ml ) in 53.3% of patients . the main concentrations of serum leptin , adiponectin , and resistin were 18.4 23.2 ng / ml , 17.5 7.4 ng / ml , and 18.7 10.5 ng / ml , respectively . women had significantly higher mean serum concentrations of leptin ( 21.1 24.3 versus 11.7 18.6 ng / ml , p = 0.002 ) , adiponectin ( 18.3 7.1 versus 15.6 7.6 ng / ml , p = 0.007 ) , leptin / resistin ratio ( 1.6 2.3 versus 0.8 1 , p = 0.006 ) , and pth ( 7.4 6.1 versus 5.5 3.5 pmol / l , p = 0.009 ) , but lower levels of 25(oh)d ( 35.3 17.6 versus 42.4 18.2 nmol / l , p = 0.009 ) , phosphate ( 0.89 0.29 versus 1.07 075 nmol / l , p = 0.003 ) , magnesium ( 0.76 0.13 versus 0.81 0.12 nmol / l , p = 0.008 ) , and egfr ( 62.7 22 versus 71.2 26.4 ml / min./1.73 m , p = 0.006 ) . mean serum concentrations of resistin , leptin / adiponectin , and adiponectin / resistin ratios , osteocalcin , calcium ( corrected for albumin ) , tsh , albumin , and haemoglobin in women and men did not differ . malnutrition defined as serum leptin concentration < 4 ng / ml in males and < 6.5 ng / ml in females was observed in 33.8% of patients the malnourished group compared to the rest of the cohort was older ( 83.6 7.8 versus 81.2 8.0 years ; p = 0.015 ) and as would be expected had higher serum levels of adiponectin ( 19.3 6.6 versus 16.5 7.5 ng / ml ; p = 0.035 ) and lower levels of leptin ( 2.9 1.3 versus 26.5 25.0 ng / ml ; p < 0.001 ) , haemoglobin ( 121.3 16.3 versus 126.3 17.4 g / l ; p = 0.021 ) , leptin / adiponectin ( 0.17 versus 2.13 ; p < 0.001 ) , and leptin / resistin ( 0.22 versus 1.94 ; p < 0.001 ) ratios ; oc levels were also lower , however , the difference did not reach statistical significance ( 15.3 9.7 versus 18.2 17.3 ng / ml ; p = 0.117 ) . patients with cervical compared to trochanteric hf had higher serum levels of adiponectin ( 18.5 7.3 versus 16.3 7.3 ng / ml , p = 0.019 ) and resistin ( 20.1 10.5 versus 16.9 ng / ml , p = 0.014 ) , lower leptin / resistin ratio ( 1.1 1.4 versus 1.7 2.6 , p = 0.025 ) , and pth concentrations ( 5.9 3.6 versus 8.0 6.9 pmol / l , p = 0.001 ) , but did not differ significantly regarding other parameters . pearson correlation analysis performed with log - transformed variables revealed that leptin correlated positively with osteocalcin ( r = 0.123 , p = 0.038 ) , bmi ( r = 0.210 , p = 0.001 ) , and haemoglobin ( r = 0.188 , p = 0.001 ) and inversely with adiponectin ( r = 0.178 , p = 0.005 ) , phosphate ( r = 0.161 , p = 0.007 ) , and age ( r = 0.154 , p = 0.009 ) . adiponectin correlated positively with pth ( r = 0.193 , p = 0.002 ) and age ( r = 0.251 , p = 0.001 ) and negatively with bmi ( r = 0.170 , p = 0.005 ) . resistin correlated positively with age ( r = 0.156 , p = 0.013 ) and negatively with serum magnesium ( r = 0.198 , p = 0.002 ) and egfr ( r = 0.126 , p = 0.044 ) . serum osteocalcin correlated positively also with leptin / adiponectin ratio ( r = 0.129 , p = 0.041 ) , leptin / resistin ratio ( r = 0.166 , p = 0.008 ) , calcium ( r = 0.169 , p = 0.004 ) , phosphate ( r = 0.129 , p = 0.003 ) , magnesium ( r = 0.124 , p = 0.038 ) , and age ( r = 0.152 , p = 0.010 ) and negatively with egfr ( r = 0.388 , p = 0.001 ) and 25(oh)d ( r = 0.127 , p = 0.037 ) . multiple regression analyses were performed to evaluate which individual adipokine or their ratios are independently associated with serum osteocalcin . as shown in table 2 , there was a significant positive correlation between serum log - leptin and log - osteocalcin before and after multiple adjustments . serum log - resistin was negatively and significantly associated with log - osteocalcin only after adjusting for age , sex , bmi , hf type , 25(oh)d , and pth . this association remained significant after all further adjustments . in the final regression when all three adipokines were used in place of one , the independent determinants of serum osteocalcin were leptin ( p = 0.040 ) , resistin ( p = 0.018 ) , age ( p = 0.018 ) , and egfr ( p < 0.001 ) . taking into account that type 2 diabetes mellitus ( dm ) , hypertension , and other cardiovascular diseases which are common in the elderly population ( table 1 ) are known to be associated with dysregulation in adipokine metabolism , we further adjusted our models for these comorbidities ( yes / no ) . these adjustments did not appreciably change the estimates for oc - leptin and oc - adiponectin associations . neither hypertension ( per se ) nor any cardiovascular disease affected the oc - resistin relationship . however , addition of type 2 dm to the models with resistin made this association nonsignificant , indicating that higher resistin levels are incorporated in type 2 dm . indeed , the patients with type 2 dm have significantly higher serum resistin concentrations ( + 29.2% ) than the rest of the cohort ( 23.0 11.2 versus 17.8 10.0 ng / ml , p = 0.010 ) . results of multivariate regression modelling testing the hypothesis that individual adipokine ratios are associated with serum osteocalcin are shown in table 3 . as it would be expected , there was a strong positive correlation between leptin / resistin ratio and serum log - osteocalcin , and it remained significant after all adjustments . there was also a positive association between leptin / adiponectin ratio and log - osteocalcin before and after adjusting for age , sex , bmi , hf type , and parameters of mineral metabolism , but this association lost significance when further adjusted for egfr and haemoglobin as well as for comorbidities . in contrast , the adiponectin / resistin ratio was significantly associated with serum log - osteocalcin only in models near fully or fully adjusted . taken together , these results indicate that leptin and resistin are independent positive and negative , respectively , determinants of serum oc . not surprisingly , the leptin / resistin ratio significantly and positively correlated with oc predicting 39.2% of the total variance in oc , and the adiponectin / resistin ratio could predict 37.4% of the variance . the partial associations between the leptin / adiponectin , adiponectin / resistin ratios and , oc confirm the independent effects of leptin and resistin on serum oc and show the influence of other factors related mainly to adiponectin . we next asked which factors are independent determinants of serum adipokine levels and their ratios . on multiple linear regression analyses and in keeping with previous results , serum oc was a significant predictor of both leptin and resistin , but not adiponectin ( table 4 ) . age was an independent determinant of each of the three adipokines . other parameters independently associated with circulating leptin were male sex , egfr , adiponectin , ( all three inversely ) , haemoglobin , and cvd ( both positively ) . serum resistin levels were independently associated with pth ( positively ) , trochanteric hf type , and serum magnesium ( both inversely ) . oc was significantly independently associated with leptin / resistin and adiponectin / resistin ratios but not with the leptin / adiponectin ratio . age , male sex ( both negatively ) , trochanteric hf type , and haemoglobin ( both positively ) were the other parameters independently associated with the leptin / resistin ratio ; the model explained 44.5% of variance in this ratio . oc was also the only independent predictor of the adiponectin / resistin ratio , but in fully adjusted model it accounted only 5.5% of variance . the main findings of this study of 294 consecutive unselected older patients with low - trauma hf are statistically significant correlations between leptin , resistin , and oc indicating complex interactions between adipocytes / monocytes / macrophages and osteoblasts . these have been demonstrated by simultaneous measurements of three major circulating adipokines ( leptin , adiponectin , and resistin ) , their ratios , and serum oc . multiple linear regression models adjusted for age , gender , bmi , hf type , key factors , or mineral metabolism ( calcium , phosphate , magnesium , 25(oh)d , pth ) , renal function , and haemoglobin showed that serum oc levels were significantly and positively associated with leptin and negatively with resistin concentrations , but not related to circulating adiponectin levels . leptin , resistin ( or leptin / resistin ratio ) , age , and egfr were the only independent predictors of serum oc levels contributing to 39.5% of oc variance . on the other hand , oc was an independent determinant of serum leptin and resistin levels , as well as leptin / resistin and adiponectin / resistin ratios . although caution is needed when interpreting results of a cross - sectional study , our data may suggest the presence of adipokine - oc loops , specifically , leptin increases and resistin decreases oc secretion by osteoblasts , whereas circulating oc influences leptin ( positive feedback loop ) and resistin ( negative feedback loop ) production . these results are in line with the current concept that efficient maintenance of metabolic homeostasis depends on interaction between adipose tissue / energy metabolism and skeleton [ 14 ] , but the directions of some associations observed in this as in other clinical studies were opposite to that reported in experimental animals ( genetically modified obese rodents ) . although adipokines , especially leptin and adiponectin , two pleiotropic hormones involved in regulation of a large variety of physiological processes , have been extensively studied in recent years , current data on their effects on bone and the adipokine - oc interactions in humans are controversial and the underlying mechanisms not fully understood . it is now well acknowledged that the effect of leptin on bone is complex and includes different pathways : central inhibition of bone formation through the hypothalamus and brainstem involving adrenergic , neuromedin u , neuropeptide y , cocaine , and amphetamine - related transcript and serotoninergic systems [ 35 , 46 , 47 ] and direct peripheral ( local ) enhancement of osteoblastic cell differentiation , proliferation , and bone mineralization [ 5 , 7 ] . results from studies of the bone phenotype in animals with leptin deficiency ( ob / ob ) and leptin receptor deficiency ( dbldb , fa / fa ) are conflicting . while several groups concluded that leptin acts as a positive regulator of bone formation [ 48 , 49 ] , other suggested a negative ( through the hypothalamus ) effect on the skeleton [ 35 , 47 ] . in humans , both positive and negative correlations between leptin and bone mineral density ( bmd ) , as well as with oc [ 1121 ] , have been reported . our finding that serum leptin is associated with oc is consistent with clinical observations that leptin positively correlated with oc and bmd [ 15 , 18 , 50 , 51 ] in different settings , whereas decrease of oc in bone predispose to hf . our results are also supported by a strong inverse association between serum leptin levels and nontraumatic fracture risk even in normal weight subjects , as well as the data that leptin enhances osteoblastogenesis in vitro [ 7 , 48 , 53 ] , exerts a positive effect in fetal bone formation and reduces bone loss in ovariectomized rats . a positive association between leptin / adiponectin ratio and oc has been reported . in our study , this association disappeared when egfr , as an independent variable , was included in the regression model , and oc was not an independent predictor of the leptin / adiponectin ratio . taken together , it appears that leptin , which is involved in multiple endocrine pathways and exerts a wide spectrum of actions , may lead to opposite effects in different metabolic conditions . the negative effects of leptin on bone may predominant over the positive ones in obesity when leptin resistance occurs or when the serum leptin concentration rises above a certain threshold [ 50 , 56 ] . in our study group , there were no obese persons and in 1/3 of patients the low serum leptin levels were indicative of malnutrition . in humans , our data suggests that in underweighted and normal weight persons , lower leptin levels are associated with decrease in oc , which in turn may further decrease leptin production . animal studies have indicated that oc regulates insulin metabolism through stimulating the expression of adiponectin in adipocytes [ 13 ] . adiponectin and its receptors are expressed on osteoblasts , and adiponectin in vitro stimulates proliferation and differentiation of osteoblasts [ 6 , 8 , 58 ] , and oc enhanced adiponectin expression in cultured adipocytes in a dose - dependent manner . experimental data in vivo ( transgenic mice models ) demonstrated all three a positive , negative or no effect of adiponectin on bone mass [ 58 , 59 ] . clinical studies reported more often a negative association [ 22 , 60 , 61 ] , and also a positive or no correlation . conflicting data on adiponectin - oc relationship [ 16 , 20 , 2230 ] together with these discrepancies suggest that the effects may differ depending on other metabolic factors and clinical features . in the present study in concordance with other human studies [ 11 , 17 , 20 , 30 ] , no correlation was observed between serum adiponectin and oc . however , we found a positive relationship between adiponectin / resistin ratio and oc , suggesting that a shift in balance towards adiponectin may increase oc production by counteracting the action of resistin . of note , this correlation was observed only when age , sex , bmi , hf type , parameters of mineral metabolism , and egfr were included , as independent variables , in the regression analysis model . this indicates that serum adiponectin / resistin ratio is positively associated with oc levels in subjects with similar above - mentioned characteristics ( e.g. , when the influence of these variables is eliminated ) . our data demonstrate a reciprocal association between resistin and oc which has not been previously described . resistin is expressed in mature human osteoblasts , and recombinant resistin increases osteoclastigenesis but only weakly affects differentiation of preosteoblasts into osteoblasts . the few clinical studies of resistin - bone relationship provided conflicting data [ 17 , 30 , 61 , 64 ] . our results are in line with observations that resistin inversely correlates with bmd in the hip , lumbar spine , and radius . oc association in our study became significant only after controlling for 25(oh)d and pth and remained significant after further adjustments for all other covariates except type 2 dm ( table 2 ) , suggesting that this relationship can be masked by parameters of mineral metabolism . indeed , our data showed that pth and magnesium were independent determinants of circulating resistin but not leptin ( table 5 ) providing a potential explanation for the masked effect . of note , after adjusting for type 2 dm , the association between oc and resistin became nonsignificant , suggesting that common signalling and metabolic pathways for oc and resistin contribute to dm . in line with other studies [ 6669 ] , we found that serum resistin levels in patients with type 2 dm were significantly higher compared to the rest of the cohort . these observations are consistent with growing evidence that resistin may be a potential mediator of dm [ 7073 ] acting , at least partially , through oc . the relationships between adipokine ratios and oc have not been systematically examined previously . in this study , after full adjustment , a significant interrelation between the leptin / resistin ratio and oc and between adiponectin / resistin ratio and oc was found . however , analyses of these ratios did not yield additional information compared to either leptin or resistin measurements . it should be pointed out that multiple regression analysis explained only 39.5% of the variance in serum oc , indicating that factors other than leptin and resistin significantly influenced the level of oc . we found that both egfr and age are independently associated with serum oc as well as leptin levels , and age is an independent determinant of resistinaemia . consistent with other studies , ours showed that deterioration of kidney function was associated with higher oc and leptin levels . there is an age - dependent decrease in proliferation and differentiation of human osteoblasts , and the highest oc levels have been reported during adolescence . however , in adults , no correlation between oc and age was found in some studies , while other described decreased oc levels with age . in about half ( 53.3% ) of our patients , the serum oc concentration was low ( lower than the low limit of the reference range ) but it was significantly and positively associated with age . it is reasonable to consider that renal dysfunction may at least partially explain this association , as we observed a strong negative correlation between age and egfr ( r = 0.313 , p = 0.001 ) and egfr was markedly decreased ( < 60 ml / min./1.73 m ) in 42.6% of our patients . the complex interplay of many metabolic , renal , and age - related factors may account for some of the discrepancies in the literature in regard to adipokine - oc interactions . it is possible that various combinations of these factors are causing distinct positive or negative effects . further complicating the matter , adiponectin and resistin ( as well as leptin ) have been shown to be neuroendocrine hormones acting directly on the brain [ 7781 ] , but in contrast to leptin , the centrally mediated effects of adiponectin and resistin on osteoblast functions are unknown . moreover , receptors for resistin and oc still remain unidentified . to develop an integrated understanding of adipokine - bone interaction , a lot there is a growing body of evidence demonstrating that in osteoporosis impaired bone metabolism , including oc production and secretion , does not exist in isolation . our data indicate the existence of bidirectional leptin - oc ( positive ) and resistin - oc ( negative ) relationships as a part of a complex energy metabolism - bone network in older patients with hf . figure 1 represents the complex interactions of oc with adipokines depicting independent significant associations between oc , circulating adipokines , their ratios , age , and renal status in older patients with hf . further examination of the role of these interactions in osteoporotic fractures and metabolic disorders was warranted . in regard to the differences in oc - adipokine interactions observed in humans and rodents , it should be noted that , firstly , the esp gene , specifically studied in knockout mouse models , is a pseudogene in humans . no functional esp gene has been identified in humans , although a close homologue of esp is expressed in human osteoblasts . secondly , in genetically modified rodents , changes in oc and adipokine levels are much larger than in clinical observations . fourthly , the effects of ageing and comorbidities have not been addressed in animal studies . the notable strength of this study is simultaneous assessment of three circulating adipokines and oc in the same cohort and adjustment for a wide range of confounding factors , the major limitations of previous studies . after bonferroni and sidak adjustments , all determinants preserved statistical significance , and in all our models ( tables 25 ) , the variance inflation factor was between 1.21 and 1.27 indicating that the amount of multicollinearity was not significant . the main limitation of our study is its cross - sectional design which precludes conclusions regarding causality . another potential limitation of this study is that only total oc and total adiponectin have been measured . the animal and in vitro studies however , other studies reported that both carboxylated and uncarboxylated forms of oc and total oc are associated with glucose metabolism and insulin resistance [ 11 , 23 , 24 , 76 ] . similarly , some studies concluded that high - molecular weight ( hmw ) adiponectin is a better predictor of insulin resistance and metabolic syndrome , while other studies did not find a significant difference between hmw and total adiponectin in this regard . we can not exclude the possibility that measurements of specific forms of oc and adiponectin may provide different results . finally , our study population represents almost exclusively elderly caucasians , and the results may have limited applicability to other age and ethnic groups . in conclusion , in older patients with hf , leptin is directly and resistin inversely associated with circulating oc , and oc is a significant independent determinant of both serum leptin ( positive ) and resistin ( negative ) concentrations . these suggest bidirectional interactions ( crosstalk ) between leptin , resistin and oc as a part of a complex homeostatic system regulating bone and energy metabolism . our data do not support an independent link between adiponectin and oc in these patients . further studies should be performed to evaluate the role of leptin - oc and resistin - oc axes in osteoporotic fractures and comorbid conditions such as cardio- and cerebrovascular diseases , diabetes , dementia , malnutrition , all of which are common in the elderly and have been shown to be associated with alterations in serum adipokine and oc levels .

introduction . experiments on genetically modified animals have discovered a complex cross - regulation between adipokines ( leptin , adiponectin ) and osteocalcin . the relationships between these molecules in human osteoporosis are still unclear . we evaluated the hypothesis of a bidirectional link between adipokines and osteocalcin . materials and methods . in a cross - sectional study of 294 older patients with osteoporotic hip fracture , we estimated serum concentrations of leptin , adiponectin , resistin , osteocalcin , parameters of mineral metabolism , and renal function . results . after adjustment for multiple potential confounders , serum osteocalcin concentration was inversely associated with resistin and positively with leptin , leptin / resistin ratio , and adiponectin / resistin ratio . in multivariate regression models , osteocalcin was an independent predictor of serum leptin , resistin , leptin / resistin , and adiponectin / resistin ratios . conclusions . our data support the bidirectional regulation between osteocalcin and adipokines , but contrary to the genetically modified animal models , in older subjects with osteoporotic hip fracture , serum osteocalcin is positively associated with leptin and inversely with resistin .

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

mainly through mouse genetic means by analysing loss - of - function models , the existence of a complex bilateral hormonal link ( crosstalk ) between bone and energy metabolism has been discovered [ 14 ] . according to the current paradigm , bone remodelling and energy metabolism are coregulated by adipocyte - derived hormones , leptin , and adiponectin , and the feedback loop between bone and energy metabolism is mediated by osteocalcin ( oc ) , an osteoblast - specific protein . the biological importance of tight connections between adipose tissue and bone remodelling is further supported by the fact that adipocytes and osteoblasts are derived from a common mesenchymal progenitor cell , leptin and adiponectin are expressed in osteoblasts [ 68 ] and oc in human adipocytes . previous human studies that have evaluated the relationship between leptin and oc yielded conflicting results , showing either no correlation [ 1116 ] , positive , or negative correlation [ 1821 ] . in the above - mentioned studies , such factors as serum calcium , phosphate , magnesium , 25-hydroxyvitamin d ( 25(oh)d ) , parathyroid hormone ( pth ) , renal status , and age , known to influence both bone metabolism and circulating adipokines , have rarely been measured and analysed . lack of assessment of several adipokines simultaneously , difference in study populations and the dual nature of leptin 's effect on the skeleton ( central antiosteogenic and peripheral osteogenic ) may also contribute to the inconsistency in human data . there are only a few studies evaluating leptin [ 36 , 37 ] and adiponectin in patients with hip fracture ( hf ) , but no research has been carried out showing the relationship between adipokines and oc in these patients . therefore , the aim of the present study was to asses in older patients with hf the association of leptin , adiponectin , and resistin , the three most widely investigated adipokines , with oc , and to examine whether oc is a significant and independent predictor of circulating adipokine levels . we also analysed the combined effect of adipokines using their ratios , since it has been suggested that metabolic functions of adipokines , especially of leptin and adiponectin , are complementary , and the leptin / adiponectin and adiponectin / resistin ratios are better clinical indicators [ 3943 ] . data were obtained from a prospective electronic database on all adult patients with fracture of the upper femur admitted to the orthopaedic ward of the canberra hospital ( canberra , australian capital territory , australia ) , a university - affiliated tertiary care centre . for assays of oc , leptin , adiponectin , and resistin serum samples were frozen in liquid nitrogen and stored at 70c , subsequently thawed and analysed in a single batch using commercially available kits . serum levels of oc were determined by electrochemiluminescence immunoassay ( elecsys 1010 ; roche diagnostics , in , usa ; analytical sensitivity 0.5 ng / ml , interassay coefficient of variation ( cv ) 2.13.1% , intraassay cv < 3% ) , leptin by enzyme - linked immunosorbent assay ( elisa ) method ( diagnostic system laboratories , webster , tx , usa ; sensitivity 0.05 ng / ml , interassay cv 3.45.5% , intraassay cv < 6% ) , total adiponectin and resistin by human elisa kits ( b - bridge international , mountain view , ca , usa ; for adiponectin sensitivity 0.5 ng / ml , intraassay cv 3.27.3% , intraassay cv < 6% ; for resistin sensitivity 0.03 ng / ml , interassay cv 4.57.2% , intraassay cv serum levels of 25(oh)d were determined by a radioimmunoassay kit ( dia sorin , stillwater , mn , usa ; sensitivity 0.7 pmol / l , interassay cv5.99.4% , intraassay cv < 11.5% ) , intact pth by solid - phase two - site chemiluminescent enzyme - linked immunometricassay on a dpc immulite 2000 analyzer ( diagnostic products , los angeles , ca , usa ; sensitivity 0.07 pmol / l , interassay cv 6.27.0% , intraassay cv < 6% ) . to assess the potential effect of multiple comparisons and the significance of multicollinearity phenomena in multivariate regression analyses , bonferroni 's and sidak 's corrections were used and the variance inflation factor was calculated . the main concentrations of serum leptin , adiponectin , and resistin were 18.4 23.2 ng / ml , 17.5 7.4 ng / ml , and 18.7 10.5 ng / ml , respectively . women had significantly higher mean serum concentrations of leptin ( 21.1 24.3 versus 11.7 18.6 ng / ml , p = 0.002 ) , adiponectin ( 18.3 7.1 versus 15.6 7.6 ng / ml , p = 0.007 ) , leptin / resistin ratio ( 1.6 2.3 versus 0.8 1 , p = 0.006 ) , and pth ( 7.4 6.1 versus 5.5 3.5 pmol / l , p = 0.009 ) , but lower levels of 25(oh)d ( 35.3 17.6 versus 42.4 18.2 nmol / l , p = 0.009 ) , phosphate ( 0.89 0.29 versus 1.07 075 nmol / l , p = 0.003 ) , magnesium ( 0.76 0.13 versus 0.81 0.12 nmol / l , p = 0.008 ) , and egfr ( 62.7 22 versus 71.2 26.4 ml / min./1.73 m , p = 0.006 ) . mean serum concentrations of resistin , leptin / adiponectin , and adiponectin / resistin ratios , osteocalcin , calcium ( corrected for albumin ) , tsh , albumin , and haemoglobin in women and men did not differ . malnutrition defined as serum leptin concentration < 4 ng / ml in males and < 6.5 ng / ml in females was observed in 33.8% of patients the malnourished group compared to the rest of the cohort was older ( 83.6 7.8 versus 81.2 8.0 years ; p = 0.015 ) and as would be expected had higher serum levels of adiponectin ( 19.3 6.6 versus 16.5 7.5 ng / ml ; p = 0.035 ) and lower levels of leptin ( 2.9 1.3 versus 26.5 25.0 ng / ml ; p < 0.001 ) , haemoglobin ( 121.3 16.3 versus 126.3 17.4 g / l ; p = 0.021 ) , leptin / adiponectin ( 0.17 versus 2.13 ; p < 0.001 ) , and leptin / resistin ( 0.22 versus 1.94 ; p < 0.001 ) ratios ; oc levels were also lower , however , the difference did not reach statistical significance ( 15.3 9.7 versus 18.2 17.3 ng / ml ; p = 0.117 ) . patients with cervical compared to trochanteric hf had higher serum levels of adiponectin ( 18.5 7.3 versus 16.3 7.3 ng / ml , p = 0.019 ) and resistin ( 20.1 10.5 versus 16.9 ng / ml , p = 0.014 ) , lower leptin / resistin ratio ( 1.1 1.4 versus 1.7 2.6 , p = 0.025 ) , and pth concentrations ( 5.9 3.6 versus 8.0 6.9 pmol / l , p = 0.001 ) , but did not differ significantly regarding other parameters . pearson correlation analysis performed with log - transformed variables revealed that leptin correlated positively with osteocalcin ( r = 0.123 , p = 0.038 ) , bmi ( r = 0.210 , p = 0.001 ) , and haemoglobin ( r = 0.188 , p = 0.001 ) and inversely with adiponectin ( r = 0.178 , p = 0.005 ) , phosphate ( r = 0.161 , p = 0.007 ) , and age ( r = 0.154 , p = 0.009 ) . serum osteocalcin correlated positively also with leptin / adiponectin ratio ( r = 0.129 , p = 0.041 ) , leptin / resistin ratio ( r = 0.166 , p = 0.008 ) , calcium ( r = 0.169 , p = 0.004 ) , phosphate ( r = 0.129 , p = 0.003 ) , magnesium ( r = 0.124 , p = 0.038 ) , and age ( r = 0.152 , p = 0.010 ) and negatively with egfr ( r = 0.388 , p = 0.001 ) and 25(oh)d ( r = 0.127 , p = 0.037 ) . in the final regression when all three adipokines were used in place of one , the independent determinants of serum osteocalcin were leptin ( p = 0.040 ) , resistin ( p = 0.018 ) , age ( p = 0.018 ) , and egfr ( p < 0.001 ) . taking into account that type 2 diabetes mellitus ( dm ) , hypertension , and other cardiovascular diseases which are common in the elderly population ( table 1 ) are known to be associated with dysregulation in adipokine metabolism , we further adjusted our models for these comorbidities ( yes / no ) . however , addition of type 2 dm to the models with resistin made this association nonsignificant , indicating that higher resistin levels are incorporated in type 2 dm . results of multivariate regression modelling testing the hypothesis that individual adipokine ratios are associated with serum osteocalcin are shown in table 3 . as it would be expected , there was a strong positive correlation between leptin / resistin ratio and serum log - osteocalcin , and it remained significant after all adjustments . there was also a positive association between leptin / adiponectin ratio and log - osteocalcin before and after adjusting for age , sex , bmi , hf type , and parameters of mineral metabolism , but this association lost significance when further adjusted for egfr and haemoglobin as well as for comorbidities . in contrast , the adiponectin / resistin ratio was significantly associated with serum log - osteocalcin only in models near fully or fully adjusted . taken together , these results indicate that leptin and resistin are independent positive and negative , respectively , determinants of serum oc . not surprisingly , the leptin / resistin ratio significantly and positively correlated with oc predicting 39.2% of the total variance in oc , and the adiponectin / resistin ratio could predict 37.4% of the variance . the partial associations between the leptin / adiponectin , adiponectin / resistin ratios and , oc confirm the independent effects of leptin and resistin on serum oc and show the influence of other factors related mainly to adiponectin . on multiple linear regression analyses and in keeping with previous results , serum oc was a significant predictor of both leptin and resistin , but not adiponectin ( table 4 ) . other parameters independently associated with circulating leptin were male sex , egfr , adiponectin , ( all three inversely ) , haemoglobin , and cvd ( both positively ) . serum resistin levels were independently associated with pth ( positively ) , trochanteric hf type , and serum magnesium ( both inversely ) . oc was significantly independently associated with leptin / resistin and adiponectin / resistin ratios but not with the leptin / adiponectin ratio . age , male sex ( both negatively ) , trochanteric hf type , and haemoglobin ( both positively ) were the other parameters independently associated with the leptin / resistin ratio ; the model explained 44.5% of variance in this ratio . oc was also the only independent predictor of the adiponectin / resistin ratio , but in fully adjusted model it accounted only 5.5% of variance . the main findings of this study of 294 consecutive unselected older patients with low - trauma hf are statistically significant correlations between leptin , resistin , and oc indicating complex interactions between adipocytes / monocytes / macrophages and osteoblasts . these have been demonstrated by simultaneous measurements of three major circulating adipokines ( leptin , adiponectin , and resistin ) , their ratios , and serum oc . multiple linear regression models adjusted for age , gender , bmi , hf type , key factors , or mineral metabolism ( calcium , phosphate , magnesium , 25(oh)d , pth ) , renal function , and haemoglobin showed that serum oc levels were significantly and positively associated with leptin and negatively with resistin concentrations , but not related to circulating adiponectin levels . leptin , resistin ( or leptin / resistin ratio ) , age , and egfr were the only independent predictors of serum oc levels contributing to 39.5% of oc variance . on the other hand , oc was an independent determinant of serum leptin and resistin levels , as well as leptin / resistin and adiponectin / resistin ratios . although caution is needed when interpreting results of a cross - sectional study , our data may suggest the presence of adipokine - oc loops , specifically , leptin increases and resistin decreases oc secretion by osteoblasts , whereas circulating oc influences leptin ( positive feedback loop ) and resistin ( negative feedback loop ) production . these results are in line with the current concept that efficient maintenance of metabolic homeostasis depends on interaction between adipose tissue / energy metabolism and skeleton [ 14 ] , but the directions of some associations observed in this as in other clinical studies were opposite to that reported in experimental animals ( genetically modified obese rodents ) . although adipokines , especially leptin and adiponectin , two pleiotropic hormones involved in regulation of a large variety of physiological processes , have been extensively studied in recent years , current data on their effects on bone and the adipokine - oc interactions in humans are controversial and the underlying mechanisms not fully understood . results from studies of the bone phenotype in animals with leptin deficiency ( ob / ob ) and leptin receptor deficiency ( dbldb , fa / fa ) are conflicting . in our study , this association disappeared when egfr , as an independent variable , was included in the regression model , and oc was not an independent predictor of the leptin / adiponectin ratio . the negative effects of leptin on bone may predominant over the positive ones in obesity when leptin resistance occurs or when the serum leptin concentration rises above a certain threshold [ 50 , 56 ] . in humans , our data suggests that in underweighted and normal weight persons , lower leptin levels are associated with decrease in oc , which in turn may further decrease leptin production . adiponectin and its receptors are expressed on osteoblasts , and adiponectin in vitro stimulates proliferation and differentiation of osteoblasts [ 6 , 8 , 58 ] , and oc enhanced adiponectin expression in cultured adipocytes in a dose - dependent manner . however , we found a positive relationship between adiponectin / resistin ratio and oc , suggesting that a shift in balance towards adiponectin may increase oc production by counteracting the action of resistin . of note , this correlation was observed only when age , sex , bmi , hf type , parameters of mineral metabolism , and egfr were included , as independent variables , in the regression analysis model . this indicates that serum adiponectin / resistin ratio is positively associated with oc levels in subjects with similar above - mentioned characteristics ( e.g. our data demonstrate a reciprocal association between resistin and oc which has not been previously described . oc association in our study became significant only after controlling for 25(oh)d and pth and remained significant after further adjustments for all other covariates except type 2 dm ( table 2 ) , suggesting that this relationship can be masked by parameters of mineral metabolism . in line with other studies [ 6669 ] , we found that serum resistin levels in patients with type 2 dm were significantly higher compared to the rest of the cohort . the relationships between adipokine ratios and oc have not been systematically examined previously . in this study , after full adjustment , a significant interrelation between the leptin / resistin ratio and oc and between adiponectin / resistin ratio and oc was found . we found that both egfr and age are independently associated with serum oc as well as leptin levels , and age is an independent determinant of resistinaemia . in about half ( 53.3% ) of our patients , the serum oc concentration was low ( lower than the low limit of the reference range ) but it was significantly and positively associated with age . further complicating the matter , adiponectin and resistin ( as well as leptin ) have been shown to be neuroendocrine hormones acting directly on the brain [ 7781 ] , but in contrast to leptin , the centrally mediated effects of adiponectin and resistin on osteoblast functions are unknown . our data indicate the existence of bidirectional leptin - oc ( positive ) and resistin - oc ( negative ) relationships as a part of a complex energy metabolism - bone network in older patients with hf . figure 1 represents the complex interactions of oc with adipokines depicting independent significant associations between oc , circulating adipokines , their ratios , age , and renal status in older patients with hf . the notable strength of this study is simultaneous assessment of three circulating adipokines and oc in the same cohort and adjustment for a wide range of confounding factors , the major limitations of previous studies . in conclusion , in older patients with hf , leptin is directly and resistin inversely associated with circulating oc , and oc is a significant independent determinant of both serum leptin ( positive ) and resistin ( negative ) concentrations . these suggest bidirectional interactions ( crosstalk ) between leptin , resistin and oc as a part of a complex homeostatic system regulating bone and energy metabolism . our data do not support an independent link between adiponectin and oc in these patients . further studies should be performed to evaluate the role of leptin - oc and resistin - oc axes in osteoporotic fractures and comorbid conditions such as cardio- and cerebrovascular diseases , diabetes , dementia , malnutrition , all of which are common in the elderly and have been shown to be associated with alterations in serum adipokine and oc levels .

immune responses induced by helminths are predominantly of the th2 type involving cytokines such as interleukin-3 ( il-3 ) , il-4 , il-5 , il-9 , il-10 , and il-13 . these cytokines mediate immune responses typically characterized by increased levels of circulating ige antibodies , eosinophils , basophils , and mast cells . during infection , the immune system is exposed to different helminth - derived molecules , including proteins , lipids , and glycoconjugates present either at the surface of the worms or in the excretory - secretory ( es ) products . interaction of helminth - derived molecules with host cells can result in a shift of the immune response , from an inflammatory towards an anti - inflammatory type of response . helminth - derived molecules can modify dendritic cells ( dcs ) function and downregulate adaptive immune responses , through the induction of a regulatory network that include regulatory t ( treg ) cells , alternatively activated macrophages ( aams ) , and regulatory b ( breg ) cells . the induced immunosuppresive network , together with cytokines produced by diverse hematopoietic and nonhematopoietic cells as integral part of immunoregulatory pathways , appears to be essential for parasite survival and its effect can be extended to other inflammatory disorders such as allergies and autoimmune diseases [ 3 , 4 ] . however , the association between helminth infections and allergy does not always have an unequivocal outcome . while certain helminth infections protect against allergic diseases ( reviewed in ) , other helminths can exacerbate this immunopathology ( reviewed in ) . here , the role of dcs , treg , and other regulatory cells in helminth - induced immunoregulation , the consequences for inflammatory diseases , and the contrasting effects of toxocara and trichinella infections on allergic manifestations are discussed . dcs are sentinels on alert for possible danger signals to immediately activate local innate immune responses and subsequently , after antigen presentation , initiate the proper adaptive immune responses . dcs play therefore an essential role in shaping the immune response and controlling the course of infection . these cells are located throughout the body forming a complex network that allows them to communicate with different populations of lymphocytes . different dc subsets may have distinct locations , where they acquire antigens to be transported to the draining lymph nodes for t - cell priming . dcs as well as other innate immune cells possess various families of pattern recognition receptors ( prrs ) such as toll - like receptors ( tlrs ) , nod - like receptors , rig - like receptors , and the c - type lectin receptors ( clrs ) that allow them to recognize a great variety of pathogen - associated molecular patterns ( pamps ) . after pathogen recognition via various prr , dcs produce molecules that induce polarization of different types of responsiveness such as th1- , th2- , th17- , or treg - related . the response of dcs to pathogens is mediated in large part via tlr , with input from other prr resulting in changes in gene expression that leads to dcs maturation . maturation of these cells refers to a transition from a resting state into a more dynamic state in which the cells present antigen in the context of mhc , express costimulatory molecules such as cd40 , cd80 , and cd86 , and secrete a broad spectrum of cytokines and chemokines . tlr - mediated responses are controlled mainly by the myd88-dependent pathway , which is used by all tlr except tlr3- and by the tir - domain - containing adapter - inducing interferon--(trif)-dependent pathway , which is used by tlr3 and tlr4 . for instance , lacto - n - fucopentaose iii ( lnfpiii ) produced by trematode schistosoma mansoni , and es-62 , a phosphorylcholine - containing protein secreted by the nematode acanthocheilonema viteae , can condition dcs to induce th2 responses through tlr4 . likewise , monoacetylated phosphatidyl serine lipids from schistosomes specifically instruct dcs to preferentially induce il-10-producing treg in a tlr2-dependent manner . this was also demonstrated in tlr2-deficient mice that showed a reduced number of cd4+cd25 + treg cells and immunopathology during schistosomiasis . studies using schistosomal antigens suggest that helminth glycans may be the conserved molecular pattern that instructs dcs via clr to drive th2-polarized responses . other recent studies demonstrate that host - like glycan antigens expressed by many helminths are recognized by dcs via lectin receptors . reported on blood group - like glycans from t. canis that bind the lectin dc - sign ( dendritic cell - specific intercellular adhesion molecule-3-grabbing nonintegrin ) , which may enable the activation of signal transduction pathways involving raf-1 and subsequent modulation of dc maturation resulting in skewing towards a th2 responses . lewis x antigen , a host - like glycan expressed on the surface of schistosomes in all life stages which is also present in secreted products such as the soluble egg antigens ( seas ) , also binds to dc - sign . dc maturation is considered to be essential for dcs to induce t - cell responses . it has become clear that dcs responding to helminth products do not mature in the conventional way upon encountering parasitic antigens but acquire a semimatured status and are still capable of inducing t - cell polarization . several studies support the findings that helminth products fail to directly activate dcs and other studies show that helminth products suppress dc maturation . for instance , sea suppresses lipopolysaccharide- ( lps- ) induced activation of immature murine dcs , as indicated by decreased mhc class ii and costimulatory molecules expression in addition to il-12 production . this resulted in increased lps - induced production of il-10 by dc after incubation by sea . pretreatment of dcs and macrophages with es-62 also inhibits their ability to produce il-12p70 in response to lps . in another study , a mixture of high molecular weight components from ascaris suum was found to reduce the lps - induced expression of mhcii , cd80 , cd86 , and cd40 molecules on mouse cd11c+ dcs and to hampered t - cell proliferative responses in vitro . fasciola hepatica tegumental antigen alone did not induce cytokine production or cell surface marker expression on murine dcs ; however , it significantly suppressed cytokine production and cell surface marker expression in dcs matured with a range of tlr and non - tlr ligands . in vitro studies on the impact of t. spiralis excretory / secretory products ( tspes ) on mice dcs revealed that these parasitic antigens suppress dc maturation induced by lps derived from different bacteria . in this study , different tlr agonists were used showing that the suppressive effect of tspes on dc maturation is restricted to tlr4 . these helminth products were also shown to interfere with the expression of several genes related to the tlr - mediated signal transduction pathways . for rat bone - marrow - derived dc it has been shown that after incubation with tspes these dcs acquire a semimatured status which is reflected in moderate upregulation of cd86 , significant upregulation of icam1 ( intercellular adhesion molecule 1 ) , and no upregulation of mhc ii , accompanied by impaired production of il-12 p70 . treg cells control peripheral immune responses and are likely to play a central role in autoimmune , infectious , and allergic diseases . three phenotypes of treg have been described to date , categorized according to their origin , function , and expression of cell surface markers : natural treg cells ( cd4+cd25+foxp3 + ) and inducible treg cells that include the il-10-producing tr1 cells and the foxp3 + t cells induced in the periphery . in spite of the complexity of regulatory cell types , cd4+cd25+foxp3 + treg cells are the most prominent population of immunoregulatory cells known so far to be induced during helminth infections . early studies had already suggested regulatory t - cell activity during chronic helminth infections in humans . reported that il-10 and transforming growth factor- ( tgf- ) production mediated the hyporesponsiveness observed in pbmc from individuals with generalized onchocercosis caused by the filarial nematode onchocerca volvulus . in a study with filariasis patients , lymphedema was associated with a deficiency in the expression of foxp3 , gitr ( glucocorticoid - induced tumour - necrosis - factor - receptor - related protein ) , tgf- , and ctla-4 ( cytotoxic t - lymphocyte antigen 4 ) , known to be expressed by treg cells , while in children infected with intestinal nematodes ( ascaris lumbricoides and trichuris trichiura ) high levels of il-10 and tgf- in addition to generalized t - cell hyporesponsiveness were found [ 27 , 28 ] . likewise , schistosome - infected individuals in kenya and gabon had higher cd4+cd25 + and cd4+cd25 + foxp3 + t - cell levels compared with uninfected individuals . one of the studies providing evidence on the suppressive effect of treg cells from helminth - infected individual is the one reported by wammes et al . . in this study , carried out in indonesia , treg cells from geohelminth - infected individuals were more effective at suppressing proliferation and ifn- production by effector t cells in response to malaria antigens and bcg than treg cells from healthy individuals . a filarial parasite of humans , brugia malayi , was found to secrete tgh-2 ( transforming growth factor homologue-2 ) , a homologue of host tgf- . since the recombinant tgh-2 can bind to the mammalian tgf- receptor , it has been suggested that it can promote the generation of regulatory t cells , as it has been shown for mammalian tgf-. in another study a significant increased expression of foxp3 and regulatory effector molecules such as tgf- , ctla-4 , pd-1 ( programmed death 1 ) and icos ( inducible costimulatory molecule ) was found in filaria - infected compared to uninfected individuals in response to live infective - stage larvae or microfilariae of brugia malayi . various studies on the role of treg cells in helminth infections have used animal models . in mice , cd25 + treg cells were shown to restrain the pathology to helminth eggs during schistosome infection and to trichuris muris in the gut . moreover , depletion of cd25 + treg cells with combined antibodies to cd25 and gitr resulted in enhanced immunity to filarial nematode litomosoides sigmodontis in mice . generation of treg cells with elevated expression of foxp3 during helminth infection has also been demonstrated . for instance , infection of balb / c mice with brugia pahangi third - stage larvae ( l3 ) resulted in expansion of a population of cd4+cd25 + t cells which was highly enriched in foxp3 and il-10 gene expression . induction of treg cells was demonstrated to be necessary to establish a chronic l. sigmodontis infection since depletion of treg cells in susceptible mice prior to infection enhanced parasitic killing and cleared the infection . in chronic infection with the gastrointestinal helminth heligmosomoides polygyrus , it was established that levels of foxp3 expression within the cd4 + t - cell population of mice mesenteric lymph nodes were significantly increased and that purified cd4+cd25 + treg cells possess suppressive activity in vitro [ 37 , 38 ] . the effect of tspes on t - cell activation in vitro was investigated using splenocytes derived from ovalbumin- ( ova)-tcr transgenic d011.10 mice that were incubated with tspes results indicate that the presence of tspes resulted in expansion of cd4+cd25 + t cells that express high levels of foxp3 + . these treg cells were shown to have suppressive activity and to produce tgf-. together these results indicate that t. spiralis secretion products can induce expansion of functional treg cells in vitro . in a rat model , the infection with t. spiralis is accompanied with the increase proportion of foxp3 + treg cells . in vitro studies showed that dcs stimulated with tspes caused strong th2 polarization , accompanied by elevated production of the regulatory cytokines il-10 and tgf- . however , unlike the mouse model described previously , conditioned rat dcs generated no increase in the proportion of cd4+cd25+foxp3 + t cells . in vivo t - cell priming with tspes stimulated dcs resulted in mixed th1/th2 cytokine response , with the dominance of the th2 type and elevated levels of regulatory cytokines . significant increase in the proportion of cd4+cd25+foxp3 + cells was found in spleen cells of recipients that received tspes stimulated dcs compared to the control value obtained from rats that received dcs cultivated in medium only . helminth infections may also lead to expansion of immunoregulatory cells other than treg cells , including alternatively activated macrophages ( aams ) and regulatory b cells . signals encountered during migration by developing macrophages determine their function at sites of inflammation or infection . among these signals , cytokines are responsible for the development of highly divergent macrophage phenotypes : classically activated and aams . reported that the homologues of the mammalian cytokine macrophage migration inhibitory factor ( mif ) expressed by brugia malayi synergized with il-4 to induce the development of suppressive aams in vitro . one pathway for this effect may be through the mif - mediated induction of il-4r expression on macrophages , amplifying in this way the potency of il-4 itself . thus , in a th2 environment , mif may prevent the classical activation of macrophages . the suppressive effect of aams on the immune response is most likely dependent on the expression of arginase-1 ( arg-1 ) as indicated by studies in which mice macrophages lacking arg-1 failed to suppress th2 responses ( reviewed in ) . b cells possess a variety of immune functions , including production of antibodies , presentation of antigens , and production of cytokines . il-10-producing regulatory b cells have great potential to regulate t - cell - mediated inflammatory responses and to downmodulate experimental autoimmune encephalomyelitis , collagen - induced arthritis , and inflammatory bowel disease . in addition , in mouse models of chronic parasitic inflammation , such as chronic schistosomiasis , il-10-producing b cells were also reported to be associated with protection against anaphylaxis . moreover , h. polygyrus - infected mice induced regulatory b cells that can downmodulate both allergy and autoimmunity in an il-10 independent manner . the helminth - induced immunosuppresive network may not only be beneficial for the parasite , but it can also have beneficial outcomes for the host , reducing allergic and autoimmune diseases [ 41 , 45 ] . epidemiological , cross - sectional studies support an inverse correlation between allergic diseases and helminth infection [ 46 , 47 ] including infections by nematode species like a. lumbricoides and necator americanus . an increased skin reactivity to house dust mites was found after antihelminthic treatment against infection with a. lumbricoides and t. trichiura . studies performed in animal model system have confirmed that helminth infection can protect against allergic disease and in particular lung - associated inflammation . for instance , s. mansoni - infected balb / c mice were protected against ova - induced experimental allergic airway inflammation ( eaai ) as indicated by reduction of eosinophils in bal , th2 cytokine production , ova - specific ige levels and reduction of the number of inflammatory cells in lungs . here , found that chronic infection with the filarial parasite litomosoides sigmodontis suppressed all pathological features of the ova - induced eaai model . additionally , these authors observed significantly increased numbers of treg cells in spleen and mediastinal lymph nodes in infected ova - treated mice compared to ova - controls animals . suppression of eaai during the course of h. polygyrus infection was shown to involve the induction of treg cells . infection with the same parasite resulted also in the inhibition of allergic response to peanut extract . several epidemiological studies have investigated the protective effect of parasitic infections in different autoimmune diseases like multiple sclerosis and type 1 diabetes . studies indicate that persons infected with chronic parasitic worm infections have lower rates of inflammatory bowel disease ( ibd ) than persons without these infections . experiments carried out using animal models of human autoimmune diseases have shown that parasites can interfere with autoimmunity . schistosoma mansoni infection has been shown to protect from type 1 diabetes and reduces the severity of eae while infection with h. polygyrus suppresses the experimental colitis . findings regarding the use of parasite antigens to suppress experimental inflammatory diseases are summarized in table 2 . although the majority of data suggest that infection with helminths is associated with a suppression of allergic and autoimmune responses , some examples provide the opposite view . epidemiological studies indicate that infection with ascaris spp , toxocara spp , fasciola hepatica , hookworms , or enterobius vermicularis has no protective effects or even enhanced allergic responses ( reviewed in ) . there are also experimental studies that show that infection with some helminths have a positive association with allergy . a study using a murine model has shown that t. canis infection results in exacerbation of eaai . other animal experiments provided evidence that parasites like nippostrongylus brasiliensis and b. malayi could also induce or exacerbate allergic responses . the links between infections and autoimmunity are complex and there is scarce evidence on the induction or exacerbation of autoimmune responses by helminths . toxocara canis and toxocara cati are roundworms of dogs and cats , respectively , that can also infect humans worldwide . after ingestion of the infectious toxocara eggs , the larvae migrate to the intestine , liver , and lungs . while in dogs and cats under the age of 6 months , the larva migrate back to the intestine ; in humans migration continues to other organs where they can persist for many years . toxocara infection results in the induction of th2 cells that make cytokines such as il-4 , il-5 , and il-13 , which induce responses to the parasite such as increased ige levels and eosinophilia ( reviewed in ) . after ingestion of trichinella infected meat , the larvae migrate to the intestine and matures to the adult stage , the parasites mate , and finally the newborn larvae ( nbl ) migrate to striated muscle cells where they become encysted . infection with t. spiralis is characterized by the induction of a th1 type of response at the beginning of the intestinal phase . when the nbl disseminate , a dominant th2 type of response develops which is essential for parasite expulsion . interestingly these helminths have a contrasting effect on inflammatory diseases , while infections with trichinella spp . can suppress ( reviewed in ) toxocara spp . studies using animal models for human autoimmune and allergic diseases indicate that trichinella infection ameliorates these immune disorders ( table 3 ) . khan et al . showed that t. spiralis infection reduces the severity of dinitrobenzenesulphonic - acid- ( dnbs- ) induced colitis in c57bl/6 mice . demonstrated that in addition to the protection exerted by the actual infection , rectal submucosal administration of t. spiralis crude muscle larvae antigen can also protect . t. spiralis infection also ameliorated autoimmune diabetes in nod mice and modulated severity of the disease in the experimental model of multiple sclerosis ( ms ) , namely , experimental autoimmune encephalomyelitis ( eae ) in dark agouti rats in a dose - dependent manner . in this study severity of eae as judged by cumulative disease index , maximal clinical score , duration of illness , and the number of mononuclear cells infiltrating the spinal cord in t. spiralis infected animals were all reduced in comparison to the uninfected eae - induced group . in a following study , these authors reported that alleviation of the disease in infected - eae rats coincided with reduced ifn- and il-17 production and increased il-4 , il-10 , and tgf- production . they suggested that mechanisms underlying the observed beneficial effect include th2 and regulatory responses provoked by the parasite . transfer of t - cell - enriched spleen cells from t. spiralis - infected rats that contained a higher proportion of cd4+cd25+foxp3 + regulatory t cells into rats in which eae was induced caused amelioration of eae , which indirectly points to the role of treg in restraining inflammatory conditions . have shown with another trichinella species , namely , with trichinella pseudospiralis , that infection results in suppression of ms in the rat . these authors used this model to compare the anti - inflammatory effects of the intestinal and late migratory phases of t. pseudospiralis infection on development of myelin - basic - protein- ( mbp- ) induced ms - like debilitation . findings from this study indicate that the late migratory phase of infection which occurred during the peak of mbp - induced debilitation significantly improved performance scores in mobility , coordination , and strength . wu et al . also reported on amelioration of clinical severity and delayed onset of eae after t. pseudospiralis infection . trichinella pseudospiralis is markedly different from t. spiralis in that it is smaller in size and that the muscle stage larvae are not surrounded by a capsule . whether the mechanisms involved in immunosuppression varies depending on trichinella species remains to be investigated . the concentrations of il-10 and tgf- were significantly increased and the recruitment of treg into draining lymph nodes was elevated as the result of t. spiralis infection . this protective effect has been recently shown to occur during acute as well as chronic phases of trichinella infection . protection against eaai to ova was stronger during the chronic phase of infection and associated with increased numbers of splenic cd4+cd25+foxp3 + treg cells with suppressive activity . adoptive transfer of cd4 + t cells from chronically infected mice with elevated numbers of treg cells in the spleen induced partial protection against eaai . the possible mechanisms by which helminths or their products could inhibit allergic responses are depicted in figure 1 . in contrast to the suppressive effect of trichinella infections on allergic diseases experimental as well as epidemiological studies indicate that toxocara infections are risk factors for allergies , including allergic asthma . studies using murine models for toxocariosis indicate that infection with t. canis leads to persistent pulmonary inflammation , eosinophilia , increase levels of circulating ige , airway hyperreactivity , and production of th-2 type cytokines . pulmonary inflammation has been shown to develop as soon as 48 hours after infection , it occurs in a dose - dependent manner , and it can persist up to 2 or 3 months . eosinophil counts also increase in the bronchoalveolar lavage ( bal ) of toxocara - infected mice [ 106 , 107 ] . relative quantification of cytokine expression in lungs of mice infected with different t. canis doses showed that while a proportional increased expression of the il-4 , il-5 , and il-10 transcripts was observed , the expression of ifn- was not different from that of uninfected controls . results from this study indicate that infection of mice with t. canis results in a dominant th2 type of immune response , independent of the inoculum size . in addition , infection of balb / c mice with 1,000 t. canis embryonated eggs results in hyperreactivity of the airways that persisted up to 30 days p.i . evaluation of parasite burden revealed that few t. canis larvae were still present in the lungs of infected mice at 60 days p.i . which could explain the persistent pulmonary inflammation observed in these mice . common features between allergic asthma and toxocariosis are the induction of a th2-cell mediated immune response including the production of high levels of ige , and eosinophilia . shares common clinical features with allergic asthma such as inflammation of the airways accompanied with wheezing , coughs , mucus hypersecretion , and bronchial hyperreactivity . in order to study the effect of toxocara infection on allergic manifestations two murine models were combined , namely , the murine model for toxocariosis described above and a murine model for allergic airway inflammation . for this study balb / c mice were infected with 500 embryonated t. canis eggs and exposed to ova sensitization followed by ova - challenge . results indicate that infection with t. canis in combination with ova treatment led to exacerbation of pulmonary inflammation ; eosinophilia ; airway hyperresponsiveness ; increase of ova specific and total ige ; increased expression of il-4 compared to mice that were only t. canis infected or ova treated . the observed exacerbation of eaai was independent of the timing of infection in relation to allergen exposure the normal or definitive hosts for t. canis are dogs whereas humans are accidental hosts for this parasite . in an accidental host the parasite does not usually develop to the adult stage and in case of toxocara spp . the continuous migration of the larvae through different organs including the lungs can cause more damage comparing to what happens in a definitive host where migration is transitory . t. spiralis can infect many different mammals including mice and humans in which the parasite completes its life cycle . and although t. spiralis pass through the lung microvascular system on its way to the skeletal muscle , it is a rapid process in which the larvae are usually not trapped in the lungs . it is likely that there are differences between parasites of humans such as t. spiralis that have evolved with their host and have developed strategies to survive without causing much damage compared to parasites such as toxocara spp . for which humans are accidental host [ 6 , 110 ] . in conclusion , helminths induce an anti - inflammatory response , which could ameliorate inflammatory diseases ; however , this is not a universal property of all helminths and different factors such as the helminth species , and whether the host is definitive or accidental , the parasite load and acute versus chronic infections may all influence the overall effect of helminth infections on inflammatory diseases . identification of the helminth molecules that induce immunosuppression and elucidation of the mechanisms involved is essential for the development of alternative strategies for prevention and/or treatment of inflammatory diseases .

macropathogens , such as multicellular helminths , are considered masters of immunoregulation due to their ability to escape host defense and establish chronic infections . molecular crosstalk between the host and the parasite starts immediately after their encounter , which influences the course and development of both the innate and adaptive arms of the immune response . helminths can modulate dendritic cells ( dcs ) function and induce immunosuppression which is mediated by a regulatory network that includes regulatory t ( treg ) cells , regulatory b ( breg ) cells , and alternatively activated macrophages ( aams ) . in this way , helminths suppress and control both parasite - specific and unrelated immunopathology in the host such as th1-mediated autoimmune and th2-mediated allergic diseases . however , certain helminths favour the development or exacerbation of allergic responses . in this paper , the cell types that play an essential role in helminth - induced immunoregulation , the consequences for inflammatory diseases , and the contrasting effects of toxocara and trichinella infection on allergic manifestations are discussed .

1. Introduction 2. Dendritic Cells 3. Regulatory T Cells 4. Other Regulatory Cells 5. Helminth Infections and Inflammatory Diseases 6. Contrasting Effect of

immune responses induced by helminths are predominantly of the th2 type involving cytokines such as interleukin-3 ( il-3 ) , il-4 , il-5 , il-9 , il-10 , and il-13 . during infection , the immune system is exposed to different helminth - derived molecules , including proteins , lipids , and glycoconjugates present either at the surface of the worms or in the excretory - secretory ( es ) products . interaction of helminth - derived molecules with host cells can result in a shift of the immune response , from an inflammatory towards an anti - inflammatory type of response . helminth - derived molecules can modify dendritic cells ( dcs ) function and downregulate adaptive immune responses , through the induction of a regulatory network that include regulatory t ( treg ) cells , alternatively activated macrophages ( aams ) , and regulatory b ( breg ) cells . however , the association between helminth infections and allergy does not always have an unequivocal outcome . while certain helminth infections protect against allergic diseases ( reviewed in ) , other helminths can exacerbate this immunopathology ( reviewed in ) . here , the role of dcs , treg , and other regulatory cells in helminth - induced immunoregulation , the consequences for inflammatory diseases , and the contrasting effects of toxocara and trichinella infections on allergic manifestations are discussed . dcs play therefore an essential role in shaping the immune response and controlling the course of infection . these cells are located throughout the body forming a complex network that allows them to communicate with different populations of lymphocytes . dcs as well as other innate immune cells possess various families of pattern recognition receptors ( prrs ) such as toll - like receptors ( tlrs ) , nod - like receptors , rig - like receptors , and the c - type lectin receptors ( clrs ) that allow them to recognize a great variety of pathogen - associated molecular patterns ( pamps ) . after pathogen recognition via various prr , dcs produce molecules that induce polarization of different types of responsiveness such as th1- , th2- , th17- , or treg - related . the response of dcs to pathogens is mediated in large part via tlr , with input from other prr resulting in changes in gene expression that leads to dcs maturation . maturation of these cells refers to a transition from a resting state into a more dynamic state in which the cells present antigen in the context of mhc , express costimulatory molecules such as cd40 , cd80 , and cd86 , and secrete a broad spectrum of cytokines and chemokines . tlr - mediated responses are controlled mainly by the myd88-dependent pathway , which is used by all tlr except tlr3- and by the tir - domain - containing adapter - inducing interferon--(trif)-dependent pathway , which is used by tlr3 and tlr4 . for instance , lacto - n - fucopentaose iii ( lnfpiii ) produced by trematode schistosoma mansoni , and es-62 , a phosphorylcholine - containing protein secreted by the nematode acanthocheilonema viteae , can condition dcs to induce th2 responses through tlr4 . reported on blood group - like glycans from t. canis that bind the lectin dc - sign ( dendritic cell - specific intercellular adhesion molecule-3-grabbing nonintegrin ) , which may enable the activation of signal transduction pathways involving raf-1 and subsequent modulation of dc maturation resulting in skewing towards a th2 responses . lewis x antigen , a host - like glycan expressed on the surface of schistosomes in all life stages which is also present in secreted products such as the soluble egg antigens ( seas ) , also binds to dc - sign . it has become clear that dcs responding to helminth products do not mature in the conventional way upon encountering parasitic antigens but acquire a semimatured status and are still capable of inducing t - cell polarization . this resulted in increased lps - induced production of il-10 by dc after incubation by sea . pretreatment of dcs and macrophages with es-62 also inhibits their ability to produce il-12p70 in response to lps . in another study , a mixture of high molecular weight components from ascaris suum was found to reduce the lps - induced expression of mhcii , cd80 , cd86 , and cd40 molecules on mouse cd11c+ dcs and to hampered t - cell proliferative responses in vitro . fasciola hepatica tegumental antigen alone did not induce cytokine production or cell surface marker expression on murine dcs ; however , it significantly suppressed cytokine production and cell surface marker expression in dcs matured with a range of tlr and non - tlr ligands . in this study , different tlr agonists were used showing that the suppressive effect of tspes on dc maturation is restricted to tlr4 . for rat bone - marrow - derived dc it has been shown that after incubation with tspes these dcs acquire a semimatured status which is reflected in moderate upregulation of cd86 , significant upregulation of icam1 ( intercellular adhesion molecule 1 ) , and no upregulation of mhc ii , accompanied by impaired production of il-12 p70 . treg cells control peripheral immune responses and are likely to play a central role in autoimmune , infectious , and allergic diseases . three phenotypes of treg have been described to date , categorized according to their origin , function , and expression of cell surface markers : natural treg cells ( cd4+cd25+foxp3 + ) and inducible treg cells that include the il-10-producing tr1 cells and the foxp3 + t cells induced in the periphery . in spite of the complexity of regulatory cell types , cd4+cd25+foxp3 + treg cells are the most prominent population of immunoregulatory cells known so far to be induced during helminth infections . in a study with filariasis patients , lymphedema was associated with a deficiency in the expression of foxp3 , gitr ( glucocorticoid - induced tumour - necrosis - factor - receptor - related protein ) , tgf- , and ctla-4 ( cytotoxic t - lymphocyte antigen 4 ) , known to be expressed by treg cells , while in children infected with intestinal nematodes ( ascaris lumbricoides and trichuris trichiura ) high levels of il-10 and tgf- in addition to generalized t - cell hyporesponsiveness were found [ 27 , 28 ] . one of the studies providing evidence on the suppressive effect of treg cells from helminth - infected individual is the one reported by wammes et al . in this study , carried out in indonesia , treg cells from geohelminth - infected individuals were more effective at suppressing proliferation and ifn- production by effector t cells in response to malaria antigens and bcg than treg cells from healthy individuals . since the recombinant tgh-2 can bind to the mammalian tgf- receptor , it has been suggested that it can promote the generation of regulatory t cells , as it has been shown for mammalian tgf-. in another study a significant increased expression of foxp3 and regulatory effector molecules such as tgf- , ctla-4 , pd-1 ( programmed death 1 ) and icos ( inducible costimulatory molecule ) was found in filaria - infected compared to uninfected individuals in response to live infective - stage larvae or microfilariae of brugia malayi . in mice , cd25 + treg cells were shown to restrain the pathology to helminth eggs during schistosome infection and to trichuris muris in the gut . in a rat model , the infection with t. spiralis is accompanied with the increase proportion of foxp3 + treg cells . in vitro studies showed that dcs stimulated with tspes caused strong th2 polarization , accompanied by elevated production of the regulatory cytokines il-10 and tgf- . however , unlike the mouse model described previously , conditioned rat dcs generated no increase in the proportion of cd4+cd25+foxp3 + t cells . in vivo t - cell priming with tspes stimulated dcs resulted in mixed th1/th2 cytokine response , with the dominance of the th2 type and elevated levels of regulatory cytokines . significant increase in the proportion of cd4+cd25+foxp3 + cells was found in spleen cells of recipients that received tspes stimulated dcs compared to the control value obtained from rats that received dcs cultivated in medium only . helminth infections may also lead to expansion of immunoregulatory cells other than treg cells , including alternatively activated macrophages ( aams ) and regulatory b cells . among these signals , cytokines are responsible for the development of highly divergent macrophage phenotypes : classically activated and aams . reported that the homologues of the mammalian cytokine macrophage migration inhibitory factor ( mif ) expressed by brugia malayi synergized with il-4 to induce the development of suppressive aams in vitro . one pathway for this effect may be through the mif - mediated induction of il-4r expression on macrophages , amplifying in this way the potency of il-4 itself . the suppressive effect of aams on the immune response is most likely dependent on the expression of arginase-1 ( arg-1 ) as indicated by studies in which mice macrophages lacking arg-1 failed to suppress th2 responses ( reviewed in ) . il-10-producing regulatory b cells have great potential to regulate t - cell - mediated inflammatory responses and to downmodulate experimental autoimmune encephalomyelitis , collagen - induced arthritis , and inflammatory bowel disease . in addition , in mouse models of chronic parasitic inflammation , such as chronic schistosomiasis , il-10-producing b cells were also reported to be associated with protection against anaphylaxis . moreover , h. polygyrus - infected mice induced regulatory b cells that can downmodulate both allergy and autoimmunity in an il-10 independent manner . the helminth - induced immunosuppresive network may not only be beneficial for the parasite , but it can also have beneficial outcomes for the host , reducing allergic and autoimmune diseases [ 41 , 45 ] . epidemiological , cross - sectional studies support an inverse correlation between allergic diseases and helminth infection [ 46 , 47 ] including infections by nematode species like a. lumbricoides and necator americanus . for instance , s. mansoni - infected balb / c mice were protected against ova - induced experimental allergic airway inflammation ( eaai ) as indicated by reduction of eosinophils in bal , th2 cytokine production , ova - specific ige levels and reduction of the number of inflammatory cells in lungs . here , found that chronic infection with the filarial parasite litomosoides sigmodontis suppressed all pathological features of the ova - induced eaai model . suppression of eaai during the course of h. polygyrus infection was shown to involve the induction of treg cells . infection with the same parasite resulted also in the inhibition of allergic response to peanut extract . findings regarding the use of parasite antigens to suppress experimental inflammatory diseases are summarized in table 2 . epidemiological studies indicate that infection with ascaris spp , toxocara spp , fasciola hepatica , hookworms , or enterobius vermicularis has no protective effects or even enhanced allergic responses ( reviewed in ) . a study using a murine model has shown that t. canis infection results in exacerbation of eaai . other animal experiments provided evidence that parasites like nippostrongylus brasiliensis and b. malayi could also induce or exacerbate allergic responses . the links between infections and autoimmunity are complex and there is scarce evidence on the induction or exacerbation of autoimmune responses by helminths . after ingestion of the infectious toxocara eggs , the larvae migrate to the intestine , liver , and lungs . while in dogs and cats under the age of 6 months , the larva migrate back to the intestine ; in humans migration continues to other organs where they can persist for many years . toxocara infection results in the induction of th2 cells that make cytokines such as il-4 , il-5 , and il-13 , which induce responses to the parasite such as increased ige levels and eosinophilia ( reviewed in ) . after ingestion of trichinella infected meat , the larvae migrate to the intestine and matures to the adult stage , the parasites mate , and finally the newborn larvae ( nbl ) migrate to striated muscle cells where they become encysted . infection with t. spiralis is characterized by the induction of a th1 type of response at the beginning of the intestinal phase . when the nbl disseminate , a dominant th2 type of response develops which is essential for parasite expulsion . interestingly these helminths have a contrasting effect on inflammatory diseases , while infections with trichinella spp . studies using animal models for human autoimmune and allergic diseases indicate that trichinella infection ameliorates these immune disorders ( table 3 ) . t. spiralis infection also ameliorated autoimmune diabetes in nod mice and modulated severity of the disease in the experimental model of multiple sclerosis ( ms ) , namely , experimental autoimmune encephalomyelitis ( eae ) in dark agouti rats in a dose - dependent manner . in this study severity of eae as judged by cumulative disease index , maximal clinical score , duration of illness , and the number of mononuclear cells infiltrating the spinal cord in t. spiralis infected animals were all reduced in comparison to the uninfected eae - induced group . in a following study , these authors reported that alleviation of the disease in infected - eae rats coincided with reduced ifn- and il-17 production and increased il-4 , il-10 , and tgf- production . transfer of t - cell - enriched spleen cells from t. spiralis - infected rats that contained a higher proportion of cd4+cd25+foxp3 + regulatory t cells into rats in which eae was induced caused amelioration of eae , which indirectly points to the role of treg in restraining inflammatory conditions . have shown with another trichinella species , namely , with trichinella pseudospiralis , that infection results in suppression of ms in the rat . these authors used this model to compare the anti - inflammatory effects of the intestinal and late migratory phases of t. pseudospiralis infection on development of myelin - basic - protein- ( mbp- ) induced ms - like debilitation . findings from this study indicate that the late migratory phase of infection which occurred during the peak of mbp - induced debilitation significantly improved performance scores in mobility , coordination , and strength . the concentrations of il-10 and tgf- were significantly increased and the recruitment of treg into draining lymph nodes was elevated as the result of t. spiralis infection . this protective effect has been recently shown to occur during acute as well as chronic phases of trichinella infection . adoptive transfer of cd4 + t cells from chronically infected mice with elevated numbers of treg cells in the spleen induced partial protection against eaai . the possible mechanisms by which helminths or their products could inhibit allergic responses are depicted in figure 1 . in contrast to the suppressive effect of trichinella infections on allergic diseases experimental as well as epidemiological studies indicate that toxocara infections are risk factors for allergies , including allergic asthma . studies using murine models for toxocariosis indicate that infection with t. canis leads to persistent pulmonary inflammation , eosinophilia , increase levels of circulating ige , airway hyperreactivity , and production of th-2 type cytokines . eosinophil counts also increase in the bronchoalveolar lavage ( bal ) of toxocara - infected mice [ 106 , 107 ] . relative quantification of cytokine expression in lungs of mice infected with different t. canis doses showed that while a proportional increased expression of the il-4 , il-5 , and il-10 transcripts was observed , the expression of ifn- was not different from that of uninfected controls . results from this study indicate that infection of mice with t. canis results in a dominant th2 type of immune response , independent of the inoculum size . in addition , infection of balb / c mice with 1,000 t. canis embryonated eggs results in hyperreactivity of the airways that persisted up to 30 days p.i . evaluation of parasite burden revealed that few t. canis larvae were still present in the lungs of infected mice at 60 days p.i . common features between allergic asthma and toxocariosis are the induction of a th2-cell mediated immune response including the production of high levels of ige , and eosinophilia . shares common clinical features with allergic asthma such as inflammation of the airways accompanied with wheezing , coughs , mucus hypersecretion , and bronchial hyperreactivity . in order to study the effect of toxocara infection on allergic manifestations two murine models were combined , namely , the murine model for toxocariosis described above and a murine model for allergic airway inflammation . results indicate that infection with t. canis in combination with ova treatment led to exacerbation of pulmonary inflammation ; eosinophilia ; airway hyperresponsiveness ; increase of ova specific and total ige ; increased expression of il-4 compared to mice that were only t. canis infected or ova treated . the observed exacerbation of eaai was independent of the timing of infection in relation to allergen exposure the normal or definitive hosts for t. canis are dogs whereas humans are accidental hosts for this parasite . in an accidental host the parasite does not usually develop to the adult stage and in case of toxocara spp . and although t. spiralis pass through the lung microvascular system on its way to the skeletal muscle , it is a rapid process in which the larvae are usually not trapped in the lungs . it is likely that there are differences between parasites of humans such as t. spiralis that have evolved with their host and have developed strategies to survive without causing much damage compared to parasites such as toxocara spp . in conclusion , helminths induce an anti - inflammatory response , which could ameliorate inflammatory diseases ; however , this is not a universal property of all helminths and different factors such as the helminth species , and whether the host is definitive or accidental , the parasite load and acute versus chronic infections may all influence the overall effect of helminth infections on inflammatory diseases . identification of the helminth molecules that induce immunosuppression and elucidation of the mechanisms involved is essential for the development of alternative strategies for prevention and/or treatment of inflammatory diseases .

coronary artery bypass surgery ( cabg ) has been considered the gold standard for treatment of unprotected left - main coronary disease ( lmca ) based on the results of several trials which have shown reductions in mortality. the current guidelines also see cabg as a class i indication for significant unprotected lmca disease in patients which are eligible for surgery . , however , a lot of the elderly patients are associated with multiple co - morbidities and at high surgical risk . though in the study of yanagawa et al . , the overall mortality of cabg declined from 6.0% ( 49/817 ) in the earliest era ( 19901996 ) to 1.9% ( 22/1132 ) in the most recent era ( 20032010 ; p < 0.001 ) , the mortality rate could be as high as 9.1% with an appreciable morbidity rate of 16.4% . elderly patients who had prior cabg are also in unfavorable anatomical conditions for repeated bypass surgery . furthermore , in some countries , religion and traditional thinking usually preclude the patients from open heart surgery . over the past 10 years , advances in percutaneous intervention ( pci ) techniques , devices and operator skills all contributed to the increased numbers of and studies on lmca pci . however , limited information is available for clinical outcomes of lmca pci in high - risk elderly patients . furthermore , coronary artery calcification , especially in the lmca , imposes a big challenge for pci , in terms of vessel rigidity and device delivery difficulties . rotational atherectomy ( ra ) , with the ability to differentially ablate calcified and fibrotic plaques , is particularly useful in these lesions . , ra has been advocated in the bare metal stent ( bms ) era , but is often under - used due to technical difficulty , cumbersome setup and no additional benefit in the reported literature . , in recent years , revival of ra use in the drug eluting stent ( des ) era in the treatment of complex lesion treatment has been witnessed . however , there have been very limited reports on using ra for lmca pci in the literature . the aim of this study was to investigate the safety and clinical efficacy of incorporating ra for plaque debulking during pci for heavily - calcified lmca lesions in actual practice . from february 2004 to march 2012 , all consecutive patients who received ra treatment for heavily - calcified lmca lesions were queried from the cath lab database for all - inclusive recruitment . the relevant clinical and angiographic characteristics at the time of index pci , as well as the clinical follow - up outcomes , were retrieved from the database , and collected after a thorough review of the medical chart records . this study protocol was approved by the institutional review board for human research of our hospital . the angiographic measurements were made on a viewing workstation with software for quantitative analysis of angiograms ( medcon / horizon / tcs , israel ) . the angiographic characterization of target lesions in the index coronary angiogram was made by reviewing the session cine thoroughly . the lmca was defined as any segment of the left main 50% diameter stenosis . the coronary artery disease ( cad ) vessel numbers were defined as the number of each of the three major coronary vessels 70% diameter stenosis . severe coronary artery calcification was defined as readily apparent radio - opacities within the vascular walls in more than one projection on the cine before contrast medium injection . in all of our patients , the merits and demerits of both pci and cabg were fully explained to both the patients themselves and their families after diagnostic angiograms were completed . the final decision to undertake pci was made after taking into account the multiple factors and personal preference . patients who signed the informed consent and subsequently completed pci with rotablation were retrospectively recruited into this study . all pcis were performed by experienced , credentialed operators using standard practice in our cath lab . patients were pretreated with aspirin and clopidogrel , or a minimum of 300 mg loading dose of clopidogrel was administered if , in rare cases , patients were not pretreated . heparin was administered to maintain an activated clotting time ( act ) of > 300 s. the decision to do ra was made at the discretion at the operator if device delivery or full lesion dilatation was deemed impossible by the heavy coronary artery calcification at the beginning of the procedure . a 0.009-inch floppy rotawire was advanced by the wire exchanging technique through a microcatheter or over - the - wire balloon . ra was carried out using the rotablator ra system ( boston scientific corporation ) , starting with a 1.25 mm or 1.5 mm burr at a speed of 180,000200,000 r / min and mostly supplemented by another burr one size larger ( figure 1 ) . each advance time was not longer than 30 s. normal saline with heparin and isosorbide dinitrate was infused during the atherectomy . ra of one or both major branches of lm was made at the discretion of the operators . if ra of both major branches was needed , usually the vessel with more critical lesions was treated first . after the blood flow in the first treated vessel was secured , rotawiring of the another branch was made and no regular wire was left in the treated vessel . after the rotablation , both major branches were rewired with usual wires and the procedure then proceeded with balloon dilatation with , or without , stent implantation to achieve optimal angiographic results with minimal residual stenosis . the use of des or bms was determined by patient option ( affordability ) , physician discretion or other co - morbidities ( impending non - cardiac surgery , drug compliance to dual antiplatelets or drug allergy ) . use of glycoprotein 2b3a antagonist , intra - aortic ballon pump ( iabp ) or intravascular ultrasound ( ivus ) were at the discretion of the operator as indicated by the clinical requirements . patients with cardiogenic shock were managed with standard practice , including iabp support , intravenous inotropes and pulmonary capillary wedge pressure monitoring . emergent coronary angiography and revascularization were intended for every patient to achieve re - opening of the culprit vessel with grade iii thrombolysis in myocardial infarction ( timi 3 ) flow as fast as possible . angiographic success was defined as achievement of a residual stenosis < 20% in the presence of timi 3 flow . procedural success was defined as achieving angiographic success without in - hospital major adverse cardiac events ( mace ) , including all - cause death , myocardial infarction ( mi ) , and repeat revascularization . target lesion revascularization ( tlr ) was defined as a repeat revascularization for a restenosis > 50% in the target segment . target vessel revascularization ( tvr ) was defined as any repeat revascularization within the treated vessel . cardiac biomarkers including creatine kinase ( ck ) , creatine kinase - mb ( ck - mb ) , and troponin - i were routinely measured in patients after the procedure . myonecrosis was defined as exceeding three times the level of ck - mb or troponin - i compared to the baseline value . clinical follow - up was carried out through reviews of medical records , or telephone contact . from february 2004 to march 2012 , all consecutive patients who received ra treatment for heavily - calcified lmca lesions were queried from the cath lab database for all - inclusive recruitment . the relevant clinical and angiographic characteristics at the time of index pci , as well as the clinical follow - up outcomes , were retrieved from the database , and collected after a thorough review of the medical chart records . this study protocol was approved by the institutional review board for human research of our hospital . the angiographic measurements were made on a viewing workstation with software for quantitative analysis of angiograms ( medcon / horizon / tcs , israel ) . the angiographic characterization of target lesions in the index coronary angiogram was made by reviewing the session cine thoroughly . the lmca was defined as any segment of the left main 50% diameter stenosis . the coronary artery disease ( cad ) vessel numbers were defined as the number of each of the three major coronary vessels 70% diameter stenosis . severe coronary artery calcification was defined as readily apparent radio - opacities within the vascular walls in more than one projection on the cine before contrast medium injection . this was the principle strictly followed at our cath lab . in all of our patients , the merits and demerits of both pci and cabg were fully explained to both the patients themselves and their families after diagnostic angiograms were completed . the final decision to undertake pci was made after taking into account the multiple factors and personal preference . patients who signed the informed consent and subsequently completed pci with rotablation were retrospectively recruited into this study . all pcis were performed by experienced , credentialed operators using standard practice in our cath lab . patients were pretreated with aspirin and clopidogrel , or a minimum of 300 mg loading dose of clopidogrel was administered if , in rare cases , patients were not pretreated . heparin was administered to maintain an activated clotting time ( act ) of > 300 s. the decision to do ra was made at the discretion at the operator if device delivery or full lesion dilatation was deemed impossible by the heavy coronary artery calcification at the beginning of the procedure . a 0.009-inch floppy rotawire was advanced by the wire exchanging technique through a microcatheter or over - the - wire balloon . ra was carried out using the rotablator ra system ( boston scientific corporation ) , starting with a 1.25 mm or 1.5 mm burr at a speed of 180,000200,000 r / min and mostly supplemented by another burr one size larger ( figure 1 ) . each advance time was not longer than 30 s. normal saline with heparin and isosorbide dinitrate was infused during the atherectomy . ra of one or both major branches of lm was made at the discretion of the operators . if ra of both major branches was needed , usually the vessel with more critical lesions was treated first . after the blood flow in the first treated vessel was secured , rotawiring of the another branch was made and no regular wire was left in the treated vessel . after the rotablation , both major branches were rewired with usual wires and the procedure then proceeded with balloon dilatation with , or without , stent implantation to achieve optimal angiographic results with minimal residual stenosis . the use of des or bms was determined by patient option ( affordability ) , physician discretion or other co - morbidities ( impending non - cardiac surgery , drug compliance to dual antiplatelets or drug allergy ) . use of glycoprotein 2b3a antagonist , intra - aortic ballon pump ( iabp ) or intravascular ultrasound ( ivus ) were at the discretion of the operator as indicated by the clinical requirements . patients with cardiogenic shock were managed with standard practice , including iabp support , intravenous inotropes and pulmonary capillary wedge pressure monitoring . emergent coronary angiography and revascularization were intended for every patient to achieve re - opening of the culprit vessel with grade iii thrombolysis in myocardial infarction ( timi 3 ) flow as fast as possible . angiographic success was defined as achievement of a residual stenosis < 20% in the presence of timi 3 flow . procedural success was defined as achieving angiographic success without in - hospital major adverse cardiac events ( mace ) , including all - cause death , myocardial infarction ( mi ) , and repeat revascularization . target lesion revascularization ( tlr ) was defined as a repeat revascularization for a restenosis > 50% in the target segment . target vessel revascularization ( tvr ) was defined as any repeat revascularization within the treated vessel . cardiac biomarkers including creatine kinase ( ck ) , creatine kinase - mb ( ck - mb ) , and troponin - i were routinely measured in patients after the procedure . myonecrosis was defined as exceeding three times the level of ck - mb or troponin - i compared to the baseline value . clinical follow - up was carried out through reviews of medical records , or telephone contact . during the study period , a total of 34 patients met the all - inclusion criterion and were recruited into this study for retrospective analysis . eighteen ( 52.9% ) patients had diabetes mellitus and twenty - two ( 64.7% ) chronic kidney disease . ten ( 29.4% ) had prior cabg , fourteen ( 41.2% ) prior pci , and nine ( 26.5% ) recent ( within four weeks ) mi . regarding clinical presentation , ten ( 29.4% ) patients presented with unstable angina , fifteen ( 44.1% ) with non - st - elevation mi ( nstemi ) and three ( 8.8% ) with st - elevation mi ( stemi ) . furthermore , the estimated glomerular filtration rate ( egfr ) was 37.8 22.1 ml / min . the mean syntax score was 50 15 , and the surgical mortality rate of the cohort according to the euroscore ii scale was 5.6 4.8% . ( a ) : severely - calcified lmca , lad and lcx could be found readily by apparent radio - opacities within the vascular walls before contrast medium injection ( as indicated by black arrows ) ; ( b ) : coronary angiography revealed lm shaft and distal true bifurcation lesions , both lad and lcx showed diffuse atherosclerosis change with heavy calcification ; ( c ) : rotaburr could successfully advance to the lad ; ( d ) : the final flow was good without residual stenosis . lad : left anterior descending artery ; lcx : left circumflex artery ; lmca : lmca : left main coronary artery ; pci : percutaneous coronary intervention . cabg : coronary artery bypass graft ; ckd : chronic kidney disease ; dm : diabetes mellitus ; egfr : estimated glomerular filtration rate ; ldl : low density lipoprotein ; lvef : left ventricular ejection fraction ; mi : myocardial infarction ; nstemi : non - st segmental elevation myocardial infarction ; pad : peripheral artery disease ; pci : percutaneous intervention ; rca : right coronary artery ; sbp : systolic blood pressure ; stemi : st segmental elevation myocardial infarction . lad : left anterior descending artery ; lcx : left circumflex artery ; lmca : left main coronary artery ; the lmca lesion characteristics are presented in table 2 . the lmca bifurcation lesions involved the ostia of both left anterior descending ( lad ) and left cirrumflex ( lcx ) coronary artery in twelve ( 35.3% ) patients . thirty - one ( 91.2% ) lesions were eccentric , and three ( 8.8% ) had thrombus . the angiographic success rate was 100% with rota burrs delivered to the target lesions in all of these patients , and there was no major procedural complication . the average number of burrs used per patient was 1.7 0.5 and the final burr size 1.6 0.2 mm . three ( 8.8% ) patients underwent ra pci without stenting . regarding the lm stenting strategy , twenty - two ( 64.7% ) were crossing - over , four ( 11.8% ) simultaneous kissing or v , two ( 5.9% ) culotte and one ( 2.9% ) crushing . des was used in twenty - three ( 67.6% ) of these patients and bms in eight ( 23.5% ) . seven patients ( 20.6% ) underwent intra - aortic balloon pump ( iabp)-assisted procedures due to cardiogenic shock , or intractable ventricular tachycardia . the baseline and post - pci diameter stenoses of lmca rate were 70% 7% and 18% 5% , respectively . bms : bare - metal stent ; des : drug - eluting stent ; iabp : intra - aortic balloon pump ; gp iib / iiia : glycoprotein iib / iiia inhibitor ; ivus : intravascular ultrasound . lmca : left main coronary artery ; mld : minimal lumen diameter ; timi : thrombolysis in myocardial infarction . the in - hospital and out - of - hospital follow - up outcomes are shown in table 5 . the interventional procedure success rate was 91.2% as three patients died during hospitalization , whom all presenting with cardiogenic shock complicating extensive anterior wall stemi on hospitalization . the average age for these succumbed patients was 82 1 years ( range 8183 years ) and all had triple vessel disease . one of them received ra plus bms for lm - lad , one ra plus des for lm - lad and the other culotte des for lm bifurcation lesion . besides these three cardiac deaths , there was one asymptomatic myonecrosis and one acute limb ischemia which may be ascribed to iabp use . there was no q - wave mi , emergent cabg , repeated pci , or cardiac tamponade during hospitalization . the incidence of out - of- hospital mace at a mean follow - up of 30.4 months ( range 299 months ) was 16.1% . among thirty - one out - of - hospital follow - up patients , 12 patients ( 38.7% ) received coronary angiographic follow - up . tlr was needed in three ( 9.7% ) and tvr in two ( 6.4% ) patients . cabg : coronary artery bypass graft ; mace : major adverse cardiac event ; mi : myocardial infarction ; nstemi : non - st segmental elevation myocardial infarction ; stemi : st segmental elevation myocardial infarction ; tlr : target lesion revascularization ; tvr : target vessel revascularization . in summary , we found in this study that pci incorporating ra for heavily calcified lmca lesions could be safely carried out with minimal complication rates in high risk elderly patients , and this procedure was associated with very favorable angiographic and clinical outcomes in actual practice . in recent years , more and more data demonstrate the safety and feasibility of pci in unprotected lmca disease . , though the major adverse cardiovascular events of pci seemed to be comparable to that of cabg in unprotected lmca disease of low or intermediate syntax scores , pci was associated with more serious adverse events in patients with a high syntax score , and it had a significantly higher tvr risk in a meta - analysis . though in the current pci guidelines , cabg remains the class i indication for unprotected lmca disease , increasingly favorable pci clinical results underscore the fact that pci for unprotected left main has been upgraded to a class iia or iib alternative to cabg in anatomically suitable patients and those with high surgical risks . , in the actual practice , religious thinking and local traditions usually prevent elderly patients with complex lm lesions from undergoing open heart surgery . on the other hand , well - performed pci was associated with short procedure time , short duration of hospitalization , and faster recovery , which are very important in high risk patients , whereas bypass surgery in elderly patients with multiple co - morbidities could be associated with very high surgical risk . , furthermore , repeat bypass surgery for patients with previous cabg is usually complicated by lack of proper arterial conduits if those were used in previous surgery and higher peri - operative morbidity and mortality , for which the independent risk factors included unstable angina , poor preoperative left ventricular function , renal insufficiency , insulin dependent diabetes and an interval shorter than one year of the initial operation . most of the previously - operated elderly patients have more than one of the above independent risk factors . therefore , pci for lmca lesions is often performed on patients with graft occlusion after cabg despite complex coronary anatomies . sometimes , pci is the only , or at least , the more favorable choice of revascularization in morbid elderly patients who could hardly tolerate repeated bypass surgery . on the other hand , the difficulties in lmca pci include the variations in main vessel and side branch vessel size , lesion location , involvement of distal bifurcation , differences in bifurcation angle , vessel calcifications , extent of other vessel involvement and occlusion of contra - lateral arteries . regarding vessel calcification , coronary artery calcification is an active and complex process that involves numerous mechanisms responsible for calcium depositions in arterial walls which progresses with ages . , though the extent of coronary artery calcification was not strongly associated with age before the ninth decade , it is not unusual that heavily calcified coronary lesions occurred in the elderly patient group . up to date , there is no effective medical therapy that can reverse the calcium deposits in the coronary vessels . very tight calcified lmca lesions may resist dilation at low balloon inflation pressures , or even rupture at the highest possible pressure . stent delivery through calcified lesions may also be difficult and stent expansion may be suboptimal due to high resistance of the calcified plaques . the revival of ra in des era , particularly in very complex heavily - calcified lesions , , underscores the importance the plaque modifications before des could be successfully delivered , precisely placed and deployed to exert the expected effects . though ra is an effective and reasonably - priced debulking modality in the cath lab , it is cumbersome to set up , more difficult to use , especially for distal lesions , and more prone to complications if not adequately prepared or performed . acute no flow , severe vessel dissection with impending acute closure , atheroembolism and transient profound hypotension are the most frequently encountered risks in rotablation . most of these are also difficult to deal with in the middle of intervening complex lesions in high risk patients . vessel perforation and entrapment of rotablator burr are less frequently met complications , but are also difficult to manage . , these are the reasons why rotablation are formidable to a lot of coronary interventionists . it is very important to prevent these events in advance while handling them adequately once they occur . on the other hand , thrombus - laden vessels , vessels with existing advanced dissections , last remaining vessels and saphenous these limitations were also present in our cath labs , in which rotablation was only used in 2.4% of all pci cases . no insurance reimbursement is probably the major cause of under - use of this device in this region and probably many others . in the current study , ra for lmca lesions was mostly done in patients with high clinical risks , including acs , depressed lv function , cardiogenic shock , chronic renal insufficiency , old age and diabetes , and in very complex coronary lesions which were heavily calcified . a significant part of patients were previously pci- or cabg - treated and many patients had triple coronary disease and true lm bifurcation lesions . ra was successfully performed and lmca pci completed with very good angiographic documented success rates in these patients , and no complications noted . though under - use , less familiarity , and resorting to ra only in heavily calcified complex lesions may increase the complication rate and decrease the angiographic and clinical outcomes , particularly in high risk patients , this , however , was not the case in our cath lab . in general , des was recommended for lm stenting , absolutely so in situations where two - stent techniques were needed . once the des is delivered and deployed after adequate debulking , good results could be anticipated . in this study , a major proportion of patients were treated with des , but a minor proportion treated with bms . this was made at the discretion of the operators , taking into consideration of patient affordability . a small number of lesions in the current study was not stented , as full expansion and timi 3 final blood flow had been achieved after ra and balloon angioplasty of these lesions without any dissection . regarding the stenting strategy for distal lmca lesions , it is determined by multiple anatomical and angiographic factors . though crossing - over technique was most often used and associated with better outcomes , , the two - stent strategy was mandatory in certain anatomies . , in the current study , a quarter of our patients were treated by the two - stent technique . this was compatible with universal practice as published in the literature . despite the complexity of our treated lesions , the angiographic success rate was 100% , which could be due to more complete lesion modification by more than one burr usage per lesion ( 1.7 0.4 ) . though there were three ( 8.8% ) in - hospital deaths in our patients , none were procedure - related and all were caused by underlying extensive myocardial infarction with cardiogenic shock . use of iabp in a considerable proportion of our procedures may argument for the high risk nature of our patients on one hand , and may account for the very high procedure success and no major complication in this group on the other hand . iabp provides temporary circulatory support during the transient cease of blood flow while intervening major epicedial coronary vessels like the left main stem . it might be especially useful in intervening left main lesions with very invasive devices like rotablation , particularly when complicated by acute no flow , profound hypotension and severe vessel dissection . however , in our cath lab , these adverse events were very low with our refined rotablation practice of giving optimal premedications ( oral dual antiplatelets , calcium channel blockers , nitrates ) before admission , large dose intra - coronary nitrate prior to initiating rotation and familiarity with this device ( quick procedure , use of two burr sizes in most cases ) . these are the reasons why iabp use was limited to certain high risk , but not most of patients . the clinical follow - up mace in patients who could be successfully discharged from hospital was 16.1% , quite similar to that in another study of ours looking at ra followed by des implantation in calcified coronary artery diseases , and to those in published studies . , it is interesting to notice that half of our patients were octogenarians or nonagenarians . dahdouh , et al . reported 85.7% of lmca lesions had moderate to severe calcification in the octogenarians . this finding suggests that increased numbers of extremely calcified lesions needed to be treated percutaneously , and this high demand dictates the incorporation of ra in pci . firstly , the sample size in this single - center , observational study was relatively small . because of the insurance reimbursement issue in our clinical practice , we had to limit ra to patients with very unfavorable angiographic characteristics which otherwise would preclude successful pci treatment if without proper debulking . despite this , use of ra in lmca pci in high risk elderly patients was quite promising and rewarding in our hands . though ivus guidance during des implantation could reduce both des thrombosis and the need for repeat revascularization , it was also underused due to no insurance coverage in our lab . even given this , the risk of stent thrombosis was negligible and tlr / tvr was not high in our high risk elderly patients . however , clinical driven angiographic follow - up is a more reasonable approach and commonly accepted worldwide . our practice was consistent with that approach in conclusion , plaque modification with ra to facilitate subsequent interventional procedures in very complex heavily - calcified lmca lesions could be safely accomplished with a very low complication rate . this appears clinically rewarding with good angiographic results and a low follow - up mace rate in high - surgical - risk elderly patients . the high procedural success rate , low tlr and very promising cumulative mace rate convince us to sustain , and even broaden , the use of this novel , but underused , device in suitable patients .

objectiveheavily calcified left - main coronary diseases ( lmca ) remain a formidable challenge for percutaneous interventions ( pci ) . this study was to investigate the safety and efficacy of using rotational atherectomy ( ra ) in treating such lesions in actual practice.methodsfrom february 2004 to march 2012 , all consecutive patients who received ra for heavily - calcified lmca lesions in our cath lab were enrolled . the relevant clinical and angiographic characteristics at the time of index pci , as well as the clinical follow - up outcomes , were retrieved and analyzed.resultsa total of 34 consecutive patients were recruited with a mean age 77.2 10.2 years . there were 82.4% presented with acute coronary syndrome and 11.8% with cardiogenic shock . chronic renal disease and diabetes were seen in 64.7% and 52.9% , respectively . triple - vessel coronary disease was found in 76.5% of them . the mean syntax score was 50 15 and euroscore ii scale 5.6 4.8 . the angiographic success rate was 100% with a procedural success rate of 91.2% . the mean number of burrs per patient was 1.7 0.5 . crossing - over stenting was used in 64.7% . most stents were drug - eluting ( 67.6% ) . intra - aortic ballon pump was used in 20.6% of the procedures . three patients died during hospitalization , all due to presenting cardiogenic shock . no major complication occurred . among 31 hospital survivors , the major adverse cardiac events ( mace ) rate was 16.1% , all due to target lesion revascularization or target vessel revascularization.conclusionsin high - surgical - risk elderly patients , plaque modification with ra in pci of heavily - calcified lmca could be safely accomplished with a minimal complication rate and low out - of - hospital mace .

Introduction Methods Patient population Angiographic characterization and measurements Statistical analysis Results Discussion Conclusions

coronary artery bypass surgery ( cabg ) has been considered the gold standard for treatment of unprotected left - main coronary disease ( lmca ) based on the results of several trials which have shown reductions in mortality. , however , a lot of the elderly patients are associated with multiple co - morbidities and at high surgical risk . , the overall mortality of cabg declined from 6.0% ( 49/817 ) in the earliest era ( 19901996 ) to 1.9% ( 22/1132 ) in the most recent era ( 20032010 ; p < 0.001 ) , the mortality rate could be as high as 9.1% with an appreciable morbidity rate of 16.4% . elderly patients who had prior cabg are also in unfavorable anatomical conditions for repeated bypass surgery . over the past 10 years , advances in percutaneous intervention ( pci ) techniques , devices and operator skills all contributed to the increased numbers of and studies on lmca pci . however , limited information is available for clinical outcomes of lmca pci in high - risk elderly patients . furthermore , coronary artery calcification , especially in the lmca , imposes a big challenge for pci , in terms of vessel rigidity and device delivery difficulties . rotational atherectomy ( ra ) , with the ability to differentially ablate calcified and fibrotic plaques , is particularly useful in these lesions . , ra has been advocated in the bare metal stent ( bms ) era , but is often under - used due to technical difficulty , cumbersome setup and no additional benefit in the reported literature . however , there have been very limited reports on using ra for lmca pci in the literature . the aim of this study was to investigate the safety and clinical efficacy of incorporating ra for plaque debulking during pci for heavily - calcified lmca lesions in actual practice . from february 2004 to march 2012 , all consecutive patients who received ra treatment for heavily - calcified lmca lesions were queried from the cath lab database for all - inclusive recruitment . the relevant clinical and angiographic characteristics at the time of index pci , as well as the clinical follow - up outcomes , were retrieved from the database , and collected after a thorough review of the medical chart records . the angiographic measurements were made on a viewing workstation with software for quantitative analysis of angiograms ( medcon / horizon / tcs , israel ) . the angiographic characterization of target lesions in the index coronary angiogram was made by reviewing the session cine thoroughly . the lmca was defined as any segment of the left main 50% diameter stenosis . the coronary artery disease ( cad ) vessel numbers were defined as the number of each of the three major coronary vessels 70% diameter stenosis . in all of our patients , the merits and demerits of both pci and cabg were fully explained to both the patients themselves and their families after diagnostic angiograms were completed . patients who signed the informed consent and subsequently completed pci with rotablation were retrospectively recruited into this study . all pcis were performed by experienced , credentialed operators using standard practice in our cath lab . heparin was administered to maintain an activated clotting time ( act ) of > 300 s. the decision to do ra was made at the discretion at the operator if device delivery or full lesion dilatation was deemed impossible by the heavy coronary artery calcification at the beginning of the procedure . ra was carried out using the rotablator ra system ( boston scientific corporation ) , starting with a 1.25 mm or 1.5 mm burr at a speed of 180,000200,000 r / min and mostly supplemented by another burr one size larger ( figure 1 ) . ra of one or both major branches of lm was made at the discretion of the operators . after the blood flow in the first treated vessel was secured , rotawiring of the another branch was made and no regular wire was left in the treated vessel . use of glycoprotein 2b3a antagonist , intra - aortic ballon pump ( iabp ) or intravascular ultrasound ( ivus ) were at the discretion of the operator as indicated by the clinical requirements . patients with cardiogenic shock were managed with standard practice , including iabp support , intravenous inotropes and pulmonary capillary wedge pressure monitoring . angiographic success was defined as achievement of a residual stenosis < 20% in the presence of timi 3 flow . procedural success was defined as achieving angiographic success without in - hospital major adverse cardiac events ( mace ) , including all - cause death , myocardial infarction ( mi ) , and repeat revascularization . target lesion revascularization ( tlr ) was defined as a repeat revascularization for a restenosis > 50% in the target segment . target vessel revascularization ( tvr ) was defined as any repeat revascularization within the treated vessel . clinical follow - up was carried out through reviews of medical records , or telephone contact . from february 2004 to march 2012 , all consecutive patients who received ra treatment for heavily - calcified lmca lesions were queried from the cath lab database for all - inclusive recruitment . the relevant clinical and angiographic characteristics at the time of index pci , as well as the clinical follow - up outcomes , were retrieved from the database , and collected after a thorough review of the medical chart records . the angiographic measurements were made on a viewing workstation with software for quantitative analysis of angiograms ( medcon / horizon / tcs , israel ) . the angiographic characterization of target lesions in the index coronary angiogram was made by reviewing the session cine thoroughly . the coronary artery disease ( cad ) vessel numbers were defined as the number of each of the three major coronary vessels 70% diameter stenosis . this was the principle strictly followed at our cath lab . in all of our patients , the merits and demerits of both pci and cabg were fully explained to both the patients themselves and their families after diagnostic angiograms were completed . patients who signed the informed consent and subsequently completed pci with rotablation were retrospectively recruited into this study . all pcis were performed by experienced , credentialed operators using standard practice in our cath lab . patients were pretreated with aspirin and clopidogrel , or a minimum of 300 mg loading dose of clopidogrel was administered if , in rare cases , patients were not pretreated . heparin was administered to maintain an activated clotting time ( act ) of > 300 s. the decision to do ra was made at the discretion at the operator if device delivery or full lesion dilatation was deemed impossible by the heavy coronary artery calcification at the beginning of the procedure . ra of one or both major branches of lm was made at the discretion of the operators . after the blood flow in the first treated vessel was secured , rotawiring of the another branch was made and no regular wire was left in the treated vessel . use of glycoprotein 2b3a antagonist , intra - aortic ballon pump ( iabp ) or intravascular ultrasound ( ivus ) were at the discretion of the operator as indicated by the clinical requirements . patients with cardiogenic shock were managed with standard practice , including iabp support , intravenous inotropes and pulmonary capillary wedge pressure monitoring . emergent coronary angiography and revascularization were intended for every patient to achieve re - opening of the culprit vessel with grade iii thrombolysis in myocardial infarction ( timi 3 ) flow as fast as possible . angiographic success was defined as achievement of a residual stenosis < 20% in the presence of timi 3 flow . procedural success was defined as achieving angiographic success without in - hospital major adverse cardiac events ( mace ) , including all - cause death , myocardial infarction ( mi ) , and repeat revascularization . target lesion revascularization ( tlr ) was defined as a repeat revascularization for a restenosis > 50% in the target segment . target vessel revascularization ( tvr ) was defined as any repeat revascularization within the treated vessel . clinical follow - up was carried out through reviews of medical records , or telephone contact . during the study period , a total of 34 patients met the all - inclusion criterion and were recruited into this study for retrospective analysis . eighteen ( 52.9% ) patients had diabetes mellitus and twenty - two ( 64.7% ) chronic kidney disease . regarding clinical presentation , ten ( 29.4% ) patients presented with unstable angina , fifteen ( 44.1% ) with non - st - elevation mi ( nstemi ) and three ( 8.8% ) with st - elevation mi ( stemi ) . the mean syntax score was 50 15 , and the surgical mortality rate of the cohort according to the euroscore ii scale was 5.6 4.8% . ( a ) : severely - calcified lmca , lad and lcx could be found readily by apparent radio - opacities within the vascular walls before contrast medium injection ( as indicated by black arrows ) ; ( b ) : coronary angiography revealed lm shaft and distal true bifurcation lesions , both lad and lcx showed diffuse atherosclerosis change with heavy calcification ; ( c ) : rotaburr could successfully advance to the lad ; ( d ) : the final flow was good without residual stenosis . lad : left anterior descending artery ; lcx : left circumflex artery ; lmca : lmca : left main coronary artery ; pci : percutaneous coronary intervention . the angiographic success rate was 100% with rota burrs delivered to the target lesions in all of these patients , and there was no major procedural complication . the average number of burrs used per patient was 1.7 0.5 and the final burr size 1.6 0.2 mm . regarding the lm stenting strategy , twenty - two ( 64.7% ) were crossing - over , four ( 11.8% ) simultaneous kissing or v , two ( 5.9% ) culotte and one ( 2.9% ) crushing . des was used in twenty - three ( 67.6% ) of these patients and bms in eight ( 23.5% ) . seven patients ( 20.6% ) underwent intra - aortic balloon pump ( iabp)-assisted procedures due to cardiogenic shock , or intractable ventricular tachycardia . the baseline and post - pci diameter stenoses of lmca rate were 70% 7% and 18% 5% , respectively . bms : bare - metal stent ; des : drug - eluting stent ; iabp : intra - aortic balloon pump ; gp iib / iiia : glycoprotein iib / iiia inhibitor ; ivus : intravascular ultrasound . the in - hospital and out - of - hospital follow - up outcomes are shown in table 5 . the interventional procedure success rate was 91.2% as three patients died during hospitalization , whom all presenting with cardiogenic shock complicating extensive anterior wall stemi on hospitalization . one of them received ra plus bms for lm - lad , one ra plus des for lm - lad and the other culotte des for lm bifurcation lesion . there was no q - wave mi , emergent cabg , repeated pci , or cardiac tamponade during hospitalization . the incidence of out - of- hospital mace at a mean follow - up of 30.4 months ( range 299 months ) was 16.1% . among thirty - one out - of - hospital follow - up patients , 12 patients ( 38.7% ) received coronary angiographic follow - up . cabg : coronary artery bypass graft ; mace : major adverse cardiac event ; mi : myocardial infarction ; nstemi : non - st segmental elevation myocardial infarction ; stemi : st segmental elevation myocardial infarction ; tlr : target lesion revascularization ; tvr : target vessel revascularization . in summary , we found in this study that pci incorporating ra for heavily calcified lmca lesions could be safely carried out with minimal complication rates in high risk elderly patients , and this procedure was associated with very favorable angiographic and clinical outcomes in actual practice . in recent years , more and more data demonstrate the safety and feasibility of pci in unprotected lmca disease . , though the major adverse cardiovascular events of pci seemed to be comparable to that of cabg in unprotected lmca disease of low or intermediate syntax scores , pci was associated with more serious adverse events in patients with a high syntax score , and it had a significantly higher tvr risk in a meta - analysis . , in the actual practice , religious thinking and local traditions usually prevent elderly patients with complex lm lesions from undergoing open heart surgery . on the other hand , well - performed pci was associated with short procedure time , short duration of hospitalization , and faster recovery , which are very important in high risk patients , whereas bypass surgery in elderly patients with multiple co - morbidities could be associated with very high surgical risk . , furthermore , repeat bypass surgery for patients with previous cabg is usually complicated by lack of proper arterial conduits if those were used in previous surgery and higher peri - operative morbidity and mortality , for which the independent risk factors included unstable angina , poor preoperative left ventricular function , renal insufficiency , insulin dependent diabetes and an interval shorter than one year of the initial operation . most of the previously - operated elderly patients have more than one of the above independent risk factors . therefore , pci for lmca lesions is often performed on patients with graft occlusion after cabg despite complex coronary anatomies . sometimes , pci is the only , or at least , the more favorable choice of revascularization in morbid elderly patients who could hardly tolerate repeated bypass surgery . very tight calcified lmca lesions may resist dilation at low balloon inflation pressures , or even rupture at the highest possible pressure . stent delivery through calcified lesions may also be difficult and stent expansion may be suboptimal due to high resistance of the calcified plaques . the revival of ra in des era , particularly in very complex heavily - calcified lesions , , underscores the importance the plaque modifications before des could be successfully delivered , precisely placed and deployed to exert the expected effects . though ra is an effective and reasonably - priced debulking modality in the cath lab , it is cumbersome to set up , more difficult to use , especially for distal lesions , and more prone to complications if not adequately prepared or performed . most of these are also difficult to deal with in the middle of intervening complex lesions in high risk patients . on the other hand , thrombus - laden vessels , vessels with existing advanced dissections , last remaining vessels and saphenous these limitations were also present in our cath labs , in which rotablation was only used in 2.4% of all pci cases . no insurance reimbursement is probably the major cause of under - use of this device in this region and probably many others . in the current study , ra for lmca lesions was mostly done in patients with high clinical risks , including acs , depressed lv function , cardiogenic shock , chronic renal insufficiency , old age and diabetes , and in very complex coronary lesions which were heavily calcified . a significant part of patients were previously pci- or cabg - treated and many patients had triple coronary disease and true lm bifurcation lesions . ra was successfully performed and lmca pci completed with very good angiographic documented success rates in these patients , and no complications noted . though under - use , less familiarity , and resorting to ra only in heavily calcified complex lesions may increase the complication rate and decrease the angiographic and clinical outcomes , particularly in high risk patients , this , however , was not the case in our cath lab . in this study , a major proportion of patients were treated with des , but a minor proportion treated with bms . this was made at the discretion of the operators , taking into consideration of patient affordability . a small number of lesions in the current study was not stented , as full expansion and timi 3 final blood flow had been achieved after ra and balloon angioplasty of these lesions without any dissection . regarding the stenting strategy for distal lmca lesions , it is determined by multiple anatomical and angiographic factors . though crossing - over technique was most often used and associated with better outcomes , , the two - stent strategy was mandatory in certain anatomies . despite the complexity of our treated lesions , the angiographic success rate was 100% , which could be due to more complete lesion modification by more than one burr usage per lesion ( 1.7 0.4 ) . though there were three ( 8.8% ) in - hospital deaths in our patients , none were procedure - related and all were caused by underlying extensive myocardial infarction with cardiogenic shock . use of iabp in a considerable proportion of our procedures may argument for the high risk nature of our patients on one hand , and may account for the very high procedure success and no major complication in this group on the other hand . however , in our cath lab , these adverse events were very low with our refined rotablation practice of giving optimal premedications ( oral dual antiplatelets , calcium channel blockers , nitrates ) before admission , large dose intra - coronary nitrate prior to initiating rotation and familiarity with this device ( quick procedure , use of two burr sizes in most cases ) . the clinical follow - up mace in patients who could be successfully discharged from hospital was 16.1% , quite similar to that in another study of ours looking at ra followed by des implantation in calcified coronary artery diseases , and to those in published studies . reported 85.7% of lmca lesions had moderate to severe calcification in the octogenarians . this finding suggests that increased numbers of extremely calcified lesions needed to be treated percutaneously , and this high demand dictates the incorporation of ra in pci . firstly , the sample size in this single - center , observational study was relatively small . because of the insurance reimbursement issue in our clinical practice , we had to limit ra to patients with very unfavorable angiographic characteristics which otherwise would preclude successful pci treatment if without proper debulking . despite this , use of ra in lmca pci in high risk elderly patients was quite promising and rewarding in our hands . though ivus guidance during des implantation could reduce both des thrombosis and the need for repeat revascularization , it was also underused due to no insurance coverage in our lab . even given this , the risk of stent thrombosis was negligible and tlr / tvr was not high in our high risk elderly patients . however , clinical driven angiographic follow - up is a more reasonable approach and commonly accepted worldwide . our practice was consistent with that approach in conclusion , plaque modification with ra to facilitate subsequent interventional procedures in very complex heavily - calcified lmca lesions could be safely accomplished with a very low complication rate . this appears clinically rewarding with good angiographic results and a low follow - up mace rate in high - surgical - risk elderly patients . the high procedural success rate , low tlr and very promising cumulative mace rate convince us to sustain , and even broaden , the use of this novel , but underused , device in suitable patients .

the surfactant complex has a distinctive composition of approximately 8085% phospholipids , 510% neutral lipids ( mostly cholesterol ) , and 510% proteins , consisting of surfactant proteins a , b , c , and d ( sp - a , sp - b , sp - c , sp - d ) . the arrangement of these components at the air liquid interface allows complex monolayers of phospholipids to form with polar head groups oriented toward the liquid phase and the hydrophobic fatty acid chains facing the air phase ( lumen ) . sp - a molecular species are hetero - oligmers composed of two distinct gene products , sp - a1 and sp - a2 , each with multiple allelic variants . sp - a is an abundant hydrophilic protein constituent of surfactant that belongs to the collagen domain and c - type lectin family . sp - a is characterized by four structural domains consisting of ( 1 ) an n - terminal cysteine rich domain ; ( 2 ) a collagen - like domain ; ( 3 ) a coiled coil neck domain ; and ( 4 ) a c - terminal ca- and carbohydrate - binding domain . sp - a monomers undergo noncovalent trimerization by interactions of their collagen domains and their coiled - coil domains , and the trimers are covalently cross - linked by disulfide bonds at the n - terminal domains . sp - a isoforms also contain cys residues that can cross - link monomers and trimers within the collagen - like domains . upon complete oligomerization , sp - a assembles into a bouquet - like structure of six trimeric subunits ( octadecamer ) . sp - a contributes to surfactant homeostasis by facilitating the formation of tubular myelin and participating in lipid recycling from the extracellular compartment . sp - a is also recognized as an important mediator of pulmonary host defense through direct binding to pathogens , glycolipids , and cell surface receptors via the carbohydrate - binding domain and collagen - like domains . the genetics of sp - a are quite complex and are likely to function in innate immunity . the human sp - a1 and sp-2 genes arose from a duplication event and are separated by < 40 kb on chromosome 10 and transcribed in opposite directions . the genes are in linkage disequilibrium and may also share cis - acting regulatory elements . there are also multiple splice forms of sp - a in the 5 utr , and additional variability in the 3 utr . four of the sp - a1 allelic variants ( 6a , 6a , 6a , 6a ) and six sp - a2 allelic variants ( 1a , 1a , 1a , 1a , 1a , and 1a ) are observed with a frequency of greater than 1% in the general population . single nucleotide polymorphisms of sp - a have been implicated in susceptibility to tuberculosis infection , pulmonary fibrosis , lung cancer , and high - altitude pulmonary edema . the study of sp - a variants has largely been confined to ectopic expression in human adenocarcinoma cell lines and transgenic mice . while many of the variants appear to adopt normal structures , several rare variants ( e.g. , sp - a2 g231v and f198s ) are not properly secreted and undergo degradation in the endoplasmic reticulum . studies of transgenic mice expressing human sp - a have further demonstrated that the 6a variant of sp - a1 ( with leu50 and trp219 ) and the 1a variant of sp - a2 ( with ala91 and lys223 ) are detectable in bronchoalveolar lavage fluid by western blotting . few high resolution mass spectrometry analyses have been performed on native sp - a , and there has been little correlation between sp - a variants and their expressed proteoforms in the human lung . furthermore , the relative quantitation of sp - a1/sp - a2 protein is limited to the use of antibodies specific for sp - a1 versus total sp - a . here , we sought to explore the capability of mass spectrometry for identification and quantitation of sp - a isoforms and common variants , and we developed a targeted mass spectrometry assay to identify individuals expressing the variants of sp - a2 at amino acid 223 ( gln223 and lys223 ) in human bronchoalveolar lavage fluid . bronchoalveolar lavage ( bal ) was performed using an approved duke university institutional review board protocol as previously described . sp - a was purified from bal fluid from normal controls and asthmatic subjects using calcium chloride precipitation and differential centrifugation as previously described . sp - a from pulmonary alveolar proteinosis patients ( app - spa ) was purified by differential centrifugation and extraction with 1-butanol , followed by affinity chromatography on mannose sepharose as previously described . genomic dna was extracted from whole blood using a paxgene blood dna kit ( qiagen ) . the region of exon 6 containing nucleotide 3625 of sp - a2 was amplified by pcr and sequenced as previously described . purified proteins and bal fluids were concentrated using an amicon ultra 10k filters . up to 2 g of sp - a was separated by 412% bis - tris nupage ( invitrogen ) followed by staining with colloidal blue stain ( invitrogen ) . bands corresponding to sp - a monomer were excised followed by in - gel digestion with sequencing grade modified trypsin ( promega ) as previously described . after in - gel digestions , peptides were lyophilized and reconstituted in 10 l of 1% ( v / v ) trifluoroacetic acid ( tfa ) and 2% acetonitrile ( acn ) . for in - solution digestion , sp - a was diluted in 50 mm ammonium bicarbonate , ph 8.0 ( ambic ) containing 0.2% rapigest sf ( waters ) and 10 mm dtt and heated at 80 c for 10 min followed by alkylation with 20 mm iodoacetamide for 30 min at room temp in the dark . proteins were digested with 1:50 ( w / w ) trypsin : substrate , or 1:50 ( w / w ) sequencing grade glu - c ( promega):substrate in the presence of 0.5 mm glu - glu , at 37 c overnight . digestions were followed by addition of 1% tfa and 2% acn and heating at 60 c for 2 h to hydrolyze the rapigest . after centrifugation , digests were transferred to maximum recovery lc vials ( waters ) . ms / ms ) was performed using a waters nanoacquity uplc and waters g1 hdms or g2 hdms operating in data - dependent acquisition mode as previously described . fifty nanograms of peptide digests were trapped on a 20 m 180 mm symmetry c18 column ( waters ) at 20 l / min for 2 min in 0.1% aqueous formic acid ( fa ) followed by separation on a 75 m 250 mm column with 1.7 m c18 bridged ethane - silicone hybrid ( beh ) particles ( waters ) using a gradient of 540% acn/0.1% fa over 30 min , a flow rate of 0.4 l / min , and a column temp of 45 c . samples were analyzed in data - dependent ( dda ) mode using a 0.9 s precursor scan ( synapt g1 ) or a 0.6 s precursor scan ( synapt g2 ) , followed by ms / ms product ion scans on the top three most intense ions using a dynamic exclusion window of 120 s. two - dimensional - lc ms / ms ( 2d - lc ms / ms ) utilized a waters nanoacquity with 2d technology uplc . five microliters of peptides recovered by in - gel digestion was trapped at 2 l / min at 97/3 ( v / v ) water / acn in 20 mm ammonium formate ( ph 10 ) on a 5 m xbridge beh130 c18 300 m 50 mm column ( waters ) . peptides were eluted from the first dimension with 10.8% , 14.0% , 16.7% , 20.4% , and 50.0% acn at 2 l / min , diluted 10-fold online with 99.8/0.1/0.1 ( v / v / v ) h2o / acn / fa and trapped on a 5 m symmetry c18 180 m 20 mm trapping column ( waters ) . second dimension separations were performed on a 1.7 m acquity beh130 c18 75 m 150 mm column ( waters ) using a linear gradient of 7 to 35% acn with 0.1% fa over 37 min , at a flow rate of 0.5 l / min and column temperature of 35 c . ms data collection was performed using a synapt g2 in dda mode as described above . ms / ms data were processed using mascot distiller v.2.2 ( matrix science ) and searched using mascot v.2.2 against a swiss - prot database with homo sapiens taxonomy ( downloaded on 10/30/2010 ; 20,259 entries ) with additional sp - a variants 6a , 6a , 6a , 1a , 1a , and 1a ) . the database also contained an equal number of reversed - sequence decoy entries for false discovery rate determination . database searches used fixed modification on cys ( carbamidomethyl ) and variable modifications on met ( oxidation ) and asn / gln ( deamidation ) and pro ( hydroxyl ) . searches allowed for up to two missed trypsin cleavages and up to five missed cleavages with glu - c specificity . ms / ms data were visualized using scaffold ( proteome software ) and annotated at a 1% peptide / protein fdr . spiketides tql peptides , containing n- and c - labeled lys or arg and carbamidomethyl cys ( c * ) , were purchased from jpt ( berlin , germany ) with the following sequences : hqilqtr , galslqgsimtvgek , eqc*vemytdgqwndr and c*vemytdgqwndr . one nmole of each peptide was resuspended by heating in 50 l of 20% ( v / v ) acetonitrile : ambic at 50 c for 45 min . a total of 12.5 l of each peptide was mixed and diluted to 250 l with ambic followed by addition of 200 ng trypsin and was digested overnight at 37 c to remove the tql tag . following digestion , formic acid was added to 1% , and the peptides were stored at 80 c . one milliliter of bal fluid per sample was concentrated as described above followed by bradford assay . the entirety of each sample ( 3050 g ) was separated by sds - page alongside 2 g of purified sp - a . after staining , the region corresponding to sp - a monomer was digested as described above . peptides were resuspended in 10 l of 1%tfa/2%acn containing 50 ng/l of trypsin - digested , immunodepleted human plasma , and 50 fmol/l of sil peptides . the trypsinized sil peptide mixture ( 50 fmol / peptide ) was analyzed on a synapt g1 , and the raw data were searched using mascot , as described above . the resulting ms / ms spectral data ( .dat format ) were used to build a spectral library in skyline . default skyline peptide and transition settings were used except for the following transition setting : monoisotopic mass and the following peptide settings : structural modifications , carbamidomethyl cys , and isotope modifications , label : c6n4arg and label : c6n2-lys . the fasta sequences corresponding to sp - a1(6a ) and sp - a2(1a ) were imported , and precursor charge states and transitions were selected based on matches to the spectral library generated from mascot search results ( table 1 ) . an initial unscheduled method was exported from skyline using a default waters collision energy profile . after a peptide retention time was established , a scheduled method was exported with a 4 min retention time window for each peptide . initial method development and was performed using a nanoacquity uplc coupled via electrospray ionization to a xevo tq mass spectrometer ( waters corporation ) . briefly , 1 l of each sample was injected directly onto a 150 m 100 mm 1.7 m acquity beh130 c18 column ( waters ) using a 23 min gradient of 340% mecn at a flow rate of 1.8 l / min with a column temperature of 35 c . a modified skyline file contained the settings as described above with the following modifications to the peptide settings : structural modifications , hydroxyp ; and max variable mods , 7 . additional peptides corresponding to the modified forms of the sp - a2-specific peptide , gdpgppgpmgppgetpcppgnnglpgapgvpger , and product ions , were selected as shown in table 1 . methods were exported with a modified waters collision energy profile for 3 + charge ions : slope , 0.0359 and y - intercept , 2.042 . analysis of a second cohort of n = 8 was performed using a waters ionkey / ms system including a nanoacquity m - class uplc and an ionkey source containing a 150 m 100 mm , 1.7 m beh130 c18 , ikey separation device interfaced to a xevo tq - s mass spectrometer ( waters corporation ) . briefly , 1 l of each sample was injected directly onto the ikey and separated using a 23 min gradient of 340% mecn at a flow rate of 3.0 l / min with a column temperature of 35 c . to determine whether mass spectrometry could be used for identification and quantitation of common sp - a variants and of the sp - a1 and sp - a2 isoforms ( figure 1a ) , we first separated several different proteins ( sp - a from normal control and alveolar proteinosis patients ( app ) ; and recombinant lys223 sp - a2 ) by sds - page , and the band containing the sp - a monomer was digested with trypsin and analyzed by lc ms / ms analysis was sufficient to sequence most of the invariant regions of sp - a , and among known variable sites , afforded sequence coverage of the tryptic peptide containing gln223 ( figure 1a ) , which is shared between major variants of sp - a1 and sp - a2 , as well as the peptide formed by cleavage after lys223 , which is found in the 1a and 1a variants of sp - a2 . in native sp - a preparations , we identified the peptide spanning thr66 , asn73 , val81 , and arg85 , which is specific to sp - a2 ; however , the corresponding sp - a1-specific peptide spanning met66 , glu73 , ile81 , and cys85 was not identified . sequence coverage was also lacking for the regions containing positions 50 ( leu or val ) , 91 ( ala or pro ) , or 219 ( arg or trp ) . the predicted tryptic peptides containing positions 50 , 91 , and 219 of sp - a are small ( 46-mers ) , which likely explains why they were not identified . ( a ) a multiple sequence alignment of sp - a1 and sp - a2 isoforms was performed , with common variants ( > 1% of the population ) vertically juxtaposed ( see figure s1 for complete alignment ) . sequence coverage of tryptic peptides ( no missed cleavages ) from gelc ms / ms analysis of sp - a are indicated as follows : solid lines indicate peptides that are common to all sp - a isoforms and variants ; dashed lines indicate variant- or isoform - determining peptides . ( b ) seven additional sp - a preparations , including native app and sp - a purified from two human asthmatics , and recombinant leu50/trp219 sp - a1 and val50/arg219 sp - a1 , were digested in solution in parallel using either trypsin or glu - c . additional variant- or isoform - determining peptides that were identified by this method versus in - gel trypsin digestion in ( a ) are shown . we explored whether additional digestion conditions might improve coverage of variable regions . in - gel digestion of sp - a with glu - c gave poor sequence coverage ( data not shown ) ; thus , an additional five native sp - a preparations , as well as two recombinant purified sp - a1 variants , were digested in solution , separately with trypsin or glu - c , and analyzed by lc these 14 analyses gave sequence coverage for all of the major variable sites of sp - a1 and sp - a2 , including all possible amino acids at positions 50 , 91 , 219 , and 223 , as well as the sp - a1- and sp - a2-specific amino acids at positions 65 , 73 , 81 , and 85 ( figure 1b ) . however , the peptides containing pro91 , arg219 , or gln223 could not be assigned to a specific isoform . except for the trp219 peptide , all of the variant- or isoform - determining peptides identified solely by in solution digestion contained missed cleavages ( figure 1b ) . thus , the identification of these sites may be highly variable based on digestion conditions . we wondered whether targeted , quantative ms could definitively determine sp - a genotype at any of the variable regions , either as a means of de novo proteotyping of sp - a variants or to validate the expression of protein - coding variants in genotyped human subjects . none of the variant - determining peptides met ideal criteria for targeted proteomics : all contained either missed cleavages , oxidizable ( met and cys ) residues or variable prolyl hydroxylation ( supporting information ) . nonetheless , we focused on the 223 position , since these peptides could be readily detected by in - gel or in - solution trypsinization without missed cleavages . notably , lys223 ( rs1965708 ) has been shown to predispose individuals to high altitude pulmonary edema , respiratory syncytial virus infection , meningococcal disease and allergic rhinitis . this minor variant of sp - a2 is heterozygously expressed in 30% of the population , whereas only 5% are homozygous for lys at the 223 position of sp - a2 . although the tryptic peptides derived from gln223 and lys223 variant proteins ( eqcvemytdgqwdnr and cvemytdgqwdnr , respectively ) can be distinguished by mass and retention time , they have identical y - series product ions that can be used for quantitation ( figure 2 ) . we developed an mrm assay using stable - isotope labeled forms of these peptides as well as hqilqtr and galslqgsimtvgek , which are conserved across all sp - a isoforms and variants ( table 1 ) . during method optimization using sil peptides , we found that direct injection of the peptides onto the lc column ( i.e. , bypassing the trapping step ) reduced chromatographic peak width , improved peak shape , and greatly improved the sensitivity of the most hydrophilic peptide ( hqilqtr ) . consequently , the targeted lc ms / ms method utilized direct injection . ms / ms spectra of 223 variant - determining peptides . ms / ms spectra were aligned for the doubly charged tryptic peptides , ( a ) eqcvemytdgqwndr and ( b ) cvemytdgqwndr , which are site - determining for sp - a variants with gln223 and lys223 , respectively . identified b - series ( red ) and y - series ( blue ) ions are labeled . we first tried to perform the mrm assay on unfractionated bal fluid , but we were unable to detect the 223 variant peptides ( data not shown ) . depth of coverage can be improved by immunodepletion of abundant plasma proteins but this method is tedious . we hypothesized that an sds - page separation might yield sufficient enrichment of sp - a from balf for targeted quantitation . to test this enrichment strategy , 1 ml of balf each from two different individuals was concentrated by centrifugal filtration , and the entirety of recovered protein ( 50 g ) was separated by sds - page alongside purified sp - a . the region corresponding to sp - a monomer was excised from the balf lanes ( not shown ) , and in - gel digests were analyzed by two - dimensional reversed - phase reversed - phase lc ms / ms ( supplemental scaffold data , supporting information ) . across the two analyses , 76 proteins were identified , with 73 being common to both samples . on the basis of number of identified spectra , sp - a was the second most abundant protein in each of these digests , demonstrating that sp - a can be easily enriched from a small volume of balf by gel - based separation . to achieve the most quantitative recovery and aid the sample stability of sp - a for analysis , we added 50 ng of digested , immunodepleted human plasma protein during the final reconstitution of the in - gel digests prior to transfer to glass sample vials as a means of reducing peptide loss due to adsorption . we also confirmed that this exogenously added protein digest did not contain any detectable native sp - a peptides by mrm ( data not shown ) . to test this optimized gelc - mrm assay , we analyzed 1 ml of balf from seven normal subjects that had been genotyped at the sp - a2 locus ( figure 3a ) . the cohort was selected to contain samples from three individuals who were homozygous for the major sp - a2 genotype ( gln223/gln223 ) , three who were homozygous for the minor sp - a2 genotype ( lys223/lys223 ) , and two who were heterozygous for these two sp - a2 genotypes ( gln223/lys223 ) . from 1 ml of balf , between 30 and 50 g of protein was recovered per sample and separated by sds - page ( figure 3a ) . following in - gel digestion and reconstitution of digests , because of the high degree of enrichment of sp - a in these samples , the levels of native peptides were as much as 10-fold higher than sil peptides , which were added at 50 fmol per injection ( supporting information ; panorama ) . to evaluate carryover , no carryover of native peptides was observed in the blank injections ( supporting information ; panorama ) . mrm - based quantitation of gln223 and lys223 variants of sp - a from human bronchoalveolar lavage fluid . ( a ) two micrograms of native purified sp - a and 3050 g of human bal protein ( concentrated from 1 ml of bal fluid ) were separated by sds - page and stained with colloidal coomassie . the approximate size of gel bands that were excised for quantitation of sp - a from bal fluid samples is shown by the dashed rectangle . ( b ) total sp - a ( per injection ) was calculated based on light\heavy ratios of the hqilqtr peptide , where the sil peptide was added at 50 fmol / injection volume ( 50 fmol/l ) . ( c ) the intensities of native eqcvemytdgqwndr and cvemytdgqwndr peptides ( sum of y6 , y8 , and y9 transitions ) in each sample were normalized to total sp - a in ( b ) . ( d ) ratio of cvem / eqcvem peptide intensities were grouped by 223 genotype . data in ( b ) and ( c ) are mean range ( n = 2 replicates per sample ) . data points in ( d ) are individual measurements ( n = 46 per group ) and horizontal lines show mean and range of values . of the two peptides targeting total sp - a , the hqilqtr peptide had higher technical reproducibility ( % cvs of < 10% for both sil and native forms based on n = 4 replicate injections of a single sample ) and much lower limit of quantitation than the galslqgsimtvgek peptide . therefore , we used the hqilqtr for single - point estimation of total sp - a quantity extracted from the gel bands . when normalized to the sil peptide standard , total levels of sp - a were very similar across samples , except for one which had roughly twice as much protein ( figure 3b ) . we used the levels of total protein , as measured by the hqilqtr peptide intensity , to normalize levels of the gln223- and lys223-determining peptides quantified from each of the seven subjects . the lys223-derived peptide was reduced approximately 50% in lys223/gln223 versus lys223/lys223 subjects and was absent from the gln223 homozygotes ( figure 3c ) . because it is shared with sp - a1 , the gln223-containing peptide was quantified in all samples ; however , its levels did appear to be highest in the gln223 homozygotes ( figure 3c ) . to try to devise a quantitative method for de novo identification of genotypes , we again compared the ratio of the intensity of lys223- versus gln223-derived peptides in these genotyped samples ( figure 3d ) : gln223 homozygotes had a ratio of 0 , whereas lys223/gln223-spa2 heterozygotes had ratios between 2 and 5 , and lys223/lys223-spa2 homozygotes had ratios of > 10 . notably , these groups do not overlap , consistent with the binary nature of the three genotypes . in addition , as this measurement is based on a ratio of two peptides , this calculation is independent of absolute protein amount and does not require normalization to the hqilqtr peptide . collectively , these data suggest that the gelc - mrm assay can identify the three genotypes of sp - a2 at the 223 position with high sensitivity and specificity . the relative amounts of sp - a1 and sp - a2 levels ( versus total sp - a ) have been shown to change with age and in disease , whereas our samples were obtained from normal controls between 18 and 35 years . since the gln223 peptide is shared with sp - a1 , a change in the relative levels of sp - a1 versus sp - a2 might skew our assay when implemented across a heterogeneous cohort . we hypothesized that this variation could be controlled for by ratioing the lys223-derived peptide to a pan - spa-2-specific peptide . there is only a single tryptic peptide , gdpgppgpmgppgetpcppgnnglpgapgvpger , that is unique to all variants of sp - a2 . in addition to having a carbamidomethylated cys due to reduction / alkylation , the observed forms of this peptide had 1 oxidized met , 1 deamidated asn and up to five hydroxylated pro residues ( supplemental scaffold data , supporting information ) . without proper site localization for many of the sites of pro hydroxylation and asn deamidation , we calculated that there could be > 40 possible forms of these modified peptides . on the other hand , we reasoned that the quantitation of these forms could be greatly simplified , since ( 1 ) all of the modified forms of the peptides had a similar retention time ; ( 2 ) the nondeamidated forms and deamidated forms of the triply charged peptide would be indistinguishable at the resolution of the triple quadrupole ms ; ( 3 ) met oxidation and pro hydroxylation give the same mass shift ; and ( 4 ) three of the most intense y - ions ( y10 , y11 , and y12 ) exist only in two possible forms , with either two or three hydroxypro modifications ( table 1 ) . we designed a simplified targeted assay to quantify the y10 , y11 , and y12 product ions for peptides having either two or three c - terminal hydroxypro modifications , and up to four additional hydroxylations / oxidations n - terminal to the site of y12 fragmentation ( 10 precursor masses ; table 1 ) . these targets were appended to our original targeted assay . to validate this modified mrm assay , we performed an additional analysis using 1 ml of balf from n = 4 individuals having the gln223/lys223 sp - a2 genotype and n = 4 individuals with the lys223/lys223 sp - a2 genotype . only one of the subjects ( a lys223 homozygous ) was shared with the previous analysis . note that we did not include any gln223/gln223 homozygotes since these individuals are readily identified by the absence of a lys223-derived peptide ( figure 3 ) . after sds - page and in - gel digestion based on the migration of app sp - a ( figure 4a ) , we analyzed tryptic digests using a waters ionkey and a xevo tq - s ms . improvements in chromatographic peak shape and sensitivity were observed ( supporting information ; panorama ) . as shown in our earlier study ( figure 3 ) , we found a clear distinction between genotypes based on the ratio of the two peptides covering the 223 variant region ( figure 4b ) , thus translating the assay between the two instrument platforms . although the absolute ratios were 1/2 of the values that were observed in the initial analysis , this could easily be explained by differences in ion suppression or ionization efficiency between the two lc importantly , this would be controlled for in any clinical translation of the assay by using protein standards . validation of mrm assay for quantitation of lys223/lys223 and gln223/lys223 sp - a2 genotypes . ( a ) 2.5 g of app sp - a and 30 g of concentrated human bal protein were separated by sds - page followed by in - gel trypsin digestion as in figure 3 . an example of the excised region of the gel is indicated by the rectangle . ( b ) the ratio of native cvemytdgqwndr / eqcvemytdgqwndr peptide intensities ( sum of y8 and y9 product ions ) were plotted as a function of 223 genotype as in panel c. ( c ) chromatograms ( sum of y10 , y11 , and y12 transitions ) from the quantitation of 10 forms of the gdpgppgpmgppgetpcppgnnglpgapgvpger peptide were overlaid . ( d ) the gdpgppgpmgppgetpcppgnnglpgapgvpger peptide intensity in each sample was normalized to total sp - a . ( e ) the intensity of the lys223-specific peptide ( cvemytdgqwndr ) was normalized to sp - a2 as determined in ( d ) . data in ( b ) , ( d ) , and ( e ) are individual measurements ( n = 8 per group ) . we next sought to determine whether we could quantify relative levels of sp - a2 using the hydroxylated sp - a2-specific peptides . the multiple forms of the hydroxylated sp - a2 peptide eluted over a distinct 3 min window , and the elution profile of most precursors had multiple peaks , consistent with mixture of hydroxylated , met oxidized , and deamidated forms with the same precursor and product ion masses ( figure 4c ) . the forms of the peptide with two or three c - terminal hydroxylations , and with one n - terminal hydroxylation / oxidation eluted as doublets , suggesting that these peptides had two major chromatographically distinguishable forms ( figure 4c ) . the number of peaks increased with the number of variable hydroxylations / oxidations on the n - terminal segment of the peptide , consistent with the existence of multiple forms of the peptide containing the same number of isobaric modifications . however , when we summed the product ion areas from all of these species and normalized to total levels of sp - a ( via peptide hqilqtr ) , there was no significant difference in the relative expression of sp - a2 between the gln223/lys223 and lys223/lys223 genotypes ( figure 4d ) . triplicate analysis of a qc pool , performed at the beginning , middle , and end of the study , showed a mean % cv of < 2% for quantitation of the hydroxylated sp - a2 peptide . finally , we normalized the intensities of the lys223-dervied peptide to the sp - a2 specific peptide and found that the homozygous minor and heterozygous genotypes were nonoverlapping ( figure 4e ) . importantly , this provides an additional method for identifying sp - a2 223 genotypes which does not utilize the pan - sp - a gln223 peptide . while a number of sp - a variants have been expressed in vitro , until now , the quantitation of nonsynonymous variants of sp - a in vivo has only been possible at the mrna level . here , we established , for the first time , a facile assay for targeted quantitation of allelic variants at position 223 of sp - a , as well as relative levels of sp - a2 versus total sp - a , using as little as 1 ml of bronchoalveolar lavage fluid . while there was no significant difference in relative sp - a2 as a function of 223 genotype , there was a slight trend toward lower sp - a2 levels versus total sp - a in the lys223/lys223 versus gln223/lys223 genotype . an analysis of a much larger cohort will be necessary to determine whether sp - a2 variants at the 223 position are expressed at significantly different levels in airway lining fluid . however , it should further be noted that the lys223 variant is shared between two alleles of sp - a2 , 1a , and 1a , that are found in > 1% of the population . the proteins encoded by these genes differ only at amino acid position 9 , which is in the signal sequence and was not observed in any of our analyses of purified sp - a . thus , additional genotyping at this snp ( rs1059046 ) would be required to fully correlate sp - a2 and lys223 variant levels with a specific allelic variant . the sp - a variant peptides had peak areas that are 1001000-fold less abundant than the hqilqtr peptide used to quantify total sp - a . thus , while mrm assays targeting best fliers may have the requisite sensitivity for targeted protein quantitation across a large dynamic range in unfractionated biofluids , targeted quantitation of variant peptides may require additional fractionation , such as the gelc - mrm approach , to achieve adequate sensitivity . with the challenges inherent in online separation of biofluid proteomes , which can be comprised of 5090% ( w / w ) albumin , gelc - mrm presents a solution for improving depth of coverage . the technique has also been successfully employed for targeted quantitation of oncogenic k - ras mutants in cancer cells and in tumor - derived fluids . gelc - mrm might not be feasible for quantitation of a target biofluid protein that has similar molecular weight as albumin , or for targeted quantitation of proteins across a broad mw range ; in those cases , immunodepletion of abundant plasma proteins may be a viable alternative . on the basis of our experience , gel - based fractionation can have a lower per - sample cost than immunodepletion and provides equal or better scalability . this exclusion of high abundances proteins by gel - based fractionation minimizes potential interfering ions and/or ion suppression , therefore increasing the robustness of the assay . selection of the appropriate gel migration location only requires a suitable protein standard or sufficient knowledge of the electrophoretic mobility of the target protein . mrm has been applied to quantitation of coding variants of only a handful of proteins ( k - ras , apoliprotein e , sp - a ) , but the potential complexity of such analyses is already apparent . for example , although targeted proteomics can be used to quantify apolipoprotein e2 ( apoe2 ) and e4 ( apoe4 ) on the basis of single variant peptides ( containing cys158 and arg112 , respectively ) , the variant sites that are found in apoe3 ( cys112 and arg158 ) are shared with apoe2 and apoe4 , respectively , making the specific quantitation of apoe3 a technical challenge . here , the quantitation of allelic variants of sp - a is further complicated by the presence of up to four copies of each peptide ( two each from sp - a1 and sp - a2 ) . in the case of sp - a , we can easily identify the gln223/gln223 homozygote based on the absence of the native lys223 peptide , but the interference from two copies of the copurifying sp - a1-derived gln223 peptide presents a unique problem for distinguishing the gln223/lys223 and gln223/gln223 alleles of sp - a2 . in a relatively homogeneous population , the ratio of the lys223-derived : gln223-derived peptide intensities are likely to suffice as a means of identifying the three sp - a2 genotypes , although a set of well - genotyped standards will be essential to transferring the method across instrument platforms and/or laboratories . the additional quantitation of a peptide specific to all common variants of sp - a2 enables relative quantitation of this isoform for the first time , as well as normalization of the lys223-dervied peptide to total sp - a2 . this avoids any potential pitfall in the assay resulting from large variation in total sp - a1:sp - a2 protein ratios that could skew stratification of sp - a2 223 genotype based on the ratio of lys223- and gln223-derived peptides . in addition to sp - a , amino acid variants of other airway lining fluid - expressed proteins , including sp - d and il-13 , have been implicated in pulmonary disease . the need to understand the implications of protein - coding , nonsynonymous single nucleotide variants ( nssnvs ) , in the lung and elsewhere , will only grow with the increasing application of large - scale and deep coverage genomic approaches ( e.g. , snp genotyping , exome sequencing , rna - seq ) . consequences of nssnvs could include improper protein translation or processing , decreased stability or increased degradation ; the presence of multiple copies of each protein necessitates the quantitation of these variants in a sequence - specific manner . until now , mass spectrometry - based studies of nssnvs have mostly focused on the searching of shotgun proteomic data using variant - containing databases . as this and a handful of other studies have shown , mrm is poised to become the preferred method for high confidence identification and quantitation of the expressed forms of proteins harboring nssnvs .

pulmonary surfactant protein a ( sp - a ) , a heterooligomer of sp - a1 and sp - a2 , is an important regulator of innate immunity of the lung . nonsynonymous single nucleotide variants of sp - a have been linked to respiratory diseases , but the expressed repertoire of sp - a protein in human airway has not been investigated . here , we used parallel trypsin and glu - c digestion , followed by lc ms / ms , to obtain sequence coverage of common sp - a variants and isoform - determining peptides . we further developed a sds - page - based , multiple reaction monitoring ( gelc - mrm ) assay for enrichment and targeted quantitation of total sp - a , the sp - a2 isoform , and the gln223 and lys223 variants of sp - a , from as little as one milliliter of bronchoalveolar lavage fluid . this assay identified individuals with the three genotypes at the 223 position of sp - a2 : homozygous major ( gln223/gln223 ) , homozygous minor ( lys223/lys223 ) , or heterozygous ( gln223/lys223 ) . more generally , our studies demonstrate the challenges inherent in distinguishing highly homologous , copurifying protein isoforms by ms and show the applicability of mrm mass spectrometry for identification and quantitation of nonsynonymous single nucleotide variants and other proteoforms in airway lining fluid .

Introduction Materials and Methods Results Discussion

the surfactant complex has a distinctive composition of approximately 8085% phospholipids , 510% neutral lipids ( mostly cholesterol ) , and 510% proteins , consisting of surfactant proteins a , b , c , and d ( sp - a , sp - b , sp - c , sp - d ) . sp - a molecular species are hetero - oligmers composed of two distinct gene products , sp - a1 and sp - a2 , each with multiple allelic variants . sp - a monomers undergo noncovalent trimerization by interactions of their collagen domains and their coiled - coil domains , and the trimers are covalently cross - linked by disulfide bonds at the n - terminal domains . four of the sp - a1 allelic variants ( 6a , 6a , 6a , 6a ) and six sp - a2 allelic variants ( 1a , 1a , 1a , 1a , 1a , and 1a ) are observed with a frequency of greater than 1% in the general population . single nucleotide polymorphisms of sp - a have been implicated in susceptibility to tuberculosis infection , pulmonary fibrosis , lung cancer , and high - altitude pulmonary edema . the study of sp - a variants has largely been confined to ectopic expression in human adenocarcinoma cell lines and transgenic mice . studies of transgenic mice expressing human sp - a have further demonstrated that the 6a variant of sp - a1 ( with leu50 and trp219 ) and the 1a variant of sp - a2 ( with ala91 and lys223 ) are detectable in bronchoalveolar lavage fluid by western blotting . few high resolution mass spectrometry analyses have been performed on native sp - a , and there has been little correlation between sp - a variants and their expressed proteoforms in the human lung . furthermore , the relative quantitation of sp - a1/sp - a2 protein is limited to the use of antibodies specific for sp - a1 versus total sp - a . here , we sought to explore the capability of mass spectrometry for identification and quantitation of sp - a isoforms and common variants , and we developed a targeted mass spectrometry assay to identify individuals expressing the variants of sp - a2 at amino acid 223 ( gln223 and lys223 ) in human bronchoalveolar lavage fluid . sp - a from pulmonary alveolar proteinosis patients ( app - spa ) was purified by differential centrifugation and extraction with 1-butanol , followed by affinity chromatography on mannose sepharose as previously described . samples were analyzed in data - dependent ( dda ) mode using a 0.9 s precursor scan ( synapt g1 ) or a 0.6 s precursor scan ( synapt g2 ) , followed by ms / ms product ion scans on the top three most intense ions using a dynamic exclusion window of 120 s. two - dimensional - lc ms / ms ( 2d - lc ms / ms ) utilized a waters nanoacquity with 2d technology uplc . ms / ms data were processed using mascot distiller v.2.2 ( matrix science ) and searched using mascot v.2.2 against a swiss - prot database with homo sapiens taxonomy ( downloaded on 10/30/2010 ; 20,259 entries ) with additional sp - a variants 6a , 6a , 6a , 1a , 1a , and 1a ) . to determine whether mass spectrometry could be used for identification and quantitation of common sp - a variants and of the sp - a1 and sp - a2 isoforms ( figure 1a ) , we first separated several different proteins ( sp - a from normal control and alveolar proteinosis patients ( app ) ; and recombinant lys223 sp - a2 ) by sds - page , and the band containing the sp - a monomer was digested with trypsin and analyzed by lc ms / ms analysis was sufficient to sequence most of the invariant regions of sp - a , and among known variable sites , afforded sequence coverage of the tryptic peptide containing gln223 ( figure 1a ) , which is shared between major variants of sp - a1 and sp - a2 , as well as the peptide formed by cleavage after lys223 , which is found in the 1a and 1a variants of sp - a2 . in native sp - a preparations , we identified the peptide spanning thr66 , asn73 , val81 , and arg85 , which is specific to sp - a2 ; however , the corresponding sp - a1-specific peptide spanning met66 , glu73 , ile81 , and cys85 was not identified . the predicted tryptic peptides containing positions 50 , 91 , and 219 of sp - a are small ( 46-mers ) , which likely explains why they were not identified . ( a ) a multiple sequence alignment of sp - a1 and sp - a2 isoforms was performed , with common variants ( > 1% of the population ) vertically juxtaposed ( see figure s1 for complete alignment ) . sequence coverage of tryptic peptides ( no missed cleavages ) from gelc ms / ms analysis of sp - a are indicated as follows : solid lines indicate peptides that are common to all sp - a isoforms and variants ; dashed lines indicate variant- or isoform - determining peptides . ( b ) seven additional sp - a preparations , including native app and sp - a purified from two human asthmatics , and recombinant leu50/trp219 sp - a1 and val50/arg219 sp - a1 , were digested in solution in parallel using either trypsin or glu - c . in - gel digestion of sp - a with glu - c gave poor sequence coverage ( data not shown ) ; thus , an additional five native sp - a preparations , as well as two recombinant purified sp - a1 variants , were digested in solution , separately with trypsin or glu - c , and analyzed by lc these 14 analyses gave sequence coverage for all of the major variable sites of sp - a1 and sp - a2 , including all possible amino acids at positions 50 , 91 , 219 , and 223 , as well as the sp - a1- and sp - a2-specific amino acids at positions 65 , 73 , 81 , and 85 ( figure 1b ) . except for the trp219 peptide , all of the variant- or isoform - determining peptides identified solely by in solution digestion contained missed cleavages ( figure 1b ) . we wondered whether targeted , quantative ms could definitively determine sp - a genotype at any of the variable regions , either as a means of de novo proteotyping of sp - a variants or to validate the expression of protein - coding variants in genotyped human subjects . this minor variant of sp - a2 is heterozygously expressed in 30% of the population , whereas only 5% are homozygous for lys at the 223 position of sp - a2 . ms / ms spectra were aligned for the doubly charged tryptic peptides , ( a ) eqcvemytdgqwndr and ( b ) cvemytdgqwndr , which are site - determining for sp - a variants with gln223 and lys223 , respectively . we hypothesized that an sds - page separation might yield sufficient enrichment of sp - a from balf for targeted quantitation . to test this enrichment strategy , 1 ml of balf each from two different individuals was concentrated by centrifugal filtration , and the entirety of recovered protein ( 50 g ) was separated by sds - page alongside purified sp - a . the region corresponding to sp - a monomer was excised from the balf lanes ( not shown ) , and in - gel digests were analyzed by two - dimensional reversed - phase reversed - phase lc ms / ms ( supplemental scaffold data , supporting information ) . to achieve the most quantitative recovery and aid the sample stability of sp - a for analysis , we added 50 ng of digested , immunodepleted human plasma protein during the final reconstitution of the in - gel digests prior to transfer to glass sample vials as a means of reducing peptide loss due to adsorption . to test this optimized gelc - mrm assay , we analyzed 1 ml of balf from seven normal subjects that had been genotyped at the sp - a2 locus ( figure 3a ) . the cohort was selected to contain samples from three individuals who were homozygous for the major sp - a2 genotype ( gln223/gln223 ) , three who were homozygous for the minor sp - a2 genotype ( lys223/lys223 ) , and two who were heterozygous for these two sp - a2 genotypes ( gln223/lys223 ) . following in - gel digestion and reconstitution of digests , because of the high degree of enrichment of sp - a in these samples , the levels of native peptides were as much as 10-fold higher than sil peptides , which were added at 50 fmol per injection ( supporting information ; panorama ) . mrm - based quantitation of gln223 and lys223 variants of sp - a from human bronchoalveolar lavage fluid . ( a ) two micrograms of native purified sp - a and 3050 g of human bal protein ( concentrated from 1 ml of bal fluid ) were separated by sds - page and stained with colloidal coomassie . ( b ) total sp - a ( per injection ) was calculated based on light\heavy ratios of the hqilqtr peptide , where the sil peptide was added at 50 fmol / injection volume ( 50 fmol/l ) . of the two peptides targeting total sp - a , the hqilqtr peptide had higher technical reproducibility ( % cvs of < 10% for both sil and native forms based on n = 4 replicate injections of a single sample ) and much lower limit of quantitation than the galslqgsimtvgek peptide . therefore , we used the hqilqtr for single - point estimation of total sp - a quantity extracted from the gel bands . collectively , these data suggest that the gelc - mrm assay can identify the three genotypes of sp - a2 at the 223 position with high sensitivity and specificity . the relative amounts of sp - a1 and sp - a2 levels ( versus total sp - a ) have been shown to change with age and in disease , whereas our samples were obtained from normal controls between 18 and 35 years . since the gln223 peptide is shared with sp - a1 , a change in the relative levels of sp - a1 versus sp - a2 might skew our assay when implemented across a heterogeneous cohort . on the other hand , we reasoned that the quantitation of these forms could be greatly simplified , since ( 1 ) all of the modified forms of the peptides had a similar retention time ; ( 2 ) the nondeamidated forms and deamidated forms of the triply charged peptide would be indistinguishable at the resolution of the triple quadrupole ms ; ( 3 ) met oxidation and pro hydroxylation give the same mass shift ; and ( 4 ) three of the most intense y - ions ( y10 , y11 , and y12 ) exist only in two possible forms , with either two or three hydroxypro modifications ( table 1 ) . to validate this modified mrm assay , we performed an additional analysis using 1 ml of balf from n = 4 individuals having the gln223/lys223 sp - a2 genotype and n = 4 individuals with the lys223/lys223 sp - a2 genotype . after sds - page and in - gel digestion based on the migration of app sp - a ( figure 4a ) , we analyzed tryptic digests using a waters ionkey and a xevo tq - s ms . validation of mrm assay for quantitation of lys223/lys223 and gln223/lys223 sp - a2 genotypes . ( a ) 2.5 g of app sp - a and 30 g of concentrated human bal protein were separated by sds - page followed by in - gel trypsin digestion as in figure 3 . the multiple forms of the hydroxylated sp - a2 peptide eluted over a distinct 3 min window , and the elution profile of most precursors had multiple peaks , consistent with mixture of hydroxylated , met oxidized , and deamidated forms with the same precursor and product ion masses ( figure 4c ) . however , when we summed the product ion areas from all of these species and normalized to total levels of sp - a ( via peptide hqilqtr ) , there was no significant difference in the relative expression of sp - a2 between the gln223/lys223 and lys223/lys223 genotypes ( figure 4d ) . triplicate analysis of a qc pool , performed at the beginning , middle , and end of the study , showed a mean % cv of < 2% for quantitation of the hydroxylated sp - a2 peptide . finally , we normalized the intensities of the lys223-dervied peptide to the sp - a2 specific peptide and found that the homozygous minor and heterozygous genotypes were nonoverlapping ( figure 4e ) . while a number of sp - a variants have been expressed in vitro , until now , the quantitation of nonsynonymous variants of sp - a in vivo has only been possible at the mrna level . here , we established , for the first time , a facile assay for targeted quantitation of allelic variants at position 223 of sp - a , as well as relative levels of sp - a2 versus total sp - a , using as little as 1 ml of bronchoalveolar lavage fluid . while there was no significant difference in relative sp - a2 as a function of 223 genotype , there was a slight trend toward lower sp - a2 levels versus total sp - a in the lys223/lys223 versus gln223/lys223 genotype . an analysis of a much larger cohort will be necessary to determine whether sp - a2 variants at the 223 position are expressed at significantly different levels in airway lining fluid . however , it should further be noted that the lys223 variant is shared between two alleles of sp - a2 , 1a , and 1a , that are found in > 1% of the population . thus , while mrm assays targeting best fliers may have the requisite sensitivity for targeted protein quantitation across a large dynamic range in unfractionated biofluids , targeted quantitation of variant peptides may require additional fractionation , such as the gelc - mrm approach , to achieve adequate sensitivity . with the challenges inherent in online separation of biofluid proteomes , which can be comprised of 5090% ( w / w ) albumin , gelc - mrm presents a solution for improving depth of coverage . gelc - mrm might not be feasible for quantitation of a target biofluid protein that has similar molecular weight as albumin , or for targeted quantitation of proteins across a broad mw range ; in those cases , immunodepletion of abundant plasma proteins may be a viable alternative . mrm has been applied to quantitation of coding variants of only a handful of proteins ( k - ras , apoliprotein e , sp - a ) , but the potential complexity of such analyses is already apparent . here , the quantitation of allelic variants of sp - a is further complicated by the presence of up to four copies of each peptide ( two each from sp - a1 and sp - a2 ) . in the case of sp - a , we can easily identify the gln223/gln223 homozygote based on the absence of the native lys223 peptide , but the interference from two copies of the copurifying sp - a1-derived gln223 peptide presents a unique problem for distinguishing the gln223/lys223 and gln223/gln223 alleles of sp - a2 . in a relatively homogeneous population , the ratio of the lys223-derived : gln223-derived peptide intensities are likely to suffice as a means of identifying the three sp - a2 genotypes , although a set of well - genotyped standards will be essential to transferring the method across instrument platforms and/or laboratories . the additional quantitation of a peptide specific to all common variants of sp - a2 enables relative quantitation of this isoform for the first time , as well as normalization of the lys223-dervied peptide to total sp - a2 . in addition to sp - a , amino acid variants of other airway lining fluid - expressed proteins , including sp - d and il-13 , have been implicated in pulmonary disease . the need to understand the implications of protein - coding , nonsynonymous single nucleotide variants ( nssnvs ) , in the lung and elsewhere , will only grow with the increasing application of large - scale and deep coverage genomic approaches ( e.g. as this and a handful of other studies have shown , mrm is poised to become the preferred method for high confidence identification and quantitation of the expressed forms of proteins harboring nssnvs .

over the last 60 years , the national health service ( nhs ) has become an intrinsic feature of the united kingdom , not only underpinning the nation s health but exemplifying some of its core values and beliefs that are still widely held today . the nhs was founded upon three core principles : to meet the needs of everyone ; to remain free at the point of delivery and that access to the nhs be based on clinical need , rather than ability to pay . these principles remain a fundamental part of the nhs yet as the years have passed , there is broad agreement that modernisation of the nhs has become a necessity . with costs soaring and demand rising exponentially ; with the need for improvements and technological developments remaining an unremitting drain on the nhs coffers and the current economic climate making protected , ring - fenced nhs budgets unsustainable in the years to come , proactive steps to reform the nhs have been taken in the shape of the health and social care act ( hsca ) 2012 . modernisation has been driven by the demands placed upon a 60-year - old health service provider . yet the drive to take the health service into the 21st century and become an economically viable and sustainable endeavour has also highlighted another deep - seated problem within the nhs : how to ensure vulnerable groups are cared for effectively , particularly with shifting demographics . the focus of this article is that of the mentally ill , and it will consider how this group fares under the changes introduced by the hsca 2012 . prior to the enactment of the hsca 2012 , the needs associated with mental health conditions had already been explicitly acknowledged as a priority . since then , a new mental health outcomes strategy was published in february 2011 , no health without mental health : a cross - government mental health outcomes strategy for people of all ages , followed by an implementation framework , published in july 2012 . the strategy aims to provide better mental health for all and to increase the number of people recovering from mental health conditions , whilst the implementation framework focuses on the provision of strong outcomes monitoring . these mental health objectives are expected to map onto the broader nhs changes under the hsca 2012 by virtue of explicit recognition within the legislation that mental ill health will be given parity alongside other physical health needs . the consolidation of these steps by the hsca 2012 is fundamental in ensuring mental health conditions are effectively recognised and responded to . achieving this will not be easy in a climate where the global burden of disease is rising , and mental health and behavioural disorders in particular account for an increasing proportion of this . provision for the mentally ill has always been stretched , struggling under the weight of systemic neglect and a lack of resources . the vulnerable , whether the mentally ill , the elderly or those who are mentally incapacitated , are particularly at risk as they are often not in a position to protect their own rights . instead , reliance is placed upon those around them and the systems they are placed within to do this for them . in the wake of the hsca 2012 , it is necessary to reflect upon whether the 2012 act offers hope to those made vulnerable through mental ill health , or whether it instead fails them , and if so , why ? this article explores this question with reference to three key policy drivers within the legislation and is structured accordingly . in the first instance , the article examines the hsca 2012 from the mental health perspective , in terms of how the restructured commissioning process operates and how it maps on to the mental health framework . attention is then given to three issues : first , whether parity between mental and physical health can in all reality have life beyond political rhetoric ; second , what impact driving up efficiency within the nhs , in terms of commissioning decisions , will have upon patients with mental health conditions and third , the extent to which the personalisation agenda can be meaningfully applied within the mental health context . these issues are considered with reference to broader policy influences within the mental health law and policy landscape . whilst the fundamental restructuring of the nhs has been the subject of recent attention with the enactment of the hsca 2012 , mental health has also been under the spotlight of reform in the past few years . the mental health act 2007 sought to respond to the challenges posed by changing psychiatric practices and the policy shift from hospital - based treatment to care in the community . over the last two decades , reliance on hospital - based care has diminished and has been replaced by the community as the dominant care environment . hospital care is now reserved largely for those requiring acute or intensive psychiatric care . to some extent resources have followed this changing pattern of care , but inevitably , service provision and delivery has been affected by the gradual shift in the mental health landscape . in parallel with the introduction of the mental health act 2007 , modifications have been made to the mental health act code of practice to reflect the legislative amendments . whilst the code is not legally binding , decision - makers are required to justify any departures from its guidance in their decision - making . the amended code features principles which are designed to promote patients interests and guide decision - making under the act . these principles are first , the purpose principle , whereby decisions under the act must be made to minimise the undesirable effects of mental disorder ; second , the least restriction principle , where decision - makers should keep to a minimum the restrictions they impose on the patient s liberty ; third , the respect principle , whereby recognition and respect should be given to the diverse needs , values and circumstances of each patient ; fourth , the participation principle that encourages patients involvement and finally , the effectiveness , efficiency and equity principle that focuses upon optimal decision - making using available resources in the most efficient way possible . in many ways , however , whilst these principles promote universally recognised values and provide an opportunity to foster better care , their literal interpretation may not always fit the actual process of implementation . ineffective implementation of core values within both hard and soft legal instruments is , perhaps , the largest source of damage for vulnerable groups and will be reflected upon throughout this article . the hsca 2012 has been heralded as the most extensive and radical reorganisation of the nhs to date and has been accompanied by significant levels of political rhetoric , speculation and controversy . the legislation had two key objectives : to improve the quality of care and outcomes for patients and to reposition the mode of provision so that health service provision becomes more patient - centred and facilitates choice . these objectives are incontrovertible ; however , many of the mechanisms that the legislation introduces to achieve these aims have generated concern amongst service users , clinicians and service providers alike . the changes introduced by the act are far reaching and for those with chronic and enduring conditions , of which all mental health conditions would likely be labelled , the hsca 2012 can be expected to wield significant weight in treatment and care planning as it becomes fully operational in the months to come . several key elements of the legislation guide its implementation : ensuring a patient - centred nhs ; promoting and supporting a clinician - led service and transferring the emphasis of measurement to clinical outcomes . however , it is conceivable that these principles have the potential to conflict with significant consequences and may have lasting implications upon the quality of delivered care . the question remains whether any one of these principles will dominate during the implementation process , and if so , which it will be . the persistent concern amongst many professional and user groups alike has been and continues to be that the political desire to make financial savings and improve the cost - effectiveness of the nhs may prove to be the overarching driver . a related concern is that the legislation represents an inevitable shift away from the ideology of universal provision , a mainstay of the old nhs , towards a stronger endorsement of expanding private sector involvement and a gradual privatisation of the health service . the reinforcement of competition principles within the health care system is likely to have a detrimental impact on the mentally vulnerable as the act opens up private sector involvement , making the process of commissioning outside of the nhs structure easier and more cost - effective . in all likelihood , this will encourage providers to be more active in lucrative areas of health care . mental health care and associated social care provision is generally seen as an unprofitable field , with long - term and often complex care and support required by individuals . the 2012 act s market - based approach may prove to be particularly damaging for the mentally ill , with resources being allocated away from the needs of this group and short - term care measures , such as acute inpatient provision , being given greater attention than the longer term health and social care needs of individuals in the community . the act also introduces a change to one of the central nhs tenets : no longer will services be exclusively operated via the nhs and its partners ; instead , any willing provider could supply services . this enables the private sector to have direct access to the central operations of the nhs , in terms of both planning and provision . although this allows for any willing provider and thus goes beyond the private sector , social enterprises may find it difficult to compete against organisations in the private sector who can afford to undercut in the race to acquire a commissioning contract . currently , the role of third sector organisations in mental health care is much more prominent and is , indeed , essential , particularly in relation to social care provision ; however , whether this will continue remains open to speculation . if third sector organisations do struggle in this new provider landscape , the mentally ill will inevitably suffer as the tailored , personal provision currently offered by many small organisations and charities is likely to be curtailed as they battle to compete . commissioning of services for mental health care and treatment services will be conducted and guided by clinical commissioning groups ( ccgs ) , which are introduced by the hsca 2012 , in a similar fashion as for all other services . the guiding principles under the hsca 2012 will be influential in how ccgs conduct their activities . in the first instance , ccgs have a duty to promote the nhs constitution and ensure patients , staff and the public are aware of the nhs constitution and their nhs constitutional rights . ccgs will also have a general duty to improve the quality of the services they provide or commission . primary medical services ( which include acute inpatient psychiatric care and secure psychiatric units ) are to be commissioned by nhs england . the focus on quality improvement goes beyond the old duty that primary care trusts ( pcts ) had under nhs act 2006 , which was to improve the quality of health care services apropos existing published standards . instead , the duty under the hsca 2012 explicitly recognises the need to consider treatment and care outcomes and the patient experience . ccgs are also required to endorse a patient - centred approach by encouraging patient involvement through shared decision - making . the implementation of this duty will be facilitated by new guidance to be published by nhs england . as part of the focus upon patient - centred provision , ccgs will now also have to operate with a view to commissioning services from more than one provider as the 2012 act also introduces a duty to enable patient choice . how viable the balancing exercise of enabling patient choice within the mental health field will be remains to be seen . the creation of patient choice relies not only upon ccg behaviour endorsing and facilitating patient choice , but the providers of these services must actually exist in mental health , the fulfilment of identified need has often presented challenges , as service provider limitations are routine . at a broader level , concern surrounds the impact this duty to facilitate patient choice may have on the market . encouraging ccgs to commission several alternative treatments from different providers may lead to more providers having a smaller market share and greater fragmentation within the health and social care service sector might result . quite how the commissioning process can effectively achieve efficiency through competition whilst also increasing patient choice is difficult to understand ; or at least , it is possible to foresee challenges and tensions developing in the attainment of this aim . patient choice is often determined through a plethora of motivating factors , not least the common desire to be close to family and friends . for many , access to psychological services is a central wish , with drug therapy being a necessity of last resort . however , as we will see later in this article , drug therapy is often deemed to offer a front - line response to patients mental health needs by general practitioners ( gps ) , and psychological services are limited in availability . ccgs will be restricted by these practical limitations , but they will also be under a duty to ensure service commissioning is subject to tender under the national health service ( procurement , patient choice and competition ) regulations 2013 . if the framework of health and social care does crumble under the weight of these different legislative objectives , those with mental health conditions may be particularly vulnerable as a fragmented health and social care service will not be beneficial to them . additional choice may inevitably be at the expense of effective integration . despite this , under the 2012 act , ccgs have a duty to promote service integration . this entails the integration of health services with health - related and social care services . the political motivation behind this duty is to improve efficiency of service provision and to reduce unnecessary costs . nonetheless , from the patient perspective , this offers an avenue for improvements in quality of life , particularly for those who need longer term support in the community . for the mentally vulnerable , effective integration of services is often particularly important , improving the implementation of treatment plans , medication compliance and ongoing community - based support . the difficulty with this duty is that as yet no guidance has been supplied to aid ccgs in the process of achieving good integration amongst and between these various services . furthermore , mental health provision is littered with countless examples of joint working failures and inadequate communication throughout the health and social care system . indeed , the ideal of achieving seamless provision is far removed from the reality for many patients , and it is often this which leads to the disjointed care that is received and the gaps in provision where patients fall through the net . the required establishment of health and wellbeing boards by each local authority may reduce the perennial problems surrounding joint working . collaboration between the board members will afford the opportunity to assess local health and social care needs , agree on spending priorities and encourage ccgs to work with seamless , joined up provision in mind . boards can extend their membership to reflect particular area needs ; this may allow a local service to be developed for local needs . the board is also required to take account of affiliated services with social care , such as , housing and education and to recognise that these services have a direct influence on the broader well - being of individuals . it is uncertain whether this will directly improve service provision , but the cross - government mental health strategy pins its hopes on the shift towards localism and local care decision - making under the 2012 act . the mental health strategy implementation framework suggests that it is this focus on local needs which can deliver the vision of improved mental health and wellbeing. the restructuring of the nhs and the changes created by the hsca 2012 to the commissioning process will take time to grow accustomed to . from a mental health perspective , the hsca 2012 offers real potential to see mental health brought from the margins of provision to feature much more prominently . it creates the possibility for a conceptual reconfiguration of health to emerge , introducing explicitly the need for parity between mental and physical health . indeed , this duty to promote health parity could create the impetus for a paradigmatic shift within health and social care provision , but just how successful the implementation of this will be remains to be seen as the high - level commitment to health parity is only one of several key objectives within the 2012 legislation . devolution of budgets down to ccgs may provide opportunities for mental health to feature more prominently within the commissioning process ; yet there are concerns that mental health needs may continue to be overlooked by ccgs when pressure to commission services efficiently whilst also increasing patient choice presents significant tensions for ccgs to overcome . we will now turn to consider three drivers within the 2012 act , exploring whether they are feasible within the mental health context or whether the legislation will prove to be detrimental to those with mental health needs . first , attention will be given to the commitment to achieving parity of physical and mental health within the health care system , followed by a consideration of how the desire to increase efficiency may influence commissioning decisions within the mental health arena and finally , consideration will be given to the move towards expanding patient choice and personalisation within the health care market . the government s draft mandate to nhs england is explicit in its message : direct recognition is to be given to the need to place mental health on the same footing as physical health . mental health conditions are now to be recognised as a clear equality issue and the nhs equality delivery system will be primed to help those providing nhs services to respond properly to it . perhaps of greatest importance is the government s recognition in the mental health implementation framework that achieving parity between physical and mental health is an absolute goal , where more still needs to be done to ensure all organisations ( both public and private ) meet their equality and inequality obligations in relation to mental health. steps are being taken to create a framework to measure outcomes and overall progress within mental health , so that improvement strategies can be created and implemented when clear underperformance is identified . clearly , making improvements for mental health provision is dependent upon good implementation . ccgs will be expected to demonstrate to nhs england that they have sufficient planned capacity and an ability to commission for improved health outcomes in mental health . owing to this shift in attitude , and indeed , reconfiguration of the conception of health within the legislation , the neglected and under - resourced mental health service may be a thing of the past . the drive to improve access to psychological therapies for patients with mental health conditions is an example of this attitudinal shift and is a welcome move . the rhetoric of achieving parity between mental and physical health is , in many ways , politically driven , though the evidence suggests that greater effort to improve mental health is needed ; mental ill health is a leading cause of suffering , economic loss and social problems and accounts for over 15% of the disease burden in developed countries . in the european union at least 83 million people ( 27% ) suffer from mental health problems ( 16.7 million in the united kingdom ) , with depression being the most common ( 812% of the adult population ) . the newly restructured system of health and social care is in its infancy , and it is still too early to say whether the steps taken to achieve parity will bear fruit . likewise , how the vulnerable will be able to protect their rights in this new health and social care environment is unknown , but it seems likely that ccgs , if motivated by market - driven policies , could lose sight of the particular needs of these vulnerable groups . in many ways , achieving parity is a deep - seated cultural issue and goes far deeper than surface - level implementation . achieving parity needs fundamental attitudinal change at institutional , organisational and individual levels . for mental health , the best hope for this change exists within the mental health implementation framework where explicit mention is made of the need to promote research into mental health and to recognise , support and strengthen academic career paths in this field . it is only by consolidating capacity , instilling aspiration and professional motivation within the mental health care framework ( both research and practice pathways ) that the cultural transformation can begin to emerge . whilst parity of mental and physical health is a clear commitment within the hsca 2012 , the introduction of competition principles will also facilitate efficiency savings . mental health needs are often complex , requiring the input of a variety of different agencies and service providers . not only can providing for this complex diet of needs be difficult , it can be expensive . both the cost and complexity of provision in mental health has been a persistent source of difficulty in the past and where tragic failures in care have occurred ; investigations have often presented a catalogue of challenges surrounding the coordination and adequate funding of care . inevitably , establishing and identifying patient need and having the resources in place to meet it are not always achievable , and it is at this point that these system failures have often occurred . the mental health care framework has very limited scope to be able to deal with increases in demand , and , traditionally , this is where the third sector has often been sought to plug the gap . it is quite possible that without any form of overarching regional oversight , a task that pcts undertook prior to the 2012 act , the commissioning process may become fragmented and uncoordinated , and ultimately , gaps in some areas may be difficult to fill as patient needs may not be recognised in the round . two separate issues in the commissioning process for mental health services exist : first , the level of clinical expertise that exists and second , whether ccgs have sufficient management experience to meet the need for equal distribution and coverage of services . these two areas raise doubts about how efficient and effective commissioning decisions will be carried out . in the first instance , there are doubts concerning gps broad clinical knowledge and expertise to identify and evaluate patient mental health needs . for many gps , the initial response to patients presenting with mid to mild mental health conditions is to prescribe medication , rather than approach treatment holistically and refer patients to psychological therapies , peer - to - peer support networks or community - based services. gps often rely heavily upon drug therapy as the first response to symptomatic presentation in patients , which adds to the sense that gps lack the depth of knowledge necessary . this is supported by recent research which reported that 30% of patients found their gp was unaware of services to support mental health recovery beyond medication . second , it is predicted that ccgs may have inadequate management expertise and from this , optimal commissioning decisions will be less likely to occur . given the sheer scale of care and social support needs that patients with mental health conditions often need , if ccgs lack membership that reflects the level of experience needed to recognise this , adequate mental health care provision is likely to be inadequate . if pockets of poor management do emerge , then mental health provision may be adversely affected . often mental health provision is not the focus , with greater attention being given to physical health needs ; yet mental health conditions account for 23% of the total burden of disease ; but in terms of nhs expenditure , only 13% of health expenditure is currently directed towards psychiatric and related services . such underinvestment is not new and despite funds being channelled through pcts at a regional level to recognised areas of need prior to the hsca 2012 , resource shortfalls have been commonplace . mental health did not gain the moniker of the cinderella service without good reason and has been struggling under the weight of systemic neglect for a considerable time . unfortunately , mental health care must compete with all other health and social care needs , of which most are far more evident and have a more tangible quality about them . whether the hsca 2012 will improve this is uncertain . management inadequacies and failures to identify needs by ccgs may not be detected as there remains some doubt about how the new nhs structure and regulatory bodies will scrutinize and oversee activities . the organisational reconfiguration reflects the mood of the government to reduce bureaucracy and complexity in the health and social care framework , to improve efficiency and to redeploy functions through bodies that are independent or at least operating at arm s length of the government . it is [ just not yet ] clear how these national bodies will interact or how they will provide coordinated and consistent governance of the nhs. the challenges facing ccgs are unlikely to reassure patients in the short term ; for mental health patients , these concerns may simply be more acute , given the complexity of typical mental health care needs which tend to stretch over a number of agencies and providers , often featuring periods of both acute need and stable chronicity . the standard and effectiveness of care received will all too often depend upon a strong framework of planned and integrated systems or pathways of care from a well - coordinated network of providers . ccgs are going to have to ensure sufficient awareness is present within the strategic planning process to take account of this , and if they do not , health conditions , including most mental health conditions , that require a complex health and social care response may suffer . it refers to a social care approach where every person in need of care and treatment will have choice and control over the shape of that support in all care settings. personalisation is characterised by shifting the power dynamic within the provider user relationship . greater emphasis is placed upon self - directed support and personal budgetary control combined with a move away from the notion that provision should follow a the personalisation agenda seeks to move the health and social care framework away from crisis management , relying upon patients identifying personal needs and making appropriate care choices to meet these needs . for this to be possible , adequate information and transparency within the system is essential . to implement the personalisation agenda , the social care system , in particular , this means that ccgs have to take seriously the need to make and implement local commissioning decisions in a way that will enable genuine choices to be made . commissioning will need to be multilayered and from a variety of providers ; it will need to be possible to manipulate services so that tailor - made packages of care can be created for individual patients . in addition to the actual availability of services , steps must be taken to facilitate patients in the decision - making process . all patients , irrespective of age , capacity or support needs , should be aided as far as possible to ensure treatment and care choices are modified and are reflective of the patient s wishes . within mental health , recovery approach. this approach is focused upon the mental health patient being afforded the opportunity to determine his own life and to be offered the support required to be able to live as independently as possible . for some time , self - directed support has been an operational feature of care in the community . the idea is founded upon flexibility , choice and control of social care funding and focuses upon giving eligible people an annual budget to spend on their own care , based upon self - designed care plans . for many , creating a care plan and then organising providers to meet these identified needs is a challenging task to undertake alone . in practice , when a plan has been formulated , social care support can be obtained from a variety of sources , including statutory social services , the private sector , the voluntary sector , community groups , neighbours , family and friends . for those who need it , assistance in devising a care plan reflective of individual need is an essential element of the process ; particularly as individual budgets are increasingly being used as a vehicle to combine several funding streams that many mental health patients may need to access in the community . payment for local authority adult social care falls within the remit for individual budgets and include integrated community equipment services , disabled facilities grants , supporting people for housing - related support , access to work and the independent living fund . glendinning s research into the effectiveness of pilot schemes conducted by the individual budgets evaluation network demonstrates some promising results for patients , whereby clear benefits can be achieved through greater choice and control over funding . however , to enable mental health patients and other chronic patients with complex social care needs to benefit from this , better integration of services and a collective willingness to embrace choice needs to be fostered . how successful the personalisation agenda and its implementation under the hsca 2012 is , is perhaps best judged by assessing the benefits to patients that have flowed from this agenda . existing research already indicates that the injection of choice and control over care options can be very positive for patients and carers alike . however , there is also evidence suggesting some groups may not be experiencing these benefits , notably , patients with mental health conditions , patients with dementia and other capacity - reducing conditions . bureaucracy and cuts in social care spending are exacerbating the situation ; patients who require significant levels of support in this process may find their experience of the personalisation agenda hampered . other associated and recurrent problems exist within the mental health system , placing further strain on the achievement of the personalisation agenda . for example , staffing shortages and service scarcities often result in extensive waiting times and inadequate response rates . as such , staffing challenges and the need for extra support by mental health patients to benefit from the personalisation agenda may in reality make this policy a largely spurious one with little practical substance . the hsca 2012 represents a significant departure from a culture of public service provision that we have become accustomed to , but does it fail the vulnerable , notably those with mental health care needs ? the need to drive efficiency up , whilst also tailoring health and social care to individual patients is , perhaps , an impossible dilemma . making systems responsive to individual need also raises the spectre of cost and waste ; meeting the 2012 act s expectations will be an exacting challenge and not for the faint - hearted . how mental health provision will fare in this new and uncharted landscape remains open ; but , inevitably , it will face its own set of problems in the months to come . does the 2012 act fail the mentally vulnerable ? time will tell , though the tensions that exist between three of the key policy drivers within the legislation , the focus of this article , suggest that where there are pressure points and the vulnerable may ultimately experience the greatest detriment . however , they can not be responsive to the more nuanced needs of patients with chronic conditions , particularly where care needs bridge both health and social care and are often required for lengthy periods of time . perhaps , the brightest ray of hope should be the recognition that parity between mental and physical health will be a clear objective . as with so many of these things , effective policy needs to be translated into a workable and user - friendly legal framework that can then be implemented . in mental health , it is the implementation stage that frequently presents the most significant challenge for decision - makers , with limitations in staffing , funding and social care placements creating bottlenecks in the system . unless these practical hurdles can be overcome , the desire to forge a new and fairer culture within health and social care , where parity between mental and physical health is the accepted benchmark , will be a very difficult one to attain . the hsca 2012 offers a very real opportunity to enable mental health to be mainstreamed into core public health priorities . but , this relies upon a determination reminiscent of aneurin bevan , the nhs will last as long as there are folk left with the faith to fight for it. it can only be hoped that there are those prepared and willing to fight to ensure the needs of vulnerable groups , such as those with mental health conditions , are met and protected and that faith in the achievement of health and social care equality endures .

although the national health service ( nhs ) is regarded as a national treasure , it is no longer immune from the colossal financial pressures brought about by global recession . economic sustainability has largely driven the reform process leading to the health and social care act ( hsca ) 2012 , however ; other considerations have also played a role in the journey to turn the health and social care service into an institution which is fit for the 21st - century needs . this article examines the impact of the hsca 2012 on those made vulnerable through mental ill health . it then considers three issues : first , whether parity between mental and physical health can have life beyond political rhetoric ; second , what impact driving up efficiency within the nhs will have upon mental health patients ; and finally , the extent to which the personalisation agenda can be meaningfully applied within the mental health context .

Introduction The HSCA 2012 the mental health perspective Parity between mental and physical health in the commissioning process: More than political rhetoric? Efficiency: The impact on mental health patients Personalisation: Mapping the agenda on to the mental health framework Conclusion

over the last 60 years , the national health service ( nhs ) has become an intrinsic feature of the united kingdom , not only underpinning the nation s health but exemplifying some of its core values and beliefs that are still widely held today . with costs soaring and demand rising exponentially ; with the need for improvements and technological developments remaining an unremitting drain on the nhs coffers and the current economic climate making protected , ring - fenced nhs budgets unsustainable in the years to come , proactive steps to reform the nhs have been taken in the shape of the health and social care act ( hsca ) 2012 . yet the drive to take the health service into the 21st century and become an economically viable and sustainable endeavour has also highlighted another deep - seated problem within the nhs : how to ensure vulnerable groups are cared for effectively , particularly with shifting demographics . the focus of this article is that of the mentally ill , and it will consider how this group fares under the changes introduced by the hsca 2012 . prior to the enactment of the hsca 2012 , the needs associated with mental health conditions had already been explicitly acknowledged as a priority . these mental health objectives are expected to map onto the broader nhs changes under the hsca 2012 by virtue of explicit recognition within the legislation that mental ill health will be given parity alongside other physical health needs . the consolidation of these steps by the hsca 2012 is fundamental in ensuring mental health conditions are effectively recognised and responded to . achieving this will not be easy in a climate where the global burden of disease is rising , and mental health and behavioural disorders in particular account for an increasing proportion of this . in the wake of the hsca 2012 , it is necessary to reflect upon whether the 2012 act offers hope to those made vulnerable through mental ill health , or whether it instead fails them , and if so , why ? this article explores this question with reference to three key policy drivers within the legislation and is structured accordingly . in the first instance , the article examines the hsca 2012 from the mental health perspective , in terms of how the restructured commissioning process operates and how it maps on to the mental health framework . attention is then given to three issues : first , whether parity between mental and physical health can in all reality have life beyond political rhetoric ; second , what impact driving up efficiency within the nhs , in terms of commissioning decisions , will have upon patients with mental health conditions and third , the extent to which the personalisation agenda can be meaningfully applied within the mental health context . these issues are considered with reference to broader policy influences within the mental health law and policy landscape . whilst the fundamental restructuring of the nhs has been the subject of recent attention with the enactment of the hsca 2012 , mental health has also been under the spotlight of reform in the past few years . the mental health act 2007 sought to respond to the challenges posed by changing psychiatric practices and the policy shift from hospital - based treatment to care in the community . to some extent resources have followed this changing pattern of care , but inevitably , service provision and delivery has been affected by the gradual shift in the mental health landscape . in parallel with the introduction of the mental health act 2007 , modifications have been made to the mental health act code of practice to reflect the legislative amendments . these principles are first , the purpose principle , whereby decisions under the act must be made to minimise the undesirable effects of mental disorder ; second , the least restriction principle , where decision - makers should keep to a minimum the restrictions they impose on the patient s liberty ; third , the respect principle , whereby recognition and respect should be given to the diverse needs , values and circumstances of each patient ; fourth , the participation principle that encourages patients involvement and finally , the effectiveness , efficiency and equity principle that focuses upon optimal decision - making using available resources in the most efficient way possible . the hsca 2012 has been heralded as the most extensive and radical reorganisation of the nhs to date and has been accompanied by significant levels of political rhetoric , speculation and controversy . the changes introduced by the act are far reaching and for those with chronic and enduring conditions , of which all mental health conditions would likely be labelled , the hsca 2012 can be expected to wield significant weight in treatment and care planning as it becomes fully operational in the months to come . however , it is conceivable that these principles have the potential to conflict with significant consequences and may have lasting implications upon the quality of delivered care . the persistent concern amongst many professional and user groups alike has been and continues to be that the political desire to make financial savings and improve the cost - effectiveness of the nhs may prove to be the overarching driver . a related concern is that the legislation represents an inevitable shift away from the ideology of universal provision , a mainstay of the old nhs , towards a stronger endorsement of expanding private sector involvement and a gradual privatisation of the health service . the reinforcement of competition principles within the health care system is likely to have a detrimental impact on the mentally vulnerable as the act opens up private sector involvement , making the process of commissioning outside of the nhs structure easier and more cost - effective . mental health care and associated social care provision is generally seen as an unprofitable field , with long - term and often complex care and support required by individuals . the 2012 act s market - based approach may prove to be particularly damaging for the mentally ill , with resources being allocated away from the needs of this group and short - term care measures , such as acute inpatient provision , being given greater attention than the longer term health and social care needs of individuals in the community . the act also introduces a change to one of the central nhs tenets : no longer will services be exclusively operated via the nhs and its partners ; instead , any willing provider could supply services . this enables the private sector to have direct access to the central operations of the nhs , in terms of both planning and provision . currently , the role of third sector organisations in mental health care is much more prominent and is , indeed , essential , particularly in relation to social care provision ; however , whether this will continue remains open to speculation . commissioning of services for mental health care and treatment services will be conducted and guided by clinical commissioning groups ( ccgs ) , which are introduced by the hsca 2012 , in a similar fashion as for all other services . the guiding principles under the hsca 2012 will be influential in how ccgs conduct their activities . in the first instance , ccgs have a duty to promote the nhs constitution and ensure patients , staff and the public are aware of the nhs constitution and their nhs constitutional rights . instead , the duty under the hsca 2012 explicitly recognises the need to consider treatment and care outcomes and the patient experience . how viable the balancing exercise of enabling patient choice within the mental health field will be remains to be seen . the creation of patient choice relies not only upon ccg behaviour endorsing and facilitating patient choice , but the providers of these services must actually exist in mental health , the fulfilment of identified need has often presented challenges , as service provider limitations are routine . encouraging ccgs to commission several alternative treatments from different providers may lead to more providers having a smaller market share and greater fragmentation within the health and social care service sector might result . quite how the commissioning process can effectively achieve efficiency through competition whilst also increasing patient choice is difficult to understand ; or at least , it is possible to foresee challenges and tensions developing in the attainment of this aim . however , as we will see later in this article , drug therapy is often deemed to offer a front - line response to patients mental health needs by general practitioners ( gps ) , and psychological services are limited in availability . ccgs will be restricted by these practical limitations , but they will also be under a duty to ensure service commissioning is subject to tender under the national health service ( procurement , patient choice and competition ) regulations 2013 . if the framework of health and social care does crumble under the weight of these different legislative objectives , those with mental health conditions may be particularly vulnerable as a fragmented health and social care service will not be beneficial to them . this entails the integration of health services with health - related and social care services . nonetheless , from the patient perspective , this offers an avenue for improvements in quality of life , particularly for those who need longer term support in the community . furthermore , mental health provision is littered with countless examples of joint working failures and inadequate communication throughout the health and social care system . indeed , the ideal of achieving seamless provision is far removed from the reality for many patients , and it is often this which leads to the disjointed care that is received and the gaps in provision where patients fall through the net . collaboration between the board members will afford the opportunity to assess local health and social care needs , agree on spending priorities and encourage ccgs to work with seamless , joined up provision in mind . it is uncertain whether this will directly improve service provision , but the cross - government mental health strategy pins its hopes on the shift towards localism and local care decision - making under the 2012 act . the mental health strategy implementation framework suggests that it is this focus on local needs which can deliver the vision of improved mental health and wellbeing. the restructuring of the nhs and the changes created by the hsca 2012 to the commissioning process will take time to grow accustomed to . from a mental health perspective , the hsca 2012 offers real potential to see mental health brought from the margins of provision to feature much more prominently . it creates the possibility for a conceptual reconfiguration of health to emerge , introducing explicitly the need for parity between mental and physical health . indeed , this duty to promote health parity could create the impetus for a paradigmatic shift within health and social care provision , but just how successful the implementation of this will be remains to be seen as the high - level commitment to health parity is only one of several key objectives within the 2012 legislation . devolution of budgets down to ccgs may provide opportunities for mental health to feature more prominently within the commissioning process ; yet there are concerns that mental health needs may continue to be overlooked by ccgs when pressure to commission services efficiently whilst also increasing patient choice presents significant tensions for ccgs to overcome . we will now turn to consider three drivers within the 2012 act , exploring whether they are feasible within the mental health context or whether the legislation will prove to be detrimental to those with mental health needs . first , attention will be given to the commitment to achieving parity of physical and mental health within the health care system , followed by a consideration of how the desire to increase efficiency may influence commissioning decisions within the mental health arena and finally , consideration will be given to the move towards expanding patient choice and personalisation within the health care market . the government s draft mandate to nhs england is explicit in its message : direct recognition is to be given to the need to place mental health on the same footing as physical health . mental health conditions are now to be recognised as a clear equality issue and the nhs equality delivery system will be primed to help those providing nhs services to respond properly to it . perhaps of greatest importance is the government s recognition in the mental health implementation framework that achieving parity between physical and mental health is an absolute goal , where more still needs to be done to ensure all organisations ( both public and private ) meet their equality and inequality obligations in relation to mental health. owing to this shift in attitude , and indeed , reconfiguration of the conception of health within the legislation , the neglected and under - resourced mental health service may be a thing of the past . the rhetoric of achieving parity between mental and physical health is , in many ways , politically driven , though the evidence suggests that greater effort to improve mental health is needed ; mental ill health is a leading cause of suffering , economic loss and social problems and accounts for over 15% of the disease burden in developed countries . in the european union at least 83 million people ( 27% ) suffer from mental health problems ( 16.7 million in the united kingdom ) , with depression being the most common ( 812% of the adult population ) . the newly restructured system of health and social care is in its infancy , and it is still too early to say whether the steps taken to achieve parity will bear fruit . likewise , how the vulnerable will be able to protect their rights in this new health and social care environment is unknown , but it seems likely that ccgs , if motivated by market - driven policies , could lose sight of the particular needs of these vulnerable groups . for mental health , the best hope for this change exists within the mental health implementation framework where explicit mention is made of the need to promote research into mental health and to recognise , support and strengthen academic career paths in this field . it is only by consolidating capacity , instilling aspiration and professional motivation within the mental health care framework ( both research and practice pathways ) that the cultural transformation can begin to emerge . whilst parity of mental and physical health is a clear commitment within the hsca 2012 , the introduction of competition principles will also facilitate efficiency savings . not only can providing for this complex diet of needs be difficult , it can be expensive . the mental health care framework has very limited scope to be able to deal with increases in demand , and , traditionally , this is where the third sector has often been sought to plug the gap . it is quite possible that without any form of overarching regional oversight , a task that pcts undertook prior to the 2012 act , the commissioning process may become fragmented and uncoordinated , and ultimately , gaps in some areas may be difficult to fill as patient needs may not be recognised in the round . two separate issues in the commissioning process for mental health services exist : first , the level of clinical expertise that exists and second , whether ccgs have sufficient management experience to meet the need for equal distribution and coverage of services . second , it is predicted that ccgs may have inadequate management expertise and from this , optimal commissioning decisions will be less likely to occur . given the sheer scale of care and social support needs that patients with mental health conditions often need , if ccgs lack membership that reflects the level of experience needed to recognise this , adequate mental health care provision is likely to be inadequate . often mental health provision is not the focus , with greater attention being given to physical health needs ; yet mental health conditions account for 23% of the total burden of disease ; but in terms of nhs expenditure , only 13% of health expenditure is currently directed towards psychiatric and related services . such underinvestment is not new and despite funds being channelled through pcts at a regional level to recognised areas of need prior to the hsca 2012 , resource shortfalls have been commonplace . mental health did not gain the moniker of the cinderella service without good reason and has been struggling under the weight of systemic neglect for a considerable time . unfortunately , mental health care must compete with all other health and social care needs , of which most are far more evident and have a more tangible quality about them . whether the hsca 2012 will improve this is uncertain . the organisational reconfiguration reflects the mood of the government to reduce bureaucracy and complexity in the health and social care framework , to improve efficiency and to redeploy functions through bodies that are independent or at least operating at arm s length of the government . it is [ just not yet ] clear how these national bodies will interact or how they will provide coordinated and consistent governance of the nhs. the challenges facing ccgs are unlikely to reassure patients in the short term ; for mental health patients , these concerns may simply be more acute , given the complexity of typical mental health care needs which tend to stretch over a number of agencies and providers , often featuring periods of both acute need and stable chronicity . ccgs are going to have to ensure sufficient awareness is present within the strategic planning process to take account of this , and if they do not , health conditions , including most mental health conditions , that require a complex health and social care response may suffer . greater emphasis is placed upon self - directed support and personal budgetary control combined with a move away from the notion that provision should follow a the personalisation agenda seeks to move the health and social care framework away from crisis management , relying upon patients identifying personal needs and making appropriate care choices to meet these needs . to implement the personalisation agenda , the social care system , in particular , this means that ccgs have to take seriously the need to make and implement local commissioning decisions in a way that will enable genuine choices to be made . this approach is focused upon the mental health patient being afforded the opportunity to determine his own life and to be offered the support required to be able to live as independently as possible . in practice , when a plan has been formulated , social care support can be obtained from a variety of sources , including statutory social services , the private sector , the voluntary sector , community groups , neighbours , family and friends . for those who need it , assistance in devising a care plan reflective of individual need is an essential element of the process ; particularly as individual budgets are increasingly being used as a vehicle to combine several funding streams that many mental health patients may need to access in the community . however , to enable mental health patients and other chronic patients with complex social care needs to benefit from this , better integration of services and a collective willingness to embrace choice needs to be fostered . how successful the personalisation agenda and its implementation under the hsca 2012 is , is perhaps best judged by assessing the benefits to patients that have flowed from this agenda . bureaucracy and cuts in social care spending are exacerbating the situation ; patients who require significant levels of support in this process may find their experience of the personalisation agenda hampered . other associated and recurrent problems exist within the mental health system , placing further strain on the achievement of the personalisation agenda . as such , staffing challenges and the need for extra support by mental health patients to benefit from the personalisation agenda may in reality make this policy a largely spurious one with little practical substance . the hsca 2012 represents a significant departure from a culture of public service provision that we have become accustomed to , but does it fail the vulnerable , notably those with mental health care needs ? the need to drive efficiency up , whilst also tailoring health and social care to individual patients is , perhaps , an impossible dilemma . how mental health provision will fare in this new and uncharted landscape remains open ; but , inevitably , it will face its own set of problems in the months to come . time will tell , though the tensions that exist between three of the key policy drivers within the legislation , the focus of this article , suggest that where there are pressure points and the vulnerable may ultimately experience the greatest detriment . however , they can not be responsive to the more nuanced needs of patients with chronic conditions , particularly where care needs bridge both health and social care and are often required for lengthy periods of time . perhaps , the brightest ray of hope should be the recognition that parity between mental and physical health will be a clear objective . in mental health , it is the implementation stage that frequently presents the most significant challenge for decision - makers , with limitations in staffing , funding and social care placements creating bottlenecks in the system . unless these practical hurdles can be overcome , the desire to forge a new and fairer culture within health and social care , where parity between mental and physical health is the accepted benchmark , will be a very difficult one to attain . the hsca 2012 offers a very real opportunity to enable mental health to be mainstreamed into core public health priorities . but , this relies upon a determination reminiscent of aneurin bevan , the nhs will last as long as there are folk left with the faith to fight for it. it can only be hoped that there are those prepared and willing to fight to ensure the needs of vulnerable groups , such as those with mental health conditions , are met and protected and that faith in the achievement of health and social care equality endures .

although an established treatment , allogeneic hematopoietic stem cell transplantation ( hsct ) is associated with several serious side effects . these are generally grouped into three categories : infectious complications , drug toxicity , and graft - versus - host disease ( gvhd ) . gvhd manifests in an acute and chronic form , each with distinct clinical signs and symptoms . although both are caused by an immunological reaction between donor - derived lymphocytes and recipient tissue , the pathophysiological mechanisms involved are different . acute gvhd usually occurs within the first three months after engraftment and it has a more rapid course . the main effector cells are donor t - cells that are transferred with the graft and become activated in response to a proinflammatory milieu and differences in cell - surface antigens . it has been shown that nave cd4 t - cells may have a particularly important role in the initiation of the gvhd process . activation of these cells by host - specific antigens elicits an immunological reaction directed against host tissue resulting in local and systemic inflammation . acute gvhd is also associated with an increased level of immunodeficiency , both directly through the immune - modulatory effect of the ongoing inflammatory process and indirectly due to the immunosuppressive agents used for treatment of this potentially life - threatening condition . with today 's methods of tissue typing , it is possible to obtain a high level of matching between donor and recipient regarding major histocompatibility antigens ( mhc ) . even though this progress has significantly improved the outcome of hsct , the incidence of acute gvhd has still been as high as 80% in some reports . one reason for this is thought to be the existence of so - called minor histocompatibility antigens . these are polymorphic , non - mhc genes , the end products of which may cause an alloreaction . severe acute gvhd is often resistant to treatment , and it is a widely accepted consensus that preventive measures significantly improve the chances of a positive outcome , as compared to efforts to treat established disease . for this purpose , a predictive test for gvhd would be of great clinical value , particularly because overtreatment with immunosuppressive agents in itself is associated with serious complications [ 7 , 8 ] . with today 's methods , there is no reliable way of predicting the risk of gvhd with any certainty . the aim of this project was to design a functional in vitro test for assessment of pretransplant risk for acute gvhd . 29 patients and their corresponding sibling donors gave their informed consent and were included in the current study . inclusion criteria for the study were a hla matched sibling donor and pbsc as stem cell source , and the apheresis was performed at karolinska university hospital . all aspects of this study were reviewed and approved by the regional ethical committee in stockholm ( approval number 2008/206 - 31 ) . blood samples were collected from patients immediately before the start of conditioning therapy and from the donors in conjunction with harvesting of the peripheral blood stem cell ( pbsc ) graft . we selected seven recipient / donor pairs where the patients had developed acute gvhd of grades ii iv , within the first three months after hsct , with manifestation in the skin and the gastrointestinal ( gi ) tract , with or without liver involvement . seven control cases were selected from those who had had no signs of gvhd and who had not received any additional immunosuppressive therapy apart from the standard gvhd prophylaxis . the remaining 15 patient / donor pairs were excluded from further studies due to established or suspected acute gvhd grade i. grading of gvhd was performed according to the glucksberg criteria . all recipients and their sibling donors were tissue - typed by allele - level pcr with sequence - specific primers . patient - donor pairs were matched regarding hla - a , hla - b , hla - c , hla - dp , hla - dq , and hla - dr . no statistical differences could be observed between the groups for the parameters shown in table 1 . fluorescein isothiocyanate ( fitc)- , phycoerythrin ( pe)- , allophycocyanin ( apc)- , bd horizon v450 ( v450)- , and pe - cy5-labelled anti - cd3 ( ucht1 ) ; apc - labelled anti - cd27 ( l128 ) ; fitc - labelled anti - cd19 ( hib19 ) ; apc - labelled anti - cd45ro ( uchl1 ) ; apc - labelled anti - cd19 ( hib19 ) ; fitc - labelled anti - cd56 ( mcam162 ) ; alexa fluor 700-labelled anti - cd4 ( rpa - t4 ) ; apc - cy7-labelled anti - cd8 ( sk1 ) ; apc - cy7-labelled anti - cd69 ( fn50 ) ; fitc - labelled anti - cd95 ( dx2 ) ; pe - cy7-labelled anti - cd3 ( sk7 ) ; pe - labelled anti - cd45ra ( hi100 ) ; fitc - labelled anti - cd28 ( cd28.2 ) ; fitc - labelled anti - cd94 ( hp-3d9 ) ; fitc - labelled anti - t - cell receptor ( tcr ) ( wt31 ) ; pe - labelled anti - tcr ( t10b9.1a-31 ) ; fitc - labelled anti - cd69 ( fn50 ) ; pe - cy7-labelled anti - ccr7 ( 3d12 ) ; bd horizon v500 ( v500)-labelled anti - cd8 ( rpa - t8 ) ; and 7-amino - actinomycin d ( 7-aad ) were purchased from bd biosciences ( franklin lakes , nj ) . pacific blue-labelled anti - cd107a ( lamp-1 ) was purchased from biolegend ( san diego , ca ) . pe - labelled anti - tcr ( b1.1 ) was purchased from ebioscience ( san diego , ca ) . fitc - labelled anti - tcr pan ( immu510 ) was purchased from beckman coulter ( fullerton , ca ) . pacific orange - labelled anti - cd8 ( 3b5 ) was purchased from invitrogen ( camarillo , ca ) . pbmcs were isolated from peripheral blood samples using density - gradient centrifugation ( 800g , 20 min ; rotina 420 [ hettich , beverly , ma , usa ] with lymphoprep [ fresenius kabi , oslo , norway ] ) . they were then cryopreserved at 196c with 10% dmso in complete rpmi-1640 medium ( hyclone [ thermo fisher scientific inc . , waltham , ma , usa ] enriched with 10% human ab - serum [ karolinska university hospital ] and 100 mg / ml streptomycin [ gibco , life technologies , paisley , uk ] ) . briefly , the cells were incubated with 1 m carboxyfluorescein succinimidyl ester ( cfse ; molecular probes , inc . the reaction was blocked with pbs containing 10% ab - serum , after which the cells were washed and resuspended in complete rpmi-1640 medium in flat - bottomed 6-well plates at 1 10 cells / ml . pbmcs from the corresponding recipients were used as stimulator cells after irradiation with 30 gy . responder and stimulator cells were added to the wells in a responder : stimulator ratio of 5 : 1 . as controls , we used responder cells that were either untreated , cfse - stained in complete medium alone , or cfse - stained in complete medium with 10 g / ml phytohemagglutinin a ( pha ; sigma - aldrich , st . the plates were incubated at 37c in an atmosphere of 5% co2 for 6 days , after which the cells were harvested , stained , and analysed by flow cytometry . cell - surface staining of thawed , unmanipulated donor pbmcs and responder cells from each mlc condition was performed as described previously . briefly , cells were incubated with the specified antibodies in pbs for 20 min at 4c , washed twice in pbs , and incubated with 7-aad for 15 min at room temperature . stained cells were analysed on a beckman coulter gallios using beckman coulter gallios acquisition software ( beckman coulter inc . , fort collins , co ) or the bd facs canto using bd facs diva software ( bd biosciences ) . the data acquired was analysed with flowjo software ( tree star inc . , cells were gated according to fluorescence - minus - one ( fmo ) samples , which were included in each experiment . lymphocytes were distinguished from monocytes by their side- and forward - scatter phenotype . supernatant samples were analysed for the levels of 26 different cytokines ( eotaxin , g - csf , gm - csf , ifn-2 , ifn- , il-10 , il-12 ( p40 ) , il-12 ( p70 ) , il-13 , il-15 , il-17 , il-1 , il-1 , il-2 , il-3 , il-4 , il-5 , il-6 , il-7 , il-8 , ip-10 , mcp-1 , mip-1 , mip-1 , tnf- , and tnf- ) . the milliplex map human cytokine / chemokine - premixed 26 plex from millipore [ millipore corporation , temecula , ca , usa ] was performed according to manufacturer 's protocol and as described before [ 1315 ] . , austin , tx , usa ] on the labscan100 ( one lambda inc . , data was analysed and displayed using graphpad prism software ( graphpad software inc . , san diego , ca ) and ibm spss statistics 23 software ( ibm , armonk , ny , usa ) . levels and changes in cell - surface markers and levels of soluble factors were compared between the non - gvhd and gvhd groups . the following markers were studied on unmanipulated donor pbmcs : cd3 , cd4 , cd8 , ccr7 , cd45ra , cd45ro , cd28 , cd69 , cd94 , cd56 , tcr , tcr , cd95 , cd19 , and cd27 . responder cells after mlc were analysed for cd3 , cd4 , cd8 , tcr , tcr , cd45ro , ccr7 , cd69 , and cd107a . whitney u test ( table 1 ; figures 13 ) and fisher 's exact test ( table 1 ) . due to sample size limitations , ( n = 7 ) based on patient characteristics after transplantation and analysed for possible differences . there was no significant difference between the non - gvhd group and the gvhd group regarding frequencies of major lymphocyte populations , that is , total t - cells ( median 55.2% versus 56.6% ; p = 0.535 ) , nk - cells ( median 10.1% versus 11.6% ; p = 0.383 ) , or b - cells ( median 15.5% ( n = 6 ) versus 6.5% ; p = 0.295 ) ( figure 1(a ) ) . in order to examine the maturation status of t - cells in the grafts the distribution of the different memory subsets of total t - cells in the two groups is shown in figure 1(b ) . no statistically significant differences between the non - gvhd and the gvhd groups were found regarding frequencies of nave ( cd45roccr7 ; median 22.1% versus 43.3% ; p = 0.165 ) , central memory ( cd45roccr7 ; median 12.6% versus 8.7% ; p = 0.306 ) , effector memory ( cd45roccr7 ; median 35.9% versus 28% ; p = 0.259 ) , or terminally differentiated t - cells ( cd45roccr7 ; median 21.7% versus 18.3% ; p = 0.620 ) . additionally , no significant differences were seen between the two categories of donors when we analysed total frequencies of cd4 and cd8 t - cells or their individual maturation status . the non - ghvd group had a higher frequency of total tcr nave memory t - cells than the gvhd group ( median 4.5% versus 0.7% ; p = 0.004 ) ( figure 2(a ) ) . after activation , t - cells transiently express certain nk - cell markers . in this material , cd94 total t - cells were found in higher frequencies in the non - gvhd group for nave memory t - cells ( median 5.2% versus 1.2% ; p = 0.018 ; figure 2(a ) ) and for terminally differentiated t - cells ( median 33.7% versus 13.8% ; p = 0.018 ; figure 2(b ) ) as compared to the gvhd group . similarly , cd56 expression on cd4 t - cells was found at higher frequencies in the non - gvhd group ( median 1.6% versus 0.6% ; p = 0.030 ) ( figure 2(c ) ) . t - cells expressing the fas receptor cd95 were observed at significantly higher frequencies in the non - gvhd group in nave memory t - cells ( median 19.6% versus 8.55% ; p = 0.026 ; figure 2(a ) ) as compared to the gvhd group . to evaluate the alloreactive capacity of donor lymphocytes towards recipient cells , a mixed lymphocyte culture ( mlc ) was performed . by using a multicolor flow cytometry panel , we wanted to detect changes in frequencies of lymphocyte subpopulations of the responding cells and their surface expression of the activation markers cd69 and cd107a after mlc . ultimately , we wanted to determine whether any changes correlated to the incidence of acute gvhd . as illustrated in figure 3(a ) , no differences in total t - cell percentages could be observed between the patient groups for all three mlc conditions . there was a change in the proportions of cd4 and cd8 t - cells after mlc when we compared the two patient groups . before the mlc , the cd4/cd8 ratio in both groups was comparable ( non - gvhd 1.44 versus gvhd 1.9 ; p = 0.165 ; figure 3(b ) ) . at day 6 , the cd4/cd8 ratio had changed in the 5 : 1 stimulated samples ( non - gvhd 1.02 versus gvhd 3.36 ; p = 0.052 ) and pha stimulated samples ( non - gvhd 0.30 versus gvhd 1.33 ; p = 0.038 ) . in both settings , the cd4/cd8 ratio skewed towards an increased proportion of cd4 t - cells in the gvhd group as compared to the non - gvhd group ( figure 3(b ) ) . the frequencies of t - cells positive for early activation marker cd69 were comparable between the two patient groups before mlc ( median non - gvhd 4% versus gvhd 6.3% ; p = 1.0 ) . however , expression of cd69 was higher for total t - cells in the non - gvhd group than in the ghvd group after an unstimulated 6-day incubation ( median 5.6% ( n = 4 ) versus 1.5% ( n = 6 ) ; p = 0.038 ) and after the 5 : 1 stimulated mlc condition ( median 5.1% ( n = 5 ) versus 1.4% ( n = 6 ) ; p = 0.009 ; figure 3(c ) ) . no difference was seen between the two patient groups in cd69 t - cell frequencies after 6-day incubation with pha . there was no significant difference in cytokine concentrations in the supernatant of all three conditions after mlc between the two patient groups ( data not shown ) . additionally , no difference in cell proliferation as measured by cfse could be observed ( data not shown ) . most studies on the effect of graft composition on outcome after hsct have focused on quantitative differences in doses of total nucleated cells , cd34cells , and t - cells . however , there have been reports indicating that frequencies of minor cell subsets and phenotypic distinctions within these populations could be connected to the alloreactive potential of the graft and may therefore affect clinical outcome [ 1825 ] . in our study we wanted to use multicolor flow cytometry to detect potential differences in frequencies of lymphocyte subsets in donor material before and after in vitro mlc in order to predict severe gvhd development . we aimed at creating a robust test that could be used in a potential clinical routine environment . as clinicians and researchers in the field know that grade i acute gvhd can easily be misdiagnosed , we only included patients without any signs of gvhd or with grade ii our results illustrate that flow cytometry on graft material using the most common phenotypic markers is not enough to detect predictive markers for gvhd ( figure 1 ) . today 's use of flow cytometers with > 2 lasers in clinical routine enables more in - depth graft analysis . the biological functions of t - cells that express the t - cell receptor ( tcr ) are not fully understood , but existing evidence points towards both proinflammatory and suppressive functions as well as antigen - presenting and cytotoxic capacities [ 2628 ] . in 2001 , using an allogeneic rat model , huang et al . showed that recipients of grafts depleted of tcr t - cells had a higher incidence of acute gvhd than those transplanted with t - cell - depleted grafts . it has also been shown that infusion of activated t - cells , in the absence of regular t - cells , can promote engraftment without causing gvhd in a mhc - mismatch situation . other animal studies have shown contradictory results , where t - cells appeared to be associated with an increased risk of gvhd [ 30 , 31 ] . to date , only some clinical studies have addressed the issue of graft t - cell content in hsct . in a report that involved 63 recipients of unrelated peripheral blood stem cell grafts , pabst et al . found a correlation between high t - cell dose and higher incidence of acute gvhd of grades ii iv , while a more recent study has shown contradicting results in a smaller , more heterogeneous cohort of patients . in our material , we found a lower frequency of nave t - cells in the peripheral blood of donors corresponding to patients who later developed acute gvhd of grades ii the timing of transfer of the t - cells in relation to the allograft has varied between studies . secondly , if the t - cells exert their effect through direct or indirect interaction with other cell types , such as regulatory t - cells and t - cells [ 26 , 27 , 34 , 35 ] , differences in ratios between the infused doses of these cells might lead to different end - results . lastly , the phenotype of t - cell subsets in the graft , particularly their activation and maturation status , could affect their function and ability to survive in vivo [ 36 , 37 ] . other minor lymphocyte populations that have attracted attention in the context of hsct are t - cells expressing nk - cell markers [ 38 , 39 ] . these can be either naturally occurring invariant nkt - cells or in vitro activated cytokine - induced killer ( cik ) cells . both cell subsets have been shown to have antitumour and tolerogenic abilities in preclinical studies [ 4043 ] . invariant nkt - cells have in several studies been shown to be able to reduce the risk for gvhd , whether they were present in high numbers in the graft or given through adoptive transfer [ 24 , 44 ] . two independent clinical trials on the use of ex vivo expanded allogeneic cik - cells , as part of treatment for relapsed haematological malignancies after hsct , have shown response rates comparable to those for regular donor lymphocyte infusions ( dli ) , but with significantly lower incidence of gvhd [ 45 , 46 ] . in the present study , we found significantly lower levels of t - cells expressing the nk - cell markers cd56 ( on cd4 t - cells ) and cd94 ( on nave and terminally differentiated t - cells ) in samples from donors before mlc in the gvhd group ( figure 2 ) . if these cells correspond to previously described inkt - cells , our study confirms that inkt - cells may indeed have a gvhd - modulatory effect . the exact mechanism for this is not known , but experiments in murine models suggest that these cells have the ability to produce il-4 and ifn- upon activation , which would help dampen the immune reaction behind gvhd [ 25 , 47 , 48 ] . unfortunately , as no nk - cell markers were included in the flow cytometry panel after mlc , it is difficult to speculate on cik - cells in this study . activation - induced cell death ( aicd ) is important in the regulation of t - cell responses and works as an inhibitory mechanism to prevent uncontrolled activation . aicd may be mediated through the interaction of cd95 ( fas ) and cd95 ligand ( fasl ) , which initiates the extrinsic pathway of programmed cell death [ 49 , 50 ] . the fas - fasl interaction is of particular importance for establishing peripheral tolerance and it has been shown to have a role in attenuation of gvhd [ 5154 ] . in our material , staining of unmanipulated donor pbmcs for cd95 revealed that samples from the gvhd group contained lower proportions of nave t - cells that expressed this receptor on their surface ( figure 2(a ) ) . surface expression of cd95 is greatly increased on t - cells upon activation , while lower expression reflects a more nave phenotype . this , together with other mechanisms , helps to protect cells from apoptosis in their resting state . it is also in accordance with the widely accepted view that acute gvhd is induced by nave t - cells , most likely due to their ability to sustain a strong response [ 56 , 57 ] . different variations of the mlc have been used to assess the alloreactive capacity of donor cells in the setting of hla - identical hscts , but the correlation to clinical results has varied . in particular , inconsistencies have been observed in matched unrelated transplantations [ 5861 ] . the common principle for these methods is incubation of donor pbmcs with inactivated cells from the recipient followed by quantitative analysis of the helper t - lymphocyte precursor subset . since these cells appeared to have a high propensity for producing il-2 , an assessment of proliferation and activity was obtained through coincubation with an il-2-dependent cell line . multicolor flow cytometry is a more direct and specific approach for quantification and categorization of lymphocyte subpopulations after allogeneic mlc . by using this sensitive technique , we could detect a shift in cd4/cd8 ratio after mlc between the patient groups . in the 5 : 1 setting , where the cells were exposed to alloantigen presentation , we could observe a trend for an increased proportion of cd4 t - cells in the gvhd group as compared to the non - gvhd group . moreover , this increased proportion of cd4 t - cells in the gvhd group was also observed in the pha setting ( figure 3(b ) ) . it would therefore appear that , after stimulation , donor cells from the gvhd group tend to skew more to a cd4 t - cell phenotype than donor cells from the non - gvhd group . additionally , we could detect a significant correlation between a decrease in the frequency of cd69 t - cells and incidence of acute gvhd grades ii iv even in this small material ( figure 3(c ) ) . cd69 is traditionally seen as one of the earliest markers emerging after activation of t - cells , though it appears to also be highly expressed by lymphocytes at mucosal sites of the human body [ 62 , 63 ] . the counter - intuitive observation in the present study , that is , low frequency of activated t - cells correlating with gvhd , might be due to an increased downregulation of cd69 in alloreactive t - cells after 6 days of activation . cd69 is known to be transiently expressed , with an expression as early as 2 hours after stimulation , but with a quick decline of expression after more than 24 hours . thus , low levels of cd69 t - cells after mlc might serve as an indicator of increased alloreactivity and risk for incidence of acute gvhd . it is possible that the 6-day mlc leads to a saturation of the system due to overstimulation . additionally , the flow cytometry panel could be expanded to include more cellular markers to identify frequencies of other cellular populations that might play important roles in this context , such as regulatory t - cells , myeloid - derived suppressor cells ( mdscs ) , and mucosal - associated - invariant t - cells ( maits ) . recent studies suggest that these may play critical roles in gvhd development after hsct and therefore warrant more extensive studies [ 6569 ] . in conclusion , our results indicate that phenotypic analysis of the donor lymphocyte subpopulations before transplantation can yield predictive information of clinical relevance . we found a positive correlation between lower frequencies of donor t - cells expressing tcr and nk - cell markers on the one hand and incidence of acute gvhd of grades ii we have also shown that a distinct relative decrease in cd69 t - cells in a flow cytometry - based allogeneic mlc can be predictive of acute gvhd . we recognise the fact that the sample groups in this pilot study are small and not perfectly matched for some clinical parameters , for example , diagnosis or conditioning regimen . . nevertheless , the findings presented here may be of value in pretransplant risk assessment and they may enable the use of a more individualized prophylactic strategy .

background . graft - versus - host disease ( gvhd ) is a serious complication after allogeneic hematopoietic stem cell transplantation ( hsct ) . we designed a functional assay for assessment of individual risk for acute gvhd . study design and methods . blood samples were collected from patients and donors before hsct . two groups of seven patients each were selected , one in which individuals developed acute gvhd grades ii iv and one in which none showed any clinical signs of gvhd . peripheral blood mononuclear cells ( pbmcs ) isolated from donors were incubated in mixed lymphocyte cultures ( mlcs ) with recipient pbmcs . the cells were characterized by flow cytometry before and after mlc . results . samples from donors in the gvhd group contained significantly lower frequencies of nave t - cells and t - cells expressing nk - cell markers cd56 and cd94 . donor samples in this group also exhibited lower frequencies of nave cd95 + t - cells compared to controls . after mlc , there were dissimilarities in the cd4/cd8 t - cell ratio and frequency of cd69 + t - cells between the two patient groups , with the non - gvhd group showing higher frequencies of cd8 + and cd69 + t - cells . conclusion . we conclude that a thorough flow cytometric analysis of donor cells for phenotype and allogeneic reactivity may be of value when assessing pretransplant risk for severe acute gvhd .

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

although an established treatment , allogeneic hematopoietic stem cell transplantation ( hsct ) is associated with several serious side effects . these are generally grouped into three categories : infectious complications , drug toxicity , and graft - versus - host disease ( gvhd ) . the main effector cells are donor t - cells that are transferred with the graft and become activated in response to a proinflammatory milieu and differences in cell - surface antigens . it has been shown that nave cd4 t - cells may have a particularly important role in the initiation of the gvhd process . even though this progress has significantly improved the outcome of hsct , the incidence of acute gvhd has still been as high as 80% in some reports . severe acute gvhd is often resistant to treatment , and it is a widely accepted consensus that preventive measures significantly improve the chances of a positive outcome , as compared to efforts to treat established disease . with today 's methods , there is no reliable way of predicting the risk of gvhd with any certainty . the aim of this project was to design a functional in vitro test for assessment of pretransplant risk for acute gvhd . 29 patients and their corresponding sibling donors gave their informed consent and were included in the current study . blood samples were collected from patients immediately before the start of conditioning therapy and from the donors in conjunction with harvesting of the peripheral blood stem cell ( pbsc ) graft . we selected seven recipient / donor pairs where the patients had developed acute gvhd of grades ii iv , within the first three months after hsct , with manifestation in the skin and the gastrointestinal ( gi ) tract , with or without liver involvement . seven control cases were selected from those who had had no signs of gvhd and who had not received any additional immunosuppressive therapy apart from the standard gvhd prophylaxis . the remaining 15 patient / donor pairs were excluded from further studies due to established or suspected acute gvhd grade i. grading of gvhd was performed according to the glucksberg criteria . all recipients and their sibling donors were tissue - typed by allele - level pcr with sequence - specific primers . fluorescein isothiocyanate ( fitc)- , phycoerythrin ( pe)- , allophycocyanin ( apc)- , bd horizon v450 ( v450)- , and pe - cy5-labelled anti - cd3 ( ucht1 ) ; apc - labelled anti - cd27 ( l128 ) ; fitc - labelled anti - cd19 ( hib19 ) ; apc - labelled anti - cd45ro ( uchl1 ) ; apc - labelled anti - cd19 ( hib19 ) ; fitc - labelled anti - cd56 ( mcam162 ) ; alexa fluor 700-labelled anti - cd4 ( rpa - t4 ) ; apc - cy7-labelled anti - cd8 ( sk1 ) ; apc - cy7-labelled anti - cd69 ( fn50 ) ; fitc - labelled anti - cd95 ( dx2 ) ; pe - cy7-labelled anti - cd3 ( sk7 ) ; pe - labelled anti - cd45ra ( hi100 ) ; fitc - labelled anti - cd28 ( cd28.2 ) ; fitc - labelled anti - cd94 ( hp-3d9 ) ; fitc - labelled anti - t - cell receptor ( tcr ) ( wt31 ) ; pe - labelled anti - tcr ( t10b9.1a-31 ) ; fitc - labelled anti - cd69 ( fn50 ) ; pe - cy7-labelled anti - ccr7 ( 3d12 ) ; bd horizon v500 ( v500)-labelled anti - cd8 ( rpa - t8 ) ; and 7-amino - actinomycin d ( 7-aad ) were purchased from bd biosciences ( franklin lakes , nj ) . pbmcs were isolated from peripheral blood samples using density - gradient centrifugation ( 800g , 20 min ; rotina 420 [ hettich , beverly , ma , usa ] with lymphoprep [ fresenius kabi , oslo , norway ] ) . briefly , the cells were incubated with 1 m carboxyfluorescein succinimidyl ester ( cfse ; molecular probes , inc . the reaction was blocked with pbs containing 10% ab - serum , after which the cells were washed and resuspended in complete rpmi-1640 medium in flat - bottomed 6-well plates at 1 10 cells / ml . the plates were incubated at 37c in an atmosphere of 5% co2 for 6 days , after which the cells were harvested , stained , and analysed by flow cytometry . briefly , cells were incubated with the specified antibodies in pbs for 20 min at 4c , washed twice in pbs , and incubated with 7-aad for 15 min at room temperature . stained cells were analysed on a beckman coulter gallios using beckman coulter gallios acquisition software ( beckman coulter inc . supernatant samples were analysed for the levels of 26 different cytokines ( eotaxin , g - csf , gm - csf , ifn-2 , ifn- , il-10 , il-12 ( p40 ) , il-12 ( p70 ) , il-13 , il-15 , il-17 , il-1 , il-1 , il-2 , il-3 , il-4 , il-5 , il-6 , il-7 , il-8 , ip-10 , mcp-1 , mip-1 , mip-1 , tnf- , and tnf- ) . levels and changes in cell - surface markers and levels of soluble factors were compared between the non - gvhd and gvhd groups . there was no significant difference between the non - gvhd group and the gvhd group regarding frequencies of major lymphocyte populations , that is , total t - cells ( median 55.2% versus 56.6% ; p = 0.535 ) , nk - cells ( median 10.1% versus 11.6% ; p = 0.383 ) , or b - cells ( median 15.5% ( n = 6 ) versus 6.5% ; p = 0.295 ) ( figure 1(a ) ) . in order to examine the maturation status of t - cells in the grafts the distribution of the different memory subsets of total t - cells in the two groups is shown in figure 1(b ) . no statistically significant differences between the non - gvhd and the gvhd groups were found regarding frequencies of nave ( cd45roccr7 ; median 22.1% versus 43.3% ; p = 0.165 ) , central memory ( cd45roccr7 ; median 12.6% versus 8.7% ; p = 0.306 ) , effector memory ( cd45roccr7 ; median 35.9% versus 28% ; p = 0.259 ) , or terminally differentiated t - cells ( cd45roccr7 ; median 21.7% versus 18.3% ; p = 0.620 ) . additionally , no significant differences were seen between the two categories of donors when we analysed total frequencies of cd4 and cd8 t - cells or their individual maturation status . the non - ghvd group had a higher frequency of total tcr nave memory t - cells than the gvhd group ( median 4.5% versus 0.7% ; p = 0.004 ) ( figure 2(a ) ) . after activation , t - cells transiently express certain nk - cell markers . in this material , cd94 total t - cells were found in higher frequencies in the non - gvhd group for nave memory t - cells ( median 5.2% versus 1.2% ; p = 0.018 ; figure 2(a ) ) and for terminally differentiated t - cells ( median 33.7% versus 13.8% ; p = 0.018 ; figure 2(b ) ) as compared to the gvhd group . similarly , cd56 expression on cd4 t - cells was found at higher frequencies in the non - gvhd group ( median 1.6% versus 0.6% ; p = 0.030 ) ( figure 2(c ) ) . t - cells expressing the fas receptor cd95 were observed at significantly higher frequencies in the non - gvhd group in nave memory t - cells ( median 19.6% versus 8.55% ; p = 0.026 ; figure 2(a ) ) as compared to the gvhd group . to evaluate the alloreactive capacity of donor lymphocytes towards recipient cells , a mixed lymphocyte culture ( mlc ) was performed . by using a multicolor flow cytometry panel , we wanted to detect changes in frequencies of lymphocyte subpopulations of the responding cells and their surface expression of the activation markers cd69 and cd107a after mlc . as illustrated in figure 3(a ) , no differences in total t - cell percentages could be observed between the patient groups for all three mlc conditions . there was a change in the proportions of cd4 and cd8 t - cells after mlc when we compared the two patient groups . before the mlc , the cd4/cd8 ratio in both groups was comparable ( non - gvhd 1.44 versus gvhd 1.9 ; p = 0.165 ; figure 3(b ) ) . at day 6 , the cd4/cd8 ratio had changed in the 5 : 1 stimulated samples ( non - gvhd 1.02 versus gvhd 3.36 ; p = 0.052 ) and pha stimulated samples ( non - gvhd 0.30 versus gvhd 1.33 ; p = 0.038 ) . in both settings , the cd4/cd8 ratio skewed towards an increased proportion of cd4 t - cells in the gvhd group as compared to the non - gvhd group ( figure 3(b ) ) . the frequencies of t - cells positive for early activation marker cd69 were comparable between the two patient groups before mlc ( median non - gvhd 4% versus gvhd 6.3% ; p = 1.0 ) . however , expression of cd69 was higher for total t - cells in the non - gvhd group than in the ghvd group after an unstimulated 6-day incubation ( median 5.6% ( n = 4 ) versus 1.5% ( n = 6 ) ; p = 0.038 ) and after the 5 : 1 stimulated mlc condition ( median 5.1% ( n = 5 ) versus 1.4% ( n = 6 ) ; p = 0.009 ; figure 3(c ) ) . no difference was seen between the two patient groups in cd69 t - cell frequencies after 6-day incubation with pha . there was no significant difference in cytokine concentrations in the supernatant of all three conditions after mlc between the two patient groups ( data not shown ) . most studies on the effect of graft composition on outcome after hsct have focused on quantitative differences in doses of total nucleated cells , cd34cells , and t - cells . however , there have been reports indicating that frequencies of minor cell subsets and phenotypic distinctions within these populations could be connected to the alloreactive potential of the graft and may therefore affect clinical outcome [ 1825 ] . in our study we wanted to use multicolor flow cytometry to detect potential differences in frequencies of lymphocyte subsets in donor material before and after in vitro mlc in order to predict severe gvhd development . as clinicians and researchers in the field know that grade i acute gvhd can easily be misdiagnosed , we only included patients without any signs of gvhd or with grade ii our results illustrate that flow cytometry on graft material using the most common phenotypic markers is not enough to detect predictive markers for gvhd ( figure 1 ) . the biological functions of t - cells that express the t - cell receptor ( tcr ) are not fully understood , but existing evidence points towards both proinflammatory and suppressive functions as well as antigen - presenting and cytotoxic capacities [ 2628 ] . showed that recipients of grafts depleted of tcr t - cells had a higher incidence of acute gvhd than those transplanted with t - cell - depleted grafts . it has also been shown that infusion of activated t - cells , in the absence of regular t - cells , can promote engraftment without causing gvhd in a mhc - mismatch situation . other animal studies have shown contradictory results , where t - cells appeared to be associated with an increased risk of gvhd [ 30 , 31 ] . to date , only some clinical studies have addressed the issue of graft t - cell content in hsct . in a report that involved 63 recipients of unrelated peripheral blood stem cell grafts , pabst et al . found a correlation between high t - cell dose and higher incidence of acute gvhd of grades ii iv , while a more recent study has shown contradicting results in a smaller , more heterogeneous cohort of patients . in our material , we found a lower frequency of nave t - cells in the peripheral blood of donors corresponding to patients who later developed acute gvhd of grades ii the timing of transfer of the t - cells in relation to the allograft has varied between studies . secondly , if the t - cells exert their effect through direct or indirect interaction with other cell types , such as regulatory t - cells and t - cells [ 26 , 27 , 34 , 35 ] , differences in ratios between the infused doses of these cells might lead to different end - results . lastly , the phenotype of t - cell subsets in the graft , particularly their activation and maturation status , could affect their function and ability to survive in vivo [ 36 , 37 ] . other minor lymphocyte populations that have attracted attention in the context of hsct are t - cells expressing nk - cell markers [ 38 , 39 ] . these can be either naturally occurring invariant nkt - cells or in vitro activated cytokine - induced killer ( cik ) cells . invariant nkt - cells have in several studies been shown to be able to reduce the risk for gvhd , whether they were present in high numbers in the graft or given through adoptive transfer [ 24 , 44 ] . two independent clinical trials on the use of ex vivo expanded allogeneic cik - cells , as part of treatment for relapsed haematological malignancies after hsct , have shown response rates comparable to those for regular donor lymphocyte infusions ( dli ) , but with significantly lower incidence of gvhd [ 45 , 46 ] . in the present study , we found significantly lower levels of t - cells expressing the nk - cell markers cd56 ( on cd4 t - cells ) and cd94 ( on nave and terminally differentiated t - cells ) in samples from donors before mlc in the gvhd group ( figure 2 ) . if these cells correspond to previously described inkt - cells , our study confirms that inkt - cells may indeed have a gvhd - modulatory effect . unfortunately , as no nk - cell markers were included in the flow cytometry panel after mlc , it is difficult to speculate on cik - cells in this study . activation - induced cell death ( aicd ) is important in the regulation of t - cell responses and works as an inhibitory mechanism to prevent uncontrolled activation . in our material , staining of unmanipulated donor pbmcs for cd95 revealed that samples from the gvhd group contained lower proportions of nave t - cells that expressed this receptor on their surface ( figure 2(a ) ) . surface expression of cd95 is greatly increased on t - cells upon activation , while lower expression reflects a more nave phenotype . it is also in accordance with the widely accepted view that acute gvhd is induced by nave t - cells , most likely due to their ability to sustain a strong response [ 56 , 57 ] . different variations of the mlc have been used to assess the alloreactive capacity of donor cells in the setting of hla - identical hscts , but the correlation to clinical results has varied . the common principle for these methods is incubation of donor pbmcs with inactivated cells from the recipient followed by quantitative analysis of the helper t - lymphocyte precursor subset . multicolor flow cytometry is a more direct and specific approach for quantification and categorization of lymphocyte subpopulations after allogeneic mlc . by using this sensitive technique , we could detect a shift in cd4/cd8 ratio after mlc between the patient groups . in the 5 : 1 setting , where the cells were exposed to alloantigen presentation , we could observe a trend for an increased proportion of cd4 t - cells in the gvhd group as compared to the non - gvhd group . moreover , this increased proportion of cd4 t - cells in the gvhd group was also observed in the pha setting ( figure 3(b ) ) . it would therefore appear that , after stimulation , donor cells from the gvhd group tend to skew more to a cd4 t - cell phenotype than donor cells from the non - gvhd group . additionally , we could detect a significant correlation between a decrease in the frequency of cd69 t - cells and incidence of acute gvhd grades ii iv even in this small material ( figure 3(c ) ) . cd69 is traditionally seen as one of the earliest markers emerging after activation of t - cells , though it appears to also be highly expressed by lymphocytes at mucosal sites of the human body [ 62 , 63 ] . the counter - intuitive observation in the present study , that is , low frequency of activated t - cells correlating with gvhd , might be due to an increased downregulation of cd69 in alloreactive t - cells after 6 days of activation . thus , low levels of cd69 t - cells after mlc might serve as an indicator of increased alloreactivity and risk for incidence of acute gvhd . additionally , the flow cytometry panel could be expanded to include more cellular markers to identify frequencies of other cellular populations that might play important roles in this context , such as regulatory t - cells , myeloid - derived suppressor cells ( mdscs ) , and mucosal - associated - invariant t - cells ( maits ) . we found a positive correlation between lower frequencies of donor t - cells expressing tcr and nk - cell markers on the one hand and incidence of acute gvhd of grades ii we have also shown that a distinct relative decrease in cd69 t - cells in a flow cytometry - based allogeneic mlc can be predictive of acute gvhd . we recognise the fact that the sample groups in this pilot study are small and not perfectly matched for some clinical parameters , for example , diagnosis or conditioning regimen . nevertheless , the findings presented here may be of value in pretransplant risk assessment and they may enable the use of a more individualized prophylactic strategy .

growing concerns over the large energy requirements needed for effective wastewater treatment has stimulated interest in the use of wastewater as a source of renewable energy . microbial fuel cells ( mfcs ) are being developed as a sustainable energy technology , as they can directly produce electricity from wastewater allowing for energy recovery to offset the costs of wastewater treatment . in an air - cathode mfc , organic matter in wastewater is oxidized by microorganisms , and electrons discharged to the anode travel through an external circuit to the cathode where they combine with oxygen , forming water . passive transfer of oxygen to the air - cathode avoids the need for energy intensive aeration of the wastewater that is currently required for typical activated sludge or aerobic membrane bioreactor processes . in addition , mfcs have lower sludge production than conventional aerobic treatment processes , which could reduce treatment costs and the challenges associated with sludge treatment and disposal . mfcs fed with domestic wastewaters have shown promising performance in terms of achieving electricity generation with simultaneous organics removal , and there continue to be improvements in mfc designs that have produced configurations more suitable for scaling up to larger systems . capital costs of the materials used in mfcs are also being reduced , for example , by using cathode catalysts such as inexpensive activated carbon . one operational aspect of using mfcs for wastewater treatment that has not been sufficiently addressed is the need to meet stringent effluent quality requirements . effluent chemical oxygen demand ( cod ) concentrations with domestic wastewater in mfcs have ranged from 23 to 164 mg / l in fed - batch tests , and 60 to 220 mg / l in continuous flow tests , depending on influent cod concentrations , reactor configurations , and cycle time or hydraulic retention time ( hrt ) . one of the reasons for these high effluent cods is likely inefficient removal of particulate organics , as biofilm reactors such as mfcs and trickling filters are more effective for soluble than particulate cod removal . thus , post - treatment or integrated processes are needed to further improve the quality of the treated wastewater to meet discharge limits . one approach to improve the overall extent of wastewater treatment has been to integrate the mfc with a membrane - based process in a single reactor . this approach has been referred to either as a membrane bioelectrochemical reactor ( mber ) or an electrochemical membrane bioreactor ( embr ) . although higher treatment efficiencies have been obtained for both acetate solutions and domestic wastewater in tests with this approach , energy consumption has only been balanced with electrical energy production when acetate was used as the substrate . the main challenges with using both mfcs and membrane processes for domestic wastewater treatment are obtaining high power production from the mfcs , while minimizing membrane fouling . using a shorter hydraulic retention time ( hrt ) in an mfc treating domestic wastewater will usually improve power production , but a shorter hrt could mean a higher organic loading rate on the membrane process , which could result in increased membrane fouling . membrane fouling control remains the biggest challenge in the use of membranes in both aerobic and anaerobic systems . in previous membrane - based mfc studies , membranes inside the mfcs fouled in 15 days , and the high maintenance costs due to cleaning processes could limit applications of integrated mfc and membrane bioreactor processes . a new approach to obtain high quality effluent with low energy requirements is proposed here based on using a second stage anaerobic fluidized bed membrane bioreactor ( afmbr ) following wastewater treatment in the mfc . the afmbr has recently been shown to be an effective approach for achieving high quality effluent when used as a post - treatment method for an anaerobic fluidized bioreactor ( afbr ) . in the afmbr , membrane fouling is controlled by using granular activated carbon ( gac ) as the fluidized particles , as these particles can scour the membrane and minimize fouling . the properties of particles used in the fluidized bed are important , as spherical plastic particles have been shown to not be as effective as gac . the use of an mfc as the primary treatment process , as opposed to an afbr , may be useful for several reasons . electrical energy is directly produced in the mfc , whereas in the afbr electricity would have to be produced in a separate process from biogas that might need to be cleaned and purified to remove hydrogen sulfide and water to improve utilization efficiencies . any hydrogen sulfide generated in situ in an mfc would be expected to be rapidly oxidized in the mfc as it is a good electron donor to the anode . there should be very little methane in the mfc effluent compared to that produced by the afbr , as organic matter is mainly converted into current or lost to aerobic degradation due to oxygen transfer across the cathode . it is important to remove dissolved methane , which can be supersaturated in these systems , to minimize its release into the atmosphere as it is a potent greenhouse gas . in this study we examined domestic wastewater treatment using a two - stage mfc - afmbr system , containing four mfcs and one afmbr , at ambient temperature . there were two separate flow lines into the afmbr , with two mfcs connected hydraulically in series ( with separate electrical circuits ) in each flow line ( figure 1 ) . the use of two mfcs in series avoided large changes in cod concentrations in each mfc , as such large cod changes have previously been shown to adversely affect current generation . each pair of mfcs had a different electrode configuration in order to compare two design approaches : using a separator electrode assembly ( sea ) , where the electrodes are sandwiched together and a separator was placed between them to prevent short circuiting and reduce oxygen crossover from the cathode ; and using a spaced electrode assembly ( spa ) , where the electrodes are kept close to each other , but with sufficient space to avoid direct contact ( no separator was used ) ( figure s1 , supporting information ( si ) ) . it has recently been shown that the spa design can reduce treatment time compared to the sea , although less energy may be recovered in the spa configuration due to the loss of organic matter to aerobic processes rather than current generation . treatment efficiency was evaluated in terms of cod and total suspended solid ( tss ) removals , and energy efficiency was quantified for both processes in terms of production and demands , under continuous flow conditions . schematic diagram ( a ) and photo ( b ) of the two - stage mfc - afmbr system . ( u = the first upstream mfc , and d = the second downstream mfc prior to the afmbr ) . the two - stage mfc - afmbr system consisted of four mfc reactors and one afmbr reactor . the four mfc reactors were arranged in two groups , each group having two mfc reactors with the same electrode configuration that were hydraulically connected in series ( figure 1 ) . single - chamber , air - cathode mfcs ( 130 ml ) were constructed as previously described . each reactor contained three brush anodes connected together externally with copper wire , and a single air - cathode . the anodes were graphite fiber brushes with a titanium wire core ( 25 mm diameter by 35 mm length ) ( mill - rose , mentor , oh ) that were heat treated at 450 c for 30 min before use . the cathode ( 35 cm projected surface area ) was made of wet - proofed carbon cloth ( 30 wt.% , # cc640wp30 , fuel cell earth , stoneham , ma ) , with a platinum catalyst ( 0.5 mg / cm ) on the water side and four ptfe diffusion layers on the air side . for the sea mfcs , the brush anodes were trimmed in half along the direction parallel to the core to prevent contact by the bristles with the cathode through the separator , as both electrodes were pressed against the separator ( si figure s1 ) . two layers of textile separator ( 46% cellulose and 54% polyester , 0.3 mm thickness , amplitude prozorb , contec inc . , spartanburg , sc ) were used in the sea reactors to prevent short - circuiting and to minimize oxygen crossover . the spa mfcs did not contain separators , so the edges of the brush anodes were set 0.8 cm from the surface of the cathodes . the afmbr ( 65 ml ) consisted of a 300 mm long by 16 mm diameter clear polyvinyl chloride ( pvc ) tube ( u.s . plastic corp . ) containing 10 g ( wet weight ) of gac ( darco mrx , 10 30 mesh , norit activated carbon ) as the fluidized bed medium and support for bacterial growth . the gac was washed with deionized water for three times prior to use to remove any residuals . the afmbr contained a submerged membrane module with eight 200 mm long polyvinylidenefluoride ( pvdf ) hollow fiber membranes ( 2.0 mm outside diameter , 0.8 mm inside diameter , 0.1 m pore size , kolon inc . , south korea ) , having a total membrane surface area of 0.004 m. a hungate tube ( 10 ml , bellco glass inc . , vineland , nj ) with the bottom cut off was glued onto the top of the pvc tube and sealed with a thick butyl rubber stopper ( 20 mm diameter , chemglass inc . , vineland , nj ) , for biogas collection and measurement . the mfcs were inoculated and fed with domestic wastewater collected from the primary clarifier of the pennsylvania state university wastewater treatment plant , and operated in continuous flow mode at an hrt of 4 h. the primary clarifier effluent was collected weekly and stored in a refrigerator ( 4 c ) to minimize cod changes over time . during tests , a container of wastewater was placed in an ice bucket to keep it cool in order to minimize degradation prior to being fed into the mfcs . the wastewater warmed in the tube when it was transferred into the bottom of the reactors using a peristaltic pump ( model no . 7523 - 90 , masterflex , vernon hills , il ) at an overall flow rate of 1560 ml / d , with 780 ml / d for each flow line . the effluent from the top of the upstream mfc reactor flowed into the bottom of the downstream mfc due to the hydraulic pressure . the effluent from the two mfcs series was delivered to the afmbr using another peristaltic pump ( as above ) at a flow rate of 1560 ml / d , producing an hrt of 1 h. the top of the membrane module was connected to the same peristaltic pump , to maintain a constant permeate flux of 16 l / m / h ( lmh ) from the afmbr . the pump was operated with a 10 min on and 1 min off cycle time , as it was previously shown that periodic relaxation of the membrane reduced trans - membrane pressure ( tmp ) . tmp was monitored continuously using a vacuum pressure gauge ( type1490 , ashcroft , stratford , ct ) . fluidization of gac was maintained with a peristaltic pump ( model no . 7523 - 80 masterflex , vernon hills , il ) at the desired flow rate of 170 ml / min , resulting in bed expansion of 7080% to a height of 210240 mm . the two - stage mfc - afmbr system was operated at room temperature ( 25 c ) . the voltage across an external resistor for the mfc circuit was measured every 20 min using a multimeter ( model 2700 ; keithley instruments , inc . ) . current was calculated using ohm s law ( i = u / r ) , with power calculated as p = iu , where u is the measured voltage ( v ) , and r the external resistance ( ) . a reference electrode [ ag / agcl ; + 200 mv vs standard hydrogen electrode ( she ) ; basi ] was inserted into the upper middle of the mfc reactor to determine the anode and cathode potentials . polarization and power curves were obtained by changing the external resistances from open circuit to 1600 , 800 , 400 , 200 , and 100 , with one day at each resistance ( six hrts ) . the averaged voltage at each external resistance was used to obtain the polarization curve . columbic efficiency ( ce ) was calculated as ce = ct / cth 100% , where ct was the total coulombs calculated by integrating the current over time ( ct = i t , where t is the time interval of one hrt ) , and cth was the theoretical amount of coulombs available based on the cod removed in the mfc over the same amount of time , calculated as cth = [ fb ( codin codout ) q t]/m , where f is faraday s constant , b = 4 is the number of electrons exchanged per mole of oxygen , codin and codout are the influent and effluent cod , q is the flow rate , t is the time interval ( hrt ) , and m = 32 is the molecular weight of oxygen . total suspended solids ( tss ) were measured using standard methods ( apha , 1998 ) . total cod ( tcod ) and soluble cod ( scod ) were measured using standard methods ( method 5220 , hach company , loveland , co ) . all samples for scod measurement were filtered through 0.45 m pore diameter syringe filters ( polyvinylidenedifluoride , pvdf , 25 mm size , restek corporation ) . conductivity and ph were measured immediately after sampling using a probe ( sevenmulti , mettler - toledo international inc . ) . the sampling points on the flow line for the chemical analyses were indicated in the si ( figure s2 ) . biogas ( 200 l samples ) of the afmbr headspace was sampled using gastight syringes ( 250 l ; hamilton samplelock syringe ) and analyzed using two gas chromatographs ( sri instruments ) for h2 , n2 , ch4 and co2 , as described previously . gas was collected and measured directly using a 10 ml glass syringe ( air - tite products co. , inc . , va ) inserted into the top of the afmbr . dissolved methane was also measured as described previously by transferring a liquid sample from the afmbr reactor into a serum bottle ( 6.5 ml , wheaton , millville , nj ) without any air contact or headspace , and sealed with a thick butyl rubber stopper ( 20 mm diameter , chemglass inc . ) . the serum bottle full of the liquid sample was then autoclaved to prevent biological activity . some liquid sample ( 1.5 ml ) was then replaced by n2 gas from this serum bottle with a syringe . after establishing gas liquid equilibrium by shaking the serum bottle for six hours at room temperature , the amount of dissolved methane was back - calculated from the measured methane amount in the headspace ( detailed information in the si ) . the start - up time needed for the sea mfcs was shorter than that required for the spas . the sea mfcs produced a stable voltage of 0.59 0.03 v ( 1000 ) after 3 days , while the spas produced 0.51 0.04 v after 3 days , and required 10 days to achieve a stable voltage of 0.58 0.01 v. stable voltage production was indicated by a deviation between the daily averaged voltage values that was < 0.006 v ( 1% of the daily averaged voltage ) over three consecutive days . there was no appreciable difference in start - up time between the upstream or downstream mfc within the individual flow paths ( data not shown ) . the power produced by the seas and spas changed over time . based on the polarization data obtained after 1 month , the sea - u mfc produced a maximum power of 0.31 mw ( 89 mw / m , normalized to the cathode projected surface area of 35 cm ) , which was comparable to that of spa - u ( 0.33 mw ) ( figure 2 ) . although the same current was produced with these two configurations , the sea - u had better cathode performance but showed poorer anode performance than the spa - u ( figure 3b and c ) . the downstream mfcs produced slightly lower maximum power than the upstream ones , with 0.28 mw for sea - d and 0.27 mw for spa - d ( figure 2 ) . the downstream mfcs generally had more positive anode potentials than the first mfcs ( figure 3b ) , likely due to the lower substrate concentrations in the downstream mfcs ( si table s1 ) , as it was shown that the anode potentials became more positive at lower substrate concentrations in a previous study . power production of the sea and spa mfcs at different time after start - up , after ( a ) 1 month and ( b ) 5 months . ( u = the first upstream mfc , and d = the second downstream mfc prior to the afmbr ) . voltage , anode potential and cathode potential of the sea and spa mfcs at different time after start - up : ( a ) voltage , ( b ) anode potential and ( c ) cathode potential at 1 month , and ( d ) voltage , ( e ) anode potential and ( f ) cathode potential at 5 month . the letters a in all electrode potentials were reported versus the ag / agcl reference electrode [ + 200 mv vs a standard hydrogen electrode ( she ) ; basi ] . after 5 months , the maximum power densities of the sea mfcs were relatively unchanged ( 0.33 mw for sea - u and 0.32 mw for sea - d ) , and the wastewater composition fed into the reactor was relatively unchanged based on the influent tcod concentrations ( 210 11 mg / l at 5 months , compared to 224 17 mg / l at 1 month ) . however , the maximum power produced by spa mfcs substantially decreased to 0.16 mw ( spa - u ) and 0.18 mw ( spa - d ) . the reason for these decreases was a large reduction in cathode potentials ( figure 3f ) , which was likely due to biofouling . while the cathodes used for the sea configuration contained a separator that covered the cathode , the spa cathodes were directly exposed to the wastewater , and thus they were more prone to fouling ( figure 3f ) . the maximum power density of 89 6 mw / m produced by the sea mfc in these continuous flow tests was lower than the maximum power densities obtained in two other studies with domestic wastewater when the mfc was operated in fed - batch mode ( 120 mw / m or 328 11 mw / m ) . the lower power density here was likely due to a lower influent cod ( 217 18 mg / l , compared to 275 71 mg / l and 303 69 mg / l ) , and operation under continuous flow conditions , where the average substrate concentration was lower than that in the fed - batch reactors at the beginning of the cycle . the different electrode configurations ( sea or spa ) did not appreciably affect the extent of cod removal . tcod removals were 28 7% for sea - u , and 34 3% for spa - u , which are comparable removals within the calculated standard deviations . the downstream mfcs had slightly lower tcod removals than the upstream mfcs , with 17 5% for sea - d and 19 fed - batch tests with domestic wastewater have shown that cod removal in mfcs is first order with respect to concentration ( unpublished data ) . thus , the reduction in cod concentration would have reduced removal rates in the downstream reactors . scod removals showed the same trends as tcod , with greater removals in the upstream reactors ( 27 10% for sea - u , 32 5% for spa - u ) than the downstream ones ( 19 5% for sea - d , and 26 7% for spa - d ) . note that these cod removals were based on the combination of all data in tests at the different external resistances used in the polarization tests , as the effluent cod concentrations in these tests did not change substantially with the different external resistances ( cod concentrations in the si , table s1 ) . these cod removals were lower than those obtained in previous studies operated in fed - batch mode using the same domestic wastewater source ( 6293% ) , due to the short hrt ( 4 h ) in this study compared to much longer fed - batch cycle times ( 1236 h ) . in additional tests , increasing the hrt to 24 h increased cod removals to 67 2% , which was about the same as that obtained in fed - batch mode with a cycle length of 24 h ( 65 1% ; 500 resistance , data not shown ) . however , a long hrt is not desirable for efficient wastewater treatment , and thus the shorter hrt was used here . the ces increased in proportion to the current ( lower resistance ) , even though the cod removals remained relatively constant with different resistances . at the maximum power density ( 0.31 0.02 mw , 0.86 0.02 ma ) , the overall ce of the sea mfcs was 18% ( 13% for sea - u and 28% for sea - d ) . over time the sea and spa mfcs had comparable overall ces at 1 month , ( range of 629% ) , but after 5 months the sea mfcs remained relatively unchanged while those of the spa mfcs decreased ( range of 420% ) with the decreased currents . ce values obtained here under continuous flow conditions were comparable to those previously reported for fed - batch conditions ( 231% ) . overall , these results suggest that the sea configuration was superior to the spa design on the basis of fixed hrts as it maintained higher power densities and ces over time with the same level of wastewater treatment . the two - stage mfc - afmbr system achieved excellent treatment levels in terms of cod and tss removals . the afmbr was first inoculated with anaerobic sludge and fed the effluent from mfcs for two weeks . after that , the membrane module was installed , and preliminary tests were conducted to optimize the design and operation of the afmbr over a period of approximately two months , with occasional system shutdown to address problems related to consistent flow and treatment . following this system optimization , a new membrane module was installed , and the mfc - afmbr system was operated continuously for 50 days in concert with the mfcs operation over the last two of the five months . tcod further decreased from the influent concentration to the afmbr of 107 10 mg / l to 16 3 mg / l in the effluent , providing an overall tcod removal for the two stages of 92.5% ( 49.1% for the mfcs , and 43.4% for the afmbr ) ( figure 4 and si table s2 ) . the effluent scod and tcod concentrations from the afmbr were identical , and therefore there was a lower overall scod removal of 86.2% ( influent scod to mfcs of 114 8 mg / l , compared to tcod of 210 11 mg / l ) ( figure 4 and si table s2 ) . a larger percent of scod was removed by the mfcs ( 50.3% ) than by the afmbr ( 35.9% ) , while particulate cod removal was 47.9% for the mfcs compared to 100% for the afmbr . changes in the forms of cod might occur through hydrolysis from particulate to soluble cod , and through biomass growth from soluble to particulate . the particulate cod removal in mfcs might be partially due to settling in the reactor chambers , while that in the afmbr was primarily due to membrane filtration . l of tss due to filtration of the wastewater through the membrane , resulting in > 99.6% tss removal ( figure 4 and si table s2 ) . these cod and tss removals are comparable to those obtained using a staged anaerobic fluidized membrane bioreactor ( saf - mbr ) treating domestic wastewater . there was little overall change in ph , as the influent ph to the mfcs of 7.6 0.1 decreased to 7.1 0.1 in the mfcs effluent , but it increased to 7.5 0.2 following treatment in the afmbr ( si table s2 ) . these ph changes might result from losses of co2 and volatile fatty acids , for example from methanogenesis processes occurring in the mfc - afmbr system . also there were no large changes in conductivity , with 1473 33 ms / cm for the mfcs influent , 1457 15 ms / cm for the mfcs effluent , and 1420 19 ms / cm for the afmbr effluent ( si table s2 ) . influent and effluent concentrations , and removals of tcod , scod and tss for the combined mfc - afmbr system . the values inside the figures were the percent of the influent concentration that was removed by the mfcs , afmbr , and the whole system . the afmbr was operated continuously for 50 days at a high membrane flux of 16 lmh , even without cleaning by backwashing or using chemicals . most of the increase in the tmp , from 0.015 to 0.035 bar , occurred during the first 8 days of operation ( figure 5 ) . thereafter , it slowly increased to 0.050 bar during the rest of the test ( figure 5 ) . liquid ( 9 ml ) was withdrawn from the afmbr every 3.5 days ( 0.16% of the total influent flow ) to removal finer material and excess suspended solids from days 8 to 50 , as suggested in a previous afmbr study . the membrane flux of 16 lmh here is higher than that previously reported for the afmbr following an afbr ( 11 lmh ) , with a pvdf hollow - fiber membrane with the same pore size as the one here ( 0.1 m ) . in that study , the tmp reached 0.25 bar in 3 days when the membrane flux was increased to 14 lmh , which is much higher than the maximum tmp observed here . the stable operation of the flux through the afmbr without appreciable membrane fouling was likely due to a combination of factors here that included the scouring effect of the gac particles on the membrane surface , intermittent filtration , and periodic removal of suspended solids . the use of mfcs as the primary treatment process likely contributed to the high flux and stable performance of the afmbr , due to the removals of cod and tss in the mfcs . the improved flux with the first - stage mfc treatment , compared to that previously obtained with a first - stage afbr treatment , suggests that mfcs might be a better first stage treatment than afbr , but this can not be concluded without direct side - by - side tests of the two different systems . the operation of the afmbr without wastewater pretreatment was not examined here as that would represent a different treatment process , and one that would not allow for electrical power generation and recovery from cod removal . energy usage for the two - stage mfc - afmbr system was calculated as previously described . all the volumetric energy densities were reported on the basis of normalizing to 1 m of wastewater treated . the energy requirements were calculated as 0.0107 kwh / m for fluidizing the gac particles , and 0.0014 kwh / m for pumping permeate through the membranes , resulting in a total electrical energy requirement for the afmbr of 0.0186 kwh / m ( table 1 ) . the electrical energy requirement for pumping liquid through the mfcs was negligible compared to that needed for the afmbr ( table 1 ) . the higher energy requirement for the afmbr was primarily due to pumping needed for liquid recirculation to maintain the gac fluidization . the energy needed for this is proportional to the total reactor flow rate and the hydraulic head loss of the system . in an afmbr reactor with a given configuration , the minimum recirculation flow rate and the hydraulic head loss are fixed , and thus the energy requirement for recirculation is inversely proportional to the permeate flow rate or the hrt . therefore , the high permeate flux and low hrt achieved for the afmbr in this study were favorable for achieving a low energy requirement of 0.0186 kwh / m . this energy requirement is lower than previous reports using the afmbr ( 0.0270.040 kwh / m ) , but there are many differences in these studies that preclude a direct comparison of these values . energy requirement = 9.8qe , where q ( m / s ) is flow rate and e ( m h2o ) is head loss . assume energy efficiency of 65% in conversion of electrical energy to pump energy . this maximum power output was quite similar to that obtained during steady operation , and therefore it represents power production that could be obtained during continuous treatment tests ( si figure s3 ) . the ratio of the electrical energy produced to that required by the mfc - afmbr system . based on the maximum power that could be produced by the sea configuration after 5 months of operation ( 0.33 mw for sea - u and 0.32 mw for sea - d , figure 2 ) , the total power that could be produced by the four mfcs coupled to the afmbr was 1.28 mw ( four times 0.32 mw ) ( table 1 ) . if all of this electrical energy was recovered , the net electrical energy available for the system operation would be 0.0197 kwh / m . this would be enough to supply the 0.0186 kwh / m required to operate the system if these values could be maintained for larger - scale systems . however , this energy balance would likely change as the size of the system increases . also , in practice there might be other energy losses that would affect overall energy recovery , that have not been included here . direct electricity production by mfcs is advantageous , compared to methanogenic reactors that require combustion of the methane to produce power , as the conversion efficiency of methane to electricity is typically only 33% . however , there will also be energy losses in converting the low voltage dc power into higher voltage dc or ac power . the additional energy that could be recovered from the methane production in the afmbr was not included in this energy balance as it would have been difficult to recover . total methane production in the afmbr was 1.67 ml / l liquid treated at ambient temperature and pressure , with most of this present as dissolved methane ( 1.5 this concentration was estimated to be 125% oversaturation relative to the concentration of methane in the afmbr headspace . the energy value of this amount of methane is 0.016 kwh / m ( methane combustion , assuming 800 kj / mol ) , equivalent to electrical energy of 0.005 kwh / m ( 33% energy recovery ) which could theoretically add 27% more energy production into the system . however , as most of this methane is dissolved , and at a low concentration , it would have been difficult to recover . the methane yield from the afmbr was 0.75 mmol / g cod removed , indicating only 5% of the cod removal in the afmbr could be attributed to methane generation . the amount of methane that theoretically could be produced in the afmbr was estimated as 17 ml / l , based on the cod removal and assuming a conversion of 0.017 mol ch4/g cod ( detailed information and calculations in the si ) . it is not clear what other processes occurred relative to cod removal as the methane production was only about /10 of that possible by methanogenesis alone . more methane might be recovered in the future with improvements in the configuration and operation of the afmbr . the two - stage mfc - afmbr system was shown to effectively treat domestic wastewater ( primary clarifier effluent ) at ambient temperatures in terms of cod and tss removals , producing a high effluent quality with a near neutral ( or net positive ) energy requirement , without the need for membrane cleaning even after 50 days of operation . tcod was reduced from 210 11 mg / l to 16 3 mg / l , resulting in 92.5% overall cod removal with > 99% removal of tss to a final effluent concentration of < 1 mg / l . the energy requirement of the afmbr is much less than that needed for aerobic mbrs with internal membranes that require air sparging to control membrane fouling . the high permeate flux ( 16 lmh ) and low hrt ( 1 h ) here resulted in an overall low energy requirement for the afmbr of only 0.0186 kwh / m . thus , the energy produced by only the mfcs ( 0.0197 kwh / m ) was theoretically sufficient here to meet the energy demands for the system operation , although the energy balance for a larger system would likely change . an additional benefit of the mfc - afmbr system should be a low sludge production rate . although sludge production was not measured here ( for an estimate , see the si ) , previous studies have shown that the sludge production by anaerobic mfc and afmbr processes are less than those of conventional aerobic processes such as activated sludge . while feasibility of the combined process was shown here , additional work will be needed to optimize the performance of the individual and combined mfc and afmbr reactors . more optimal treatment could likely be obtained by adjusting the hrts of the two systems , and examining benefits of treatment rates compared to electrical power production . the current findings were sufficient to show that the two - stage mfc - afmbr system is useful for treatment of low strength wastewater even at ambient temperatures . the robustness of the system at other temperatures , particularly lower ones , would need to be investigated . however , mfcs have been shown to function over a wide range of temperatures . the assessment of nitrogen , phosphorus , and pathogen removals will be examined in future studies to see to what extent the mfc - afmbr system can be used for these wastewater components . other issues to address are capture and use(or destruction ) of the methane to avoid its release into the air , and efficient use of the electricity produced by mfcs . following these optimization studies , it should be possible to better evaluate the economics of the system compared to traditional treatment systems .

microbial fuel cells ( mfcs ) are a promising technology for energy - efficient domestic wastewater treatment , but the effluent quality has typically not been sufficient for discharge without further treatment . a two - stage laboratory - scale combined treatment process , consisting of microbial fuel cells and an anaerobic fluidized bed membrane bioreactor ( mfc - afmbr ) , was examined here to produce high quality effluent with minimal energy demands . the combined system was operated continuously for 50 days at room temperature ( 25 c ) with domestic wastewater having a total chemical oxygen demand ( tcod ) of 210 11 mg / l . at a combined hydraulic retention time ( hrt ) for both processes of 9 h , the effluent tcod was reduced to 16 3 mg / l ( 92.5% removal ) , and there was nearly complete removal of total suspended solids ( tss ; from 45 10 mg / l to < 1 mg / l ) . the afmbr was operated at a constant high permeate flux of 16 l / m2/h over 50 days , without the need or use of any membrane cleaning or backwashing . total electrical energy required for the operation of the mfc - afmbr system was 0.0186 kwh / m3 , which was slightly less than the electrical energy produced by the mfcs ( 0.0197 kwh / m3 ) . the energy in the methane produced in the afmbr was comparatively negligible ( 0.005 kwh / m3 ) . these results show that a combined mfc - afmbr system could be used to effectively treat domestic primary effluent at ambient temperatures , producing high effluent quality with low energy requirements .

Introduction Materials and Methods Results and Discussion

growing concerns over the large energy requirements needed for effective wastewater treatment has stimulated interest in the use of wastewater as a source of renewable energy . microbial fuel cells ( mfcs ) are being developed as a sustainable energy technology , as they can directly produce electricity from wastewater allowing for energy recovery to offset the costs of wastewater treatment . passive transfer of oxygen to the air - cathode avoids the need for energy intensive aeration of the wastewater that is currently required for typical activated sludge or aerobic membrane bioreactor processes . one operational aspect of using mfcs for wastewater treatment that has not been sufficiently addressed is the need to meet stringent effluent quality requirements . effluent chemical oxygen demand ( cod ) concentrations with domestic wastewater in mfcs have ranged from 23 to 164 mg / l in fed - batch tests , and 60 to 220 mg / l in continuous flow tests , depending on influent cod concentrations , reactor configurations , and cycle time or hydraulic retention time ( hrt ) . the main challenges with using both mfcs and membrane processes for domestic wastewater treatment are obtaining high power production from the mfcs , while minimizing membrane fouling . using a shorter hydraulic retention time ( hrt ) in an mfc treating domestic wastewater will usually improve power production , but a shorter hrt could mean a higher organic loading rate on the membrane process , which could result in increased membrane fouling . in previous membrane - based mfc studies , membranes inside the mfcs fouled in 15 days , and the high maintenance costs due to cleaning processes could limit applications of integrated mfc and membrane bioreactor processes . a new approach to obtain high quality effluent with low energy requirements is proposed here based on using a second stage anaerobic fluidized bed membrane bioreactor ( afmbr ) following wastewater treatment in the mfc . the afmbr has recently been shown to be an effective approach for achieving high quality effluent when used as a post - treatment method for an anaerobic fluidized bioreactor ( afbr ) . the use of an mfc as the primary treatment process , as opposed to an afbr , may be useful for several reasons . electrical energy is directly produced in the mfc , whereas in the afbr electricity would have to be produced in a separate process from biogas that might need to be cleaned and purified to remove hydrogen sulfide and water to improve utilization efficiencies . there should be very little methane in the mfc effluent compared to that produced by the afbr , as organic matter is mainly converted into current or lost to aerobic degradation due to oxygen transfer across the cathode . in this study we examined domestic wastewater treatment using a two - stage mfc - afmbr system , containing four mfcs and one afmbr , at ambient temperature . treatment efficiency was evaluated in terms of cod and total suspended solid ( tss ) removals , and energy efficiency was quantified for both processes in terms of production and demands , under continuous flow conditions . schematic diagram ( a ) and photo ( b ) of the two - stage mfc - afmbr system . the two - stage mfc - afmbr system consisted of four mfc reactors and one afmbr reactor . the mfcs were inoculated and fed with domestic wastewater collected from the primary clarifier of the pennsylvania state university wastewater treatment plant , and operated in continuous flow mode at an hrt of 4 h. the primary clarifier effluent was collected weekly and stored in a refrigerator ( 4 c ) to minimize cod changes over time . the effluent from the two mfcs series was delivered to the afmbr using another peristaltic pump ( as above ) at a flow rate of 1560 ml / d , producing an hrt of 1 h. the top of the membrane module was connected to the same peristaltic pump , to maintain a constant permeate flux of 16 l / m / h ( lmh ) from the afmbr . the two - stage mfc - afmbr system was operated at room temperature ( 25 c ) . columbic efficiency ( ce ) was calculated as ce = ct / cth 100% , where ct was the total coulombs calculated by integrating the current over time ( ct = i t , where t is the time interval of one hrt ) , and cth was the theoretical amount of coulombs available based on the cod removed in the mfc over the same amount of time , calculated as cth = [ fb ( codin codout ) q t]/m , where f is faraday s constant , b = 4 is the number of electrons exchanged per mole of oxygen , codin and codout are the influent and effluent cod , q is the flow rate , t is the time interval ( hrt ) , and m = 32 is the molecular weight of oxygen . total suspended solids ( tss ) were measured using standard methods ( apha , 1998 ) . biogas ( 200 l samples ) of the afmbr headspace was sampled using gastight syringes ( 250 l ; hamilton samplelock syringe ) and analyzed using two gas chromatographs ( sri instruments ) for h2 , n2 , ch4 and co2 , as described previously . after establishing gas liquid equilibrium by shaking the serum bottle for six hours at room temperature , the amount of dissolved methane was back - calculated from the measured methane amount in the headspace ( detailed information in the si ) . the sea mfcs produced a stable voltage of 0.59 0.03 v ( 1000 ) after 3 days , while the spas produced 0.51 0.04 v after 3 days , and required 10 days to achieve a stable voltage of 0.58 0.01 v. stable voltage production was indicated by a deviation between the daily averaged voltage values that was < 0.006 v ( 1% of the daily averaged voltage ) over three consecutive days . based on the polarization data obtained after 1 month , the sea - u mfc produced a maximum power of 0.31 mw ( 89 mw / m , normalized to the cathode projected surface area of 35 cm ) , which was comparable to that of spa - u ( 0.33 mw ) ( figure 2 ) . the downstream mfcs generally had more positive anode potentials than the first mfcs ( figure 3b ) , likely due to the lower substrate concentrations in the downstream mfcs ( si table s1 ) , as it was shown that the anode potentials became more positive at lower substrate concentrations in a previous study . voltage , anode potential and cathode potential of the sea and spa mfcs at different time after start - up : ( a ) voltage , ( b ) anode potential and ( c ) cathode potential at 1 month , and ( d ) voltage , ( e ) anode potential and ( f ) cathode potential at 5 month . after 5 months , the maximum power densities of the sea mfcs were relatively unchanged ( 0.33 mw for sea - u and 0.32 mw for sea - d ) , and the wastewater composition fed into the reactor was relatively unchanged based on the influent tcod concentrations ( 210 11 mg / l at 5 months , compared to 224 17 mg / l at 1 month ) . the reason for these decreases was a large reduction in cathode potentials ( figure 3f ) , which was likely due to biofouling . the maximum power density of 89 6 mw / m produced by the sea mfc in these continuous flow tests was lower than the maximum power densities obtained in two other studies with domestic wastewater when the mfc was operated in fed - batch mode ( 120 mw / m or 328 11 mw / m ) . the lower power density here was likely due to a lower influent cod ( 217 18 mg / l , compared to 275 71 mg / l and 303 69 mg / l ) , and operation under continuous flow conditions , where the average substrate concentration was lower than that in the fed - batch reactors at the beginning of the cycle . at the maximum power density ( 0.31 0.02 mw , 0.86 0.02 ma ) , the overall ce of the sea mfcs was 18% ( 13% for sea - u and 28% for sea - d ) . over time the sea and spa mfcs had comparable overall ces at 1 month , ( range of 629% ) , but after 5 months the sea mfcs remained relatively unchanged while those of the spa mfcs decreased ( range of 420% ) with the decreased currents . the two - stage mfc - afmbr system achieved excellent treatment levels in terms of cod and tss removals . after that , the membrane module was installed , and preliminary tests were conducted to optimize the design and operation of the afmbr over a period of approximately two months , with occasional system shutdown to address problems related to consistent flow and treatment . following this system optimization , a new membrane module was installed , and the mfc - afmbr system was operated continuously for 50 days in concert with the mfcs operation over the last two of the five months . tcod further decreased from the influent concentration to the afmbr of 107 10 mg / l to 16 3 mg / l in the effluent , providing an overall tcod removal for the two stages of 92.5% ( 49.1% for the mfcs , and 43.4% for the afmbr ) ( figure 4 and si table s2 ) . the effluent scod and tcod concentrations from the afmbr were identical , and therefore there was a lower overall scod removal of 86.2% ( influent scod to mfcs of 114 8 mg / l , compared to tcod of 210 11 mg / l ) ( figure 4 and si table s2 ) . a larger percent of scod was removed by the mfcs ( 50.3% ) than by the afmbr ( 35.9% ) , while particulate cod removal was 47.9% for the mfcs compared to 100% for the afmbr . these cod and tss removals are comparable to those obtained using a staged anaerobic fluidized membrane bioreactor ( saf - mbr ) treating domestic wastewater . there was little overall change in ph , as the influent ph to the mfcs of 7.6 0.1 decreased to 7.1 0.1 in the mfcs effluent , but it increased to 7.5 0.2 following treatment in the afmbr ( si table s2 ) . these ph changes might result from losses of co2 and volatile fatty acids , for example from methanogenesis processes occurring in the mfc - afmbr system . also there were no large changes in conductivity , with 1473 33 ms / cm for the mfcs influent , 1457 15 ms / cm for the mfcs effluent , and 1420 19 ms / cm for the afmbr effluent ( si table s2 ) . influent and effluent concentrations , and removals of tcod , scod and tss for the combined mfc - afmbr system . the values inside the figures were the percent of the influent concentration that was removed by the mfcs , afmbr , and the whole system . the afmbr was operated continuously for 50 days at a high membrane flux of 16 lmh , even without cleaning by backwashing or using chemicals . liquid ( 9 ml ) was withdrawn from the afmbr every 3.5 days ( 0.16% of the total influent flow ) to removal finer material and excess suspended solids from days 8 to 50 , as suggested in a previous afmbr study . the membrane flux of 16 lmh here is higher than that previously reported for the afmbr following an afbr ( 11 lmh ) , with a pvdf hollow - fiber membrane with the same pore size as the one here ( 0.1 m ) . the stable operation of the flux through the afmbr without appreciable membrane fouling was likely due to a combination of factors here that included the scouring effect of the gac particles on the membrane surface , intermittent filtration , and periodic removal of suspended solids . the use of mfcs as the primary treatment process likely contributed to the high flux and stable performance of the afmbr , due to the removals of cod and tss in the mfcs . the improved flux with the first - stage mfc treatment , compared to that previously obtained with a first - stage afbr treatment , suggests that mfcs might be a better first stage treatment than afbr , but this can not be concluded without direct side - by - side tests of the two different systems . the operation of the afmbr without wastewater pretreatment was not examined here as that would represent a different treatment process , and one that would not allow for electrical power generation and recovery from cod removal . energy usage for the two - stage mfc - afmbr system was calculated as previously described . the energy requirements were calculated as 0.0107 kwh / m for fluidizing the gac particles , and 0.0014 kwh / m for pumping permeate through the membranes , resulting in a total electrical energy requirement for the afmbr of 0.0186 kwh / m ( table 1 ) . the electrical energy requirement for pumping liquid through the mfcs was negligible compared to that needed for the afmbr ( table 1 ) . therefore , the high permeate flux and low hrt achieved for the afmbr in this study were favorable for achieving a low energy requirement of 0.0186 kwh / m . this energy requirement is lower than previous reports using the afmbr ( 0.0270.040 kwh / m ) , but there are many differences in these studies that preclude a direct comparison of these values . the ratio of the electrical energy produced to that required by the mfc - afmbr system . based on the maximum power that could be produced by the sea configuration after 5 months of operation ( 0.33 mw for sea - u and 0.32 mw for sea - d , figure 2 ) , the total power that could be produced by the four mfcs coupled to the afmbr was 1.28 mw ( four times 0.32 mw ) ( table 1 ) . if all of this electrical energy was recovered , the net electrical energy available for the system operation would be 0.0197 kwh / m . this would be enough to supply the 0.0186 kwh / m required to operate the system if these values could be maintained for larger - scale systems . the additional energy that could be recovered from the methane production in the afmbr was not included in this energy balance as it would have been difficult to recover . total methane production in the afmbr was 1.67 ml / l liquid treated at ambient temperature and pressure , with most of this present as dissolved methane ( 1.5 this concentration was estimated to be 125% oversaturation relative to the concentration of methane in the afmbr headspace . the energy value of this amount of methane is 0.016 kwh / m ( methane combustion , assuming 800 kj / mol ) , equivalent to electrical energy of 0.005 kwh / m ( 33% energy recovery ) which could theoretically add 27% more energy production into the system . the methane yield from the afmbr was 0.75 mmol / g cod removed , indicating only 5% of the cod removal in the afmbr could be attributed to methane generation . the amount of methane that theoretically could be produced in the afmbr was estimated as 17 ml / l , based on the cod removal and assuming a conversion of 0.017 mol ch4/g cod ( detailed information and calculations in the si ) . more methane might be recovered in the future with improvements in the configuration and operation of the afmbr . the two - stage mfc - afmbr system was shown to effectively treat domestic wastewater ( primary clarifier effluent ) at ambient temperatures in terms of cod and tss removals , producing a high effluent quality with a near neutral ( or net positive ) energy requirement , without the need for membrane cleaning even after 50 days of operation . tcod was reduced from 210 11 mg / l to 16 3 mg / l , resulting in 92.5% overall cod removal with > 99% removal of tss to a final effluent concentration of < 1 mg / l . the energy requirement of the afmbr is much less than that needed for aerobic mbrs with internal membranes that require air sparging to control membrane fouling . the high permeate flux ( 16 lmh ) and low hrt ( 1 h ) here resulted in an overall low energy requirement for the afmbr of only 0.0186 kwh / m . thus , the energy produced by only the mfcs ( 0.0197 kwh / m ) was theoretically sufficient here to meet the energy demands for the system operation , although the energy balance for a larger system would likely change . an additional benefit of the mfc - afmbr system should be a low sludge production rate . the current findings were sufficient to show that the two - stage mfc - afmbr system is useful for treatment of low strength wastewater even at ambient temperatures . the assessment of nitrogen , phosphorus , and pathogen removals will be examined in future studies to see to what extent the mfc - afmbr system can be used for these wastewater components . other issues to address are capture and use(or destruction ) of the methane to avoid its release into the air , and efficient use of the electricity produced by mfcs .

the transition from student to working nurse has long been recognized as difficult and has attracted the attention of researchers for decades [ 15 ] . various studies have shown that new graduates are inadequately prepared for their role as nurses , and they are ineffectually oriented to the work place [ 68 ] . the nursing discipline and nurse education are currently experiencing the compound pressure of expectations , which may lead to deteriorating conditions for nursing practice . the clinical field , under increasing pressure to be profitable , efficient , and effective , is demanding education programmes that produce graduate nurses who are work ready from day one . professional discussions of the deteriorating conditions for nursing as a caring profession have also cited the apparent movement among nurses away from both a wish for human contact and a willingness to comfort the sick and suffering for its own sake [ 10 , 11 ] . such studies indicate that the conditions for nursing remain challenging in terms of increased pressure in the work environment and new nurses ' readiness and willingness to care for patients . this situation inspired us to investigate empirically how new nurses are taking on the nursing role in hospitals . until the 1980s , nurse education in many western countries was based predominantly on an apprenticeship model , in which student nurses were salaried members of the nursing work force in hospitals [ 1214 ] . the three - year programme in schools of nursing had no formal academic recognition . in the early 1990s , major reforms in many countries redefined both the process and the product of nurse education . the aim was to equip student nurses with the necessary knowledge and skills to assume the role of graduates , and to achieve academic recognition [ 6 , 15 , 16 ] . efforts to improve the quality of study programmes and subsequent nursing practice led to a shift towards research - based teaching . however , this drift towards academia seems to have gone too far , resulting in complaints about weak practical skills and the lack of caring abilities among recently trained nurses [ 17 , 18 ] . new nurses ' qualifications can not be viewed solely in terms of education policy and reform . changes in the organization and delivery of health care have a great impact on nurse education . the current nursing culture in hospital wards is characterized by a stronger treatment culture and greater demands for efficiency than in previous years . in all western countries , several reforms have led to higher stress levels in hospital wards , more specialized medical treatments and technologies , and shorter patient stays [ 6 , 7 , 20 ] . a related factor in norway is that the health - care system has undergone substantial organizational changes in the past 1015 years . it is evident from the literature that new nurses do not experience a supportive practice environment and regularly encounter unrealistic expectations [ 8 , 22 ] . boswell and wilhoit found that nurses may need as long as one year after graduation to feel competent . many health - care organizations have introduced mentorship strategies as a way of increasing work readiness , minimizing the effects of reality shock , and reducing graduate nurse attrition [ 8 , 22 ] . kramer used the term reality shock in nursing to describe how school - bred values conflict with work - world values , a concept based on the foundational tenets of culture shock . reports of this transition in nursing have proliferated in recent years [ 5 , 7 , 16 , 19 , 24 ] . the term transition refers to the processes of learning and adjustment that a new staff member undergoes to acquire the skills , knowledge , and values required to become an effective member of the health - care team [ 7 , 25 ] . this process involves shifting from one set of expected positional behaviours to another in a specific social system : it incorporates a socialization process or rites of passage , in which the graduates absorb and adopt the language , culture , and rules of the work place . in a canadian study , duchscher emphasized that it was the quality of the transitional experience that was likely to influence graduate nurses ' self - confidence and retention in nursing . that study provided insight into the socialization process of nurses by asking them to reflect on their first six months in the profession . a clear conflict emerged between the nurses ' desire to deliver good nursing and their lack of competence , time , and energy to do so . the expectations encountered by new graduates in norway today are usually the same as those from before the 1990s , despite the changes in nurse education and in the content and context of hospital nursing . graduates are offered some form of orientation programme , usually lasting two or three weeks . although the content varies , these programmes predominantly focus on patients ' diagnoses , hospital routines , and medical treatments characteristic of the ward . graduates in hospitals are supposed to have a mentor among the clinical nursing staff , who provides some support during the first four or five months in the ward . however , because of the hectic clinical setting , new nurses spend much of their time on their own . research conducted into this transition using qualitative methodologies has investigated the nature of the transition process and the values of new nurses [ 16 , 24 ] , but little attention has been given to the reality of new nurses ' clinical practice . missing from the literature is an insider 's perspective on new nurses ' experience when they take on the nursing role in a hospital setting . knowledge of new graduates ' experiences in their daily work situations should help improve understanding of the challenges they face in their role as nurses . in this paper , we present the findings of a study that focused on the question : what opportunities and limitations do newly qualified nurses encounter when taking on the nursing role ? an ethnographic design was used , drawing upon observations , interviews , and the analysis of documents . this was a qualitative design , monitoring the nurses in their encounters with colleagues , physicians , and patients in clinical situations . the combination of different data sources is often highly productive , and the resulting analysis and findings can be more complete when a number of different information sources are used to explore a question . the setting was one gynaecological and one orthopaedic ward of equal size ( 30 beds ) in a norwegian university hospital , during one year of fieldwork . a meeting was held to give the participants and their colleagues information about the study . written information was also distributed to all new nurses in the wards . for nurses to be included , they had to have been qualified for about three months when the study started . although the sample selected was a convenience sample , it was also purposeful , because the participants were ideal in terms of having some experience of being a new nurse . the sample comprised 13 new graduates ; all were women , in their early to mid - twenties ( mean age 25 years ) , and from norwegian backgrounds . reflection notes written by the new graduates about their work and caring experiences were included as data . the researcher ( msb ) observed the nurses ' activities at meetings , in staff rooms , in patient rooms , in ancillary rooms , in the tea room , and in the corridors at different times during the day and evening . the nurses ' shifts provided the starting points and the structure of the fieldwork . during observations , the researcher wore the same uniform as the nurses in order to merge into the setting and make it easier to be accepted as a natural participant in the ward . observational notes were written out in full immediately after each period of observation so that no valuable information would be lost or forgotten . we followed the guidelines of lincoln and guba for establishing the trustworthiness of data . to establish the credibility of the data , the informants were encouraged to share their perspectives on different situations with the researcher . the informants were also asked to read through some of the transcripts , and they were encouraged to comment on their accuracy , correct them as required , and provide supplementary explanations if they felt it was appropriate . although we acknowledge the criticisms concerning participant checking , we felt that the participants themselves should establish the validity of the interpretations . the dependability of the analysis required sufficient arguments and documentation in the text so that the reader could follow the analysis . finally , the study fulfils the criterion of confirmability when its findings can be applied to contexts beyond the study situation . the analysis proceeded in parallel with the data collection , as the researchers read through the transcripts repeatedly . initially , the task was to find concepts that would make sense of the data . according to benner , the nurses ' world can never be delineated completely , because although it is historical , contextual , and multifaceted , it can only be grasped in finite , situated terms . we followed the principle of analysing abundant data on a few informants and have provided thick descriptions . the main analysis was performed after the end of the year of fieldwork . for this purpose the process started with quite open questions , for example , what do new graduates do ? we wished to create new ways of looking at the nurses ' work in order to interpret everyday situations with a higher degree of abstraction . in this process , more specific questions developed , consistent with themes such as the nurse 's responsibility or being busy . other themes increased in importance as they recurred in the nurses ' actions and statements . we searched for similarities and contrasts in the ways in which the individual and the group as a whole experienced their relationships with colleagues and patients in different situations . because the researcher was an external person , with no employment links to the hospital , the participants could feel free to be honest in dialogues about their work and their work environment . the participants were informed that they were free to withdraw from the study at any time without any explanation . according to common practice , the nurses always asked the patients ' permission to bring a researcher into their rooms . in this section , the two central findings are presented from the perspectives of tina and kari . their experiences represent typical challenges that the nurses reported throughout the entire body of material . the patients in the ward have many complex diseases and they need regular observation day and night , in one room , a young woman lay with her eyes closed and her face towards us . tina quietly approached the bedside and bent down , whispering : are you awake ? can i get you something to drink ? the patient only grunted in reply and did not open her eyes . her face was pale and she had a kidney bowl on her chest holding some absorbent tissue . while looking at the intravenous catheter and checking the intravenous injection , tina kept an eye on the patient 's face . a glass of fresh water was on the bedside table , and tina carefully supported the patient and helped her take a small mouthful . after helping the patient into a comfortable position , tina said that she would be back again soon . on the way out , i noticed a soft light in the single room , a glow from the lamp beside the bed , positioned in such a way that it would not bother the patient . i asked tina about this patient afterwards , and she told me that the patient had a serious disease , hyperemesis gravidarum , which meant that she was constantly sick with nausea and vomiting . the patient would have to stay in bed for months or possibly for her entire pregnancy , and she was not supposed to have anything to drink or eat . i really feel sorry for this patient , so young and being so sick day after day , she said . later in the afternoon , and in between phone calls and her responsibilities for other patients i have to follow up and see if she is all right , and whether she has vomited . she helped the patient and offered her a special moisture stick in a glass of iced water to cleanse her mouth , which would give her a sense of tasting water . tina 's caring and sensitive attitude when taking care of the patient was in marked contrast with her much more determined and brisk manner of walking as soon as she was back in the corridor . involvement in patient situations gave tina an opportunity to be close to a person who had complex care needs . tina was pleased because she felt that she had displayed the clinical skills required for the medical treatment , while at the same time comforting and calming the patient . she was also aware that being responsible for a number of patients at the same time generated a kind of anxiety within her . she said that this was particularly so when she did not have enough time for all her patients during a shift : on night duty , i have got more time for the patients . i can be there when the day - time 's hectic rhythms are not in force any more . kari said she had started to feel more secure in her position , but that acting as a team leader was still an overwhelming responsibility . this included all the hands - on work , such as medications , wound care , and many quite specialized procedures . after the early morning report , she sat down to look through trays of paperwork , talk to secretaries , fetch laboratory results , and check physicians ' orders and medications . i could hear kari and two other nurses in the staff room talking about the stress of getting an overview , while they were waiting for the physician 's round . no , she said , but the physician coming to perform the doctor 's round would be late . then , why did she persist with the paperwork for so long ? when i asked kari about this afterwards , she told me that the paperwork concerned the need for getting the essential data about the patients . she added that she was reluctant to begin the patients ' morning care if she was going to be interrupted in the middle of these activities , and that it was often difficult to finish an assignment that had been started . in the middle of giving a bed bath to one patient , i often have to leave the patient to give another patient premedication or join the physician 's rounds . the physician seemed busy , barely greeting kari before he headed for the first patient 's room . at the first patient 's bedside , the physician asked kari about the records and started to explain the details of the disease to the patient . kari attempted to add something , but the physician turned away and attended briefly to the other patient in the room , before leaving . kari followed , but she stopped by the first patient 's bed to say , by the time she was back in the corridor , the physician was already in the next room ; he finished his whole round in five minutes . after the round , kari prepared some medications , and then she went to visit two patients , checking their intravenous lines and some dressings . she asked the patients how they were feeling , and she seemed to create good relationships with the patients . back in the corridor , she told me that she would have no time to follow up the patients this morning as she had planned , apart from talking with the patient who looked worried after receiving the physician 's information . at the end of the day , i asked kari about her working relationships with the physicians . she told me that they were not always like that some are quite friendly , but for the most part , cooperating with them is frustrating . then , she blurted out : it is all too much running after them on our time . in kari 's case , it was clear that the situation required balance between attending to the patients ' needs and acquiring the necessary overview before the physician 's round . at the same time , she had learned to recognize and take into account patients ' particular illness - related problems , and how to communicate her observations to the physician . the experience of being the team leader gave kari the opportunity to learn what can only be mastered in practice . despite the difficulties of her situation , kari felt satisfied when she had made sure that the patients were satisfied , and when , as team leader , she had got everything done , written the reports , ordered the blood tests , and signed patients out and arranged for social workers and physiotherapists , and everything else that has to be done . the new nurses were unprepared for the myriad of feelings provoked by this responsibility while trapped in administrative routines ; they had not been aware of the extent of the work load and its consequences for the quality of their care . one nurse , who considered herself well acquainted with the ward from her student days , noticed a marked difference between being a student and bearing the responsibility of being a nurse . she said that the main difference between training and being on the job is in fact the responsibility . the new nurses had high expectations of their own capabilities and felt that they should be able to handle difficult situations . however , there was a discrepancy between their perceptions of their own skills and knowledge and their actual ability to draw on these to assume the responsibility required . one nurse wrote in a narrative that she was very much aware that there would be a lot of responsibility , but i feel there 's even more . i feel that it 's more and more like nursing on an acute ward as far as the responsibility is concerned . many of the patients in the gynaecological ward were women close to their own age , with serious and sometimes life - threatening diseases . when asked to discuss this , the nurses said that the responsibility was difficult when there were time constraints and you feel that you do not have the resources you need , or the energy itself and the knowledge to tackle what is expected of you . then it gets tough , and that happens quite frequently . despite the conflict between patient care and administrative tasks , the new nurses were enthusiastic about learning to do their job well . for many of them , a positive aspect of working in a surgical ward was the variety in their work : this was one of the main reasons that they had begun their careers in a hospital . one nurse was pleased because she had learned that she wanted to be an angel , but also that this is where it 's all happening . she added : i want the best of both worlds , to mean something to the patients , but also to learn more . another nurse said that 's perhaps what gives most of the meaning to being a nurse , that 's what we work for . she told me about a time when she was happy because a patient told her in the morning that the painkiller she had given him had helped and that he had slept well for the rest of the night . they seemed to want intimacy to be there for the patients but they also wanted excitement . they found it exciting to learn more about technical procedures and to manage new situations with the patients , especially when they could concentrate on what they were doing without interruption . the general impression derived from the data was that the new nurses experienced little support or had few opportunities to discuss the challenging situations they encountered in their work . the bustle in the wards meant that conversations often took place in passing , for example , on the way to the morning care routine . the new graduates ' relational experiences with the patients received little attention , and the patients ' situations were only rarely the subject of discussion in the care environment . resolving the conflict inherent in scheduling and organizing their attention to patients was a challenge for the new nurses , and the fragmentation of their work left them feeling that their more experienced colleagues neither understood nor respected their ideas about nursing . the level of support for the new graduates in this respect varied ; for some , it was negligible . one nurse said if there 's anyone who needs guidance , it 's us , the newcomers . if only we could talk more with the experienced nurses , then there would be some guidance at least . although the quality of mentorship varied , it was perceived as useful when it included opportunities to work together , formal meetings and completion of paperwork . the most readily available support was given by the other nurses in the team ; such support often involved a mix of informal input , such as discussing a shift before going home , and more structured support , such as help filling out a critical - incident statement . the new nurses seemed to have developed a defensive attitude in response to this busyness and lack of support : you just get on with your job as part of the system . although each graduate nurse was supposed to have a mentor for the first three or four months , the system did not work well because their shifts were not coordinated . the empirical findings of this study both conflict and concur with other relevant research findings . our study showed that the new nurses were keenly aware of their patients ' needs and wanted to give them the best care possible , and that they were concerned about creating good relationships with their patients . this patient - centred caring was also observed in duchscher 's study , in which it was associated with increased knowledge . however , when rognstad et al . studied students ' educational and occupational motivations , they found a high degree of self - centredness . the students were described as typical representatives of the what 's in it for me generation , whose main concern was to fulfil their own wishes and needs . she found that nursing students had an ambivalent attitude : they were motivated towards nursing because they wanted to help the sick , but at the same time , helping others increased their self - understanding and accentuated their own importance . in this respect , tveit encourages us to perceive self - centredness among students and new nurses as a potential strength . as in the studies of both tveit and maben et al . , the new nurses in our study wanted to make a difference to their patients . the nurses ' positive energy and interest in improving their skills did not seem to be fully exploited , or valued by their more experienced colleagues . . found that their participants ' attempts to put their ideals into practice were often hindered by processes designated organizational sabotage and professional sabotage . the new nurses were sometimes anxious about whether they could meet the profession 's expectations , especially when they were responsible for a large number of patients , and particularly on days when they were team leaders . whereas existing studies have described anxiety ( fear of making mistakes ) and a desire for ongoing feedback among new nurses [ 16 , 35 , 36 ] , the nurses in our study did not seem to worry constantly about making mistakes . they felt a lack of energy and knowledge and wanted support , but they seemed to manage the challenge of assuming responsibility . as team leaders , the nurses experienced different styles of physician cooperation , and felt that they were in an insecure position because the physicians seemed to expect the nurses to be ready for them at any time . this finding confirms those of duchscher , dyess and sherman , and martin and wilson , in that the new nurses in their studies frequently experienced less than ideal communication with physicians . the lack of professional confidence that new nurses feel in challenging situations can be heightened when another professional is brusque or expresses displeasure . however , as leever et al . pointed out , the collaboration between nurses and physicians in a hospital is crucial to the care of individual patients . although the nurses in our study did not directly confirm the findings of kramer in terms of massive transition , there was a perceptible psychological shift , which seemed to mark the adjustment from being a student to becoming a staff nurse . this adjustment was mainly associated with the recognition that the main difference between training and being on the job was the responsibility , in that the new nurses had become answerable for all their decisions in practice , a factor also identified by maben and clark and dearmun . the new graduates wanted to manage , despite the difficulties , and they , therefore , accepted work loads and responsibilities that could provoke feelings of stress . in an effort to conform to the implicit value of being seen to be doing a good job and to play the organizational game , the nurses became aware of the importance of appearing to be busy according to lindberg - sand , this aspect of reality shock can be understood as a profound transition experience . she suggested that new nurses ' experiences of responsibility are interwoven with the aspect of time , so that the feeling of being responsible increased in tandem with the speed with which the nurses fulfilled their duties . in this context , the new graduates in our study seemed to have adapted to the implicit requirement to be efficient in order to avoid trouble and to earn the respect of their leader and other staff members . unacknowledged value conflicts and role ambiguities may lie behind the unrealistic expectations held by senior nurses . ellerton and gregor and maben et al . described an incongruence between the personal values of new graduates and the established values in clinical practice settings and questioned the effect on nurses of not having the energy or capacity to realize their ideal of nursing . these conflicting expectations emerged clearly in our study and illustrate the pressures felt by new nurses . according to duchscher and cowin , the primary challenge for new nurses lies in their struggle to construct a new professional sense of self - confidence that fuses the ideals of their education with the realities of their practice context . the new nurses in our study were attracted to a job with possibilities for excitement , and for them , working in a surgical ward was about learning more . they saw the job as giving them an opportunity to take care of patients and to learn how to be a good nurse . however , it became apparent that there was no support tailored to their individual learning needs , leading to a dissonance between the support and supervision that they felt they needed . as recent graduates , they had expected that the more experienced nurses would provide guidance as they developed their clinical skills , assist them with decision making , and help them meet their patients ' needs and the organizational requirements of the hospital . wenger argues for an interaction between the nurses ' participation in practical work and a dialogue about this work . it is through practice that students and new graduates can work and form opinions about that work . our findings support olson 's conclusion that collegiality is highly valued by new nurses today , and that mutually supportive and empathetic relationships with colleagues play an important role in how well new nurses adapt to the culture of nursing . this study explored the perceptions and experience of the reality of practice among 13 new graduate nurses in a norwegian hospital . although small , the sample provided an opportunity for observation , in - depth interviews , repeat interviews , and ongoing journal reflections , all of which enriched the study . the questions addressed to the informants were constructed during the fieldwork , and they should be recognized as coming from within the author 's personal and professional paradigm of thought . this subjectivity indirectly instilled a conceptual bias in the fieldwork process , although rigorous attempts were made to ensure that the informants directed the type of data collected . the context of the two surgical wards differed , but this issue was not addressed in this study . however , incorporating these contextual differences would not have increased the validity of the data . all data were collected by the main author . at the time of the study , the author had been a practising acute - care nurse for five years and had previous involvement as a nurse educator . as in all qualitative research , the author 's practice experience necessarily influenced both the process and the content of the study . this is a strength of the study , because it offers an insider 's perspective on the views of new graduates . the research presented in this paper shows that newly graduated nurses are likely to enter nursing practice with empathy for their patients and enthusiasm for their profession . however , this potential in new nurses and their readiness to learn seem to be hindered by organizational and professional limitations , including time pressures and a lack of support . for a transition to be optimal , it must take place in a well - structured ward , with a supportive environment characterized by initiatives that will meet the needs of new nurses during their first year of practice . such initiatives should include support that extends beyond clinical orientation and basic unit - specific mentoring programmes . the work experiences of new nurses provide substantial information about improvement opportunities at the ward level . these factors have an important impact on new nurses ' work practices and on future nurse retention . further research in this area is required to explore the experience of being constrained within the professional nursing role , the kinds of feelings this issue engenders in newly graduated nurses , and the effects on their future careers .

the transition from student to working nurse has long been recognized as challenging . this paper presents the findings of research into the opportunities and limitations encountered by newly qualified nurses when taking on the nursing role . the study had an ethnographic design . observation , interviews , and document analysis were used to gain insight into nurses ' daily work from the perspective of recently graduated nurses . thirteen nurses were monitored closely during their first year in a hospital setting in norway . these new nurses generally entered the field with empathy for their patients , enthusiasm for the profession , and readiness to learn more about being a good nurse . however , their more experienced colleagues seemed to neither respect nor nurture this attitude . the new nurses experienced heavier responsibilities than expected , fragmentation of patient care , and stressful interactions with colleagues . the lack of a supportive work environment and role models increased the new nurses ' experience of overwhelming responsibility in their daily work situations . the nurses learned to cope the hard way , despite the organizational culture , not because of it . adjusting the profession 's expectations of new nurses , and offering good role models and more comprehensive support programmes , would markedly ease the transition for new nurses .

1. Introduction 2. Background 3. Methods 4. Findings 5. Discussion 6. Conclusions

the transition from student to working nurse has long been recognized as difficult and has attracted the attention of researchers for decades [ 15 ] . various studies have shown that new graduates are inadequately prepared for their role as nurses , and they are ineffectually oriented to the work place [ 68 ] . the nursing discipline and nurse education are currently experiencing the compound pressure of expectations , which may lead to deteriorating conditions for nursing practice . the clinical field , under increasing pressure to be profitable , efficient , and effective , is demanding education programmes that produce graduate nurses who are work ready from day one . such studies indicate that the conditions for nursing remain challenging in terms of increased pressure in the work environment and new nurses ' readiness and willingness to care for patients . this situation inspired us to investigate empirically how new nurses are taking on the nursing role in hospitals . until the 1980s , nurse education in many western countries was based predominantly on an apprenticeship model , in which student nurses were salaried members of the nursing work force in hospitals [ 1214 ] . the aim was to equip student nurses with the necessary knowledge and skills to assume the role of graduates , and to achieve academic recognition [ 6 , 15 , 16 ] . however , this drift towards academia seems to have gone too far , resulting in complaints about weak practical skills and the lack of caring abilities among recently trained nurses [ 17 , 18 ] . new nurses ' qualifications can not be viewed solely in terms of education policy and reform . in all western countries , several reforms have led to higher stress levels in hospital wards , more specialized medical treatments and technologies , and shorter patient stays [ 6 , 7 , 20 ] . a related factor in norway is that the health - care system has undergone substantial organizational changes in the past 1015 years . it is evident from the literature that new nurses do not experience a supportive practice environment and regularly encounter unrealistic expectations [ 8 , 22 ] . kramer used the term reality shock in nursing to describe how school - bred values conflict with work - world values , a concept based on the foundational tenets of culture shock . the term transition refers to the processes of learning and adjustment that a new staff member undergoes to acquire the skills , knowledge , and values required to become an effective member of the health - care team [ 7 , 25 ] . this process involves shifting from one set of expected positional behaviours to another in a specific social system : it incorporates a socialization process or rites of passage , in which the graduates absorb and adopt the language , culture , and rules of the work place . in a canadian study , duchscher emphasized that it was the quality of the transitional experience that was likely to influence graduate nurses ' self - confidence and retention in nursing . that study provided insight into the socialization process of nurses by asking them to reflect on their first six months in the profession . a clear conflict emerged between the nurses ' desire to deliver good nursing and their lack of competence , time , and energy to do so . the expectations encountered by new graduates in norway today are usually the same as those from before the 1990s , despite the changes in nurse education and in the content and context of hospital nursing . although the content varies , these programmes predominantly focus on patients ' diagnoses , hospital routines , and medical treatments characteristic of the ward . however , because of the hectic clinical setting , new nurses spend much of their time on their own . research conducted into this transition using qualitative methodologies has investigated the nature of the transition process and the values of new nurses [ 16 , 24 ] , but little attention has been given to the reality of new nurses ' clinical practice . missing from the literature is an insider 's perspective on new nurses ' experience when they take on the nursing role in a hospital setting . knowledge of new graduates ' experiences in their daily work situations should help improve understanding of the challenges they face in their role as nurses . in this paper , we present the findings of a study that focused on the question : what opportunities and limitations do newly qualified nurses encounter when taking on the nursing role ? an ethnographic design was used , drawing upon observations , interviews , and the analysis of documents . this was a qualitative design , monitoring the nurses in their encounters with colleagues , physicians , and patients in clinical situations . the combination of different data sources is often highly productive , and the resulting analysis and findings can be more complete when a number of different information sources are used to explore a question . the setting was one gynaecological and one orthopaedic ward of equal size ( 30 beds ) in a norwegian university hospital , during one year of fieldwork . a meeting was held to give the participants and their colleagues information about the study . for nurses to be included , they had to have been qualified for about three months when the study started . although the sample selected was a convenience sample , it was also purposeful , because the participants were ideal in terms of having some experience of being a new nurse . the sample comprised 13 new graduates ; all were women , in their early to mid - twenties ( mean age 25 years ) , and from norwegian backgrounds . reflection notes written by the new graduates about their work and caring experiences were included as data . the researcher ( msb ) observed the nurses ' activities at meetings , in staff rooms , in patient rooms , in ancillary rooms , in the tea room , and in the corridors at different times during the day and evening . the nurses ' shifts provided the starting points and the structure of the fieldwork . during observations , the researcher wore the same uniform as the nurses in order to merge into the setting and make it easier to be accepted as a natural participant in the ward . the informants were also asked to read through some of the transcripts , and they were encouraged to comment on their accuracy , correct them as required , and provide supplementary explanations if they felt it was appropriate . finally , the study fulfils the criterion of confirmability when its findings can be applied to contexts beyond the study situation . according to benner , the nurses ' world can never be delineated completely , because although it is historical , contextual , and multifaceted , it can only be grasped in finite , situated terms . we wished to create new ways of looking at the nurses ' work in order to interpret everyday situations with a higher degree of abstraction . other themes increased in importance as they recurred in the nurses ' actions and statements . we searched for similarities and contrasts in the ways in which the individual and the group as a whole experienced their relationships with colleagues and patients in different situations . because the researcher was an external person , with no employment links to the hospital , the participants could feel free to be honest in dialogues about their work and their work environment . the participants were informed that they were free to withdraw from the study at any time without any explanation . according to common practice , the nurses always asked the patients ' permission to bring a researcher into their rooms . in this section , the two central findings are presented from the perspectives of tina and kari . their experiences represent typical challenges that the nurses reported throughout the entire body of material . while looking at the intravenous catheter and checking the intravenous injection , tina kept an eye on the patient 's face . a glass of fresh water was on the bedside table , and tina carefully supported the patient and helped her take a small mouthful . on the way out , i noticed a soft light in the single room , a glow from the lamp beside the bed , positioned in such a way that it would not bother the patient . i asked tina about this patient afterwards , and she told me that the patient had a serious disease , hyperemesis gravidarum , which meant that she was constantly sick with nausea and vomiting . the patient would have to stay in bed for months or possibly for her entire pregnancy , and she was not supposed to have anything to drink or eat . later in the afternoon , and in between phone calls and her responsibilities for other patients i have to follow up and see if she is all right , and whether she has vomited . she helped the patient and offered her a special moisture stick in a glass of iced water to cleanse her mouth , which would give her a sense of tasting water . tina was pleased because she felt that she had displayed the clinical skills required for the medical treatment , while at the same time comforting and calming the patient . she said that this was particularly so when she did not have enough time for all her patients during a shift : on night duty , i have got more time for the patients . kari said she had started to feel more secure in her position , but that acting as a team leader was still an overwhelming responsibility . this included all the hands - on work , such as medications , wound care , and many quite specialized procedures . after the early morning report , she sat down to look through trays of paperwork , talk to secretaries , fetch laboratory results , and check physicians ' orders and medications . i could hear kari and two other nurses in the staff room talking about the stress of getting an overview , while they were waiting for the physician 's round . she added that she was reluctant to begin the patients ' morning care if she was going to be interrupted in the middle of these activities , and that it was often difficult to finish an assignment that had been started . the physician seemed busy , barely greeting kari before he headed for the first patient 's room . after the round , kari prepared some medications , and then she went to visit two patients , checking their intravenous lines and some dressings . she asked the patients how they were feeling , and she seemed to create good relationships with the patients . she told me that they were not always like that some are quite friendly , but for the most part , cooperating with them is frustrating . at the same time , she had learned to recognize and take into account patients ' particular illness - related problems , and how to communicate her observations to the physician . the experience of being the team leader gave kari the opportunity to learn what can only be mastered in practice . despite the difficulties of her situation , kari felt satisfied when she had made sure that the patients were satisfied , and when , as team leader , she had got everything done , written the reports , ordered the blood tests , and signed patients out and arranged for social workers and physiotherapists , and everything else that has to be done . the new nurses were unprepared for the myriad of feelings provoked by this responsibility while trapped in administrative routines ; they had not been aware of the extent of the work load and its consequences for the quality of their care . one nurse , who considered herself well acquainted with the ward from her student days , noticed a marked difference between being a student and bearing the responsibility of being a nurse . she said that the main difference between training and being on the job is in fact the responsibility . the new nurses had high expectations of their own capabilities and felt that they should be able to handle difficult situations . however , there was a discrepancy between their perceptions of their own skills and knowledge and their actual ability to draw on these to assume the responsibility required . when asked to discuss this , the nurses said that the responsibility was difficult when there were time constraints and you feel that you do not have the resources you need , or the energy itself and the knowledge to tackle what is expected of you . then it gets tough , and that happens quite frequently . despite the conflict between patient care and administrative tasks , the new nurses were enthusiastic about learning to do their job well . for many of them , a positive aspect of working in a surgical ward was the variety in their work : this was one of the main reasons that they had begun their careers in a hospital . she added : i want the best of both worlds , to mean something to the patients , but also to learn more . another nurse said that 's perhaps what gives most of the meaning to being a nurse , that 's what we work for . she told me about a time when she was happy because a patient told her in the morning that the painkiller she had given him had helped and that he had slept well for the rest of the night . they seemed to want intimacy to be there for the patients but they also wanted excitement . they found it exciting to learn more about technical procedures and to manage new situations with the patients , especially when they could concentrate on what they were doing without interruption . the general impression derived from the data was that the new nurses experienced little support or had few opportunities to discuss the challenging situations they encountered in their work . the bustle in the wards meant that conversations often took place in passing , for example , on the way to the morning care routine . the new graduates ' relational experiences with the patients received little attention , and the patients ' situations were only rarely the subject of discussion in the care environment . resolving the conflict inherent in scheduling and organizing their attention to patients was a challenge for the new nurses , and the fragmentation of their work left them feeling that their more experienced colleagues neither understood nor respected their ideas about nursing . the level of support for the new graduates in this respect varied ; for some , it was negligible . the most readily available support was given by the other nurses in the team ; such support often involved a mix of informal input , such as discussing a shift before going home , and more structured support , such as help filling out a critical - incident statement . the new nurses seemed to have developed a defensive attitude in response to this busyness and lack of support : you just get on with your job as part of the system . although each graduate nurse was supposed to have a mentor for the first three or four months , the system did not work well because their shifts were not coordinated . the empirical findings of this study both conflict and concur with other relevant research findings . our study showed that the new nurses were keenly aware of their patients ' needs and wanted to give them the best care possible , and that they were concerned about creating good relationships with their patients . however , when rognstad et al . she found that nursing students had an ambivalent attitude : they were motivated towards nursing because they wanted to help the sick , but at the same time , helping others increased their self - understanding and accentuated their own importance . in this respect , tveit encourages us to perceive self - centredness among students and new nurses as a potential strength . , the new nurses in our study wanted to make a difference to their patients . the nurses ' positive energy and interest in improving their skills did not seem to be fully exploited , or valued by their more experienced colleagues . the new nurses were sometimes anxious about whether they could meet the profession 's expectations , especially when they were responsible for a large number of patients , and particularly on days when they were team leaders . whereas existing studies have described anxiety ( fear of making mistakes ) and a desire for ongoing feedback among new nurses [ 16 , 35 , 36 ] , the nurses in our study did not seem to worry constantly about making mistakes . they felt a lack of energy and knowledge and wanted support , but they seemed to manage the challenge of assuming responsibility . as team leaders , the nurses experienced different styles of physician cooperation , and felt that they were in an insecure position because the physicians seemed to expect the nurses to be ready for them at any time . this finding confirms those of duchscher , dyess and sherman , and martin and wilson , in that the new nurses in their studies frequently experienced less than ideal communication with physicians . the lack of professional confidence that new nurses feel in challenging situations can be heightened when another professional is brusque or expresses displeasure . however , as leever et al . pointed out , the collaboration between nurses and physicians in a hospital is crucial to the care of individual patients . although the nurses in our study did not directly confirm the findings of kramer in terms of massive transition , there was a perceptible psychological shift , which seemed to mark the adjustment from being a student to becoming a staff nurse . this adjustment was mainly associated with the recognition that the main difference between training and being on the job was the responsibility , in that the new nurses had become answerable for all their decisions in practice , a factor also identified by maben and clark and dearmun . the new graduates wanted to manage , despite the difficulties , and they , therefore , accepted work loads and responsibilities that could provoke feelings of stress . in an effort to conform to the implicit value of being seen to be doing a good job and to play the organizational game , the nurses became aware of the importance of appearing to be busy according to lindberg - sand , this aspect of reality shock can be understood as a profound transition experience . she suggested that new nurses ' experiences of responsibility are interwoven with the aspect of time , so that the feeling of being responsible increased in tandem with the speed with which the nurses fulfilled their duties . in this context , the new graduates in our study seemed to have adapted to the implicit requirement to be efficient in order to avoid trouble and to earn the respect of their leader and other staff members . unacknowledged value conflicts and role ambiguities may lie behind the unrealistic expectations held by senior nurses . these conflicting expectations emerged clearly in our study and illustrate the pressures felt by new nurses . according to duchscher and cowin , the primary challenge for new nurses lies in their struggle to construct a new professional sense of self - confidence that fuses the ideals of their education with the realities of their practice context . the new nurses in our study were attracted to a job with possibilities for excitement , and for them , working in a surgical ward was about learning more . they saw the job as giving them an opportunity to take care of patients and to learn how to be a good nurse . as recent graduates , they had expected that the more experienced nurses would provide guidance as they developed their clinical skills , assist them with decision making , and help them meet their patients ' needs and the organizational requirements of the hospital . wenger argues for an interaction between the nurses ' participation in practical work and a dialogue about this work . our findings support olson 's conclusion that collegiality is highly valued by new nurses today , and that mutually supportive and empathetic relationships with colleagues play an important role in how well new nurses adapt to the culture of nursing . this study explored the perceptions and experience of the reality of practice among 13 new graduate nurses in a norwegian hospital . although small , the sample provided an opportunity for observation , in - depth interviews , repeat interviews , and ongoing journal reflections , all of which enriched the study . the questions addressed to the informants were constructed during the fieldwork , and they should be recognized as coming from within the author 's personal and professional paradigm of thought . however , incorporating these contextual differences would not have increased the validity of the data . at the time of the study , the author had been a practising acute - care nurse for five years and had previous involvement as a nurse educator . this is a strength of the study , because it offers an insider 's perspective on the views of new graduates . the research presented in this paper shows that newly graduated nurses are likely to enter nursing practice with empathy for their patients and enthusiasm for their profession . however , this potential in new nurses and their readiness to learn seem to be hindered by organizational and professional limitations , including time pressures and a lack of support . for a transition to be optimal , it must take place in a well - structured ward , with a supportive environment characterized by initiatives that will meet the needs of new nurses during their first year of practice . the work experiences of new nurses provide substantial information about improvement opportunities at the ward level . these factors have an important impact on new nurses ' work practices and on future nurse retention . further research in this area is required to explore the experience of being constrained within the professional nursing role , the kinds of feelings this issue engenders in newly graduated nurses , and the effects on their future careers .

the level of the genetic diversity is pivotal for world food security and survival of human civilization on earth . historically , humans exploited plant species for their livelihoods that resulted in domestication of many of them as improved cultivars to produce food for the better supply of the human diet . presently , out of 150 plant species cultivated in agriculture , twelve provide about 75% of human food and four produce 50% of human diet . according to food and health organization report , ~800 million people in the developing countries are suffering from food deficiency that underlies an attention to improve agricultural production to eliminate or , at least , reduce the feeding problems . the narrow genetic base of modern crop cultivars is the serious obstacle to sustain and improve crop productivity due to rapid vulnerability of genetically uniform cultivars by potentially new biotic and abiotic stresses . however , plant germplasm resources worldwide , comprising of wild plant species , modern cultivars , and their crop wild relatives , are the important reservoirs of natural genetic variations , originated from a number of historical genetic events as a respond to environmental stresses and selection through crop domestication [ 1 , 5 ] . the efficient exploiting these ex situ conserved genetic diversities is vital to overcome future problems associated with narrowness of genetic base of modern cultivars . however , many agriculturally important variations such as productivity and quality , tolerance to environmental stresses , and some of forms of disease resistance are controlled by polygenes and multifactorial that greatly depends on genetic environmental ( g e ) interactions [ 1 , 6 ] . these complex traits are referred to as quantitative trait loci ( qtls ) , and it is challenging to identify qtls based on only traditional phenotypic evaluation . identification of qtls of agronomic importance and its utilization in a crop improvement further requires mapping of these qtls in a genome of crop species using molecular markers [ 1 , 6 ] . this was the major breakthrough and accomplishment in many crops in genomics era since the end of the 20th century , and now extended to flourish in the 21st century . in this review , we provide a brief description for the concept of genetic mapping ; then , as a flourish of the crop genomics era , we thoroughly review one of the powerful genetic mapping tools for crops , linkage disequilibrium ( ld)-based association study , as a high - resolution , broader allele coverage , and cost effective gene tagging approach in plant germplasm resources . this provides an opportunity to widely dissect and exploit existing natural variations for crop improvement . the main goal of genetic mapping is to detect neutrally inherited markers in close proximity to the genetic causatives or genes controlling the complex quantitative traits . genetic mapping can be done mostly in two ways : ( 1 ) using the experimental populations ( also referred to as biparental mapping populations ) that is called qtl - mapping as well as genetic mapping or gene tagging , and ( 2 ) using the diverse lines from the natural populations or germplasm collections that is called ld - mapping or association mapping . the details of the traditional qtl - mapping approach has recently been reviewed by collard et al . , and further basic description of the approach here would be a redundant . for detailed concept , models and methodologies , problems , and perspectives of linkage analysis , here , we briefly outline linkage mapping procedure for the sake of highlighting the merits of the alternative approach - association mapping . so that such a linkage analysis can be done [ 68 ] , firstly , the experimental populations such as f2 , back cross ( bc ) , double haploid ( dh ) , recombinant inbred line ( ril ) , and near isogenic line ( nil ) populations , derived from the genetic hybridization of two parental genotypes with an alternative trait of interest , need to be developed . secondly , these experimental populations including a large number of progenies or lines are measured for the segregation of a trait of interest in the different environmental conditions . thirdly , a set of polymorphic dna markers , differentiating the parental genotypes and segregating in a mapping population , need to be identified and genotyped . for that , usual practice is that , first , the parental genotypes are screened , and if markers are polymorphic over the parents , then , all individuals of a mapping population are genotyped with these polymorphic molecular markers . once genotypic data of a mapping population is ready , marker data is used to construct the framework linkage maps , representing the order ( position ) and linkage ( a relative genetic distance in cm ) of used molecular markers along the linkage groups or segments of particular chromosomes . consequently , these markers ordered along the linkage map are statistically correlated with phenotypic characteristics of individuals of a mapping population , and qtl regions affecting a trait of interest , along with closely positioned marker tags to that qtl , are identified . one can imagine these linkage marker maps as a road map , marker tags as the labels directing to specific places , and qtls to a community / neighborhood ( with specific function ) on the map . the precision of qtl - mapping largely depends on the genetic variation ( or genetic background ) covered by a mapping population , the size of a mapping population , and a number of marker loci used . once qtls affecting a trait of interest accurately tagged using above - outlined approach , marker tags are the most effective tools in a crop improvement that allows the mobilization of the genes of interest from donor lines to the breeding material through marker - assisted selection ( mas ) . although traditional qtl - mapping will continue being an important tool in gene tagging of crops , it is a now classical approach and overall is very costly [ 1 , 9 ] , and has low resolution with simultaneous evaluation of only a few alleles in a longer research time scale . in linkage mapping , the major limitation , hampering the fine mapping , is associated with the availability of only a few meiotic events to be used that occurred since experimental hybridization in a recent past . these limitations , however , can be reduced with the use of association mapping . turning the gene - tagging efforts from biparental crosses to natural population of lines ( or germplasm collections ) , and from traditional qtl - mapping to linkage disequilibrium ( ld)-based association study became a powerful tool in mapping of the genes of interest . this leads to the most effective utilization of ex situ conserved natural genetic diversity of worldwide crop germplasm resources . ld refers to a historically reduced ( nonequilibrium ) level of the recombination of specific alleles at different loci controlling particular genetic variations in a population . this ld can be detected statistically , and has been widely applied to map and eventually clone a number of genes underlying the complex genetic traits in humans [ 1316 ] . the advantages of population - based association study , utilizing a sample of individuals from the germplasm collections or a natural population , over traditional qtl - mapping in biparental crosses primarily are due to ( 1 ) availability of broader genetic variations with wider background for marker - trait correlations ( i.e. , many alleles evaluated simultaneously ) , ( 2 ) likelihood for a higher resolution mapping because of the utilization of majority recombination events from a large number of meiosis throughout the germplasm development history , ( 3 ) possibility of exploiting historically measured trait data for association , and ( 4 ) no need for the development of expensive and tedious biparental populations that makes approach timesaving and cost - effective [ 1719 ] . although the overall approach of population - based association mapping in plants varies based on the methodology chosen ( see below sections ) , assuming structured population samples , the performance of association mapping includes the following steps ( see figure 1 ) : ( 1 ) selection of a group of individuals from a natural population or germplasm collection with wide coverage of genetic diversity ; ( 2 ) recording or measuring the phenotypic characteristics ( yield , quality , tolerance , or resistance ) of selected population groups , preferably , in different environments and multiple replication / trial design ; ( 3 ) genotyping a mapping population individuals with available molecular markers ; ( 4 ) quantification of the extent of ld of a chosen population genome using a molecular marker data ; ( 5 ) assessment of the population structure ( the level of genetic differentiation among groups within a sampled population individuals ) and kinship ( coefficient of relatedness between pairs of each individuals within a sample ) ; and ( 6 ) based on information gained through quantification of ld and population structure , correlation of phenotypic and genotypic / haplotypic data with the application of an appropriate statistical approach that reveals marker tags positioned within close proximity of targeted trait of interest . consequently , a specific gene(s ) controlling a qtl of interest can be cloned using the marker tags and annotated for an exact biological function ( figure 1 ) . as a starting point for association mapping , it is important to gain knowledge of the patterns of ld for genomic regions of the target organisms and the specificity of the extent of ld among different populations or groups to design and conduct unbiased association mapping [ 20 , 21 ] genetic linkage generally refers to coinheritance of different loci within a genetic distance on the chromosome . there are two terms used in population genetics , linkage equilibrium ( le ) , and linkage disequilibrium ( ld ) to describe linkage relationships ( co - occurrence ) of alleles at different loci in a population . le is a random association of alleles at different loci and equals the product of allele frequencies within haplotypes , meaning that at random combination of alleles at each locus its haplotypes ( combination of alleles ) frequency has equal value in a population . in contrast , ld is a nonrandom association of alleles at different loci , describing the condition with nonequal ( increased or reduced ) frequency of the haplotypes in a population at random combination of alleles at different loci . ld is not the same as linkage , although tight linkage may generate high levels of ld between alleles . usually , there is significant ld between more distant sites or sites located in different chromosomes , caused by some specific genetic factors [ 9 , 2224 ] that will be discussed in below sections . ( gpd ) or gametic disequilibrium ( gld ) [ 11 , 25 ] in the literature that describes the same nonrandom association of haplotypes within unrelated populations with a distantly shared ancestry , assuming hardy - weinberg equilibrium ( hwe ) . the concept of ld was first described by jennings in 1917 , and its quantification ( d ) was developed by lewtonin in 1964 . the simplified explanation of the commonly used ld measure , d or d ( standardized version of d ) , is the difference between the observed gametic frequencies of haplotypes and the expected gametic haplotype frequencies under linkage equilibrium ( d = pab papb = pabpab pabpab ) . besides d , a various different measures of ld ( d , r , d , d * , f , x ( 2 ) , and ) have been developed to quantify ld [ 25 , 2729 ] . the detail formulae and description of ld quantification was well explained by a number of review papers [ 10 , 25 , 26 ] with a number of hypothetical scenarios for ld and le . the merits , sensitivity , comparison , appropriate statistical tests , and calculation methodology for these ld measures with the utilization of biallelic or multiallelic loci have been extensively described in the literature in detail [ 10 , 26 , 30 , 31 ] , and have recently been reviewed by gupta et al . . hence here we highlight only some of key utility properties of ld measures to provide a brief understanding the merits of ld in association mapping . choosing the appropriate ld measures really depends on the objective of the study , and one performs better than other in particular situations and cases ; however , d and r is the most commonly used measures of ld [ 25 , 26 ] . d is informative for the comparisons of different allele frequencies across loci and strongly inflated in a small sample size and low - allele frequencies ; therefore , intermediate values of d is dangerous for comparative analyses of different ld studies and should be verified with the r before using for quantification of the extent of ld . the r , the square of the correlation coefficient between the two loci have more reliable sampling properties than d with the cases of low allele frequencies . the r is affected by both mutation and recombination while d is affected by more mutational histories ( it might indicate minimal historic recombination when high d values used ) [ 10 , 25 , 26 , 31 ] . considering the objective , the most appropriate ld quantification measure needed for association mapping is r that is also an indicative of marker - trait correlations [ 25 , 26 , 32 ] . the r value varies from 0 to 1 , and it will be equal to 1 when only two haplotypes are present . the r value of equal to 0.1 ( 10% ) or above considered the significant threshold for the rough estimates of ld to reveal association between pairs of loci . it is noteworthy to briefly mention here that the estimation of above described gld ( commonly used in association mapping ) between different loci ordered within gametes assumes that a targeted population or sampled germplasm is randomly mating and under hwe . nevertheless , many natural populations violate hwe due to different genetic events ( bottleneck , mutation , admixture , artificial selection , population structure , etc . ) occurred in history of a population , and are under hardy - weinberg disequilibrium ( hwd ) . a concept of zygotic disequilibrium ( zld ) was introduced for such a nonequilibrium population that measures ld between different loci of gametes . zld , being defined as a deviation of joint zygotic frequencies from the expected values of zero zygotic associations [ 35 , 36 ] , has a power to measure nonrandom associations at both gametic and zygotic level [ 34 , 37 ] . it shares the most of statistical properties of gld , and the results of gld and zld are mostly in agreement , yet zld detects more extensive ld than determined by gld . the statistical models of zld measures for biallelic and multilocus data , its application for natural populations , and inference the genetic and demographic events from the comparisons of gld and zld results as well as implication for whole genome association studies ( wgas ) were excellently addressed and described by a number of studies [ 3537 ] . one of such efficient methodology is the maximum likelihood estimate ( mle ) using an expectation maximization algorithm . several computer software packages are available and can be utilized for calculation of ld using variety type of molecular markers . these software packages were extensively listed and described in the review by gupta et al . . graphical display of pairwise ld between two loci is very useful to estimate the ld patterns measured using a large number of molecular markers . pairwise ld can be depicted as a color - code triangle plot ( figure 2 ) based on significant pairwise ld level ( r , and p - value as well as d ) that helps to visualize the block of loci ( red blocks ) in significant ld . the large red blocks of haplotypes along the diagonal of the triangle plot indicate the high level of ld between the loci in the blocks , meaning that there has been a limited or no recombination since ld block formations . there is freely available specific computer software , graphical overview of linkage disequilibrium ( gold ) , to depict the structure and pattern of ld . some other software packages measuring ld such as trait analysis by association , evolution and linkage ( tassel ) [ 33 , 40 ] and powermarker have also similar graphical display features . the strong block - like ld structures are of a great interest in association mapping which simplifies ld mapping efforts of complex traits . ld blocks are very useful in association mapping when sizes are calculated , which suggest the needs for the minimum number of markers to efficiently cover the genome - wide haplotype blocks in association mapping . to estimate the size of these ld blocks , the r values ( alternatively , d can also be used ) usually plotted against the genetic ( cm ) or weighted ( bp ) distance referred to as a ld decay plot one can estimate an average genome - wide decay of ld by plotting ld values obtained from a data set covering an entire genome ( i.e. , with more or less evenly spaced markers across all chromosomes in a genome ) against distance . alternatively , the extent of ld for particular region ( gene or chromosome ) can be estimated from an ld decay plot generated using dataset obtained from a region of interest . when such a ld decay plot generated , usual practice is to look for distance point where ld value ( r ) decreases below 0.1 or half strength of d ( d = 0.5 ) based on curve of nonlinear logarithmic trend line ( see , e.g. , [ 33 , 43 , 44 ] ) . this gives the rough estimates of the extent of ld for association study , but for more accurate estimates , highly significant threshold ld values ( r 0.2 ) are also used as a cutoff point . the decrease of the ld within the genetic distance indicates that the portion of ld is conserved with linkage and proportional to recombination [ 22 , 25 ] . there are many genetic and demographic factors that play a role in the shaping of the haplotypic ld blocks in a genome [ 9 , 22 , 23 , 25 , 26 ] . although mutation and recombination are one of the strong impact factors influencing ld , generally , factors affecting ld can be grouped into two categories : ( 1 ) factors that increasing ld , and ( 2 ) factors that decreasing ld . the increase of ld is observed with new mutation , mating system ( self - pollination ) , genetic isolation , population structure , relatedness ( kinship ) , small founder population size or genetic drift , admixture , selection ( natural , artificial , and balancing ) , epistasis , and genomic rearrangements [ 25 , 26 ] . the decrease of ld is observed with high recombination and mutation rate , recurrent mutations , outcrossing , and gene conversions [ 25 , 26 ] . however , more often there is a significant ld between pairs of loci located far from each other or even in different chromosomes that might cause spurious correlations in association mapping . these long stretched ld or ld between unlinked loci indicate the existence of other ld generating factors than linkage itself in a genome [ 9 , 22 , 23 ] . one of those factors is selection that generate ld between unlinked loci through a hitchhiking effect ( high - frequency sweeping and fixation of alleles flanking a favored variant ) , and epistatic selection or the so - called coadapted genes that is the result of coselection of loci during breeding for multiple traits , common in traditional crop breeding programs worldwide . the population structure ( existences of distinctly clustered subdivisions in a population ) and population admixture are the main factors to create such an ld between unlinked loci . this primarily happens due to the occurrence of distinct allele frequencies with different ancestry in an admixed or structured population . theoretically , relatedness generates ld between linked loci , yet it might also generate ld between unlinked loci pairs when predominant parents exist in germplasm groups . there is evidence that relatedness caused ld between linked and unlinked loci in an equal proportion in maize germplasm . the high ratio value of linked to unlinked loci in ld is good indicative to draw conclusion about the role of ld generating factor(s ) such as selection or population stratification ( cryptic relatedness ) [ 9 , 22 , 23 ] . the other factors such as genetic drift or bottlenecks might have also generated ld in a genome [ 2224 ] , which is evidenced by nonuniform distribution of ld in chromosomes . knowing these factors that are increasing or decreasing ld in a genome , obvious question one might ask is whether increased or decreased level of ld is favored in association mapping ? very extensive level of ld ( means ld persists within a long distance ) , theoretically , reduces a number of markers needed for association mapping , but makes resolution lower ( coarse mapping ) . in contrast , less extensive level of ld ( means that ld quickly decays within a short distance ) requires many markers to tag a gene of interest , but in high resolution ( fine mapping ) . hence , choosing a population with low or high level of ld depends on the objective of association mapping study . furthermore , increased ld level due to ld between unlinked loci is not salutary in association mapping since it tends to cause spurious marker - trait associations . ld generated by selection , population structure , relatedness , and genetic drift might be theoretically useful for association mapping in specific situations and population groups that reduces number of markers needed for association mapping [ 9 , 22 ] , but requires serious attention to control factors affecting ld ( e.g. , population structure and relatedness ) to perform unbiased population - based association mapping in plants [ 41 , 47 ] ( see next sections ) . there are other factors affecting ld referred to as a whole ascertainment bias that are associated with an assayed sample and data characteristics . some of these factors leading to inaccurate estimate of ld were well reviewed by gupta et al . . one of such factors largely affecting the ld and leading inaccurate estimates is the presence of minor alleles ( also referred as to rare alleles that are present in only 5 to 10% individuals of the sample ) in a dataset . minor alleles are problematic in ld quantification as they largely inflate ld values ( in particular the d and p - values ) [ 43 , 4850 ] . the r is also very sensitive and has a large variance with rare alleles [ 43 , 51 ] . hence in the quantification of ld and association mapping , markers with minor allele frequency of 510% ( varied from study to study ) are ( 1 ) removed before analysis ( see , e.g. , [ 17 , 18 , 43 , 44 , 51 ] ) , ( 2 ) pooled into common allelic class ( see , e.g.,[44 , 46 ] ) , and ( 3 ) replaced with missing values ( see , e.g. , [ 52 , 53 ] ) . the quantification methodology of ld , perfectly suitable for biallelic codominant type of markers ( majorly , single nucleotide polymorphisms ( snps ) and now largely extended to multiallelic simple sequence repeats - ssrs ) , has been well developed and used in human , animal , and plant populations ( for reviews see [ 25 , 2730 ] ) . ld quantification using dominant markers ( such as random amplified polymorphic dnas - rapd , and amplified fragment length polymorphisms - aflps ) is poorly explored and usually subject to wrong perception and interpretation . however , many underrepresented plant species , like forest trees , or other crops with limited genomic information largely rely on dominant type of markers such as rapds and aflps . furthermore , even with codominant , and multiallelic ssr markers , there is a great challenge with assigning correct allelic relationships ( identity by decent ) of multiple band amplicons when diverse , reticulated , and polyploid germplasm resources , lacking historical pedigree information , are genotyped . to avoid such a challenging cases , ( 1 ) one might select only single band ssr loci and code a dataset as a codominant marker type , yet such a single band ssrs are usually not many in polyploid crop genomes and yield also multiple bands when very diverse germplasm resources are genotyped ; ( 2 ) alternatively , multiple - band ssrs with unknown allelic relationship may be scored as a dominant marker taking each band as an independent marker locus ( uniquely ) with a clear size band separation ( see , e.g. , [ 52 , 56 ] ) . could a dominant marker data be used for ld quantification ? there are some reports where ld level of natural forest tree populations has been measured using dominant markers ( aflps ) and commonly used statistical approach ( see , e.g. , ) . there are also a number of reports where dominantly coded ( present versus absent ) marker data of rapd , rflp , aflp , candidate gene ( caps ) , and ssrs were successfully used in genome - wide ld analyses and ld - based association mapping in plants ( see , e.g. , [ 1719 , 56 , 58 , 59 , 60 ] ) , demonstrating the feasibility of dominantly coded molecular data in revealing of haplotypic associations . although a dominant type of coding has limited statistical power compared to codominant markers in population - based analyses because of missing heterozygote information , previous studies suggested that it can be successfully applied to the clustering of individuals and grouping of populations using a bayesian approach when a large number of loci are genotyped [ 61 , 62 ] . dominant - type markers can be a useful tool to estimate the kinship coefficients between individuals . recently , li et al . investigated the use of dominant markers in estimation of ld in diploid species and developed appropriate em algorithm . based on their conclusion from the comparative data simulation of dominant versus codominant markers , the dominant - type markers could effectively be used in ld analysis with preferentially large number of marker loci and population sample sizes of 200 for high heterozygous ( proportion of alternative alleles ( present versus absent ) in a data set , i.e. , 0.5 versus 0.5 ) marker data or with even larger sample size 400 for low - heterozygous ( i.e. , 0.9 versus 0.1 ) dominant markers . it is also recommended that a mixture of codominant and dominant markers should be used to better characterization of a genetic structure of a population . ld quantification and ld - based association mapping have been a research objective in plants beginning with the model organism as arabidopsis , and now extended to crops as maize , barley , durum wheat , spring wheat , rice , sorghum , sugarcane , sugar beet , soybean , and grape , as well as in forest tree species , and forage grasses . sequenced 0.51 kb long 13 fragments from a 250 kb region surrounding the flowering time fri gene in a 20 global sample of a. thaliana , highly selfing model plant species . later , investigation of the same authors with markers surrounding the disease resistance locus rpm1 in a globally - derived set of 96 arabidopsis accessions revealed that a genome - wide ld extended up to 50250 kb . ld blocks extended up to 50100 cm in local michigan arabidopsis populations . these long - stretched lds in local arabidopsis population were explained as a genetic bottleneck or founder effect through introduction a. thaliana into north america in recent past ( 200 years ago ) . in contrast , in other study that targeted the region surrounding another disease resistance gene rps5 , tian et al . likewise , ld quickly decays within 1050 kb distance around the clavata 2 region of arabidopsis . recently , ehrenreich et al . reported the ld decay within ~10 kb in extensive sequence analysis of 600-bp fragments of the regions more axillary growth 2 ( max2 ) and more axillary growth 3 ( max3 ) in a panel of 96 accessions from a restricted geographic range in central europe . in their genome - wide survey of 1347 fragments of 600-bp lengths , plagnol et al . reported that ld completely disappears after ~100 kb , which is comparable to that observed in human . in maize ( zea mays l. ) , a highly outcrossing crop species , very rapid genome - wide ld decay was determined . tenaillon et al . first reported the extent of ld for maize , genotyping of 21 loci of chromosome 1 over the 25 individuals of the exotic landrace and united states maize germplasm . an average ld decay was determined to occur within 400 bp with r = 0.2 and extended up to 1000 bp ( ~1 kb ) in a group of us inbred lines . later , remington et al . also reported a very rapid decline of ld in their survey of 6 genes ( 1.210 kb long ) in 102 inbred lines , including tropical and semitropical lines with a wide genetic diversity . for these surveyed genes , ld declined generally within 2002000 bp with r = 0.1 except sugary1 ( su1 ) loci , where ld remained significant ( r = 0.3 0.4 ) for 10 kb distance . this was explained by strong selective episodes in su1 gene . in the same study , remington et al . found higher level of ld with 47 ssr markers compared to those obtained from snp data . this result was explained by different mutation rate of these two marker systems that tends to capture different historic information . ( i.e. , restricted to particular regions , meaning that high ld stretches interspersed with regions of low ld ) extended up to 3 kb . examined the genetic diversity and ld in a cross section of 147 european and united states elite inbred material with 100 ssrs . they reported an average significant ( p < .5 ) ld block size of 26 cm for flint group , or 41 cm for dent group with nonuniform distribution of ld among 10 chromosomes . they showed a very long stretched ld blocks up to 105 cm in chromosome 2 and up to 103 cm in chromosome 7 in flint and dent groups , respectively . obtaining of different result from earlier studies was explained due to using ( 1 ) much higher marker density , and ( 2 ) both related and unrelated inbred lines . in another study , the same authors examined 72 european elite inbred lines with 452 aflp and 93 ssr markers and reported much shorter average ld block sizes for aflp ( 4 cm ) , but extensive ld for ssr ( 30 - 31 cm ) in both flint and dent germplasm groups . this suggested a potential for exploiting both markers in association mapping , but with the favor of ssrs over aflps because of power of detecting ld . recently , andersen et al . reported that ld is persisted over entire 3.5 kb phenylalanine ammonia lyase ( pal ) gene with the r > 0.2 in a survey of 32 european maize inbred lines . in the selfing tetraploid wheat ( triticum durum desf . ) , maccaferri et al . quantified ld in a 134 durum wheat accessions that extended up to 10 and 20 cm with d = 0.67 and 0.43 , respectively . in hexaploid wheat ( triticum aestivum l ) , almost completely self - pollinating species , strong ld was determined to occur on average within < 1 and ~5 cm for region on chromosome 2d and centromeric region 5a that was surveyed with 36 ssr markers in a 95 cultivars of winter wheat . investigated the genome - wide ld among 43 us wheat elite cultivars and breeding lines representing seven us wheat market classes using 242 ssrs distributed throughout the wheat genome . for this germplasm collection , a genome - wide ld estimates were generally less than 1 cm for the genetically linked loci pairs . most of the ld regions observed were between loci less than 10 cm apart , suggesting ld is likely to vary widely among wheat populations . tommasini et al . reported that ld on chromosome 3b extended up to 0.5 cm in 44 varieties or 30 cm in 240 ril populations of winter wheat , surveyed with 91 ssr and sts markers . this suggested usefulness of cultivar germplasm over biparental mapping population in association mapping . in rice ( oryza staiva l ) , a selfing species , garris et al . examined the ld surrounding disease resistance locus xa5 using 21 ssrs in a survey of 114 rice accessions . agrama and eizenga investigated ld patterns in a worldwide collection of oryza staiva , and its wild relatives using 176 ssr markers . although it was not specifically indicated , ld decay plot suggests a long range ld declining ~50 cm with d = 0.5 in the international and us interestingly , ld persisted over an average of 225 cm distance with significant d > 0.5 in a wild accessions . in contrast , many other studies reported a less extent of ld in wild and landrace ( broad - based ) germplasm and high extent of ld in cultivar ( narrow - based ) germplasm resources in plants [ 9 , 43 ] . reported that ld in o. rufipogon decays within 5 kb , while it declines at 50 kb in o. sativa ssp . observed that the extent of ld is greatest in temperate japonica ( > 500 kb ) , followed by tropical japonica ( ~150 kb ) and indica ( ~75 kb ) that was revealed by using unlinked snps . ld extends over a shorter distance in o. rufipogon ( 40 kb ) than in any of the o. sativa groups assayed in their study . ld also has been extensively quantified another highly self - pollinated crop , barley ( hordeum vulgare l ) , where the extent of ld varied from 10 cm to 50 cm range depending on assayed set of a germplasm [ 17 , 77 ] . measured ld in four genes surrounding hardness locus ( ha ) in three different gene pools and reported a long stretched ld extended up to at least 212 kb in inbred barley and 98 kb in landrace barley germplasm . in contrast to these long range lds observed in barley germplasm , morrell et al . reported a rapid decay of ld detected within 300 bp in their study of 18 nuclear genes ( average length of 1 361.1 bp ) in 25 diverse wild barley accessions . in that , ld completely disappeared within a 1200 bp distance . this demonstrates another example of variability of ld quantification across germplasm resources , breeding material , and regions tested . furthermore , genome - wide ld has been quantified for many other plant species that extended up to 10 cm in sugar cane ( saccharum ) , 1050 cm in soybean ( glycine max ) [ 79 , 80 ] , 3 cm in sugar beet ( beta vulgaris l ) , 50 cm in sorghum ( sorghum bicolor ) , 510 cm in grape ( vitis vinifera l ) , 1634 kb in poplar ( populus trichocarpa ) , < 500 bp in european aspen ( poplus termula ) , 2000 bp in loblolly pine ( pinus taeda ) , 1000 bp in douglas - fir ( pseudostuga menziensii ) , 100200 bp in norway spruce ( picea abies ) , 200 - 1 , 200 bp in silage maize ( zea mays l ) [ 87 , 88 ] , and 5002000 bp in ryegrass ( lolium perenne ) [ 8991 ] . also , ld quantification for other important crops , perhaps , is in progress . in this context , recently , we have quantified ld level for improved varieties and landrace stock germplasm of cotton ( gossypium hirsutum l ) . survey of 200 microsatellite markers in 335 g. hirsutum variety germplasm demonstrated that a genome - wide averages of ld extended up to genetic distance of 25 cm with r > 0.1 . likewise , our another companion study using 95 core set microsatellite markers in a total of 286 exotic g. hirsutum revealed that a genome - wide averages of ld decays within the genetic distance at < 10 cm in the landrace stocks germplasm and > 30 cm in photoperiodic variety germplasm , providing evidence of the potential for association mapping of agronomically important traits in cotton ( abdurakhmonov et al . important information and implication for association mapping gained from above studies are that : ( 1 ) ld more quickly declines in outcrossing plant species than highly self - pollinating plants , enabling high resolution mapping of a trait of interest in outbreeder plant germplasm . at the same time , ld rapidly declines in crop variety groups ( even in selfing species ) compared to populations derived from biparental crosses , which provides an advantage of discovery more polymorphisms in the variety germplasms than biparental populations of self - pollinated crops ; ( 2 ) the extent of ld varies across the genomic regions , among population samples and between species with the examples of localized ld ; ( 3 ) ld measures differ per marker systems used as a reflection of capturing of different historic information in a genome due to different mutation rate ( e.g. , snp versus ssr or aflp versus ssr ) ; ( 4 ) an estimate of genome - wide averages for the extent of ld in plant germplasm may not adequately reflect ld patterns of specific regions or specific population groups . each of these specific regions or population groups should additionally be explored for the extent of ld in order to conduct successful association mapping of variants within regions or populations of interest ; ( 5 ) ld blocks in narrow - based germplasm groups are longer than broad - based germplasm groups in plants [ 9 , 43 ] . this suggests an opportunity perform coarse mapping with less number of markers in narrow - based plant germplasm and then fine mapping in broad - based plant germplasm , assuming that genetic causations is sufficiently similar across germplasm groups . this also suggest an opportunity develop a set of mapping populations with the required amount of ld and diversity for high - resolution mapping through directed crossing between selected broad- and narrow - based germplasm groups ; and ( 6 ) confounding population characteristics and biological behavior have serious impact on pattern and structure of ld in plant germplasm resources that need to be taken into consideration in conducting unbiased association mapping . there are many types of different methodologies that have been developed and initially are widely used for association mapping studies in human ( comprehensively reviewed by schulze and mcmahon ) , yet perfectly applicable without change or case - to - case modifications for wide range of organisms , including plants . lately , some considerably successful achievements have been made to develop powerful , more precise , and unbiased population - based association - mapping methodology for plants . here , we provide a brief overview for a basic concept and ideology of widely used pioneer methodologies for association mapping , and then highlight the latest developments in the methodology and experimental design of association mapping in plant population with the examples of association mapping of useful traits in crop species . the classical methodology and design of association mapping is case and control ( also referred to as case - control ) approach that identifies the causative gene tags in the comparison of allele frequencies in a sample of unrelated affected ( referred to as cases ) individuals and a sample of uninfected or healthy individuals ( referred to as controls ) [ 93 , 94 ] . this design requires an equal numbers of unrelated and unstructured case - control samples for accurate mapping . the pearson chi - square test , fisher 's exact test , or yates continuity correction can be used for a comparison of allele frequencies and detection of an association between a disease phenotype and marker . although favored , the random sampling individuals from a population do not provide the equal representation of case and controls in the mapping population since cases in the population are usually low , thus requires special efforts to collect the cases . case and control approach is seriously affected by the existence of population structure and stratification that caught the attention of scientist . falk and rubinstein developed a haplotype relative risk ( hrr ) approach that minimizes , but not eliminates population stratification issues in association mapping . in that , first , a pseudocontrol group ( containing combination of two alleles that are not transmitted to affected offspring ) is created ; then , the marker allele frequencies in case and pseudocontrol groups are correlated . to efficiently eliminate the confounding effects coming from population structure and stratification , spielman et al . developed transmission disequilibrium test ( tdt ) method that compares transmission versus nontransmission of marker alleles to affected offspring by using chi - square test , assuming a linkage between marker and trait . the tdt design requires genotyping of markers from three individuals : one heterozygous parent , one homozygous parent , and one affected offspring . although hrr performs better with unstructured sample than tdt because of its power to completely eliminate spurious association with good experimental design , later is widely used as a tool for unbiased fine mapping of traits in the presence of linkage with a biallelic , one marker model that can accommodate pedigree structure [ 30 , 93 ] . nonetheless , initial tdt approach had issues with the use of multiallelic markers , multiple markers , missing parental information , extended ( larger ) pedigrees , and complex quantitative traits . to address these issues , a variety of extensions of tdt approach were developed and applied for multiallelic markers ( i.e. , gtdt , etdt , mc - tm ) [ 98102 ] , multiple markers [ 103105 ] , missing parental information ( curtis - test , s - tdt , sdt , 1-tdt , c - tdt or rc - tdt ) [ 96 , 106110 ] , which were reviewed by schulze and mcmahon in detail . shortly after publication of various extensions of tdt to multiallelic and multiple markers , the extensions for x - linked genes , such as xs - tdt or xrc - tdt were developed and applied . tdt approach was also extended to pedigrees of any size as a pdt approach [ 112 , 113 ] that was demonstrated more powerful than tdt , and s - tdt or sdt under the assumption of high disease prevalence [ 93 , 114 ] . further , there were many studies to extend the tdt approaches to qtl and covariates . one of the comprehensive approaches , qtdt was developed with its three different extensions for quantitative traits for any pedigree structure [ 115 , 116 ] . these family - based association - mapping approaches have their other improvements using more powerful statistical and robust algorithmic procedures , such as likelihood - base statistics and em algorithm ( tdtlike , lrt , em - lrt ) [ 117119 ] . the unified family - based association test package ( fbat ) incorporating some of tdt is also developed [ 120122 ] to deal with wide types of experimental designs . control and family - based designs , referred to as identity by decent ( ibd ) mapping , haplotypes - sharing analysis ( hsa ) , and decay of haplotypes sharing ( dhs ) , involves the analysis of haplotypes by testing the length of haplotypes in the data sample , assuming affected individuals will have a longer haplotypes than controls . although family - based association mapping methodology is effective to control confounding effects of a sample and remove spurious associations , it is less powerful design and have its disadvantageous sides compared to case - control that led to develop the methodologies with better controlling of population structure and stratification . such an improved methodology for a case and control design or random samples from a population involves the use of additional markers that have neutral effect ( null loci ) to the trait of interest in the analysis . this approach is referred to as the genomic control ( gc ) that finds confounding effects of a population and corrects it , thus enabling to remove spurious associations [ 127 , 128 ] . although gc is powerful then tdt , it will not remove spurious associations in highly structured populations . put it as methods like genomic control , which simply rescale p - values without changing the ranking of loci are not likely to be useful in genome - wide scans where the existence of true positives is not in doubt . sa first searches a population for closely related clusters / subdivisions using bayesian approach , and then uses the clustering matrices ( q ) in association mapping ( by a logistic regression ) to correct the false associations . population structure and shared coancestry coefficients between individuals of subdivisions of a population can be effectively estimated with structure program using several models for linked and unlinked markers . similar type of methodology measuring and using the population subdivisions ( k ) in association mapping referred to as mixture model was proposed by several other studies [ 131 , 132 ] . however , sa incorporating only population structure information in the analysis is not good enough itself when highly structured population with some degree of related individuals used in the association mapping . developed new methodology , a mixed linear model ( mlm ) that combines both population structure information ( q - matrix ) and level of pairwise relatedness coefficientskinship ( k - matrix ) in the analysis . to perform mlm : ( 1 ) q - matrix is generated using structure , ( 2 ) the pairwise relatedness coefficients between individuals of a mapping population ( k - matrix ) measured using spagedi software , and ( 3 ) then both q- and k - matrices are used in association mapping to control spurious associations . although computationally intensive , mlm approach found to be effective in removing the confounding effects of the population in association mapping . extensively tested the mlm approach of yu et al . in their global set of 95 highly structured arabidopsis population and came to overall agreement with better performance of q + k mlm model than any of the other tests that used k- or q - matrix alone . however , they also noted that ( 1 ) k matrix would alone be good enough if a kinship estimated as a proportion of shared haplotypes for each pair of individuals ( as denoted k * ) ; ( 2 ) the replacement of q - matrix ( from the computational intensive structure analysis ) with p - matrix ( from more robust principal component analysis ) performed similarly to mlm of yu et al . , thus suggesting a potential for future replacements ; ( 3 ) removing of the confounding effects will also subject to remove true associations with biological effect , which is strongly correlated with population structure that requires a caution ; and ( 4 ) in a small and highly structured population , the causations with major effect should be expected to be found and , perhaps , larger samples and adequate marker densities are needed for genome - wide dissection of the most traits of interest segregating in an association mapping population . there are other types of mixed models for association mapping that have its own advantages to control population confounding effects and tag a genetic causative of a trait of interest . one of such mixed models utilizes a sample with pedigree information to measure a pedigree - based relatedness and incorporates it directly in qtl - mapping and association mapping [ 59 , 137 , 138 ] . this type of mixed model for known pedigree population combines haplotype effects with pedigree - based structure of variance - covariance relatedness matrix and random polygenic effect that control the population structure [ 59 , 139 ] . the efficiency of pedigree population for association mapping depends on the population size of pedigree founders ( i.e. , pedigree population obtained from just two parents will not provide significant level of ld ) and the level of relatedness of the founders . latter is very important and may still lead to spurious association due to initial population structure ( mostly unknown ) coming from founders that needs to be analyzed also by using structure . however , as stated by malosetti et al . and others the pedigree - based mixed model is highly appropriate in association mapping in crops due to ( 1 ) plant breeding programs have already generated many useful pedigree populations that contain ld useful for association studies but can not be used as an independent ld - mapping population , and ( 2 ) many historical trait data sets in plant breeding are unbalanced that have been collected over multiple - years , and multienvironmental trials . at the same time , issues with obtaining the fine - grained pedigree information and difficulty of finding population structure of narrow - based elite cultivars are the concern in pedigree - based mixed model . there is another mixed model that combines the bayesian variable selection for mapping multiple qtls and ld mapping method , incorporating estimates of population structure , but not relatedness . authors stated that incorporation of multiple qtl effects and population structure efficiently reduces spurious association and useful for future whole genome associations , with the development of more complex models dealing with differences of ld and effect of qtl alleles between populations . the other mixed model approach combines qtl and ld analyses of distinct studies . in that , qtls or candidate genes with already annotated biological function(s ) are used as a priori information in association mapping [ 140 , 141 ] . this is one of the effective alternative strategies in association mapping that allow reducing the total amount of marker genotyping ( because of preselecting of markers restricted to qtl region ) in less number of individuals . the power of association mapping is the probability of detecting the true associations within the mapping population size that really depends on ( 1 ) the extent and evolution of the ld in a population , ( 2 ) the complexity and mode of gene action of the trait of interest , ( 3 ) sample size and experimental design . the power can be increased utilizing the better data ( knowledgeable experimental design and accurate measurements ) and increasing the sample size . in qtl mapping studies , there are specific statistical approaches to estimate the false - positive level of the obtained strong ( p - value ) associations ( control for type i error ) such as a permutation test or false discovery rate ( fdr ) . a statistical approach within the bayesian framework is used test the reliability of obtained significance ( p - values ) in association mapping because of possibility of getting unreliable values due to ( 1 ) overestimation of effects ( selection bias ) , ( 2 ) association coming from neglecting confounding effects of a sample , ( 3 ) poor experimental design , and ( 4 ) instability of genetic effects across different environments . ball developed a methodology , combining the bayesian and non - bayesian approaches , that determines the bayes factors guiding to properly design the experiments with given power to detect reliable effects . to detect the reliable effects in association mapping , experiments should be designed at least with the bayes factor of 20 that may require much larger sample sizes . bayes factor provides stronger evidence than conventional p - values . if given bayes factor value ( say b = 20 ) reached with larger sample than the original experimental design , then , the original results indicate a very weak evidence to provide the real effects . at this point , requirement for larger sample size might make association mapping disadvantageous over a traditional qtl - mapping . however , the sample size for association mapping can be decreased keeping the high power with ( 1 ) preselecting a priori known qtl regions or candidate genes ( from qtl - mapping and expression analyses ) , ( 2 ) using the large populations with samples longer ld block that require a less number of markers to find useful associations , ( 3 ) an alternative experimental design ( i.e. , tdt ) , and ( 4 ) choosing the single marker from the haplotypes of interest that would cut also marker number and so genotyping cost . bayes factor can be calculated using r function of ld.design from lddesign package the pioneer association studies in plants were performed by beer et al . in oat , and by virk et al . in rice . associated 13 qtl with rflp loci using 64 oat varieties and landraces , yet without considering the population structure that resulted in more increased associations than what were obtained in separate analysis of subpopulations . virk et al . predicted 6 trait values using rapd markers in rice germplasm . later , association mapping was extended to sea beet , barley , maize , wheat , potato , more examples in rice , and arabidopsis that have utilized population level of ld considering a population structure . reported association of alfp markers with bolting gene in sea beat . in barley , various traits such as yield , yield stability , heading date , flowering time , plant height , rachilla length , resistance to mildew and leaf rust were associated with many different types of molecular markers [ 17 , 18 , 157 , 158 ] . in maize , flowering time and plant height [ 43 , 69 ] were associated using snp and ssrs . following these pioneer studies of association mapping in maize , several other traits such as phenotypic variation in flowering time , endosperm color , starch production , maysin and chlorogenic acid accumulation , cell wall digestibility , and forage quality were associated using snp markers of candidate genes [ 71 , 87 , 88 , 149153 ] . in wheat , breseghello and sorrells reported first association mapping of kernel size and milling quality in a collection of usa winter wheat using ssrs . following this work , association mapping of a high molecular - weight glutenin and blotch resistance were reported that utilized snps , ssrs , and sts markers . in rice , association mapping has not widely been applied yet due to highly structured population of rice ( due to high selfing ) [ 58 , 133 ] . however , zhang et al . successfully used association mapping for multiple agronomic traits using discriminant analysis ( da ) with ssr and aflp markers . recently , iwata et al . associated rflp markers with width and length of milled rice grains in a set of 332 rice germplasm using their multiple qtl model considering the population structure . association mapping approach was also successfully applied in tetraploid potato where resistance to wilt disease , bacterial blight , phytophtora that utilized a pedigree - based mixed model . to date association mapping has also been extended to long lifespan plant species , forest tree populations , where associations of polymorphisms in cinnamoil coa reductase ( ccr ) with earlywood microfibril angle trait , and polymorphisms a putative stress response gene with wood density and wood growth rate were reported . there are also the examples of association mapping successes for cold tolerance , flowering time , water - soluble carbohydrate content , and forage quality in forges species that have recently been reviewed by dobrowolski and forster ( table 1 ) [ 87 , 88 , 161 ] . association mapping of traits in arabidopsis also has been reported and overall suitability of the approach well documented . later zhao et al . revisited to these association results with their mixed model approach and reproved some of previously reported associations ( with phyc ) , but challenged the power of these associations detected by using they put it as clearly , none of these polymorphisms would have been picked up in a genome - wide scan while noting the use of different sample and trait measurements in the original studies . they also reported one of the significant flowering time associated polymorphisms in clf gene in their genome - wide analysis using mlm . flowering time ( in fri gene ) and pathogen resistance ( in rpm1 , rps5 , and rps2 genes ) associated polymorphisms were also reported . recently , ehrenreich et al . reported polymorphisms of candidate genes ( sps1 , max2 , and max3 ) associated with branching architecture in a survey of 36 genes involved in branch development that were genotyped in a panel of 96 arabidopsis accessions from central europe . table 1 summarizes the ld and association mapping efforts in plants including some of very recent whole genome association mapping studies . as one can see , within the frame of above highlighted association studies in plants , various association mapping methodologies ( table 1 ) , molecular markers ( both dominant and co - dominant markers ) , and plant germplasm resources ( including landrace stocks , elite germplasm , and experimental populations e.g . , rils ) have been utilized . identifying of the most appropriate approach and marker systems , therefore , is challenging and might be irrelevant case - to - case basis . choosing the appropriate association mapping depends on ( 1 ) the extent and evolution of the linkage disequilibrium in a population , ( 2 ) the level of population structure and stratification , ( 3 ) availability of pedigree information , ( 4 ) complexity of the trait of interest under study , and ( 5 ) availability of the genomic information and resources . based on reported studies , gc is favored approach when population structure is suspected , but failed to be detected ; however , mlm considering both relatedness and population structure and pedigree - based mixed model or multiple qtl model performs well in most cases with highly structured and stratified population although one still might argue based on his own experience , knowledge , and type of gemplasm used . according to stich et al . , sa and mlm models do not explicitly correct for ld caused by selection and genetic drift , the major factors causing ld in plant germplasm and breeding materials . hence stich et al . suggested use of family based association approach with breeding materials . however , again the choice of methodology greatly depends on the germplasm used for mapping . the germplasm materials used for association mapping were comprehensively discussed by breseghello and sorrells . thus the association mapping methodology , initially developed by the human geneticists , has found its successive application in plant germplasm resources , in particular after recent improvements in minimization of spurious associations . the examples of association mapping studies performed in various plant germplasm resources including model plant arabidopsis and extended to various crop germplasm largely demonstrate the flourish of crop genomics era with the utilization of powerful ld - based association mapping tool . this is also a good indicative of the potential utilization of this technology with the other crops and plant species in the future . currently , a number of such studies are , perhaps , in progress in many laboratories worldwide . the near - future completion of genome sequencing projects of crop species , powered with more cost - effective sequencing technologies , will certainly create a basis for application of whole genome - association studies , accounting for rare and common copy number variants ( cnv ) ( for review see , e.g. , ) and epigenomics details of the trait of interest in plants , which is widely being applied in human genetics with great success . this will provide with more powerful association mapping tool(s ) for crop breeding and genomics programs in tagging true functional associations conditioning genetic diversities , and consequently , its effective utilization .

compared to the conventional linkage mapping , linkage disequilibrium ( ld)-mapping , using the nonrandom associations of loci in haplotypes , is a powerful high - resolution mapping tool for complex quantitative traits . the recent advances in the development of unbiased association mapping approaches for plant population with their successful applications in dissecting a number of simple to complex traits in many crop species demonstrate a flourish of the approach as a powerful gene tagging tool for crops in the plant genomics era of 21st century . the goal of this review is to provide nonexpert readers of crop breeding community with ( 1 ) the basic concept , merits , and simple description of existing methodologies for an association mapping with the recent improvements for plant populations , and ( 2 ) the details of some of pioneer and recent studies on association mapping in various crop species to demonstrate the feasibility , success , problems , and future perspectives of the efforts in plants . this should be helpful for interested readers of international plant research community as a guideline for the basic understanding , choosing the appropriate methods , and its application .

1. INTRODUCTION 2. GENETIC MAPPING OF CAUSATIVE VARIANTS 3. ASSOCIATION MAPPING AS AN ALTERNATIVE APPROACH 4. LINKAGE DISEQUILIBRIUM (LD) 5. ASSOCIATION STUDIES IN PLANTS 6. CONCLUSIONS

however , plant germplasm resources worldwide , comprising of wild plant species , modern cultivars , and their crop wild relatives , are the important reservoirs of natural genetic variations , originated from a number of historical genetic events as a respond to environmental stresses and selection through crop domestication [ 1 , 5 ] . identification of qtls of agronomic importance and its utilization in a crop improvement further requires mapping of these qtls in a genome of crop species using molecular markers [ 1 , 6 ] . this was the major breakthrough and accomplishment in many crops in genomics era since the end of the 20th century , and now extended to flourish in the 21st century . in this review , we provide a brief description for the concept of genetic mapping ; then , as a flourish of the crop genomics era , we thoroughly review one of the powerful genetic mapping tools for crops , linkage disequilibrium ( ld)-based association study , as a high - resolution , broader allele coverage , and cost effective gene tagging approach in plant germplasm resources . the main goal of genetic mapping is to detect neutrally inherited markers in close proximity to the genetic causatives or genes controlling the complex quantitative traits . genetic mapping can be done mostly in two ways : ( 1 ) using the experimental populations ( also referred to as biparental mapping populations ) that is called qtl - mapping as well as genetic mapping or gene tagging , and ( 2 ) using the diverse lines from the natural populations or germplasm collections that is called ld - mapping or association mapping . , and further basic description of the approach here would be a redundant . for detailed concept , models and methodologies , problems , and perspectives of linkage analysis , here , we briefly outline linkage mapping procedure for the sake of highlighting the merits of the alternative approach - association mapping . secondly , these experimental populations including a large number of progenies or lines are measured for the segregation of a trait of interest in the different environmental conditions . in linkage mapping , the major limitation , hampering the fine mapping , is associated with the availability of only a few meiotic events to be used that occurred since experimental hybridization in a recent past . turning the gene - tagging efforts from biparental crosses to natural population of lines ( or germplasm collections ) , and from traditional qtl - mapping to linkage disequilibrium ( ld)-based association study became a powerful tool in mapping of the genes of interest . this ld can be detected statistically , and has been widely applied to map and eventually clone a number of genes underlying the complex genetic traits in humans [ 1316 ] . , many alleles evaluated simultaneously ) , ( 2 ) likelihood for a higher resolution mapping because of the utilization of majority recombination events from a large number of meiosis throughout the germplasm development history , ( 3 ) possibility of exploiting historically measured trait data for association , and ( 4 ) no need for the development of expensive and tedious biparental populations that makes approach timesaving and cost - effective [ 1719 ] . although the overall approach of population - based association mapping in plants varies based on the methodology chosen ( see below sections ) , assuming structured population samples , the performance of association mapping includes the following steps ( see figure 1 ) : ( 1 ) selection of a group of individuals from a natural population or germplasm collection with wide coverage of genetic diversity ; ( 2 ) recording or measuring the phenotypic characteristics ( yield , quality , tolerance , or resistance ) of selected population groups , preferably , in different environments and multiple replication / trial design ; ( 3 ) genotyping a mapping population individuals with available molecular markers ; ( 4 ) quantification of the extent of ld of a chosen population genome using a molecular marker data ; ( 5 ) assessment of the population structure ( the level of genetic differentiation among groups within a sampled population individuals ) and kinship ( coefficient of relatedness between pairs of each individuals within a sample ) ; and ( 6 ) based on information gained through quantification of ld and population structure , correlation of phenotypic and genotypic / haplotypic data with the application of an appropriate statistical approach that reveals marker tags positioned within close proximity of targeted trait of interest . as a starting point for association mapping , it is important to gain knowledge of the patterns of ld for genomic regions of the target organisms and the specificity of the extent of ld among different populations or groups to design and conduct unbiased association mapping [ 20 , 21 ] genetic linkage generally refers to coinheritance of different loci within a genetic distance on the chromosome . there are two terms used in population genetics , linkage equilibrium ( le ) , and linkage disequilibrium ( ld ) to describe linkage relationships ( co - occurrence ) of alleles at different loci in a population . the merits , sensitivity , comparison , appropriate statistical tests , and calculation methodology for these ld measures with the utilization of biallelic or multiallelic loci have been extensively described in the literature in detail [ 10 , 26 , 30 , 31 ] , and have recently been reviewed by gupta et al . hence here we highlight only some of key utility properties of ld measures to provide a brief understanding the merits of ld in association mapping . choosing the appropriate ld measures really depends on the objective of the study , and one performs better than other in particular situations and cases ; however , d and r is the most commonly used measures of ld [ 25 , 26 ] . d is informative for the comparisons of different allele frequencies across loci and strongly inflated in a small sample size and low - allele frequencies ; therefore , intermediate values of d is dangerous for comparative analyses of different ld studies and should be verified with the r before using for quantification of the extent of ld . the statistical models of zld measures for biallelic and multilocus data , its application for natural populations , and inference the genetic and demographic events from the comparisons of gld and zld results as well as implication for whole genome association studies ( wgas ) were excellently addressed and described by a number of studies [ 3537 ] . pairwise ld can be depicted as a color - code triangle plot ( figure 2 ) based on significant pairwise ld level ( r , and p - value as well as d ) that helps to visualize the block of loci ( red blocks ) in significant ld . ld blocks are very useful in association mapping when sizes are calculated , which suggest the needs for the minimum number of markers to efficiently cover the genome - wide haplotype blocks in association mapping . although mutation and recombination are one of the strong impact factors influencing ld , generally , factors affecting ld can be grouped into two categories : ( 1 ) factors that increasing ld , and ( 2 ) factors that decreasing ld . however , more often there is a significant ld between pairs of loci located far from each other or even in different chromosomes that might cause spurious correlations in association mapping . one of those factors is selection that generate ld between unlinked loci through a hitchhiking effect ( high - frequency sweeping and fixation of alleles flanking a favored variant ) , and epistatic selection or the so - called coadapted genes that is the result of coselection of loci during breeding for multiple traits , common in traditional crop breeding programs worldwide . very extensive level of ld ( means ld persists within a long distance ) , theoretically , reduces a number of markers needed for association mapping , but makes resolution lower ( coarse mapping ) . hence , choosing a population with low or high level of ld depends on the objective of association mapping study . ld generated by selection , population structure , relatedness , and genetic drift might be theoretically useful for association mapping in specific situations and population groups that reduces number of markers needed for association mapping [ 9 , 22 ] , but requires serious attention to control factors affecting ld ( e.g. , population structure and relatedness ) to perform unbiased population - based association mapping in plants [ 41 , 47 ] ( see next sections ) . hence in the quantification of ld and association mapping , markers with minor allele frequency of 510% ( varied from study to study ) are ( 1 ) removed before analysis ( see , e.g. , [ 17 , 18 , 43 , 44 , 51 ] ) , ( 2 ) pooled into common allelic class ( see , e.g.,[44 , 46 ] ) , and ( 3 ) replaced with missing values ( see , e.g. to avoid such a challenging cases , ( 1 ) one might select only single band ssr loci and code a dataset as a codominant marker type , yet such a single band ssrs are usually not many in polyploid crop genomes and yield also multiple bands when very diverse germplasm resources are genotyped ; ( 2 ) alternatively , multiple - band ssrs with unknown allelic relationship may be scored as a dominant marker taking each band as an independent marker locus ( uniquely ) with a clear size band separation ( see , e.g. there are also a number of reports where dominantly coded ( present versus absent ) marker data of rapd , rflp , aflp , candidate gene ( caps ) , and ssrs were successfully used in genome - wide ld analyses and ld - based association mapping in plants ( see , e.g. although a dominant type of coding has limited statistical power compared to codominant markers in population - based analyses because of missing heterozygote information , previous studies suggested that it can be successfully applied to the clustering of individuals and grouping of populations using a bayesian approach when a large number of loci are genotyped [ 61 , 62 ] . ld quantification and ld - based association mapping have been a research objective in plants beginning with the model organism as arabidopsis , and now extended to crops as maize , barley , durum wheat , spring wheat , rice , sorghum , sugarcane , sugar beet , soybean , and grape , as well as in forest tree species , and forage grasses . obtaining of different result from earlier studies was explained due to using ( 1 ) much higher marker density , and ( 2 ) both related and unrelated inbred lines . likewise , our another companion study using 95 core set microsatellite markers in a total of 286 exotic g. hirsutum revealed that a genome - wide averages of ld decays within the genetic distance at < 10 cm in the landrace stocks germplasm and > 30 cm in photoperiodic variety germplasm , providing evidence of the potential for association mapping of agronomically important traits in cotton ( abdurakhmonov et al . important information and implication for association mapping gained from above studies are that : ( 1 ) ld more quickly declines in outcrossing plant species than highly self - pollinating plants , enabling high resolution mapping of a trait of interest in outbreeder plant germplasm . at the same time , ld rapidly declines in crop variety groups ( even in selfing species ) compared to populations derived from biparental crosses , which provides an advantage of discovery more polymorphisms in the variety germplasms than biparental populations of self - pollinated crops ; ( 2 ) the extent of ld varies across the genomic regions , among population samples and between species with the examples of localized ld ; ( 3 ) ld measures differ per marker systems used as a reflection of capturing of different historic information in a genome due to different mutation rate ( e.g. each of these specific regions or population groups should additionally be explored for the extent of ld in order to conduct successful association mapping of variants within regions or populations of interest ; ( 5 ) ld blocks in narrow - based germplasm groups are longer than broad - based germplasm groups in plants [ 9 , 43 ] . this also suggest an opportunity develop a set of mapping populations with the required amount of ld and diversity for high - resolution mapping through directed crossing between selected broad- and narrow - based germplasm groups ; and ( 6 ) confounding population characteristics and biological behavior have serious impact on pattern and structure of ld in plant germplasm resources that need to be taken into consideration in conducting unbiased association mapping . here , we provide a brief overview for a basic concept and ideology of widely used pioneer methodologies for association mapping , and then highlight the latest developments in the methodology and experimental design of association mapping in plant population with the examples of association mapping of useful traits in crop species . although hrr performs better with unstructured sample than tdt because of its power to completely eliminate spurious association with good experimental design , later is widely used as a tool for unbiased fine mapping of traits in the presence of linkage with a biallelic , one marker model that can accommodate pedigree structure [ 30 , 93 ] . nonetheless , initial tdt approach had issues with the use of multiallelic markers , multiple markers , missing parental information , extended ( larger ) pedigrees , and complex quantitative traits . to perform mlm : ( 1 ) q - matrix is generated using structure , ( 2 ) the pairwise relatedness coefficients between individuals of a mapping population ( k - matrix ) measured using spagedi software , and ( 3 ) then both q- and k - matrices are used in association mapping to control spurious associations . however , they also noted that ( 1 ) k matrix would alone be good enough if a kinship estimated as a proportion of shared haplotypes for each pair of individuals ( as denoted k * ) ; ( 2 ) the replacement of q - matrix ( from the computational intensive structure analysis ) with p - matrix ( from more robust principal component analysis ) performed similarly to mlm of yu et al . , thus suggesting a potential for future replacements ; ( 3 ) removing of the confounding effects will also subject to remove true associations with biological effect , which is strongly correlated with population structure that requires a caution ; and ( 4 ) in a small and highly structured population , the causations with major effect should be expected to be found and , perhaps , larger samples and adequate marker densities are needed for genome - wide dissection of the most traits of interest segregating in an association mapping population . and others the pedigree - based mixed model is highly appropriate in association mapping in crops due to ( 1 ) plant breeding programs have already generated many useful pedigree populations that contain ld useful for association studies but can not be used as an independent ld - mapping population , and ( 2 ) many historical trait data sets in plant breeding are unbalanced that have been collected over multiple - years , and multienvironmental trials . this is one of the effective alternative strategies in association mapping that allow reducing the total amount of marker genotyping ( because of preselecting of markers restricted to qtl region ) in less number of individuals . the power of association mapping is the probability of detecting the true associations within the mapping population size that really depends on ( 1 ) the extent and evolution of the ld in a population , ( 2 ) the complexity and mode of gene action of the trait of interest , ( 3 ) sample size and experimental design . a statistical approach within the bayesian framework is used test the reliability of obtained significance ( p - values ) in association mapping because of possibility of getting unreliable values due to ( 1 ) overestimation of effects ( selection bias ) , ( 2 ) association coming from neglecting confounding effects of a sample , ( 3 ) poor experimental design , and ( 4 ) instability of genetic effects across different environments . to detect the reliable effects in association mapping , experiments should be designed at least with the bayes factor of 20 that may require much larger sample sizes . however , the sample size for association mapping can be decreased keeping the high power with ( 1 ) preselecting a priori known qtl regions or candidate genes ( from qtl - mapping and expression analyses ) , ( 2 ) using the large populations with samples longer ld block that require a less number of markers to find useful associations , ( 3 ) an alternative experimental design ( i.e. to date association mapping has also been extended to long lifespan plant species , forest tree populations , where associations of polymorphisms in cinnamoil coa reductase ( ccr ) with earlywood microfibril angle trait , and polymorphisms a putative stress response gene with wood density and wood growth rate were reported . association mapping of traits in arabidopsis also has been reported and overall suitability of the approach well documented . table 1 summarizes the ld and association mapping efforts in plants including some of very recent whole genome association mapping studies . as one can see , within the frame of above highlighted association studies in plants , various association mapping methodologies ( table 1 ) , molecular markers ( both dominant and co - dominant markers ) , and plant germplasm resources ( including landrace stocks , elite germplasm , and experimental populations e.g . choosing the appropriate association mapping depends on ( 1 ) the extent and evolution of the linkage disequilibrium in a population , ( 2 ) the level of population structure and stratification , ( 3 ) availability of pedigree information , ( 4 ) complexity of the trait of interest under study , and ( 5 ) availability of the genomic information and resources . the examples of association mapping studies performed in various plant germplasm resources including model plant arabidopsis and extended to various crop germplasm largely demonstrate the flourish of crop genomics era with the utilization of powerful ld - based association mapping tool . this is also a good indicative of the potential utilization of this technology with the other crops and plant species in the future .

due in part to the aging of the us population , the number of individuals with multiple concurrent chronic diseases has increased significantly during the past several decades.14 the presence of multiple comorbid conditions makes clinical decision - making and patient management challenging , increases the risk for adverse outcomes , and is associated with higher medical care costs.59 coronary artery disease disproportionately affects older individuals and is associated with a high burden of additional comorbidities.7,10 although disease states such as diabetes , hypertension , heart failure , and atrial fibrillation share independent associations with increased mortality in patients with acute myocardial infarction ( ami),1114 it is unclear whether these and other important comorbidities cluster together , or if comorbid disease burden is associated with variability in treatment or reduced survival.4,15,16 moreover , data are lacking on how these disease patterns , and patient - related outcomes , may have changed during recent years . the objectives of the present study were to examine nearly 2-decade - long trends ( 19902007 ) in the prevalence of six important chronic conditions , namely atrial fibrillation , diabetes , heart failure , hypertension , myocardial infarction , and stroke , singly and in combination , in 9581 patients hospitalized with ami . these comorbid illnesses were selected for examination on the basis of established associations with the development of ami or adverse ami - related prognosis and their relative frequency in this patient population.13,17,18 we examined the clinical correlates of multiple comorbid conditions , evaluated whether the presence of multiple concurrent chronic diseases influences hospital and posthospital admission mortality in patients hospitalized with ami , and determined whether the prognosis associated with these chronic conditions has changed during the years under study . the worcester heart attack study is an ongoing population - based investigation that is examining long - term trends in the incidence , hospital , and postdischarge case - fatality rates of ami among residents of the worcester metropolitan area ( 2000 census estimate : 478,000 ) hospitalized at all 16 greater worcester medical centers on an approximate biennial basis beginning in 1975.1923 the periods under study were selected because of the availability of grant funding and for purposes of examining changes in our principal study outcomes over an approximate alternate yearly basis . fewer hospitals ( n = 11 ) have been included during recent study years due to hospital closures , mergers , and conversion to chronic care facilities . of the current hospitals , three are considered to be tertiary care / university - based medical centers . the details of this study have been described previously.1923 in brief , computerized printouts for patients admitted to all greater worcester hospitals with primary and/or secondary discharge diagnosis codes consistent with the presence of possible ami were obtained , and international classification of disease ( icd ) codes in which ami may have been diagnosed were reviewed for purposes of sample selection ( icd-9 codes 410414 , 786.5 ) . cases of possible ami treated at all greater worcester medical centers were independently validated according to predefined criteria for ami.2123 this study was approved by the institutional review board at the university of massachusetts medical school . patients who satisfied the diagnostic criteria for ami and were residents of the worcester metropolitan area were included in this population - based investigation . patients could have been included more than once in this investigation if they had been rehospitalized for ami during the different years under study , with subsequent reclassification of the order of the present ami ( eg , initial versus prior ) . patients who developed symptoms of ami after hospital admission or after an interventional procedure or surgery were excluded because we wanted to study ami that occurred de novo in the broader community setting . information about demographic characteristics , comorbidities , and clinical data was abstracted from the hospital medical records of patients with confirmed ami by trained study physicians and nurses . information was collected about patient age , sex , race , marital status , body mass index , comorbidities , ami type ( q wave versus non - q wave ) , hospital treatment practices , inhospital complications , and hospital discharge status . discharged hospital patients were followed on an annual basis through review of records for additional hospitalizations and search of death certificates at state and local divisions of vital statistics . some form of additional follow - up after being discharged from the hospital through either review of postdischarge medical records or death certificates was obtained for more than 99% of discharged hospital survivors from the cohorts included to date with follow - up completed through 2008 . the presence of prior atrial fibrillation , diabetes , heart failure , hypertension , and stroke was identified based on review of hospital medical records and physician s progress notes . these comorbidities were considered to be present if they had been previously diagnosed in hospitalized patients . patients with missing data on previously diagnosed atrial fibrillation ( 2% ) were excluded from the analyses . multiple comorbidities were defined as the presence of one or more of these previously diagnosed comorbid conditions ; further categorization into cutpoints of any two , any three , and at least four of these comorbidities was also carried out . in addition , common clusters of comorbidities among the six conditions examined were identified . for purposes of these latter analyses , a common cluster was defined as any combination of two or more comorbidities present in at least 2% of the study population . the primary outcomes of interest were 30-day post hospital admission total death rates and 1-year all - cause mortality . the overall prevalence of multiple comorbidities , and changes over time therein , were calculated and compared using the chi - square test for trends . in examining whether these trends were affected by other patient demographic and clinical characteristics , a multinomial logistic regression analysis was used to adjust for potential confounding factors , with patients without any of the comorbidities examined serving as the reference group . these factors were chosen based on findings from prior studies including age ( < 65 , 6574 , 7584 , 85 years ) , sex , race ( white versus nonwhite ) , marital status ( single , married , divorced , widowed ) , type of ami ( q wave versus non - q wave ) , and clinical complications that patients may have developed during their acute hospitalization ( eg , heart failure , atrial fibrillation , cardiogenic shock ) . study years were grouped into five periods ( 19901991 , 19931995 , 19971999 , 20012003 , and 20052007 ) for ease of analysis . in examining the influence of multiple comorbid conditions on posthospital admission , all - cause death rates , age - sex adjusted survival curves for 30-day and 1-year mortality according to number of comorbidities in addition , multivariable - adjusted cox regression models yielded estimated hazard ratios and 95% confidence intervals for the risk of dying at selected time points according to the number of cardiovascular comorbidities that had been previously diagnosed ( a list of controlling variables is included in the footnotes of the tables ) . proportional hazard assumptions were checked graphically for all variables by plotting a cox model versus kaplan - meier curves , and the assumptions of these models were satisfied ( data not shown ) . interaction terms between number of comorbidities present and study period were included in the multivariable - adjusted regression models for the purpose of examining whether any observed associations may have changed significantly over time . all analyses were performed using stata software ( version 11.0 ; stata corporation , college station , tx ) . information about demographic characteristics , comorbidities , and clinical data was abstracted from the hospital medical records of patients with confirmed ami by trained study physicians and nurses . information was collected about patient age , sex , race , marital status , body mass index , comorbidities , ami type ( q wave versus non - q wave ) , hospital treatment practices , inhospital complications , and hospital discharge status . discharged hospital patients were followed on an annual basis through review of records for additional hospitalizations and search of death certificates at state and local divisions of vital statistics . some form of additional follow - up after being discharged from the hospital through either review of postdischarge medical records or death certificates was obtained for more than 99% of discharged hospital survivors from the cohorts included to date with follow - up completed through 2008 . the presence of prior atrial fibrillation , diabetes , heart failure , hypertension , and stroke was identified based on review of hospital medical records and physician s progress notes . these comorbidities were considered to be present if they had been previously diagnosed in hospitalized patients . patients with missing data on previously diagnosed atrial fibrillation ( 2% ) were excluded from the analyses . multiple comorbidities were defined as the presence of one or more of these previously diagnosed comorbid conditions ; further categorization into cutpoints of any two , any three , and at least four of these comorbidities was also carried out . in addition , common clusters of comorbidities among the six conditions examined were identified . for purposes of these latter analyses , a common cluster was defined as any combination of two or more comorbidities present in at least 2% of the study population . the primary outcomes of interest were 30-day post hospital admission total death rates and 1-year all - cause mortality . the overall prevalence of multiple comorbidities , and changes over time therein , were calculated and compared using the chi - square test for trends . in examining whether these trends were affected by other patient demographic and clinical characteristics , a multinomial logistic regression analysis was used to adjust for potential confounding factors , with patients without any of the comorbidities examined serving as the reference group . these factors were chosen based on findings from prior studies including age ( < 65 , 6574 , 7584 , 85 years ) , sex , race ( white versus nonwhite ) , marital status ( single , married , divorced , widowed ) , type of ami ( q wave versus non - q wave ) , and clinical complications that patients may have developed during their acute hospitalization ( eg , heart failure , atrial fibrillation , cardiogenic shock ) . study years were grouped into five periods ( 19901991 , 19931995 , 19971999 , 20012003 , and 20052007 ) for ease of analysis . in examining the influence of multiple comorbid conditions on posthospital admission , all - cause death rates , age - sex adjusted survival curves for 30-day and 1-year mortality according to number of comorbidities were created . in addition , multivariable - adjusted cox regression models yielded estimated hazard ratios and 95% confidence intervals for the risk of dying at selected time points according to the number of cardiovascular comorbidities that had been previously diagnosed ( a list of controlling variables is included in the footnotes of the tables ) . proportional hazard assumptions were checked graphically for all variables by plotting a cox model versus kaplan - meier curves , and the assumptions of these models were satisfied ( data not shown ) . interaction terms between number of comorbidities present and study period were included in the multivariable - adjusted regression models for the purpose of examining whether any observed associations may have changed significantly over time . all analyses were performed using stata software ( version 11.0 ; stata corporation , college station , tx ) . the study sample consisted of 9581 residents of the worcester metropolitan area hospitalized with independently validated ami at all greater worcester medical centers in the 10 study years between 1990 and 2007 . the average age of this patient population was 70 years , 57% were men , and the majority ( 93% ) were caucasian . overall , a total of 7379 ( 77% ) patients had one or more of the comorbid conditions examined previously diagnosed at the time of their index hospitalization for ami . in our study population , 35% of patients had a single comorbidity previously diagnosed , 25% had two , 12% had three , and 5% had four or more of these clinically important comorbid conditions present . the number of comorbidities previously diagnosed in hospitalized patients was relatively stable between 1990 ( mean 1.2 , median 1 ) and 2007 ( mean 1.6 , median 1 ) . however , significant changes were noted at the extremes of our study population distribution ; the proportion of participants in whom no comorbid illnesses were present declined by nearly one - half ( 31% to 16% ) , whereas the number of participants with four or more comorbidities diagnosed previously more than doubled ( 3% to 7% ) between 1990 and 2007 ( figure 1 , p for trend < 0.05 ) . these increasing trends remained statistically significant after adjusting for several potentially confounding factors in a multinomial logistic regression analysis ( figure 2 ) . compared with patients hospitalized during our earliest study years ( 19901991 ) , patients hospitalized during the most recent periods under study ( 19971999 , 20012003 , and 20052007 ) were significantly more likely to report having multiple comorbidities present . older individuals , women , nonwhite , and widowed patients were more likely to have multiple comorbidities previously diagnosed as compared with middle - aged persons , men , white , and other patients hospitalized with ami ( table 1 ) . patients with a non - q wave ami and those who developed serious clinical complications of ami , including heart failure and atrial fibrillation , were more often affected by multiple comorbidities as compared with respective comparison groups ( table 1 ) . in order to determine the frequency and prognostic impact of specific comorbidity patterns ( or clusters ) , as well as changes over time therein , we examined the magnitude of all possible comorbidity clusters in our study population and how the presence of specific comorbidity combinations related to short- and long - term prognosis . as shown in figure 3 , the most common comorbidity cluster was hypertension and diabetes , which occurred in one out of every eight patients hospitalized with ami . hypertension , diabetes , and heart failure was the next most common comorbidity combination , affecting approximately 5% of study patients . both of these comorbidity clusters , in addition to the combination of hypertension and atrial fibrillation , increased in prevalence over the study period ( figure 3 , p for trend < 0.05 ) . these increasing trends remained statistically significant after adjusting for a number of potentially confounding demographic and clinical factors in a multinominal logistic regression model ( data not shown ) . in examining the relationship between number of comorbidities and all - cause mortality , having multiple comorbidities previously diagnosed prior to hospitalization for ami was significantly associated with poorer short- and long - term survival ( table 2 , figure 4 ) . after adjustment for several potentially confounding demographic ( eg , age , sex ) and clinical factors ( eg , hospital clinical complications ) of prognostic importance , patients with two or more comorbidities were significantly more likely to have died within 30 days or at 1 year after hospital admission ( table 2 ) . the risk of dying within 30 days and at 1 year after admission for ami was directly related to the number of comorbidities present . the prognosis associated with having multiple comorbidities among patients hospitalized with ami did not change significantly over time ( p = 0.57 for interaction ) . patients with ami and several of the most common comorbidity clusters were at higher risk for dying within 30 days and at 1 year after hospital admission , even after adjustment for several potentially confounding factors ( table 3 ) . in particular , patients with the following combinations : hypertension + heart failure , hypertension + stroke , hypertension + diabetes + heart failure , or hypertension + diabetes + stroke , had greater odds of dying within 30 days or by 1 year after hospitalization for ami than patients without these comorbid conditions . overall , a total of 7379 ( 77% ) patients had one or more of the comorbid conditions examined previously diagnosed at the time of their index hospitalization for ami . in our study population , 35% of patients had a single comorbidity previously diagnosed , 25% had two , 12% had three , and 5% had four or more of these clinically important comorbid conditions present . the number of comorbidities previously diagnosed in hospitalized patients was relatively stable between 1990 ( mean 1.2 , median 1 ) and 2007 ( mean 1.6 , median 1 ) . however , significant changes were noted at the extremes of our study population distribution ; the proportion of participants in whom no comorbid illnesses were present declined by nearly one - half ( 31% to 16% ) , whereas the number of participants with four or more comorbidities diagnosed previously more than doubled ( 3% to 7% ) between 1990 and 2007 ( figure 1 , p for trend < 0.05 ) . these increasing trends remained statistically significant after adjusting for several potentially confounding factors in a multinomial logistic regression analysis ( figure 2 ) . compared with patients hospitalized during our earliest study years ( 19901991 ) , patients hospitalized during the most recent periods under study ( 19971999 , 20012003 , and 20052007 ) were significantly more likely to report having multiple comorbidities present . older individuals , women , nonwhite , and widowed patients were more likely to have multiple comorbidities previously diagnosed as compared with middle - aged persons , men , white , and other patients hospitalized with ami ( table 1 ) . patients with a non - q wave ami and those who developed serious clinical complications of ami , including heart failure and atrial fibrillation , were more often affected by multiple comorbidities as compared with respective comparison groups ( table 1 ) . in order to determine the frequency and prognostic impact of specific comorbidity patterns ( or clusters ) , as well as changes over time therein , we examined the magnitude of all possible comorbidity clusters in our study population and how the presence of specific comorbidity combinations related to short- and long - term prognosis . as shown in figure 3 , the most common comorbidity cluster was hypertension and diabetes , which occurred in one out of every eight patients hospitalized with ami . hypertension , diabetes , and heart failure was the next most common comorbidity combination , affecting approximately 5% of study patients . both of these comorbidity clusters , in addition to the combination of hypertension and atrial fibrillation , increased in prevalence over the study period ( figure 3 , p for trend < 0.05 ) . these increasing trends remained statistically significant after adjusting for a number of potentially confounding demographic and clinical factors in a multinominal logistic regression model ( data not shown ) . in examining the relationship between number of comorbidities and all - cause mortality , having multiple comorbidities previously diagnosed prior to hospitalization for ami was significantly associated with poorer short- and long - term survival ( table 2 , figure 4 ) . after adjustment for several potentially confounding demographic ( eg , age , sex ) and clinical factors ( eg , hospital clinical complications ) of prognostic importance , patients with two or more comorbidities were significantly more likely to have died within 30 days or at 1 year after hospital admission ( table 2 ) . the risk of dying within 30 days and at 1 year after admission for ami was directly related to the number of comorbidities present . the prognosis associated with having multiple comorbidities among patients hospitalized with ami did not change significantly over time ( p = 0.57 for interaction ) . patients with ami and several of the most common comorbidity clusters were at higher risk for dying within 30 days and at 1 year after hospital admission , even after adjustment for several potentially confounding factors ( table 3 ) . in particular , patients with the following combinations : hypertension + heart failure , hypertension + stroke , hypertension + diabetes + heart failure , or hypertension + diabetes + stroke , had greater odds of dying within 30 days or by 1 year after hospitalization for ami than patients without these comorbid conditions . in this community - wide investigation of central massachusetts residents hospitalized with an independently validated ami at all metropolitan worcester medical centers between 1990 and 2007 , the prevalence of previously diagnosed atrial fibrillation , heart failure , diabetes mellitus , and hypertension increased over time in hospitalized patients , as did the odds of being diagnosed with multiple comorbidities . although the median number of comorbidities that were previously diagnosed in this patient population did not increase between 1990 and 2007 , a dramatic increase in the percentage of hospitalized patients having four or more comorbid illnesses was observed , as was a concomitant decline in the proportion of patients without any of the comorbidities examined . the prevalence of several comorbidity combinations , most notably hypertension and diabetes , increased among ami patients during the years under study . an increasing number of comorbid diseases was inversely associated with survival at 30 days and 1 year after admission to all central massachusetts medical centers . in addition , despite increases in the proportion of patients with the highest burden of comorbidity , the adverse prognosis associated with having multiple comorbidities did not change significantly over the nearly 2-decade - long period under study . consistent with the published literature , we observed a high prevalence of the important cardiovascular comorbidities of atrial fibrillation , heart failure , prior mi , diabetes mellitus , hypertension , and stroke in our patient population.13,2426 the proportion of ami patients affected by these comorbidities increased during the period under study , with the exception of prior mi and stroke . our results confirm the findings of other contemporary investigations that have studied patients hospitalized with ami , such as those from national registry of myocardial infarction-2 , which have also shown that the burden of atrial fibrillation , heart failure , diabetes mellitus , hypertension , and stroke have increased in this large multiregional population.10 given the strong relationship between age , sex , and adiposity with the cardiovascular conditions under study , the observed trends are likely related to the increasing number of elderly , women , and obese patients hospitalized with ami in our study population.17,22,27 a major focus of the present investigation was to examine the magnitude of cardiometabolic comorbidities in patients hospitalized with ami . the vast majority ( > 80% ) of patients in our study sample were affected by at least one of the comorbidities under study , and more than half were affected by two or more comorbid diseases ; indeed , approximately one in every eight patients had four or more of the cardiovascular comorbidities examined . although limited data on this topic exist , these observations validate our clinical impression that cardiovascular multimorbidity is the rule rather than the exception in patients hospitalized with ami.4,15,16 notably , rates of multimorbidity in our study were similar to , but slightly higher than , rates observed in similarly aged individuals included in prior investigations.7,8 there are several possible explanations for the high rates of multimorbidity observed in our hospitalized patient population . first , in light of the strong pathophysiological associations between diseases such as hypertension and heart failure , causal comorbidity has likely contributed to clustering of these diseases in our middle - aged and elderly patient population . complicating comorbidity , or the strong causal relationship between one disease and another , for example , between hypertension and stroke or obesity and diabetes , may also explain the frequent clustering of comorbidities in our study sample.28 the prevalence of patients without important cardiovascular comorbidities has decreased significantly , while the prevalence of patients with multiple comorbidities has increased significantly during the years under study in this population - based investigation . reasons for the increasing rates of multimorbidity in our sample likely include enhanced risk factor and disease screening as well as improved patient and provider awareness of the importance of these chronic conditions between 1990 and 2007.17 however , the strong association between age , obesity , and several of the examined comorbidities , both singly and in combination , suggests that the increasing age and adiposity of patients hospitalized with ami noted over time may also have contributed to observed trends . this hypothesis is supported by our observation that the proportion of patients affected by specific comorbidity clusters that are strongly related to advancing age and obesity , such as hypertension and diabetes , increased most significantly over the study period . similarly , because women and nonwhite patients with ami were more likely to have multiple comorbidities , the increasing proportion of these patients in our community sample may have also contributed to increasing rates of multimorbidity noted during the years under study . as has been reported previously , patients who reported having any of the cardiovascular comorbidities under study ( with the exception of hypertension ) were more likely to die during the first year after admission for ami . we observed that having multiple comorbidities present was significantly associated with poorer survival at 30 days and 1 year after hospital admission for ami and that the overall risk of dying increased linearly with the number of comorbidities present . these data suggest that the concomitant occurrence of multiple comorbid diseases exerts a more significant adverse effect on the short - term and long - term health of patients with an ami than when they occur singly.7,16 our findings are supported by several prior investigations , including one involving more than 100,000 medicare beneficiaries , which showed that the odds of clinical complications increased exponentially with increasing number of comorbidities ( one comorbidity = four complications per 1000 beneficiaries ; four comorbidities = 34 complications per 1000 beneficiaries).8 our observation that multimorbidity is associated with poor short - term and better long - term survival is likely related to the fact that health care providers have little help in adjusting care for patients with multiple conditions , given that clinical trials and , in turn , practice guidelines , focus almost exclusively on patients with a single disease entity . rates of complications from ami treatment , such as bleeding,29 may be higher in patients with multiple comorbidities than in patients without any comorbid illness , due to factors such as drug drug interactions or reduced drug clearance . our results suggest that further population - based data on the use and impact of contemporary ami treatments in patients with multiple comorbidities are needed . the strengths of the present study include its community - based design , its large sample of patients hospitalized with ami , and its almost 2-decade - long perspective into changing trends in several clinically relevant endpoints . first , although trends in the death rates of patients with ami may have been influenced by cardiac treatments , our study did not examine the role and impact of hospital treatment practices , given the observational nature of this investigation , and our nonrandomized study design did not allow for adjustment for differences in these practices . given that the majority of our study participants were white , our findings may lack generalizability to other ethnic or racial groups . finally , physician thresholds for diagnosing several of the comorbid conditions under study may have changed over time , though it is unknown whether such a trend has occurred in our study population . the strengths of the present study include its community - based design , its large sample of patients hospitalized with ami , and its almost 2-decade - long perspective into changing trends in several clinically relevant endpoints . first , although trends in the death rates of patients with ami may have been influenced by cardiac treatments , our study did not examine the role and impact of hospital treatment practices , given the observational nature of this investigation , and our nonrandomized study design did not allow for adjustment for differences in these practices . given that the majority of our study participants were white , our findings may lack generalizability to other ethnic or racial groups . finally , physician thresholds for diagnosing several of the comorbid conditions under study may have changed over time , though it is unknown whether such a trend has occurred in our study population . in this study of almost 10,000 patients hospitalized with ami over a recent 2-decade period , we observed that the odds of having multiple cardiovascular comorbidities previously diagnosed in these high - risk patients increased significantly over time . presence of multimorbidity was associated with reduced short- and long - term survival and the poor prognosis associated with multimorbidity persisted over the period under study . our findings highlight the need for additional contemporary data to help guide the monitoring and treatment of patients with cardiovascular disease and multiple concurrent medical illnesses , as well as the importance of skilled and coordinated cardiovascular care for these high - risk patients .

backgroundthe objectives of this community - based study were to examine the overall and changing ( 19902007 ) frequency and impact on 30-day and 1-year death rates from multiple cardiovascular comorbidities in adults from a large central new england metropolitan area hospitalized with acute myocardial infarction ( ami).methodsthe study population consisted of 9581 patients hospitalized with ami at all 11 medical centers in the metropolitan area of worcester , ma , during 10 annual periods between 1990 and 2007 . the comorbidities examined included atrial fibrillation , diabetes , heart failure , hypertension , and stroke.resultsthirty-five percent of participants had a single diagnosed cardiovascular comorbidity , 25% had two , 12% had three , and 5% had four or more comorbidities . between 1990 and 2007 , the proportion of patients without any of these comorbidities decreased significantly , while the proportion of patients with multiple comorbidities increased significantly during the years under study . an increasing number of comorbidities was associated with higher 30-day and 1-year postadmission death rates in patients hospitalized with ami.conclusionpatients hospitalized with ami carry a significant burden of comorbid cardiovascular disease that adversely impacts their 30-day and longer - term survival . increased attention to the management of ami patients with multiple cardiovascular comorbidities is warranted .

Introduction Methods Data collection Data analysis Results Prevalence of comorbidities Factors associated with multiple comorbidities Comorbidity clustering Mortality associated with multiple comorbidities and clusters of comorbidities Discussion Study strengths and limitations Conclusion

due in part to the aging of the us population , the number of individuals with multiple concurrent chronic diseases has increased significantly during the past several decades.14 the presence of multiple comorbid conditions makes clinical decision - making and patient management challenging , increases the risk for adverse outcomes , and is associated with higher medical care costs.59 coronary artery disease disproportionately affects older individuals and is associated with a high burden of additional comorbidities.7,10 although disease states such as diabetes , hypertension , heart failure , and atrial fibrillation share independent associations with increased mortality in patients with acute myocardial infarction ( ami),1114 it is unclear whether these and other important comorbidities cluster together , or if comorbid disease burden is associated with variability in treatment or reduced survival.4,15,16 moreover , data are lacking on how these disease patterns , and patient - related outcomes , may have changed during recent years . the objectives of the present study were to examine nearly 2-decade - long trends ( 19902007 ) in the prevalence of six important chronic conditions , namely atrial fibrillation , diabetes , heart failure , hypertension , myocardial infarction , and stroke , singly and in combination , in 9581 patients hospitalized with ami . these comorbid illnesses were selected for examination on the basis of established associations with the development of ami or adverse ami - related prognosis and their relative frequency in this patient population.13,17,18 we examined the clinical correlates of multiple comorbid conditions , evaluated whether the presence of multiple concurrent chronic diseases influences hospital and posthospital admission mortality in patients hospitalized with ami , and determined whether the prognosis associated with these chronic conditions has changed during the years under study . the worcester heart attack study is an ongoing population - based investigation that is examining long - term trends in the incidence , hospital , and postdischarge case - fatality rates of ami among residents of the worcester metropolitan area ( 2000 census estimate : 478,000 ) hospitalized at all 16 greater worcester medical centers on an approximate biennial basis beginning in 1975.1923 the periods under study were selected because of the availability of grant funding and for purposes of examining changes in our principal study outcomes over an approximate alternate yearly basis . the presence of prior atrial fibrillation , diabetes , heart failure , hypertension , and stroke was identified based on review of hospital medical records and physician s progress notes . multiple comorbidities were defined as the presence of one or more of these previously diagnosed comorbid conditions ; further categorization into cutpoints of any two , any three , and at least four of these comorbidities was also carried out . for purposes of these latter analyses , a common cluster was defined as any combination of two or more comorbidities present in at least 2% of the study population . the overall prevalence of multiple comorbidities , and changes over time therein , were calculated and compared using the chi - square test for trends . in examining whether these trends were affected by other patient demographic and clinical characteristics , a multinomial logistic regression analysis was used to adjust for potential confounding factors , with patients without any of the comorbidities examined serving as the reference group . these factors were chosen based on findings from prior studies including age ( < 65 , 6574 , 7584 , 85 years ) , sex , race ( white versus nonwhite ) , marital status ( single , married , divorced , widowed ) , type of ami ( q wave versus non - q wave ) , and clinical complications that patients may have developed during their acute hospitalization ( eg , heart failure , atrial fibrillation , cardiogenic shock ) . in examining the influence of multiple comorbid conditions on posthospital admission , all - cause death rates , age - sex adjusted survival curves for 30-day and 1-year mortality according to number of comorbidities in addition , multivariable - adjusted cox regression models yielded estimated hazard ratios and 95% confidence intervals for the risk of dying at selected time points according to the number of cardiovascular comorbidities that had been previously diagnosed ( a list of controlling variables is included in the footnotes of the tables ) . the presence of prior atrial fibrillation , diabetes , heart failure , hypertension , and stroke was identified based on review of hospital medical records and physician s progress notes . multiple comorbidities were defined as the presence of one or more of these previously diagnosed comorbid conditions ; further categorization into cutpoints of any two , any three , and at least four of these comorbidities was also carried out . for purposes of these latter analyses , a common cluster was defined as any combination of two or more comorbidities present in at least 2% of the study population . in examining whether these trends were affected by other patient demographic and clinical characteristics , a multinomial logistic regression analysis was used to adjust for potential confounding factors , with patients without any of the comorbidities examined serving as the reference group . these factors were chosen based on findings from prior studies including age ( < 65 , 6574 , 7584 , 85 years ) , sex , race ( white versus nonwhite ) , marital status ( single , married , divorced , widowed ) , type of ami ( q wave versus non - q wave ) , and clinical complications that patients may have developed during their acute hospitalization ( eg , heart failure , atrial fibrillation , cardiogenic shock ) . in examining the influence of multiple comorbid conditions on posthospital admission , all - cause death rates , age - sex adjusted survival curves for 30-day and 1-year mortality according to number of comorbidities were created . in addition , multivariable - adjusted cox regression models yielded estimated hazard ratios and 95% confidence intervals for the risk of dying at selected time points according to the number of cardiovascular comorbidities that had been previously diagnosed ( a list of controlling variables is included in the footnotes of the tables ) . interaction terms between number of comorbidities present and study period were included in the multivariable - adjusted regression models for the purpose of examining whether any observed associations may have changed significantly over time . the study sample consisted of 9581 residents of the worcester metropolitan area hospitalized with independently validated ami at all greater worcester medical centers in the 10 study years between 1990 and 2007 . in our study population , 35% of patients had a single comorbidity previously diagnosed , 25% had two , 12% had three , and 5% had four or more of these clinically important comorbid conditions present . the number of comorbidities previously diagnosed in hospitalized patients was relatively stable between 1990 ( mean 1.2 , median 1 ) and 2007 ( mean 1.6 , median 1 ) . however , significant changes were noted at the extremes of our study population distribution ; the proportion of participants in whom no comorbid illnesses were present declined by nearly one - half ( 31% to 16% ) , whereas the number of participants with four or more comorbidities diagnosed previously more than doubled ( 3% to 7% ) between 1990 and 2007 ( figure 1 , p for trend < 0.05 ) . compared with patients hospitalized during our earliest study years ( 19901991 ) , patients hospitalized during the most recent periods under study ( 19971999 , 20012003 , and 20052007 ) were significantly more likely to report having multiple comorbidities present . older individuals , women , nonwhite , and widowed patients were more likely to have multiple comorbidities previously diagnosed as compared with middle - aged persons , men , white , and other patients hospitalized with ami ( table 1 ) . patients with a non - q wave ami and those who developed serious clinical complications of ami , including heart failure and atrial fibrillation , were more often affected by multiple comorbidities as compared with respective comparison groups ( table 1 ) . as shown in figure 3 , the most common comorbidity cluster was hypertension and diabetes , which occurred in one out of every eight patients hospitalized with ami . hypertension , diabetes , and heart failure was the next most common comorbidity combination , affecting approximately 5% of study patients . both of these comorbidity clusters , in addition to the combination of hypertension and atrial fibrillation , increased in prevalence over the study period ( figure 3 , p for trend < 0.05 ) . in examining the relationship between number of comorbidities and all - cause mortality , having multiple comorbidities previously diagnosed prior to hospitalization for ami was significantly associated with poorer short- and long - term survival ( table 2 , figure 4 ) . the prognosis associated with having multiple comorbidities among patients hospitalized with ami did not change significantly over time ( p = 0.57 for interaction ) . in particular , patients with the following combinations : hypertension + heart failure , hypertension + stroke , hypertension + diabetes + heart failure , or hypertension + diabetes + stroke , had greater odds of dying within 30 days or by 1 year after hospitalization for ami than patients without these comorbid conditions . in our study population , 35% of patients had a single comorbidity previously diagnosed , 25% had two , 12% had three , and 5% had four or more of these clinically important comorbid conditions present . the number of comorbidities previously diagnosed in hospitalized patients was relatively stable between 1990 ( mean 1.2 , median 1 ) and 2007 ( mean 1.6 , median 1 ) . however , significant changes were noted at the extremes of our study population distribution ; the proportion of participants in whom no comorbid illnesses were present declined by nearly one - half ( 31% to 16% ) , whereas the number of participants with four or more comorbidities diagnosed previously more than doubled ( 3% to 7% ) between 1990 and 2007 ( figure 1 , p for trend < 0.05 ) . compared with patients hospitalized during our earliest study years ( 19901991 ) , patients hospitalized during the most recent periods under study ( 19971999 , 20012003 , and 20052007 ) were significantly more likely to report having multiple comorbidities present . older individuals , women , nonwhite , and widowed patients were more likely to have multiple comorbidities previously diagnosed as compared with middle - aged persons , men , white , and other patients hospitalized with ami ( table 1 ) . patients with a non - q wave ami and those who developed serious clinical complications of ami , including heart failure and atrial fibrillation , were more often affected by multiple comorbidities as compared with respective comparison groups ( table 1 ) . as shown in figure 3 , the most common comorbidity cluster was hypertension and diabetes , which occurred in one out of every eight patients hospitalized with ami . hypertension , diabetes , and heart failure was the next most common comorbidity combination , affecting approximately 5% of study patients . both of these comorbidity clusters , in addition to the combination of hypertension and atrial fibrillation , increased in prevalence over the study period ( figure 3 , p for trend < 0.05 ) . in examining the relationship between number of comorbidities and all - cause mortality , having multiple comorbidities previously diagnosed prior to hospitalization for ami was significantly associated with poorer short- and long - term survival ( table 2 , figure 4 ) . after adjustment for several potentially confounding demographic ( eg , age , sex ) and clinical factors ( eg , hospital clinical complications ) of prognostic importance , patients with two or more comorbidities were significantly more likely to have died within 30 days or at 1 year after hospital admission ( table 2 ) . the prognosis associated with having multiple comorbidities among patients hospitalized with ami did not change significantly over time ( p = 0.57 for interaction ) . in particular , patients with the following combinations : hypertension + heart failure , hypertension + stroke , hypertension + diabetes + heart failure , or hypertension + diabetes + stroke , had greater odds of dying within 30 days or by 1 year after hospitalization for ami than patients without these comorbid conditions . in this community - wide investigation of central massachusetts residents hospitalized with an independently validated ami at all metropolitan worcester medical centers between 1990 and 2007 , the prevalence of previously diagnosed atrial fibrillation , heart failure , diabetes mellitus , and hypertension increased over time in hospitalized patients , as did the odds of being diagnosed with multiple comorbidities . although the median number of comorbidities that were previously diagnosed in this patient population did not increase between 1990 and 2007 , a dramatic increase in the percentage of hospitalized patients having four or more comorbid illnesses was observed , as was a concomitant decline in the proportion of patients without any of the comorbidities examined . the prevalence of several comorbidity combinations , most notably hypertension and diabetes , increased among ami patients during the years under study . an increasing number of comorbid diseases was inversely associated with survival at 30 days and 1 year after admission to all central massachusetts medical centers . in addition , despite increases in the proportion of patients with the highest burden of comorbidity , the adverse prognosis associated with having multiple comorbidities did not change significantly over the nearly 2-decade - long period under study . consistent with the published literature , we observed a high prevalence of the important cardiovascular comorbidities of atrial fibrillation , heart failure , prior mi , diabetes mellitus , hypertension , and stroke in our patient population.13,2426 the proportion of ami patients affected by these comorbidities increased during the period under study , with the exception of prior mi and stroke . our results confirm the findings of other contemporary investigations that have studied patients hospitalized with ami , such as those from national registry of myocardial infarction-2 , which have also shown that the burden of atrial fibrillation , heart failure , diabetes mellitus , hypertension , and stroke have increased in this large multiregional population.10 given the strong relationship between age , sex , and adiposity with the cardiovascular conditions under study , the observed trends are likely related to the increasing number of elderly , women , and obese patients hospitalized with ami in our study population.17,22,27 a major focus of the present investigation was to examine the magnitude of cardiometabolic comorbidities in patients hospitalized with ami . the vast majority ( > 80% ) of patients in our study sample were affected by at least one of the comorbidities under study , and more than half were affected by two or more comorbid diseases ; indeed , approximately one in every eight patients had four or more of the cardiovascular comorbidities examined . although limited data on this topic exist , these observations validate our clinical impression that cardiovascular multimorbidity is the rule rather than the exception in patients hospitalized with ami.4,15,16 notably , rates of multimorbidity in our study were similar to , but slightly higher than , rates observed in similarly aged individuals included in prior investigations.7,8 there are several possible explanations for the high rates of multimorbidity observed in our hospitalized patient population . complicating comorbidity , or the strong causal relationship between one disease and another , for example , between hypertension and stroke or obesity and diabetes , may also explain the frequent clustering of comorbidities in our study sample.28 the prevalence of patients without important cardiovascular comorbidities has decreased significantly , while the prevalence of patients with multiple comorbidities has increased significantly during the years under study in this population - based investigation . reasons for the increasing rates of multimorbidity in our sample likely include enhanced risk factor and disease screening as well as improved patient and provider awareness of the importance of these chronic conditions between 1990 and 2007.17 however , the strong association between age , obesity , and several of the examined comorbidities , both singly and in combination , suggests that the increasing age and adiposity of patients hospitalized with ami noted over time may also have contributed to observed trends . this hypothesis is supported by our observation that the proportion of patients affected by specific comorbidity clusters that are strongly related to advancing age and obesity , such as hypertension and diabetes , increased most significantly over the study period . similarly , because women and nonwhite patients with ami were more likely to have multiple comorbidities , the increasing proportion of these patients in our community sample may have also contributed to increasing rates of multimorbidity noted during the years under study . as has been reported previously , patients who reported having any of the cardiovascular comorbidities under study ( with the exception of hypertension ) were more likely to die during the first year after admission for ami . we observed that having multiple comorbidities present was significantly associated with poorer survival at 30 days and 1 year after hospital admission for ami and that the overall risk of dying increased linearly with the number of comorbidities present . these data suggest that the concomitant occurrence of multiple comorbid diseases exerts a more significant adverse effect on the short - term and long - term health of patients with an ami than when they occur singly.7,16 our findings are supported by several prior investigations , including one involving more than 100,000 medicare beneficiaries , which showed that the odds of clinical complications increased exponentially with increasing number of comorbidities ( one comorbidity = four complications per 1000 beneficiaries ; four comorbidities = 34 complications per 1000 beneficiaries).8 our observation that multimorbidity is associated with poor short - term and better long - term survival is likely related to the fact that health care providers have little help in adjusting care for patients with multiple conditions , given that clinical trials and , in turn , practice guidelines , focus almost exclusively on patients with a single disease entity . rates of complications from ami treatment , such as bleeding,29 may be higher in patients with multiple comorbidities than in patients without any comorbid illness , due to factors such as drug drug interactions or reduced drug clearance . our results suggest that further population - based data on the use and impact of contemporary ami treatments in patients with multiple comorbidities are needed . the strengths of the present study include its community - based design , its large sample of patients hospitalized with ami , and its almost 2-decade - long perspective into changing trends in several clinically relevant endpoints . first , although trends in the death rates of patients with ami may have been influenced by cardiac treatments , our study did not examine the role and impact of hospital treatment practices , given the observational nature of this investigation , and our nonrandomized study design did not allow for adjustment for differences in these practices . the strengths of the present study include its community - based design , its large sample of patients hospitalized with ami , and its almost 2-decade - long perspective into changing trends in several clinically relevant endpoints . first , although trends in the death rates of patients with ami may have been influenced by cardiac treatments , our study did not examine the role and impact of hospital treatment practices , given the observational nature of this investigation , and our nonrandomized study design did not allow for adjustment for differences in these practices . in this study of almost 10,000 patients hospitalized with ami over a recent 2-decade period , we observed that the odds of having multiple cardiovascular comorbidities previously diagnosed in these high - risk patients increased significantly over time . presence of multimorbidity was associated with reduced short- and long - term survival and the poor prognosis associated with multimorbidity persisted over the period under study . our findings highlight the need for additional contemporary data to help guide the monitoring and treatment of patients with cardiovascular disease and multiple concurrent medical illnesses , as well as the importance of skilled and coordinated cardiovascular care for these high - risk patients .

rhabdomyosarcomas ( rms ) are the most frequent soft tissue sarcoma in children and rms cells resemble those in the early stages of myogenic differentiation , . histologically there are two major subtypes , embryonal ( erms ) accounting for 6070% of cases and alveolar ( arms ) . approximately 70% of arms harbor fusion genes that join the 5 sequence of pax3 or pax7 to the 3 sequence of foxo1 gene , . the encoded fusion proteins play an oncogenic role in the development of arms in association with co - operating events , notably overexpression of mycn that is a transcriptional target of the fusion protein and genomically amplified in approximately 20% of cases , , , . the presence of pax3-foxo1 has been correlated with aggressive clinical behavior in several studies , , , and gene expression profiles and genomic imbalances are similar in fusion gene negative arms and erms , . micrornas ( mirnas ) are small ( 2022 nt ) rna molecules that can alter cellular processes including proliferation , differentiation and apoptosis . mirnas inhibit protein synthesis of specific genes by degrading specific mrna species or hindering translation . specific mrna degradation involves base - pairing to partially complementary sequences within the 3 utr or other less frequently studied mechanisms including their binding to the 5 utr and gene promoters regions . each mirna potentially regulates hundreds of target gene products and it is suggested that the entire protein coding genome is regulated by mirnas . individual mirnas have been ascribed oncogenic or tumor suppressor roles in cancer and their roles can clearly be cell context specific . roles for individual mirnas in rms have been described such as mir-1 , mir-206 , mir-133a / b and mir-378 that maintain an undifferentiated myogenic state , , , , , , and mir-29 that is described with a tumor suppressor role . however , a comprehensive view of how mirnas shape the gene expression and biological features of a large number of rms has not been described but could contribute to identification of prognostic markers / signatures and ultimately therapeutic targets , as indicated by previous studies of other tumor types , , . here we derived mirna expression profiling data from high - throughput sequencing analyses of 64 primary rms samples and took a systems biology approach to integrate this with parallel gene expression profiling data . this allowed us to identify co - expression mrna networks ( modules ) with assigned biological functions that correlated with both patient outcome data and the presence of pax3/7-foxo1 . this led us to demonstrate that pax3-foxo1 regulates the expression of many mirnas , including mir-9 - 5p that was indirectly altered by pax3-foxo1 and shown to be of clinical and functional relevance . sixty - four samples ( 36 arms and 28 erms ) were available from rms patients as part of a previously described cohort . clinico - pathological features of the cases used in this study for mirna profiling are summarized in supplementary table s1a . samples from a similarly treated cohort of 154 rms patients were used for mirna validation analyses and their clinico - pathological details are summarized in supplementary table s1b . centres were collected through the children 's cancer and leukaemia group ( local research ethics committee protocol nos . 1836 and 2015 and multi - regional research ethics committee 06/4/71 with consent , where required ) . human cell lines derived from embryonal ( rh2 , rh36 , rd , rmsym , ruch2 , ruch3 , ct10 ) and alveolar ( rh3 , rh4 , rh5 , rh18 , rh41 , rms , scmcrmz , cw9019 ) were cultured and validated by dna fingerprinted as previously described , . rna extraction was performed using trizol reagent following manufacturer 's instructions ( invitrogen carlsbad , ca ) . size selection and library preparation was performed by fasteris , plan - les ouates , switzerland including duplicates for quality control using 5 g of rna in a maximum volume of 1015 l that was sent to the company . briefly , small rna fragments were purified by acrylamide gel purification and 3 and 5 adapters were ligated to the ends of single stranded small rna molecules . reverse transcription and pcr amplification was performed to generate the dna colonies template library , which was diluted to 10 nm . the mirna expression profiling was performed by high - throughput sequencing analysis using a solexa / illumina genome analyzer ii . each sample specific sequence was then retrieved using a bar - coding system made of 4 nucleotides . the solexa fasta - q format sequence of each sample was aligned to 2 databases : mature mirna sequence ( mirbase release 21 ) and est ( ncbi ) . the mature mirna sequence was extended with ns at each end to allow the alignment with segments of rna one or two nucleotides longer than the wild type sequence . all the alignments were performed using novoalign 2.06.09 with default parameters , after removing the 3 adapter and cutting the sequence to 30 nt . first of all the sequence with low solexa quality score ( which did not passed the cut - off in the novoalign software ) and those having less than 5 reads in at least 2 samples were filtered out from the analysis . when multiple alignments with the same score were detected , the sequence was randomly assigned to one of them . after the alignment each sequence was assigned to an rna class based on entrez gene type . the variance of the counts data were stabilized using a power transformation function and normalized by applying a non - linear function ( lowess ) . differentially expressed mirnas were identified by fitting a linear model ( limma ) and adjusting for multiple testing by computing fdr values from p - values using the benjamini - hochberg procedure ( limma package ) . clinical correlations between levels of mirna expression and patient outcome data were performed by fitting a cox proportional hazards regression model and adjusting for multiple testing , as described above . rna gene expression profiling data for 64 rms samples parallel to those profiled for mirnas were obtained and pre - analyzed as previously described . probe sets were filtered in three steps : 1 ) probe sets with log intensity below 6 across all the sample were removed ; 2 ) if multiple probe sets per gene were present , only the one with the highest coefficient of variation was retained ; 3 ) the first 8000 most variable probe sets associated to a unique entrez i d were selected for further analysis . the samples underwent weighted gene co - expression network analysis ( wgcna ) using the r package version 0.96 . briefly , a co - expression similarity measure was calculated by raising the absolute correlation coefficient between genes to a selected power . then using a soft thresh - holding procedure modules were defined by cutting the breaches using a high cut - off of 0.99 and a minimum number of genes within the resulting dendrogram equal or above 30 . the expression of all the genes within each module was summarized as eigengene , representation of the first component was obtained by singular value decomposition of standardized values . association between modules expression and clinico - pathological parameters were evaluated using the kruskal - wallis rank sum test applied to both the eigengene and the gene expression levels . in particular , for the gene expression , the statistical values were obtained by testing the association of each gene with a specific parameter and averaged within each module . therefore , high mean values of the module were interpreted as a larger number of genes within that module associated to the parameter tested . cox 's regression model was used to correlation module expression with patient overall survival ( os ) or progression free survival ( pfs ) . finally , correlation of mirna expression levels with the modules was assessed calculating pearson correlation considering module eigengenes . mirnas were associated to the modules if they showed an absolute correlation coefficient above 0.4 and p value < 0.001 . mir-9 - 5p knockdown in the rh30 cell line was achieved by transfecting the cells with miscript mirna inhibitor using hiperfect reagent ( qiagen gmbh , hilden , germany ) . levels of expression during experiments and analyses of levels in primary rms samples were assayed where appropriate using taqman microrna assays according to the manufacturer 's instructions ( applied biosystems , ca , usa ) . briefly , rna was reverse transcribed using taqman microrna reverse transcription ( rt ) kit ( applied biosystems , ca , usa ) with specific - mirna primers , then the product used in the real - time pcr reaction in the abi prism 7900ht sequence detection system ( applied biosystems , ca , usa ) . expression data were analyzed by the comparative threshold cycle ( ct ) method accordingly to user bulletin # 2 ( applied biosystems ) . results were expressed in term of the ct value and obtained as follows : ct mirna = average ( ct u6 ; ctrun48 ) ct mirna , where ct mirna , ct u6 and ctrun48 represents the comparative threshold cycle for mirna and the two endogenous controls u6 and run48 , respectively . all experiments were performed in triplicate and differences between groups were evaluated using the mann - whitney test . sirna against pax3-foxo1 was synthesized by thermo fisher scientific ( ma , usa ) and rh4 cells were transfected with this using lipofectamine rnaimax ( invitrogen , ca , usa ) according to the manufacturer 's instructions . cells were cultured for 48 h before total rna and mirna was extracted using mirneasy mini kit ( qiagen , hilden , germany ) . to analyze pax3-foxo1 levels after rna interference , cdna was synthesized using high capacity cdna reverse transcription kit ( applied biosystems , ca , usa ) and quantitative rt - pcr was performed using viia 7 real - time pcr system ( applied biosystems , ca , usa ) . primers and probes designed for analysis of pax3-foxo1 were as follows : forward 5-gaacccaccattggcaat-3 , probe 5-cctctcacctcagaattcaattcgtcataatctg-3 , reverse 5-tctgcacacgaatgaacttgct-3. to analyze mirna expression after pax3-foxo1 knock - down in rh4 cells , cdna was synthesized using taqman microrna reverse transcription kit and megaplex primer pools ( applied biosystems , ca , usa ) . quantification of mirnas was performed on taqman array human microrna a+b cards set v3.0 using viia 7 real - time pcr system according to the manufacturer 's instructions and analyzed with viia 7 software ( applied biosystems , ca , usa ) . at 72 h post transfection , cells were re - suspended in dmem plus 1% fcs and placed in triplicate into cell culture inserts ( bd , nj , usa ) before being submerged into specially adapted 24 well plates ( bd , nj , usa ) containing dmem plus 10% fcs . 24 h later , cells that had not migrated were removed using a cotton bud , and cells that had migrated to the base of the inserts were fixed overnight in 100% methanol at 20 c and then stained for 2 min with 2% crystal violet solution ( sigma - aldrich , mo , usa ) . cells were photographed at 10 magnification using a leica dm irb microscope with a leica 420c camera attached ( leica microsystems , milton keynes , uk ) ( 4 fields of view ) and counted manually in photoshop cs3 . viability was assessed at 72 h post transfection using cell titer aqueous one reagent ( promega , wi , usa ) as previously described . mirna expression profiling was performed by high - throughput sequencing analysis of 68 samples ( 3 skeletal muscle , 1 myoblast and 64 rms patient samples ( supplementary table s1 ) ) . on average , around 2,069,412 tags were sequenced for each sample ( range : 753,6147,390,288 ) . after sequence alignment and filtering , each tag was assigned to a rna class based on entrez gene type and 78% and 73% were shown to be mirnas in rms and skeletal muscle , respectively , confirming their successful enrichment ( supplementary fig . since mirnas are often included in transcription clusters , their expression is more coordinated when located in close proximity compare to those further apart . direct comparisons of levels of expression across molecular ( fusion positive versus negative rms ) , histological ( skeletal muscle versus rms ) , and clinical ( metastatic m1 versus non metastatic m0 ) entities produced lists of mirnas significantly over or under - expressed in these groups ( supplementary tables s2 - 5 ) . correlation between mirna expression and overall survival ( os ) and progression free survival ( pfs ) was also assessed by cox proportional hazard regression model ( supplementary tables s6 - 9 ) . unsupervised hierarchical clustering analysis performed using mirnas with at least 100 tags in 50% of the samples showed the presence of distinct patterns of expression across rms samples ( fig . 1 ) . notably , mirna expression patterns mostly distinguished rms histological subtypes showing sets of mirnas positively and negatively associated with fusion gene status . the well - known muscle specific mirnas including mir-1 , mir-133 and mir-206 showed higher expressed in skeletal muscle versus rms subtypes . application of class prediction analysis based on nearest shrunken centroid classifier showed that mirna expression can distinguish fusion positive and negative patients with around 89% accuracy ( supplementary fig . these data suggest a strong interplay between fusion protein and mirna expression that potentially contributes to the malignant phenotype of rms . based on the principle that co - expressed genes are commonly associated with similar biological function / pathways , we employed wgcna using parallel gene expression data from the 64 rms patients and identified 12 groups of co - expressing genes / modules . each module was color coded and tested for go term enrichment in order to associate each module with biological functions ( table 1 ) . consistent modular gene structure was also seen using a larger population of 132 publicly available rms samples suggesting that these modules are a good representation of the biological characteristic of rms tumors ( data not shown ) . in addition , an eigengene per module was generated for each patient to summarize the expression of all the genes contained in each module . these values were used to correlate modules to each other as well as to clinico - pathological features ( fig . 2 ) . modules did not show significant correlation with irs stage , metastasis , tumor size or age of patient at diagnosis ( data not shown ) . however notably , the turquoise , green / yellow , pink and purple modules showed a significant correlation fusion status and histological subtype ( fig . the association between modules and overall survival ( os ) or progression free survival ( pfs ) was also tested . the pink and turquoise modules showed a significant correlation with the os and pfs ( supplementary table s10 ) . as mirnas are known to modulate many of their targets through mrna transcript degradation , , we correlated the expression of mirnas to the module eigengenes in order to evaluate their level of association . 3 represents a heatmap of the correlation between module eigengenes and a subset of mirnas selected based on their coefficient values ( absolute value above 0.4 ) and significance ( p value < 0.001 ) . the power of this approach is demonstrated by well - known myogenic mirs ( myomirs ) -1 , -206 and -133a that are most highly expressed in more differentiated skeletal muscle . these myomirs correlated positively with yellow ( mir-1 , direct pearson ( dp ) 0.59 ; mir-206 , dp 0.49 ; mir-133a , dp 0.58 ) , and purple modules ( mir-1 , dp 0.73 ; mir-206 , dp 0.46 ; mir-133a , dp 0.70 ) ( fig . are myod - associated myomirs most highly expressed early in myogenic differentiation and show a negative correlations with the yellow and purple modules ( mir-221 , dp-0.24 , -0.043 ; mir-222 , dp-0.2 , -0.041 , respectively ) . both modules had functional annotations linked to skeletal muscle development . additionally mir-1 and -133a showed inverse correlation with green module ( mir-1 , dp-0.31 ; mir-133a , dp-0.37 ) that was linked to cell cycle , indicating that low levels of these myomirs correlated with genes involved in cell cycle progression . these myomirs / gene expression correlations associated with differentiation processes strongly support the ability of the approach to identify key roles for mirnas in rms . consistent with mirna expression accurately distinguishing fusion positive from negative patients , four of the 12 modules correlated with fusion gene positivity ( turquoise , purple , green / yellow , and pink ) with turquoise correlating most strongly ( fig . mirnas were also overexpressed in pax3/7-foxo1 positive cases compared to rms without the fusion gene ( supplementary table s3 ) . based on these results , we hypothesized that the fusion protein could regulate mirna expression . to identify mirnas altered by pax3-foxo1 , rna interference ( rnai ) was used to reduce expression levels of the fusion gene in the cell line rh4 ( supplementary fig . s3a ) , and corresponding mirnas levels screened for in duplicate ( supplementary table s11 ) . to prioritize mirnas of likely relevance in rms patients for further investigation we identified those that were reduced by knockdown of the fusion protein , were most strongly positively correlated with the turquoise and purple modules and that were also differentially overexpressed comparing fusion gene positive with negative rms . this identified mirnas as candidates for further investigation ( table 2 ) , including mir-9 - 5p that was additionally correlated with metastatic behavior and poor outcome ( supplementary tables s4-s9 ) . mir-9 - 5p expression levels were analyzed by quantitative rt - pcr in a larger cohort of rms patients ( n = 154 , supplementary table s1b ) and , consistent with the sequencing data ( supplementary table s3 ) , results showed that levels of mir-9 - 5p were significantly higher in fusion gene positive versus negative rms ( wilcoxon test p < 0.0001 ) , skeletal muscle and myoblasts ( wilcoxon test p < in addition , fusion gene positive rms cell lines had significantly higher mir-9 - 5p levels than erms cell lines ( wilcoxon test p = 0.04 ) ( supplementary fig . mir-9 - 5p levels decreased by reducing pax3-foxo1 expression ( table 2 , supplementary table s11 ) , we examined available data for pax3-foxo1 and mycn ( walters et al . no sites were identified for pax3-foxo1 but mycn binding sites were found consistent with those reported for myc and mycn in neuroblastoma . based on mycn being a downstream transcriptional target of pax3-foxo1 , , we also tested the effects of decreasing mycn levels on mir-9 - 5p levels in a pax3-foxo1 positive cell line . modules associated with the pax3-foxo1 fusion genes ( turquoise and purple ) positively correlated with mir-9 - 5p expression . significantly higher levels of mir-9 - 5p expression were noted in metastatic versus non - metastatic considering both all rms and fusion positive rms ( supplementary tables s4 and s5 ) . significantly lower os and pfs in all rms was associated with high mir-9 - 5p expression ( supplementary tables s6 and s8 ) and also within fusion positive rms cases ( supplementary tables s7 and s9 ) . univariate analyses , including only pax3-foxo1 cases , also revealed significant correlations with outcome ( supplementary table s12 ) , although the sample size was very limited . univariate analyses of the quantitative rt - pcr data for the second validation cohort ( n = 154 , supplementary table s1b ) using a cox regression model with mir-9 - 5p as a continuous variable revealed significant correlations in fusion gene positive rms with os ( p = 0.037 , hr = 1.242 , ci 95% 1.0131.522 ) and pfs ( p = 0.034 , hr = 1.239 , ci 95% 1.0161.511 ) . in pax3-foxo1 cases ( n = 45 ) , a significant correlation with os was also observed ( p = 0.012 , hr = 1.445 , ci 95% 1.0841.925 ) and a trend with pfs ( p = 0.055 , hr = 1.29 , ci 95% 0.9951.676 ) . no correlations with mir-9 - 5p levels and survival were observed in fusion gene negative patients ( os ; p = 0.65 , hr = 0.971 , ci 95% 0.8531.104 . pfs ; p = 0.65 , hr = 0.994 , ci 95% 0.8531.104 ) . kaplan - meier analyses were consistent with these results : log - rank test identified significant correlations with both os ( p = 0.003 ) ( fig . 4c ) and pfs ( p = 0.004 ) ( supplementary fig . 3c ) when rms fusion positive patients ( n = 57 ) were split into three groups based on their levels of mir-9 - 5p expression ( percentiles 033% , 3466% , 67100% ) . when the highest mir-9 - 5p expressers were compared to the lowest in multivariate analysis ( mva ) , mir-9 - 5p retained its independent predictive value for os ( p = 0.028 , hr = 4.4 , ci 95% 1.216.6 ) ( fig . 4d ) and pfs ( p = 0.012 , hr = 3.74 , ci 95% 1.3410.44 ) ( supplementary fig . when mva analyses was repeated considering only the 45 pax3-foxo1 , high mir-9 - 5p expression remained an independent predictive variable correlating with outcome ( p = 0.004 , hr = 4.55 , ci 95% 1.6312.76 ) correlating with outcome . due to the significant correlations of high mir-9 - 5p levels with metastatic disease and poor survival as well as strong negative correlations with a module associated with processes in cell adhesion ( pink module ) ( fig 3 ) , we tested whether mir-9 - 5p functionally influenced the migratory behavior of rms cells . cell migration in the rh30 cell line was assessed 72 h after transfection with a synthetic inhibitor against mir-9 - 5p by placing cells into a transwell migratory assay for 24 h. at the end point of experiments , levels of mir-9 - 5p were decreased on average by 70% compared to control treated cells ( fig . there was significant reduction in levels of migration in cells with reduced mir-9 - 5p levels versus the controls ( p < 0.001 , fig . 4f ) but no effect was on cell viability , as assessed by the mts metabolic assay ( fig . here we describe the sequencing analyses of mirnas in a large series ( n = 64 ) of well - characterized rms samples . we were broadly able to distinguish tumor from skeletal muscle and also fusion gene positive from fusion gene negative rms . this is consistent with a previous study of sarcomas that included only 7 rms cases in which cluster analyses of mirna data separated skeletal muscle from rms and other types of sarcoma and placed 3 alveolar rms in a different group from one embryonal case and 3 adult pleomorphic cases . furthermore , our integrated analyses of mirna expression data with parallel gene expression levels using wcgna identified co - expression modules that we were able to assign with biological functions and also correlate with clinical outcome data and fusion gene status . given that mirnas regulate the expression of many genes in a dynamic and cell context specific manner , these results provide a valuable framework and resource for further investigations of rms . reassuringly , modules identified as associated with skeletal muscle development ( fig . 3 ) included correlations with mirnas that have been previously described as mirnas involved in myogenesis ( myomirs ) and also shown to functionally impact on the phenotype of rms cell lines . specifically these included mirs -1 , -206 , -133a and mir-378 that correlated positively with muscle associated modules ( yellow and purple , table 1 , fig . 3 ) , , , , , , . in addition , we previously showed that lower levels of mir-206 expression correlated with poorer outcome in samples from rms patients that was linked to their undifferentiated myogenic phenotype , indicating relevance to the clinical behavior of patients . mir-221 and mir-222 are also described as myomirs and their overexpression and reduction is reported to enhance and decrease myod protein levels , respectively . as predicted from this , our analyses of mirs-221 - 3p and -222 - 3p inversely correlated their expression with the skeletal muscle associated modules ( yellow and purple ) in contrast to mirs -1 , -133 and -378 which were positively correlated to these functional modules . mir-128 - 3p shows a similar expression pattern in these modules to mirs -1 , -133 and -378 and is described with a clear role in muscle development . a definitive role for mirs -221 , -222 and 128 - 3p in rms remains to be determined , as well as other mirnas correlated with the modules defined with myogenic functions , such as mir-96 - 3p , mir-152 - 3p and mir-156 - 5p . interestingly , mir-1 , 133 , 206 , 378 and 128 - 3p negatively , and mir-221 and -222 positively , correlated with gene expression processes for cell adhesion , chemotaxis and extracellular organisation ( pink module ) . this is consistent with the differentiation status of rms cells shown in oncogenic kras ( g12d ) induced zebrafish models of rms where embryonal rms cells recapitulate normal myogenesis and their associated migratory and proliferative capacity . also , targets of these mirnas in these networks are likely to contribute to these invasive and migratory phenotypes in rms , for example mir-206 targets the met tyrosine - kinase receptor which is highly expressed in rms and enhances rms cell migration . similarly , mir-221 and -222 have strong evidence to support that they target timp3 and mmp1 proteins known to influence the extracellular matrix ( http://mirtarbase.mbc.nctu.edu.tw/index.php ) . the myomirs -1 , -133a and mir-378 also inversely correlated with modules linked to cell cycle progression ( green and red ) , consistent with their role in cell cycle arrest when overexpressed in rms , and the requirement of this prior to normal myogenic differentiation . these cell cycle related modules were positively correlated with other mirnas that may play a role in rms , including mir-106b , that is overexpressed and associated with proliferation in medulloblastoma , mir-32 that when down - regulated in mesenchymal stem cells inhibits cell cycle progression and mir-130b that enhances profileration through inactivating hippo signaling in gliomas . mir-19a-3p , as part of the polycistronic mirna cluster mir-17 - 92 , also positively correlated with these cell cycle modules as well as a module associated with fusion genes ( turquoise ) . the latter and red modules were both linked to sonic hedgehog ( shh ) signaling . together this is consistent with high expression of the mir17 - 92 cluster being genomically amplified in around 20% of rms and mycn driven , mostly in those with pax7-foxo1 fusion genes , and possible involvement of the cluster in proliferation through shh signaling , . the strongest positive and negative correlations between genes and mirna expression levels were associated with fusion gene positive rms cases ( fig . 2 , fig . in addition , the turquoise and pink modules linked to fusion gene positivity were also most significantly associated with poor outcome ( supplementary table s10 ) . this is consistent with several studies , including our own , demonstrating the prognostic value of the presence of a fusion gene in rms , , , . our analyses indicates that the fusion protein impacts on the expression of mirnas and through screening changes to mirna levels following reduction of pax3-foxo1 expression in a cell line , we were able to show that the fusion protein alters the expression levels of micrornas . interestingly , mir-9 - 5p was one of the top hits in the screen , was highly correlated with the modules associated with the presence of the fusion gene and it was more highly expressed in fusion gene positive rms compared to fusion gene negative rms ( table 2 ) . although no pax3-foxo1 binding sites were identified near mir-9 - 5p , we showed mir-9 - 5p to be indirectly driven by pax3-foxo1 via mycn . myc / mycn have previously been shown to bind to the mir-9 locus and activate expression in other cell types . mycn is known to be a direct transcriptional target of the fusion protein and to operate in a positive feedback loop mechanism . this overlap in mirna targets for pax3-foxo1 and mycn is to the gene expression changes identified through altering mycn levels and pax3-foxo1 expression in rms that show considerable overlap . taken together , this indicates that the fusion protein together with mycn , and other transcription factors downstream of pax3-foxo1 , such as myod1 , regulate the expression of mirnas and their many target genes , to massively re - programme gene expression in rms cells towards a tumorigenic phenotype . the contribution of these altered levels of mirnas is predicted to impact on the phenotype of fusion positive rms in ways that include the developmental , myogenic , immunological and cell adhesion processes indicated in the co - expression modules ( fig . mir-9 - 5p levels are shown to contribute to the clinical and biological behavior of rms . mir-9 - 5p levels were higher in metastatic versus non - metastatic cases and correlated with a poor outcome of patients , including within fusion gene positive group . through reducing mir-9 - 5p we deduce a role for mir-9 - 5p in enhancing rms cell migration and this cellular phenotype potentially facilitates metastastic behavior . this is similar to the roles for mir-9 - 5p in the proliferation , differentiation and migration of neural progenitor cells but mir-9 - 5p may alter different gene targets dependent on cell context . also , aberrant mir-9 - 5p levels have been indicated in several cancer types where it may be either overexpressed or underexpressed . overexpression has been described in hodgkin 's lymphoma , brain tumors , neuroblastoma and gastric cancers , in the latter affecting proliferation via the mir-9 target cdx2 . reduced levels of mir-9 - 5p play a role in ovarian cancer cells where this , in part , enhances cell proliferation through nfkappab1 . furthermore , in normal epithelial and breast cancer cells , mir-9 - 5p induces epithelial to mesenchymal transition by targeting e cadherin ( cdh1 ) . recently , reducing mir-9 - 5p in an osteosarcoma cell line and identification of high levels of expression in primary tumors suggests a role for this mirna in regulating the progression of osteosarcoma . this role in osteosarcomas in addition to rms described here , indicates a role for mir-9 - 5p in mesenchymal malignancies further to that previously described epithelial and neural cancers . mirnas altered by the fusion protein , including indirectly mir-9 - 5p ( table 2 , supplementary tables s3 and s11 ) , are likely to play an important role in rms . furthermore , the expression levels and the functional framework presented have assigned likely functions for mirnas in rms for further investigation . these could identify additional tumor biomarkers and , depending on their expression in bodily fluids , useful markers for disease monitoring , as exemplified by the detection of the myomir mir-206 in serum from rms patients . achieving good in vivo delivery for therapeutically targeting or introducing mirnas however , further understanding of mirnas in rms may ultimately enable targeting their key roles via regulation of multiple genes and pathways in ways that could improve outcome for patients .

rhabdomyosarcomas ( rms ) in children and adolescents are heterogeneous sarcomas broadly defined by skeletal muscle features and the presence / absence of pax3/7-foxo1 fusion genes . micrornas are small non - coding rnas that regulate gene expression in a cell context specific manner . sequencing analyses of micrornas in 64 rms revealed expression patterns separating skeletal muscle , fusion gene positive and negative rms . integration with parallel gene expression data assigned biological functions to 12 co - expression networks / modules that reassuringly included myogenic roles strongly correlated with micrornas known in myogenesis and rms development . modules also correlated with clinical outcome and fusion status . regulation of micrornas by the fusion protein was demonstrated after pax3-foxo1 reduction , exemplified by mir-9 - 5p . mir-9 - 5p levels correlated with poor outcome , even within fusion gene positive rms , and were higher in metastatic versus non - metastatic disease . mir-9 - 5p reduction inhibited rms cell migration . our findings reveal micrornas in a regulatory framework of biological and clinical significance in rms .

Introduction Materials and methods Results Discussion

rhabdomyosarcomas ( rms ) are the most frequent soft tissue sarcoma in children and rms cells resemble those in the early stages of myogenic differentiation , . the encoded fusion proteins play an oncogenic role in the development of arms in association with co - operating events , notably overexpression of mycn that is a transcriptional target of the fusion protein and genomically amplified in approximately 20% of cases , , , . the presence of pax3-foxo1 has been correlated with aggressive clinical behavior in several studies , , , and gene expression profiles and genomic imbalances are similar in fusion gene negative arms and erms , . micrornas ( mirnas ) are small ( 2022 nt ) rna molecules that can alter cellular processes including proliferation , differentiation and apoptosis . individual mirnas have been ascribed oncogenic or tumor suppressor roles in cancer and their roles can clearly be cell context specific . roles for individual mirnas in rms have been described such as mir-1 , mir-206 , mir-133a / b and mir-378 that maintain an undifferentiated myogenic state , , , , , , and mir-29 that is described with a tumor suppressor role . however , a comprehensive view of how mirnas shape the gene expression and biological features of a large number of rms has not been described but could contribute to identification of prognostic markers / signatures and ultimately therapeutic targets , as indicated by previous studies of other tumor types , , . here we derived mirna expression profiling data from high - throughput sequencing analyses of 64 primary rms samples and took a systems biology approach to integrate this with parallel gene expression profiling data . this allowed us to identify co - expression mrna networks ( modules ) with assigned biological functions that correlated with both patient outcome data and the presence of pax3/7-foxo1 . this led us to demonstrate that pax3-foxo1 regulates the expression of many mirnas , including mir-9 - 5p that was indirectly altered by pax3-foxo1 and shown to be of clinical and functional relevance . the variance of the counts data were stabilized using a power transformation function and normalized by applying a non - linear function ( lowess ) . rna gene expression profiling data for 64 rms samples parallel to those profiled for mirnas were obtained and pre - analyzed as previously described . the samples underwent weighted gene co - expression network analysis ( wgcna ) using the r package version 0.96 . briefly , a co - expression similarity measure was calculated by raising the absolute correlation coefficient between genes to a selected power . association between modules expression and clinico - pathological parameters were evaluated using the kruskal - wallis rank sum test applied to both the eigengene and the gene expression levels . mir-9 - 5p knockdown in the rh30 cell line was achieved by transfecting the cells with miscript mirna inhibitor using hiperfect reagent ( qiagen gmbh , hilden , germany ) . levels of expression during experiments and analyses of levels in primary rms samples were assayed where appropriate using taqman microrna assays according to the manufacturer 's instructions ( applied biosystems , ca , usa ) . expression data were analyzed by the comparative threshold cycle ( ct ) method accordingly to user bulletin # 2 ( applied biosystems ) . results were expressed in term of the ct value and obtained as follows : ct mirna = average ( ct u6 ; ctrun48 ) ct mirna , where ct mirna , ct u6 and ctrun48 represents the comparative threshold cycle for mirna and the two endogenous controls u6 and run48 , respectively . 24 h later , cells that had not migrated were removed using a cotton bud , and cells that had migrated to the base of the inserts were fixed overnight in 100% methanol at 20 c and then stained for 2 min with 2% crystal violet solution ( sigma - aldrich , mo , usa ) . mirna expression profiling was performed by high - throughput sequencing analysis of 68 samples ( 3 skeletal muscle , 1 myoblast and 64 rms patient samples ( supplementary table s1 ) ) . after sequence alignment and filtering , each tag was assigned to a rna class based on entrez gene type and 78% and 73% were shown to be mirnas in rms and skeletal muscle , respectively , confirming their successful enrichment ( supplementary fig . direct comparisons of levels of expression across molecular ( fusion positive versus negative rms ) , histological ( skeletal muscle versus rms ) , and clinical ( metastatic m1 versus non metastatic m0 ) entities produced lists of mirnas significantly over or under - expressed in these groups ( supplementary tables s2 - 5 ) . unsupervised hierarchical clustering analysis performed using mirnas with at least 100 tags in 50% of the samples showed the presence of distinct patterns of expression across rms samples ( fig . notably , mirna expression patterns mostly distinguished rms histological subtypes showing sets of mirnas positively and negatively associated with fusion gene status . the well - known muscle specific mirnas including mir-1 , mir-133 and mir-206 showed higher expressed in skeletal muscle versus rms subtypes . application of class prediction analysis based on nearest shrunken centroid classifier showed that mirna expression can distinguish fusion positive and negative patients with around 89% accuracy ( supplementary fig . based on the principle that co - expressed genes are commonly associated with similar biological function / pathways , we employed wgcna using parallel gene expression data from the 64 rms patients and identified 12 groups of co - expressing genes / modules . the power of this approach is demonstrated by well - known myogenic mirs ( myomirs ) -1 , -206 and -133a that are most highly expressed in more differentiated skeletal muscle . both modules had functional annotations linked to skeletal muscle development . these myomirs / gene expression correlations associated with differentiation processes strongly support the ability of the approach to identify key roles for mirnas in rms . consistent with mirna expression accurately distinguishing fusion positive from negative patients , four of the 12 modules correlated with fusion gene positivity ( turquoise , purple , green / yellow , and pink ) with turquoise correlating most strongly ( fig . mirnas were also overexpressed in pax3/7-foxo1 positive cases compared to rms without the fusion gene ( supplementary table s3 ) . based on these results , we hypothesized that the fusion protein could regulate mirna expression . to identify mirnas altered by pax3-foxo1 , rna interference ( rnai ) was used to reduce expression levels of the fusion gene in the cell line rh4 ( supplementary fig . to prioritize mirnas of likely relevance in rms patients for further investigation we identified those that were reduced by knockdown of the fusion protein , were most strongly positively correlated with the turquoise and purple modules and that were also differentially overexpressed comparing fusion gene positive with negative rms . this identified mirnas as candidates for further investigation ( table 2 ) , including mir-9 - 5p that was additionally correlated with metastatic behavior and poor outcome ( supplementary tables s4-s9 ) . mir-9 - 5p expression levels were analyzed by quantitative rt - pcr in a larger cohort of rms patients ( n = 154 , supplementary table s1b ) and , consistent with the sequencing data ( supplementary table s3 ) , results showed that levels of mir-9 - 5p were significantly higher in fusion gene positive versus negative rms ( wilcoxon test p < 0.0001 ) , skeletal muscle and myoblasts ( wilcoxon test p < in addition , fusion gene positive rms cell lines had significantly higher mir-9 - 5p levels than erms cell lines ( wilcoxon test p = 0.04 ) ( supplementary fig . mir-9 - 5p levels decreased by reducing pax3-foxo1 expression ( table 2 , supplementary table s11 ) , we examined available data for pax3-foxo1 and mycn ( walters et al . based on mycn being a downstream transcriptional target of pax3-foxo1 , , we also tested the effects of decreasing mycn levels on mir-9 - 5p levels in a pax3-foxo1 positive cell line . modules associated with the pax3-foxo1 fusion genes ( turquoise and purple ) positively correlated with mir-9 - 5p expression . significantly higher levels of mir-9 - 5p expression were noted in metastatic versus non - metastatic considering both all rms and fusion positive rms ( supplementary tables s4 and s5 ) . significantly lower os and pfs in all rms was associated with high mir-9 - 5p expression ( supplementary tables s6 and s8 ) and also within fusion positive rms cases ( supplementary tables s7 and s9 ) . univariate analyses of the quantitative rt - pcr data for the second validation cohort ( n = 154 , supplementary table s1b ) using a cox regression model with mir-9 - 5p as a continuous variable revealed significant correlations in fusion gene positive rms with os ( p = 0.037 , hr = 1.242 , ci 95% 1.0131.522 ) and pfs ( p = 0.034 , hr = 1.239 , ci 95% 1.0161.511 ) . no correlations with mir-9 - 5p levels and survival were observed in fusion gene negative patients ( os ; p = 0.65 , hr = 0.971 , ci 95% 0.8531.104 . 3c ) when rms fusion positive patients ( n = 57 ) were split into three groups based on their levels of mir-9 - 5p expression ( percentiles 033% , 3466% , 67100% ) . when the highest mir-9 - 5p expressers were compared to the lowest in multivariate analysis ( mva ) , mir-9 - 5p retained its independent predictive value for os ( p = 0.028 , hr = 4.4 , ci 95% 1.216.6 ) ( fig . when mva analyses was repeated considering only the 45 pax3-foxo1 , high mir-9 - 5p expression remained an independent predictive variable correlating with outcome ( p = 0.004 , hr = 4.55 , ci 95% 1.6312.76 ) correlating with outcome . due to the significant correlations of high mir-9 - 5p levels with metastatic disease and poor survival as well as strong negative correlations with a module associated with processes in cell adhesion ( pink module ) ( fig 3 ) , we tested whether mir-9 - 5p functionally influenced the migratory behavior of rms cells . cell migration in the rh30 cell line was assessed 72 h after transfection with a synthetic inhibitor against mir-9 - 5p by placing cells into a transwell migratory assay for 24 h. at the end point of experiments , levels of mir-9 - 5p were decreased on average by 70% compared to control treated cells ( fig . there was significant reduction in levels of migration in cells with reduced mir-9 - 5p levels versus the controls ( p < 0.001 , fig . here we describe the sequencing analyses of mirnas in a large series ( n = 64 ) of well - characterized rms samples . we were broadly able to distinguish tumor from skeletal muscle and also fusion gene positive from fusion gene negative rms . this is consistent with a previous study of sarcomas that included only 7 rms cases in which cluster analyses of mirna data separated skeletal muscle from rms and other types of sarcoma and placed 3 alveolar rms in a different group from one embryonal case and 3 adult pleomorphic cases . furthermore , our integrated analyses of mirna expression data with parallel gene expression levels using wcgna identified co - expression modules that we were able to assign with biological functions and also correlate with clinical outcome data and fusion gene status . given that mirnas regulate the expression of many genes in a dynamic and cell context specific manner , these results provide a valuable framework and resource for further investigations of rms . reassuringly , modules identified as associated with skeletal muscle development ( fig . 3 ) included correlations with mirnas that have been previously described as mirnas involved in myogenesis ( myomirs ) and also shown to functionally impact on the phenotype of rms cell lines . as predicted from this , our analyses of mirs-221 - 3p and -222 - 3p inversely correlated their expression with the skeletal muscle associated modules ( yellow and purple ) in contrast to mirs -1 , -133 and -378 which were positively correlated to these functional modules . a definitive role for mirs -221 , -222 and 128 - 3p in rms remains to be determined , as well as other mirnas correlated with the modules defined with myogenic functions , such as mir-96 - 3p , mir-152 - 3p and mir-156 - 5p . interestingly , mir-1 , 133 , 206 , 378 and 128 - 3p negatively , and mir-221 and -222 positively , correlated with gene expression processes for cell adhesion , chemotaxis and extracellular organisation ( pink module ) . also , targets of these mirnas in these networks are likely to contribute to these invasive and migratory phenotypes in rms , for example mir-206 targets the met tyrosine - kinase receptor which is highly expressed in rms and enhances rms cell migration . the myomirs -1 , -133a and mir-378 also inversely correlated with modules linked to cell cycle progression ( green and red ) , consistent with their role in cell cycle arrest when overexpressed in rms , and the requirement of this prior to normal myogenic differentiation . these cell cycle related modules were positively correlated with other mirnas that may play a role in rms , including mir-106b , that is overexpressed and associated with proliferation in medulloblastoma , mir-32 that when down - regulated in mesenchymal stem cells inhibits cell cycle progression and mir-130b that enhances profileration through inactivating hippo signaling in gliomas . mir-19a-3p , as part of the polycistronic mirna cluster mir-17 - 92 , also positively correlated with these cell cycle modules as well as a module associated with fusion genes ( turquoise ) . together this is consistent with high expression of the mir17 - 92 cluster being genomically amplified in around 20% of rms and mycn driven , mostly in those with pax7-foxo1 fusion genes , and possible involvement of the cluster in proliferation through shh signaling , . the strongest positive and negative correlations between genes and mirna expression levels were associated with fusion gene positive rms cases ( fig . in addition , the turquoise and pink modules linked to fusion gene positivity were also most significantly associated with poor outcome ( supplementary table s10 ) . this is consistent with several studies , including our own , demonstrating the prognostic value of the presence of a fusion gene in rms , , , . our analyses indicates that the fusion protein impacts on the expression of mirnas and through screening changes to mirna levels following reduction of pax3-foxo1 expression in a cell line , we were able to show that the fusion protein alters the expression levels of micrornas . interestingly , mir-9 - 5p was one of the top hits in the screen , was highly correlated with the modules associated with the presence of the fusion gene and it was more highly expressed in fusion gene positive rms compared to fusion gene negative rms ( table 2 ) . although no pax3-foxo1 binding sites were identified near mir-9 - 5p , we showed mir-9 - 5p to be indirectly driven by pax3-foxo1 via mycn . mycn is known to be a direct transcriptional target of the fusion protein and to operate in a positive feedback loop mechanism . this overlap in mirna targets for pax3-foxo1 and mycn is to the gene expression changes identified through altering mycn levels and pax3-foxo1 expression in rms that show considerable overlap . taken together , this indicates that the fusion protein together with mycn , and other transcription factors downstream of pax3-foxo1 , such as myod1 , regulate the expression of mirnas and their many target genes , to massively re - programme gene expression in rms cells towards a tumorigenic phenotype . the contribution of these altered levels of mirnas is predicted to impact on the phenotype of fusion positive rms in ways that include the developmental , myogenic , immunological and cell adhesion processes indicated in the co - expression modules ( fig . mir-9 - 5p levels are shown to contribute to the clinical and biological behavior of rms . mir-9 - 5p levels were higher in metastatic versus non - metastatic cases and correlated with a poor outcome of patients , including within fusion gene positive group . through reducing mir-9 - 5p we deduce a role for mir-9 - 5p in enhancing rms cell migration and this cellular phenotype potentially facilitates metastastic behavior . this is similar to the roles for mir-9 - 5p in the proliferation , differentiation and migration of neural progenitor cells but mir-9 - 5p may alter different gene targets dependent on cell context . also , aberrant mir-9 - 5p levels have been indicated in several cancer types where it may be either overexpressed or underexpressed . reduced levels of mir-9 - 5p play a role in ovarian cancer cells where this , in part , enhances cell proliferation through nfkappab1 . furthermore , in normal epithelial and breast cancer cells , mir-9 - 5p induces epithelial to mesenchymal transition by targeting e cadherin ( cdh1 ) . recently , reducing mir-9 - 5p in an osteosarcoma cell line and identification of high levels of expression in primary tumors suggests a role for this mirna in regulating the progression of osteosarcoma . this role in osteosarcomas in addition to rms described here , indicates a role for mir-9 - 5p in mesenchymal malignancies further to that previously described epithelial and neural cancers . mirnas altered by the fusion protein , including indirectly mir-9 - 5p ( table 2 , supplementary tables s3 and s11 ) , are likely to play an important role in rms . furthermore , the expression levels and the functional framework presented have assigned likely functions for mirnas in rms for further investigation . these could identify additional tumor biomarkers and , depending on their expression in bodily fluids , useful markers for disease monitoring , as exemplified by the detection of the myomir mir-206 in serum from rms patients . achieving good in vivo delivery for therapeutically targeting or introducing mirnas however , further understanding of mirnas in rms may ultimately enable targeting their key roles via regulation of multiple genes and pathways in ways that could improve outcome for patients .

the online version of this article ( doi:10.1007/s00249 - 015 - 1017-x ) contains supplementary material , which is available to authorized users . the presence of coexisting liquid - ordered and liquid - disordered lipid phases has been implicated in diverse biological processes with high sensitivity to perturbations in membrane composition , tension , curvature , and temperature ( lingwood et al . lipid phase dynamics may inherently provide nanoscale clustering and facilitate lipid - raft - mediated processes ( veatch et al . however , trace membrane additives can disturb the lipid phase dynamics and the associated biological processes . some additives possess rotational asymmetry , preferentially localize to the phase boundary , reduce the phase miscibility , decrease the miscibility transition temperature ( tmis ) , and act as linactants , 1d analogs of surfactants ( trabelsi et al . other membrane additives are rotationally symmetric , preferentially localize into one particular lipid phase , and may increase or decrease tmis . broadly , the effects of membrane additives are quantified by the change to the lipid phase tmis . the difference between a sample temperature and tmis determines fundamental equilibrium qualities such as the correlation length of the coexisting phases ( honerkamp - smith et al . 2008 ; palmieri and safran 2013a ) and the dynamics of the phase mobility ( honerkamp - smith et al . 2012 ; palmieri and safran 2013b ) . numerous molecules have demonstrated potent abilities to alter membrane phase miscibility without necessarily containing a dependence on the rotation of the molecule within the bilayer , including cholesterol ( dietrich et al . 2001 ; zhao et al . 2007a ; levental et al . 2009 ; heberle et al . 1991 ; gray et al . 2013 ) , insecticides ( jorgensen et al . 1991 ) , n - propyl gallate ( zhao et al . 2007b ) , c - reactive protein ( senz et al . 2010 ) , and flavonoids ( ostroumova et al . other molecules have been hypothesized to prefer the boundary between two lipid phases because of their molecular structure ; for example , hybrid lipids contain one liquid - order - preferring tail and one liquid - disorder - preferring tail ( brewster et al . 2009 ; hassan - zadeh et al . 2014 ; li and gorfe 2014 ) , and asymmetrically lipidated proteins , such as n - ras and h - ras , have shown preference for partitioning at the lipid phase boundary ( nicolini et al . further , molecules such as the nonsteroidal anti - inflammatory drug indomethacin may contain both a net phase preference and significant molecular structure asymmetry , although the nanoscale partitioning has yet to be studied ( zhou et al . previous studies have focused on theoretical predictions and experimental examination of systems with a significant fraction of the membrane consisting of phase - polarized particles ( > 10 mol% ) ( brewster et al . 2009 ; yamamoto et al . 2010 ; yamamoto and safran 2011 ; palmieri and safran 2013a , b ; hassan - zadeh et al . 2014 ) , whereas this manuscript focuses on small composition changes that can greatly affect membrane phase dynamics and provide a means for live cells to efficiently regulate lipid phase - dependent processes . monte carlo simulations of 2d ising models with conserved order parameters have been used to demonstrate key membrane properties ( frazier et al . 2007 ; yethiraj and weisshaar 2007 ; honerkamp - smith et al . 2011 , 2012 ) . within the ising model , a particle s phase preference ( ) is quantified for liquid - disordered ( black , = 1 ) or liquid - ordered ( white , = 1 ) phases , although the particular phase associated with the color and sign of is arbitrary in this symmetric system . the internal energy between two particles ( ji , j ) is equal to j for nearest neighbors and zero otherwise , and it can be calculated from the sum of all particle interactions in the hamiltonian ( h ) according to:1\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h = -\sum\limits _ { \langle i , j \rangle } { j_{i , j } \sigma_{i } \sigma_{j } } .$$\end{document}h=-i , jji , jij . when this model contains 50 % white and 50 % black particles on a square lattice , it exhibits critical behavior with \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$t_{\text{mis } } = 2j/(k_{\text{b } } \ln ( 1 + \sqrt 2 ) ) $ $ \end{document}tmis=2j/(kbln(1 + 2 ) ) , where kb is boltzmann s constant . isolated plasma membrane vesicles from mast cells happen to be of a composition that demonstrates near - critical behavior with tmis = 22 c ( veatch et al . 2008 ) , corresponding to a value of j = 0.26 kcal / mol . to relate the complex molecular composition of near - critical membranes to the two - state ising model , each pixel from the model represents the mean composition of each state . for example , the white particles in the ising model represent the cell membrane s average liquid - ordered phase composed of lipids with longer , more saturated acyl tails , greater sphingomyelin content , and slightly higher cholesterol content than the black particles . this model permits the observation of complex phase behaviors without explicit incorporation of the molecular details or concentrations of the membrane , as long as the average phase segregation preferences are represented by the value of j. in this manuscript , we demonstrate the influence of trace additives in a two - component nearest neighbor model on a square lattice with conserved order parameters as a means of simulating how additives of differing molecular structure could influence lipid phase mixing . instead of modifying the value of j to represent changes in membrane composition , a third particle type was incorporated in these simulations to provide greater structural detail of the additive s effect on phase separation and additive partitioning . in a ternary lipid membrane , for example , the additive could be a fourth type of molecule or more of one molecule type that was already present in the membrane . simulated additives were either rotationally symmetric gray particles or rotational asymmetric phase - polarized particles with gray values of g or polarizations of p , respectively ( fig . 1 ) . generally , the addition of a third type of particle to the system results in a change to the phase diagram , new phase transitions , and variations from critical behavior . simulations performed with additives of g = 0 were analogous to the blume - emery - griffiths model ( blume et al . 1971 ) , for which the phase transitions were modeled against an additive concentration with mean - field approximations . however , the additive concentrations explored here are consistent with the composition fluctuations in living and model membranes , including variations in content of cholesterol ( dietrich et al . 2001 ; pokorny et al . 2010 ) , fluorophores ( veatch et al . 2007a ; frazier et al . 2007 ) , and a variety of other biologically active molecules ( jorgensen et al . 2010 ; gray et al . 2013 ; ostroumova et al . 2014 ) . while real membranes have been demonstrated to be near critical in composition ( veatch et al . 2007b , 2008 ) , they have not been shown to be any closer to critical than the additive - included simulations shown here . further , the experimental method for observing plasma membrane extracts involved the incorporation of fluorescent lipid additives ( < 0.25 mol% ) ( veatch et al . 2008 ) , which altered the membrane composition and presumably its phase behavior but did not diminish the significance or confidence in the result that the membranes were near critical in composition.fig . 1 white , liquid - ordered ( = 1 ) ; black , liquid - disordered ( = 1 ) ; gray particles ( = g ) were rotationally symmetric . phase - polarized additives , or janus additives , were rotationally asymmetric with a polarized phase preference ( || = p ) white , liquid - ordered ( = 1 ) ; black , liquid - disordered ( = 1 ) ; gray particles ( = g ) were rotationally symmetric . phase - polarized additives , or janus additives , were rotationally asymmetric with a polarized phase preference ( || = p ) this work focuses on small perturbations to the well - understood two - state ising model and demonstrates how trace additives would affect the characteristic size of the co - existing single - phase domains ( ) and tmis . for 3 mol% additives , tmis and both changed monotonically with increasing additive concentration , while tmis changed linearly . rotationally symmetric particles of g < 1 and rotationally asymmetric particles of all values of p caused a decrease in both tmis and . however , rotationally symmetric additives of g > 1 resulted in an increase in both tmis and . the morphological detail provided by this model yields the distributions of additives within the system , which is unavailable from mean - field models . the resulting distributions of the additives at the boundary between phases were quantified , and the applicability of this analysis for assessing experimental membrane additives or cellular perturbations is discussed . non - local particle exchanges were allowed via a monte carlo algorithm conserving the system composition ( e.g. , 49.5 % white , 49.5 % black , and 1 % additive ) and equilibrated for 10 global equilibration sweeps on a 512 512 square grid with biperiodic boundary conditions . an additional 10 global sweeps were performed over which an average of the system parameters was reported , e.g. , the correlation length . the symmetry of this configuration with equal fractions of white and black particles resulted in the additives having the same effect , regardless of the sign of g or p. further details are provided in the supplemental material including a movie of 50 different time points of the equilibrated system.fig . 2increasing the mole fraction of phase - polarized particles caused an increase in the phase miscibility and a decrease of t mis . a with p = 1 , phase - polarized additives are displayed as red squares in these images , and zoomed - in regions show the individual additives in fig . blue outlined images indicate where t = t mis with j set such that t mis of the additive - free system \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$(t_{mis}^{\prime})$$\end{document}(tmis ) is 22 c . increasing the concentration of phase - polarized particles with p = 1 resulted in a decrease of b and c t mis . t mis = t mis\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$t_{mis}^{\prime}$$\end{document}tmis. uncertainty of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$t_{mis}^{\prime}$$\end{document}tmis was approximately 0.6 c , and uncertainty of was smaller than the symbol unless otherwise indicated increasing the mole fraction of phase - polarized particles caused an increase in the phase miscibility and a decrease of t mis . a with p = 1 , phase - polarized additives are displayed as red squares in these images , and zoomed - in regions show the individual additives in fig . blue outlined images indicate where t = t mis with j set such that t mis of the additive - free system \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$(t_{mis}^{\prime})$$\end{document}(tmis ) is 22 c . increasing the concentration of phase - polarized particles with p = 1 resulted in a decrease of b and c t mis . t mis = t mis\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$t_{mis}^{\prime}$$\end{document}tmis. uncertainty of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$t_{mis}^{\prime}$$\end{document}tmis was approximately 0.6 c , and uncertainty of was smaller than the symbol unless otherwise indicated the correlation length of the phases was calculated from the azimuthal average of the 2d spatial correlation of the system configuration ( i ) where all white particles were valued at 1 , black particles at 0 , and additives at ( g/2 + 0.5 ) . unless otherwise stated , if p 0 , then g = 0 , and vice versa . the 2d correlation function ( c ) as a function of the position \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\left ( { \bar{r } } \right)$$\end{document}r can be calculated by2\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$c\left ( { \bar { r } } \right ) = \frac{{\left\langle { i\left ( { \overline{r } } \right)i\left ( { \overline{r } + \overline{r } } \right ) } \right\rangle } } { { \left\langle { i\left ( { \overline{r } } \right ) } \right\rangle^{2 } } } , \,$$\end{document}cr=irir+rir2,where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\langle \ , \rangle$$\end{document} represents the average over all 2d space \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ ( { \bar { r } } ) $ $ \end{document}(r ) . computationally , this was expedited by the use of fast fourier transforms ( ffts ) with3\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$c\left ( { \bar { r } } \right ) = \frac{{{\text{fft}}^ { - 1 } \left ( { \left| { { \text{fft}}\left ( i \right ) } \right|^{2 } } \right)}}{n},$$\end{document}cr=fft-1ffti2n , and normalization ( n ) ( veatch et al . 2012 ) . the 1d correlation function ( c ) was computed from4\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$c\left ( r \right ) = \frac{1}{2\pi } \int\limits_{0}^{2\pi } { c\left ( { r,\theta } \right){\text{d}}\theta } , $ $ \end{document}cr=1202cr,d,and the correlation length ( ) was calculated by nonlinear least square fitting of cfit to c(r ) where5\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$c_{\text{fit } } = c_{0 } \frac{{{\text{e}}^ { - r/\xi } } } { { r^{\eta } } } $ $ \end{document}cfit = c0e - r/rand the critical exponent equals 0.25 , which is exact for the ising model . the critical exponents for the three - component systems and finite systems are not equal to that of the ising model ; however , additionally allowing to change did not significantly change fitting results . changes to tmis tmis was determined by measuring the shift in as a function of temperature and by ascertaining the maximum of the specific heat versus temperature ( veatch et al . the finite size effects that alter the perceived transition temperature were consistent for all of these simulations on the same size system , which permitted this simplistic calculation of tmis and its broad applicability to systems of other sizes . a demonstration of the independence of these results on system size is presented in the supplemental material ( fig . changes in phase miscibility with an increasing additive fraction are visibly obvious in images of the system configuration ( figs . 2 , s5 , s6 ) . tmis decreased linearly with increasing fractions of phase - polarized particles , or janus particles , as predicted from mean - field models ( yethiraj and weisshaar 2007 ; brewster et al . the miscibility temperature is reduced for any phase polarization ( p ) , while rotationally symmetric gray particles either increased or decreased tmis depending on their gray value ( g ) ( figs . 3 , s6 ) . increasing p for the phase - polarized additives resulted in a greater decrease in tmis until p > 3 , upon which the additives segregated into a third phase , separate from the white and black particles , and reduced the white and black particle mixing . gray particles with g < 1 encouraged phase miscibility by decreasing the differences between the ordered and disordered phases , analogous to the incorporation of n - alcohols into membranes ( gray et al . 2013 ) . however , when g > 1 , the opposite effect dominates ; the difference between the phases was increased when g > 1 and tmis was increased , analogous to the addition of -lysin to membranes ( pokorny et al . gray particles with g > 1 were no longer of in - between phase and were more liquid - order preferring than white particles , i.e. , super-white.fig . 3 a and b t mis and either increased or decreased upon additive addition depending on the additive properties . t mis = t mis\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$t_{mis}^{\prime}$$\end{document}tmis where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$t_{mis}^{\prime}$$\end{document}tmis = 22 c . measurement of is limited by the system size , resulting in large uncertainties for situations with > 100 pixels ( fig . t mis and changes are symmetrical for negative values of g or p a and b t mis and either increased or decreased upon additive addition depending on the additive properties . t mis = t mis\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$t_{mis}^{\prime}$$\end{document}tmis where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$t_{mis}^{\prime}$$\end{document}tmis = 22 c . measurement of is limited by the system size , resulting in large uncertainties for situations with > 100 pixels ( fig . t mis and changes are symmetrical for negative values of g or p when p or g > 3 , the additives condensed and formed a distinct phase excluding white or black particles ( fig . s6 ) , and the white - black miscibility temperature became less affected by the additives ( fig . 3 ) . for p or g > 3 , simulations were unable to capture a diverse set of configurations for the additive aggregation because of the improbable rearrangement of the condensed phases , analogous to the kinetically trapped configurations that are commonly experienced experimentally . aggregates of phase - polarized particles at p = 4 demonstrated rotational ordering of the additives ( fig . a particle was declared to be at the phase boundary if it was adjacent to two white and two black particles . the ratio of additives at the phase boundary to all additives with no adjacent additives was calculated ( fig . as p increased , the fraction of phase - polarized particles at the phase boundary increased ; as g increased , the fraction of gray particles at the phase boundary decreased . lowering the temperature would have made phase - polarized particles more likely to be found at the phase boundary if the phase morphology had stayed consistent . however , increasing temperature from 8.5 to 36 c resulted in 2.3 0.4 times more phase boundary ( fig . s8 ) , which was dependent on both simulation size and additive properties , and generally resulted in a higher probability for the additives being located at the phase boundary for higher temperatures . additive partitioning was normalized to the amount of phase boundary in order to calculate the fold enhancement of the additive to the phase boundary relative to the white or black particles ( fig . additives more concentrated at the phase boundary than the g = 1 particles preferentially partitioned at the phase boundary , whereas additives less concentrated at the phase boundary than the g = 1 particles preferentially partitioned away from the phase boundary , which is consistent with the sign of tmis ( fig . 3 ) . although there is not a local energy difference for the g = 0 additive if it is immersed in a single phase or at the phase boundary , there is an energy difference for the system that encourages g < 1 additives to be concentrated at the phase boundary . at colder temperatures , the phase - polarized additives especially concentrated at the phase boundary because of the decreased entropic drive for mixing.fig . 4 a the probability that an additive was found at the phase boundary varied with the additive properties and temperature . the fraction of additives at the boundary was calculated as the ratio of the number of additives surrounded by two white and two black particles versus the total number of additives , excluding additives adjacent to other additives . b the additive locations were normalized by the amount of phase boundary in each condition to yield the fold enhancement of the additive at the phase boundary versus generic white particles . uncertainty was calculated as the standard deviation of different time points and smaller than the symbol unless otherwise indicated a the probability that an additive was found at the phase boundary varied with the additive properties and temperature . the fraction of additives at the boundary was calculated as the ratio of the number of additives surrounded by two white and two black particles versus the total number of additives , excluding additives adjacent to other additives . b the additive locations were normalized by the amount of phase boundary in each condition to yield the fold enhancement of the additive at the phase boundary versus generic white particles . uncertainty was calculated as the standard deviation of different time points and smaller than the symbol unless otherwise indicated these results apply to additives in systems that are near critical compositions . it is important to note that these results would vary greatly for compositions that are far from critical compositions because of the increased phase boundary line tension and the increased likelihood for boundary - preferring particles to localize at the boundary in such systems . in all the prior described simulations , only additives with however , real membrane additives would likely possess both a net phase preference ( g 0 ) and a rotational phase asymmetry ( p 0 ) . to predict how changing both g and p for a membrane additive would affect the miscibility transition temperature of the membrane , simulations were performed for additives with rotational asymmetry and a net phase preference at 1 mol% ( fig . the changes to the miscibility transition temperature were dependent on both the g and p value of the additive in a cumulative effect . for small values of g and p ( i.e. , either g or p 1 ) , tmis as a function of g and p was approximately equal to the sum of the change in miscibility temperature from 1 mol% gray particles of gray value g plus the change from 1 mol% polarized particles of polarization p ( fig . 5b).fig . 5 a changes to the miscibility transition temperature were induced by the incorporation of 1 mol% additives with both net and rotationally asymmetric phase preferences , i.e. , dependent on both g and p values , as shown in the inset . b for small values of g and p , t mis as a function of g and p , t mis ( g , p ) , is approximately equal to t mis ( g , 0 ) + t mis ( 0 , p ) a changes to the miscibility transition temperature were induced by the incorporation of 1 mol% additives with both net and rotationally asymmetric phase preferences , i.e. , dependent on both g and p values , as shown in the inset . b for small values of g and p , t mis as a function of g and p , t mis ( g , p ) , is approximately equal to t mis ( g , 0 ) + t mis ( 0 , p ) taken together , these idealized membrane additives represent different means by which molecules may alter the lipid phases in a membrane . a membrane - associated molecule may be experimentally quantified with both a p value and g value to describe the effects of the additive molecular structure on lipid phase dynamics in a particular membrane . g is analogous to the partition constant and quantifies the molecule s preference for one phase over the other . p quantifies the molecule s phase asymmetry and preference to partition at the phase boundary . the effects of a particular membrane additive are determined entirely by the membrane s additive - free miscibility temperature ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$t_{mis}^{\prime}$$\end{document}tmis ) or internal energy between two particles ( j ) of the near - critical membrane without a dependence on the particular molecular details of the individual membrane components . the temperatures reported throughout this article could be alternately represented as reduced temperatures ( tr ) , where tr = ( t \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$t_{mis}^{\prime}$$\end{document}tmis)/ \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$t_{mis}^{\prime}$$\end{document}tmis. upon rescaling , these results would be directly applicable to other membranes with different values of j to predict tmis for different \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$t_{mis}^{\prime}$$\end{document}tmis. further , experimental use of membranes of varying \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$t_{mis}^{\prime}$$\end{document}tmis would permit the exploration of different tr values within experimentally limited temperature ranges . for example , increasing the fatty acid tail length in the saturated , liquid - order preferring lipid of a ternary mixture typically increases \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$t_{mis}^{\prime}$$\end{document}tmis and j , while decreasing the additive s g value and enhancing the additive s phase boundary preference . similarly , the p and g values for a membrane additive in a complex cellular membrane could be predicted by measuring the additive 's effects on synthetic model membranes of greatly different molecular composition , but similar \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$t_{mis}^{\prime}$$\end{document}tmis and j. values of g and p that are greater than one represent the phase preferences for the additives that are stronger that then constituents of the phases themselves . for example , if dipalmitoylphosphatidylcholine ( dppc ) was added to a dioleoylphosphatidylcholine ( dopc)/dimyristoylphosphatidylcholine ( dmpc)/cholesterol bilayer , it would likely have a g > 1 since dppc has a stronger preference for the liquid - ordered phase than dopc , dmpc , or cholesterol . similarly , it is feasible that a particular phase - preferring component of a hybrid molecule could have a stronger phase preference than average constituents of that phase , i.e. , p > , the palmitoyl group of h - ras or palmitoyloleoylphosphatidylcholine ( popc ) could prefer the liquid - ordered phase in a dopc / dmpc / cholesterol bilayer even more than dopc , dmpc , or cholesterol . the quantification of p and g values of cholesterol , for example , requires knowledge of the effects cholesterol addition has on lipid phase separation and the likelihood of cholesterol to be found on the phase boundary . cholesterol preferentially partitions into liquid - ordered phases , thereby indicating a value of g > 0 . the addition of cholesterol to a membrane typically encourages phase miscibility , indicating that cholesterol has a value of g < 1 . the value of g for cholesterol will vary with the surrounding membrane lipids ; however , quantification of cholesterol s effects on the tmis for near - critical membranes composed of dopc , dppc , and cholesterol displayed approximately 15 c reduction in tmis with the addition of 5 mol% cholesterol ( veatch et al . 2007b ) , which implies a g value of 0.3 for cholesterol in this system ( i.e. , fig . the focused quantification of changes in tmis with variations in cholesterol concentration in near - critical ternary systems would provide greater certainty in the determination of g and p. similar analysis on the addition of the fluorescent lipid dii - c12 into membranes composed of dopc , dppc , and cholesterol at molar ratios of 35:35:30 demonstrates how a more complex mechanism may also be present ( veatch et al . in this system , tmis increased linearly with increasing dii - c12 concentrations up to 0.1 mol% with reduced effects on tmis at higher dii - c12 concentrations . this difference may be due to a particularly drastic effect of dii - c12 pushing the membrane toward or away from a critical composition ( veatch et al . 2007a ) or specific interactions between dii - c12 and the other lipids in the system . the theoretical framework presented here provides a basis for the interpretation of experimental data for the lipid phase and boundary preference of membrane - associated molecules . similarly , this framework provides a means of predicting the lipid - raft association and lipid phase - dependent clustering in complex systems through the extension of results from model membrane studies . this ability to quantify the phase preference of membrane additives may prove to be a valuable parameter for the therapeutic development and evaluation of the therapeutic mechanisms . supplementary material 1 ( avi 12307 kb ) supplementary material 2 ( docx 19959 kb )

the addition of trace molecules into membranes can significantly alter the morphology of the co - existing liquid phases and lipid phase transition temperature . membrane additives may affect lipid phase dynamics through preferentially partitioning to the boundary between lipid phases or preferentially mixing into one lipid phase . the characteristic differences between these mechanisms are demonstrated here in a minimalistic nearest neighbor model to provide a framework for how slight changes to membrane composition may affect lipid - phase - dependent processes , such as lipid - raft formation , immunological signaling , and molecular sorting preceding endocytosis with coexisting liquid phases . within the low mole fractions explored here ( 3 mol% ) , increasing the additive concentration linearly changed the phase miscibility temperature . rotationally asymmetric janus particles reduced the miscibility transition temperature for all fractions and degree of phase polarization . rotationally symmetric additives , however , either increased or decreased the phase miscibility temperature depending on the phase preference of the additive . while most experimental molecules may contain aspects of both of these idealized additives , this model provides a broad framework to quantify the effects of membrane additives in regard to lipid phase preference , lipid - raft association , and contribution to lipid phase - dependent molecular sorting.electronic supplementary materialthe online version of this article ( doi:10.1007/s00249 - 015 - 1017-x ) contains supplementary material , which is available to authorized users .

Electronic supplementary material Introduction Methods Results Discussion Electronic supplementary material

the online version of this article ( doi:10.1007/s00249 - 015 - 1017-x ) contains supplementary material , which is available to authorized users . the presence of coexisting liquid - ordered and liquid - disordered lipid phases has been implicated in diverse biological processes with high sensitivity to perturbations in membrane composition , tension , curvature , and temperature ( lingwood et al . lipid phase dynamics may inherently provide nanoscale clustering and facilitate lipid - raft - mediated processes ( veatch et al . however , trace membrane additives can disturb the lipid phase dynamics and the associated biological processes . some additives possess rotational asymmetry , preferentially localize to the phase boundary , reduce the phase miscibility , decrease the miscibility transition temperature ( tmis ) , and act as linactants , 1d analogs of surfactants ( trabelsi et al . other membrane additives are rotationally symmetric , preferentially localize into one particular lipid phase , and may increase or decrease tmis . broadly , the effects of membrane additives are quantified by the change to the lipid phase tmis . numerous molecules have demonstrated potent abilities to alter membrane phase miscibility without necessarily containing a dependence on the rotation of the molecule within the bilayer , including cholesterol ( dietrich et al . 2010 ) , and flavonoids ( ostroumova et al . other molecules have been hypothesized to prefer the boundary between two lipid phases because of their molecular structure ; for example , hybrid lipids contain one liquid - order - preferring tail and one liquid - disorder - preferring tail ( brewster et al . 2014 ; li and gorfe 2014 ) , and asymmetrically lipidated proteins , such as n - ras and h - ras , have shown preference for partitioning at the lipid phase boundary ( nicolini et al . further , molecules such as the nonsteroidal anti - inflammatory drug indomethacin may contain both a net phase preference and significant molecular structure asymmetry , although the nanoscale partitioning has yet to be studied ( zhou et al . previous studies have focused on theoretical predictions and experimental examination of systems with a significant fraction of the membrane consisting of phase - polarized particles ( > 10 mol% ) ( brewster et al . 2014 ) , whereas this manuscript focuses on small composition changes that can greatly affect membrane phase dynamics and provide a means for live cells to efficiently regulate lipid phase - dependent processes . this model permits the observation of complex phase behaviors without explicit incorporation of the molecular details or concentrations of the membrane , as long as the average phase segregation preferences are represented by the value of j. in this manuscript , we demonstrate the influence of trace additives in a two - component nearest neighbor model on a square lattice with conserved order parameters as a means of simulating how additives of differing molecular structure could influence lipid phase mixing . instead of modifying the value of j to represent changes in membrane composition , a third particle type was incorporated in these simulations to provide greater structural detail of the additive s effect on phase separation and additive partitioning . generally , the addition of a third type of particle to the system results in a change to the phase diagram , new phase transitions , and variations from critical behavior . 1971 ) , for which the phase transitions were modeled against an additive concentration with mean - field approximations . however , the additive concentrations explored here are consistent with the composition fluctuations in living and model membranes , including variations in content of cholesterol ( dietrich et al . 2007b , 2008 ) , they have not been shown to be any closer to critical than the additive - included simulations shown here . 2008 ) , which altered the membrane composition and presumably its phase behavior but did not diminish the significance or confidence in the result that the membranes were near critical in composition.fig . phase - polarized additives , or janus additives , were rotationally asymmetric with a polarized phase preference ( || = p ) white , liquid - ordered ( = 1 ) ; black , liquid - disordered ( = 1 ) ; gray particles ( = g ) were rotationally symmetric . phase - polarized additives , or janus additives , were rotationally asymmetric with a polarized phase preference ( || = p ) this work focuses on small perturbations to the well - understood two - state ising model and demonstrates how trace additives would affect the characteristic size of the co - existing single - phase domains ( ) and tmis . for 3 mol% additives , tmis and both changed monotonically with increasing additive concentration , while tmis changed linearly . however , rotationally symmetric additives of g > 1 resulted in an increase in both tmis and . the morphological detail provided by this model yields the distributions of additives within the system , which is unavailable from mean - field models . the resulting distributions of the additives at the boundary between phases were quantified , and the applicability of this analysis for assessing experimental membrane additives or cellular perturbations is discussed . 2increasing the mole fraction of phase - polarized particles caused an increase in the phase miscibility and a decrease of t mis . a with p = 1 , phase - polarized additives are displayed as red squares in these images , and zoomed - in regions show the individual additives in fig . increasing the concentration of phase - polarized particles with p = 1 resulted in a decrease of b and c t mis . uncertainty of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$t_{mis}^{\prime}$$\end{document}tmis was approximately 0.6 c , and uncertainty of was smaller than the symbol unless otherwise indicated increasing the mole fraction of phase - polarized particles caused an increase in the phase miscibility and a decrease of t mis . a with p = 1 , phase - polarized additives are displayed as red squares in these images , and zoomed - in regions show the individual additives in fig . increasing the concentration of phase - polarized particles with p = 1 resulted in a decrease of b and c t mis . changes to tmis tmis was determined by measuring the shift in as a function of temperature and by ascertaining the maximum of the specific heat versus temperature ( veatch et al . the finite size effects that alter the perceived transition temperature were consistent for all of these simulations on the same size system , which permitted this simplistic calculation of tmis and its broad applicability to systems of other sizes . tmis decreased linearly with increasing fractions of phase - polarized particles , or janus particles , as predicted from mean - field models ( yethiraj and weisshaar 2007 ; brewster et al . the miscibility temperature is reduced for any phase polarization ( p ) , while rotationally symmetric gray particles either increased or decreased tmis depending on their gray value ( g ) ( figs . increasing p for the phase - polarized additives resulted in a greater decrease in tmis until p > 3 , upon which the additives segregated into a third phase , separate from the white and black particles , and reduced the white and black particle mixing . gray particles with g < 1 encouraged phase miscibility by decreasing the differences between the ordered and disordered phases , analogous to the incorporation of n - alcohols into membranes ( gray et al . however , when g > 1 , the opposite effect dominates ; the difference between the phases was increased when g > 1 and tmis was increased , analogous to the addition of -lysin to membranes ( pokorny et al . 3 a and b t mis and either increased or decreased upon additive addition depending on the additive properties . t mis and changes are symmetrical for negative values of g or p a and b t mis and either increased or decreased upon additive addition depending on the additive properties . s6 ) , and the white - black miscibility temperature became less affected by the additives ( fig . for p or g > 3 , simulations were unable to capture a diverse set of configurations for the additive aggregation because of the improbable rearrangement of the condensed phases , analogous to the kinetically trapped configurations that are commonly experienced experimentally . aggregates of phase - polarized particles at p = 4 demonstrated rotational ordering of the additives ( fig . as p increased , the fraction of phase - polarized particles at the phase boundary increased ; as g increased , the fraction of gray particles at the phase boundary decreased . lowering the temperature would have made phase - polarized particles more likely to be found at the phase boundary if the phase morphology had stayed consistent . s8 ) , which was dependent on both simulation size and additive properties , and generally resulted in a higher probability for the additives being located at the phase boundary for higher temperatures . additive partitioning was normalized to the amount of phase boundary in order to calculate the fold enhancement of the additive to the phase boundary relative to the white or black particles ( fig . additives more concentrated at the phase boundary than the g = 1 particles preferentially partitioned at the phase boundary , whereas additives less concentrated at the phase boundary than the g = 1 particles preferentially partitioned away from the phase boundary , which is consistent with the sign of tmis ( fig . although there is not a local energy difference for the g = 0 additive if it is immersed in a single phase or at the phase boundary , there is an energy difference for the system that encourages g < 1 additives to be concentrated at the phase boundary . at colder temperatures , the phase - polarized additives especially concentrated at the phase boundary because of the decreased entropic drive for mixing.fig . the fraction of additives at the boundary was calculated as the ratio of the number of additives surrounded by two white and two black particles versus the total number of additives , excluding additives adjacent to other additives . b the additive locations were normalized by the amount of phase boundary in each condition to yield the fold enhancement of the additive at the phase boundary versus generic white particles . uncertainty was calculated as the standard deviation of different time points and smaller than the symbol unless otherwise indicated a the probability that an additive was found at the phase boundary varied with the additive properties and temperature . the fraction of additives at the boundary was calculated as the ratio of the number of additives surrounded by two white and two black particles versus the total number of additives , excluding additives adjacent to other additives . b the additive locations were normalized by the amount of phase boundary in each condition to yield the fold enhancement of the additive at the phase boundary versus generic white particles . it is important to note that these results would vary greatly for compositions that are far from critical compositions because of the increased phase boundary line tension and the increased likelihood for boundary - preferring particles to localize at the boundary in such systems . in all the prior described simulations , only additives with however , real membrane additives would likely possess both a net phase preference ( g 0 ) and a rotational phase asymmetry ( p 0 ) . to predict how changing both g and p for a membrane additive would affect the miscibility transition temperature of the membrane , simulations were performed for additives with rotational asymmetry and a net phase preference at 1 mol% ( fig . the changes to the miscibility transition temperature were dependent on both the g and p value of the additive in a cumulative effect . , either g or p 1 ) , tmis as a function of g and p was approximately equal to the sum of the change in miscibility temperature from 1 mol% gray particles of gray value g plus the change from 1 mol% polarized particles of polarization p ( fig . 5 a changes to the miscibility transition temperature were induced by the incorporation of 1 mol% additives with both net and rotationally asymmetric phase preferences , i.e. b for small values of g and p , t mis as a function of g and p , t mis ( g , p ) , is approximately equal to t mis ( g , 0 ) + t mis ( 0 , p ) a changes to the miscibility transition temperature were induced by the incorporation of 1 mol% additives with both net and rotationally asymmetric phase preferences , i.e. b for small values of g and p , t mis as a function of g and p , t mis ( g , p ) , is approximately equal to t mis ( g , 0 ) + t mis ( 0 , p ) taken together , these idealized membrane additives represent different means by which molecules may alter the lipid phases in a membrane . a membrane - associated molecule may be experimentally quantified with both a p value and g value to describe the effects of the additive molecular structure on lipid phase dynamics in a particular membrane . the effects of a particular membrane additive are determined entirely by the membrane s additive - free miscibility temperature ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$t_{mis}^{\prime}$$\end{document}tmis ) or internal energy between two particles ( j ) of the near - critical membrane without a dependence on the particular molecular details of the individual membrane components . the temperatures reported throughout this article could be alternately represented as reduced temperatures ( tr ) , where tr = ( t \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$t_{mis}^{\prime}$$\end{document}tmis)/ \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$t_{mis}^{\prime}$$\end{document}tmis. for example , increasing the fatty acid tail length in the saturated , liquid - order preferring lipid of a ternary mixture typically increases \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$t_{mis}^{\prime}$$\end{document}tmis and j , while decreasing the additive s g value and enhancing the additive s phase boundary preference . similarly , the p and g values for a membrane additive in a complex cellular membrane could be predicted by measuring the additive 's effects on synthetic model membranes of greatly different molecular composition , but similar \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$t_{mis}^{\prime}$$\end{document}tmis and j. values of g and p that are greater than one represent the phase preferences for the additives that are stronger that then constituents of the phases themselves . similarly , it is feasible that a particular phase - preferring component of a hybrid molecule could have a stronger phase preference than average constituents of that phase , i.e. the quantification of p and g values of cholesterol , for example , requires knowledge of the effects cholesterol addition has on lipid phase separation and the likelihood of cholesterol to be found on the phase boundary . the addition of cholesterol to a membrane typically encourages phase miscibility , indicating that cholesterol has a value of g < 1 . the value of g for cholesterol will vary with the surrounding membrane lipids ; however , quantification of cholesterol s effects on the tmis for near - critical membranes composed of dopc , dppc , and cholesterol displayed approximately 15 c reduction in tmis with the addition of 5 mol% cholesterol ( veatch et al . the focused quantification of changes in tmis with variations in cholesterol concentration in near - critical ternary systems would provide greater certainty in the determination of g and p. similar analysis on the addition of the fluorescent lipid dii - c12 into membranes composed of dopc , dppc , and cholesterol at molar ratios of 35:35:30 demonstrates how a more complex mechanism may also be present ( veatch et al . the theoretical framework presented here provides a basis for the interpretation of experimental data for the lipid phase and boundary preference of membrane - associated molecules . similarly , this framework provides a means of predicting the lipid - raft association and lipid phase - dependent clustering in complex systems through the extension of results from model membrane studies . this ability to quantify the phase preference of membrane additives may prove to be a valuable parameter for the therapeutic development and evaluation of the therapeutic mechanisms .

the postsynaptic sites of excitatory glutamatergic synapses in the brain , dendritic spines , are small protrusions that extend from dendrites and are associated with structural plasticity . while the role of dendritic spines in models of learning and memory and synaptic plasticity continues to gain support , alterations in dendritic spine density and morphology have been consistently documented in numerous disorders associated with intellectual disabilities [ 2 , 3 ] . one such disease associated with both intellectual disability and dendritic spine pathology is rett syndrome ( rtt ; mim 312750 ) . rtt is an x chromosome - linked disorder that affects approximately 1 : 10,00015,000 females worldwide and is the leading cause of severe intellectual disabilities in females . in addition to a reduction in the size of neuronal cell bodies , a decrease in dendritic complexity of pyramidal cells was described in several brain regions [ 57 ] . previous work in our laboratory using postmortem brain tissue from female rtt individuals demonstrated that hippocampal ca1 pyramidal neurons have lower spine density than age - matched female unaffected individuals . mutations in mecp2 , the gene encoding methyl - cpg - binding protein-2 , have been identified in ~90% of rtt individuals [ 1012 ] . using an in vitro organotypic slice culture system , we demonstrated that expression of mecp2 missense mutations commonly found in rtt individuals caused a significant reduction of dendritic spine density in hippocampal pyramidal neurons , especially of the more mature mushroom type spines . consistent with these findings , neurons generated in vitro from induced pluripotent stem cells ( ipscs ) derived from skin fibroblasts of rtt patients showed lower dendritic spine density than control neurons . while these observations shed light on the role of mecp2 in the formation and/or maintenance of excitatory synapses on dendritic spines , further characterizing the available mouse models of rtt will allow defining phenotypic endpoints to evaluate novel pharmacological interventions . the most studied mouse models of rtt either lack the mecp2 protein by deletion of mecp2 exons 3 and 4 ( bird line ) or express a nonfunctional mutant protein due to deletion of mecp2 exon 3 ( jaenisch line ) (; reviewed in [ 16 , 17 ] ) . quantitative confocal microscopy of lucifer yellow - labeled neurons from both of these mecp2-deficient mouse strains revealed lower dendritic spine density in several brain regions , including pyramidal neurons of the ca1 region of the hippocampus ; it should be noted that these observations were made in postnatal - day 21 mice ( before overt rtt - like symptoms ) , and that they are consistent with the lower density of excitatory synapses estimated from vglut1-psd95 puncta observed in the hippocampal ca1 region of 2-week - old mecp2 null mice . symptomatic mecp2 null mice at 5 weeks , however , showed comparable density of vglut1-psd95 puncta to that in wildtype littermates . to better understand the role of mecp2 on dendritic spine maintenance , we analyzed dendritic spine density and morphology by confocal microscopy and excitatory synapses by electron microscopy in hippocampal ca1 pyramidal neurons of presymptomatic ( p7 and p15 ) and symptomatic ( p4060 ) male mecp2 mutant mice and their age - matched wildtype littermates . our observations indicate that dendritic spine density is lower only at postnatal - day 7 ( p7 ) , while it does not differ at p15 or later when symptoms are well established . consistently , stereological analyses at the em level revealed comparable density of asymmetric spine synapses between symptomatic mecp2 mutant and wildtype littermates . these results raise caution regarding the use of dendritic spine density in hippocampal neurons as a phenotypic endpoint for the evaluation of therapeutic interventions in symptomatic mecp2-deficient mice . in addition , these observations demonstrate that proper mecp2 function is required for the early development of dendritic spines in ca1 pyramidal neurons and that a secondary compensatory mechanism seems to take place in symptomatic mecp2 mutant mice . breeding pairs of mice lacking exon 3 of the x chromosome - linked mecp2 gene ( b6.cg-mecp2 , jaenisch strain ; c57bl/6 background ) were purchased from the mutant mouse regional resource center at ucdavis . a colony was established at uab by breeding wildtype males with heterozygous mecp2 mutant females , following the original breeding scheme , which is recommended by the supplier . hemizygous male mice of the mecp2 mutant strain are healthy until 5 - 6 weeks of age , when they begin acquiring rtt - like motor symptoms , such as hypoactivity , hind limb elevation , and reflex impairments . for the present studies , the experimental subjects were homozygous mecp2 mutant males ( called mecp2 mutants ) and wildtype male mice that were littermates of postnatal - day 7 ( p7 ) , p15 , and between p40 and p60 . animals were handled and housed according to the committee on laboratory animal resources of the national institutes of health . all experimental protocols were annually reviewed at the university of alabama at birmingham and at the universit di torino and approved by each institution 's respective institutional animal care and use committee . mice that were postnatal - day ( p7 ) were euthanized by cervical dislocation , and the brain was immersed in 4% paraformaldehyde ( in 0.1 m phosphate buffer ) ; hippocampi were later dissected and kept in fixative for an additional 2 hours . p15 or p4060 mice were deeply anesthetized with ketamine ( 100 mg / kg ) and euthanized by transcardiac perfusion with ~200 ml of 4% paraformaldehyde ( in 0.1 m phosphate buffer ) . after dissection , brains and hippocampi were rinsed several times in 0.1 m phosphate buffered saline ( pbs ) and cut into 100 m thick coronal sections with a mcilwain tissue chopper , which were further rinsed in 0.1 m pbs and stored at 4c until dii labeling . to visualize dendritic spines by laser - scanning confocal microscopy , coronal sections containing the hippocampus were stained with the lipophilic fluorescent dye 1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate ( dii ; invitrogen , carlsbad , ca , usa ) by particle - mediated labeling ( diolistics ) [ 20 , 21 ] . first , dii was diluted in dimethyl chloride ( methylene chloride ; sigma , st . then , 20 mg of 1.1 m tungsten particles ( bio - rad ; hercules , ca , usa ) were placed on top of a pre - cleaned glass slide and spread out evenly with two pre - cleaned razor blades . the dii solution was added onto the tungsten particles and allowed to completely evaporate . to prevent clumping of the dii / tungsten mixture , razor blades were used to break apart the mixture . additionally , a small amount of polyvinylpyrrollidone ( 1020 l pvp made in fresh in 100% ethanol ; bio - rad ) was added to the dii / tungsten mixture to further prevent particle clumping and improve their coating to the tefzel tubing . the dii - coated tungsten particles were then added to a glass tube with 3 ml of water and sonicated for 1 hr . after sonication , the solution was vortexed and then aspirated and coated onto tefzel tubing for 15 mins . after 15 mins , the solution was removed and the tubing was allowed to dry for 15 mins . dii - coated tungsten bullets were shot only once onto individual hippocampal slices with a custom - modified helios hand - held gene gun ( bio - rad ) using 75 psi he pressure through a 40 m pore size filter . after labeling with dii bullets , slices were rinsed and stored in pbs for 1530 min at room temperature in the dark to allow diffusion of dii . slices were finally washed with pbs and mounted on glass slides with vectashield ( vector laboratories , burlingame , ca , usa ) . figure 1(a ) shows tungsten bullets and the resulting dii fluorescence in a representative section of perfusion - fixed hippocampus stained by diolistics . it should be noted , from examining table 1 , for mice that were younger in age , the total dendritic length examined is smaller than that obtained for the older mice . this discrepancy is presumably a result of older mice having an abundant number of cells and with longer dendrites . high - resolution images of spiny apical secondary or tertiary dendrites showing adequate dii labeling were acquired from ca1 stratum radiatum in a fluoview fv-300 laser - scanning confocal microscope ( olympus , center valley , pa , usa ) using an oil immersion 60 x 1.45 na objective lens ( planapo , olympus ) , with additional 3x digital zoom . dii was excited with an hene green laser ( 543 nm ) , and its fluorescence detected with a cube containing a 555 nm dichroic mirror and a 585 40 nm emission filter ( semrock , lake forest , il , usa ) . series of optical sections in the z - axis were acquired with 0.1 m intervals through individual apical dendritic branches . figure 1(b ) shows representative examples of a ca1 pyramidal neuron and a segment of apical dendrite stained with dii . dendritic spines were identified as small protrusions that extended less than 3 m from the parent dendrite and counted manually in maximum - intensity projections of confocal z - stacks using imagej software ( w. s. rasband , imagej , us national institutes of health , bethesda , maryland , usa ; http://rsb.info.nih.gov/ij/ , 19972009 ) ; protrusions that were more than 3 m were classified as filopodia and rarely seen except in the p7 mice . care was taken to ensure that each spine was counted only once by following its projection course through the stack of z - sections . spine density was calculated by quantifying the number of spines per dendritic segment and normalized to 10 m of dendrite length . microscope calibrations were performed using 1.07 m fluorescent latex microspheres ( molecular probes , eugene , or , usa ) , which yielded a lateral resolution of 0.09 m per pixel . counting and measuring of individual spines was conducted in a blind fashion , as the genotype was unknown to the person performing the image analysis . the categorization of different morphological spine types was performed as described [ 22 , 23 ] . briefly , geometrical dimensions of individual spines were measured in maximum - intensity projections of the z - stacks using imagej and used to calculate the l / n and h / n ratios , where l is spine length , h is the maximum head width , and n is the maximum neck width . then , spine types were grouped as mature - shaped spines , which included type - i ( stubby ) and type - ii ( mushroom ) shaped spines , or immature - shaped thin ( type - iii ) spines , following published criteria . table 1 shows the spine density and proportions of spine types in each genotype , as well as the total number of dendritic spines counted and measured and the total dendritic length analyzed . male mecp2 mutant mice and wildtype littermates ( 3 per group ) were anaesthetized with an intraperitoneal injection of chloral hydrate and transcardially perfused with ice cold 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 m phosphate buffer ( pb ; ph 7.4 ) . after perfusion , brains were left in the same fixative overnight at 4c , washed several times in 0.1 m pb , and then postfixed with 1% osmium tetroxide ( in 0.1 m cacodylate buffer ) for 1 hour . for resin embedding , tissues were dehydrated in a graded series of ethanol ( 30100% ) and infiltrated with an epon - araldite mixture . ultrathin serial sections ( 70 nm ) were cut with an ultramicrotome ( leica ultracut , wetzlar , germany ) and collected on single slot copper grids coated with a pioloform solution . the grids were counterstained with uranyl acetate and lead citrate and imaged in a jem-1010 electron microscope ( jeol , japan ) equipped with a side - mounted ccd camera with 1376 1032 pixels ( mega view iii ; soft imaging system gmbh , muenster , germany ) . asymmetric synapses were identified by the presence of at least three vesicles within the profile adjacent to the presynaptic membrane and the presence of a clear psd . unbiased stereological estimation of asymmetric spine synapses ( presumptive excitatory synapses ) was performed as previously described by using the double dissector method . in this method , images of two serial sections are acquired , the first of which is designated as the reference section and the second as the look - up section . twenty - five pairs of nonoverlapping electron micrographs were acquired for each animal in ca1 stratum radiatum at 20,000 magnification . within an unbiased counting frame that represented 28 m , the number of synapses that were present in the reference section but absent in the look - up section were counted . the cross - section area of dendritic spines and presynaptic terminals was analyzed on the same digital images using imagej as previously described . averages of multiple measurements are presented as mean standard error of the mean ( sem ) . data were statistically analyzed using unpaired student 's t - test using prism software ( graphpad software , inc . , san diego , ca , usa ) . probability values lower than 0.05 were considered statistically significant ( i.e. , p < 0.05 , less than 5% probability that the observations are due to chance ) . when lower than this cut - off value , the actual p values are given in section 3 ( rather than just the statement less than ) to provide readers with more detailed information regarding the outcome of the statistical analyses . cumulative frequencies plots were first analyzed using the normal distribution kolmogorov - smirnov ( k - s ) fitting test , and then k - s two - sample tests for subsequent paired comparisons . we previously showed that pyramidal neurons from rat hippocampus overexpressing human mecp2 carrying single point missense mutations commonly found in rtt individuals have lower dendritic spine density than control neurons . consistent with lower dendritic spine density in female rtt individuals , such spine loss in mutant mecp2-expressing neurons was observed after 4 days of over expression in vitro in organotypic slice cultures . in addition , 3-week - old mecp2-deficient mice have lower spine density than age - matched wildtypes , but older symptomatic mecp2 mutant mice show comparable density of vglut1-psd95 puncta to that in wildtype littermates . to address a potential developmental progression of this dendritic spine phenotype in mecp2 mutant mice ( mecp2 ) , we performed quantitative analyses of dendritic spines in dii - labeled hippocampal sections from mutant and age - matched wildtype littermates by laser - scanning confocal microscopy . figure 2(a ) shows representative examples of maximum - intensity projections of confocal stacks from segments of secondary apical dendrites of ca1 pyramidal neurons used for quantitative analyses . dendritic spine density in ca1 pyramidal neurons of p7 mecp2 mutants was lower compared to age - matched wildtype littermates ( wildtype 3.65 0.54 spines per 10 m of dendritic length , n = 5 dendrite segments from 3 mice , versus mecp2 2.23 0.40 spines/10 m , n = 10 segments/3 mice ; p = 0.030 ; figures 2(a ) and 2(d ) ) . on the other hand , dendritic spine density in p15 mecp2 mutant mice was not statistically different compared to age - matched wildtype littermates ( wildtype 5.39 1.20 spines/10 m , n = 7 segments/3 mice versus mecp2 5.87 0.43 spines/10 m , n = 9 segments/3 mice ; p = 0.359 ; figures 2(b ) and 2(d ) ) . despite differences in spine density in p7 mice , the proportions of the three main morphological types of spines did not differ between mecp2 and age - matched wildtype littermates ( stubby : wildtype 0.49 0.07 versus mecp2 0.46 0.08 ; p = 0.396 , mushroom : wildtype 0.24 0.02 versus mecp2 0.16 0.04 ; p = 0.110 , thin : wildtype 0.27 0.05 versus mecp2 0.25 0.06 ; p = 0.401 ) . similarly , the proportions of morphological spine types were comparable in p15 mice of both genotypes ( stubby : wildtype 0.39 0.01 versus mecp2 0.33 0.03 ; p = 0.087 , mushroom : wildtype 0.47 0.03 versus mecp2 0.50 0.03 ; p = 0.244 , thin : wildtype 0.14 0.03 versus mecp2 0.16 0.02 ; p = 0.269 ) . dendritic spine density in ca1 pyramidal neurons of p4060 mecp2 mutant mice exhibiting characteristic rtt - like symptoms were not statistically different than in age - matched wildtype littermates ( wildtype 14.39 0.77 spines/10 m of dendritic length , n = 35 segments/5 mice versus mecp2 15.82 0.86 spines/10 m , n = 29 segments/5 mice ; p = 0.110 ; figures 2(c ) and 2(d ) ) . we next determined if the proportion of the three major morphological types of dendritic spines were affected in symptomatic mecp2 mutant mice . the proportion of stubby spines and mushroom spines , larger spines considered to be more stable , was comparable between the genotypes ( stubby : wildtype 0.51 0.02 versus mecp2 0.53 0.02 ; p = 0.211 ; mushroom : wildtype 0.33 0.01 versus mecp2 0.35 0.01 ; p = 0.06 ) . on the other hand , the proportion of the more motile and immature thin spines was lower in symptomatic mecp2 mutant mice ( wildtype 0.16 0.01 versus mecp2 0.11 0.01 ; p = 0.005 ) . electron microscopy of asymmetric synapses on dendritic spines ( presumptive excitatory ) within ca1 stratum radiatum of symptomatic mecp2 mutant mice was conducted to determine if mecp2 alters density of excitatory synapses . ultrastructural observations revealed a phenotype consistent with the above confocal microscopy results ( figure 3(a ) ) . unbiased stereological analyses revealed that the density of asymmetric spine synapses in symptomatic mecp2 mutant mice ( 2.12 0.11 synapses per m ; n = 3 mice ) was not significantly different than in wildtype littermates ( 2.06 0.05 synapses/m , intriguingly , the cross - sectional area of individual spines ( figure 3(c ) ) and presynaptic terminals ( figure 3(d ) ) were smaller in mecp2 mutant mice ( kolmogorov - smirnov test ; p < 0.01 ) , but only for the smallest asymmetric spine synapses . it should be noted that these spines have dimensions below the resolution of diffraction - limited light microscopy and thus are not detectable in our dendritic spine density measurements relying on confocal microscopy ( figure 2(b ) ) . quantitative confocal microscopy of dii - labeled dendrites revealed that ca1 pyramidal neurons from p7 mecp2 mutant mice had lower spine density compared to age - matched wildtype littermates . these differences in spine density were not observed neither in slightly older but still presymptomatic mecp2 mutant mice at p15 nor in p4060 mice that express the full spectrum of rtt - like symptoms , consistent with an analysis of the density of vglut1-psd95 puncta in area ca1 . stereological analyses at the electron microscopy level revealed that the density of asymmetric spine synapses is comparable in p4060 fully symptomatic mecp2 mutant mice compared to age - matched wildtype littermates . the only significant difference between genotypes during the symptomatic stage was a lower proportion of immature thin spines and a smaller cross - sectional area of individual spines and presynaptic terminals but only for the smallest asymmetric spine synapses in mecp2 mutant mice . it should be noted that the smallest asymmetric spine synapses analyzed at the em level have dimensions below the resolution of diffraction - limited light microscopy and thus are not detectable in our measurements using confocal microscopy . our observations during the symptomatic stage demonstrate that mecp2 loss - of - function causes subtle structural modifications of excitatory ca3-ca1 synapses without major changes in excitatory synapse density , as we showed in an independent em study using random single ultrathin sections . these data demonstrate that proper mecp2 function is required for early development of dendritic spines in ca1 pyramidal neurons and that a compensatory mechanism that normalizes spine density seems to occur later in development . a recurrent theme in cytological studies of postmortem rtt brains is the observation of significant differences in the fine structure of dendrites . studies of neurons from cortical regions of the brain have shown impaired dendritic branching in individuals with rtt . in addition , qualitative observations of pyramidal neurons of the motor cortex of rtt individuals described segments of dendrites that were bare of spines . using quantitative confocal microscopy of dii - labeled hippocampal sections , we recently showed that ca1 pyramidal neurons have lower dendritic spine density in female rtt individuals than in age - matched unaffected females . our results on hippocampal dendritic spine density show that male mecp2 mutant mice at the symptomatic stage do not recapitulate the human phenotype observed in autopsy material from female rtt individuals . while many factors could contribute to these findings ( including gender differences , disease severity , seizure disorder , and x - chromosomal inactivation ratio ) , these results suggest that hippocampal neuron spine density in this particular mouse model is not a phenotype with sufficient face validity for rtt . however , these mice still yield relevant information on the role of mecp2 in the cns . further postmortem studies in more individuals with rtt as well as other mecp2-associated conditions are needed to better understand the consequences on dendritic spine density and morphology . if such studies are correlated with a detailed clinical history , they will further yield a better understanding of the contribution of other factors to the spine phenotype , such as specific mecp2 mutations , disease severity and progression , and life - long medications . our previous in vitro studies in rat hippocampal neurons in primary culture demonstrated that either shrna - mediated knockdown of endogenous mecp2 protein levels or overexpression of human mecp2 missense mutations common in rtt patients ( r106w or t158 m ) reduced dendritic length and branching during early neuronal development . furthermore , using a postnatal rat hippocampal slice culture preparation , we observed that the knockdown of endogenous mecp2 protein levels resulted in reduced dendritic spine density , especially of mature spines ; however , overexpression of rtt - associated human mecp2 missense mutations led to a dramatic reduction in dendritic spine density . consistently , a recent report described that neurons derived from ipscs obtained from reprogramming of skin fibroblasts of rtt patients have reduced dendritic spine density . while these exciting observations on patient - derived neurons provide additional evidence of the importance of dendritic spines in the neuropathology of rtt , it should be made clear that no causative biochemical underpinning has yet been established . transgenic mice that either lack mecp2 or express a mutant nonfunctional mecp2 peptide fragment are excellent experimental models to help determine the synaptic defects that contribute to rtt . these mice recapitulate several behavioral features of rtt and display many defects in dendritic structure and synaptic transmission and plasticity ( reviewed in [ 16 , 17 ] ) . however , they have also yielded varying results in terms of dendritic spine alterations . in contrast with the present observations , a quantitative study of two different mecp2 mutant lines ( mecp2 and mecp2 ) described that pyramidal neurons from hippocampal area ca1 and layers ii - iii of the motor cortex have lower dendritic spine densities than their control wildtype littermates at approximately 3 weeks of age . the parsimonious explanation for this apparent discrepancy is that these studies used mice of different strains and the genetic background of the different mouse lines contributed to the divergent observations on dendritic spine density . while both studies employed commercially available mecp2 mice , the genetic background is different : we used mecp2 mice on a pure c57bl/6 background inbred for more than 10 generations , while belichenko et al . used two different lines ( mecp2 and mecp2 ) on a mixed genetic background . for example , neurite outgrowth is significantly different in two mouse strains where nogo - a was knocked out . when comparing two different mecp2 transgenic mouse lines maintained on different genetic backgrounds , belichenko et al . described significant differences in spine density between age - matched wildtype mice of the two different strains , strongly suggesting that genetic background contributes to the phenotype under study , for example , dendritic spine density and morphology . we show here that hippocampal pyramidal neurons exhibit a dendritic spine phenotype only in neonatal ( p7 ) mutant mice , well before excitatory synapse expansion , while spine density in mutants recovers to wildtype levels a week later ( p15 ) and is maintained at wildtype levels throughout the symptomatic stage ( p4060 ) . this developmental progression of the dendritic spine phenotype is also reflected in the density of vglut1-psd95 puncta , which is lower in area ca1 of 2-week - old mecp2 null mice , but comparable to wildtype levels at 5 weeks of age . together with our dendritic spine observations , those vglut1-psd95 puncta results are consistent with the present em analyses in symptomatic mecp2 mutant mice , which revealed comparable densities of asymmetric spine synapses in stratum radiatum of area ca1 of both genotypes . altogether , these data demonstrate that proper mecp2 functioning is required for dendritic spine formation during early postnatal development , and that a secondary compensatory mechanism seems to take place in symptomatic mecp2 mutant mice . a couple of possibilities exist as to the extent of the compensatory mechanisms necessary to bring spine density to wildtype levels . one possibility is that enhanced hippocampal network activity in mecp2 mutants promotes dendritic spine formation . a second possibility is that deranged homeostatic plasticity promotes spinogenesis , while still affecting pyramidal neuron function . despite the lack of differences in dendritic spine density in fully symptomatic mecp2 mutant mice , we observed a significant , yet small , reduction in the proportion of immature thin spines in mecp2 mutants compared to age - matched wildtype animals . what consequence this might have on hippocampal synaptic function remains debatable ; however , it is hypothesized that thin spines represent learning spines because of their constant changing in response to neuronal activity ( e.g. , ltp and ltd ) , while mushroom spines are considered more mature and stable memory spines . considering that dendritic spines are highly sensitive to the levels of neuronal activity , the shift in the proportion of morphological spine types could also reflect a response to the heightened neuronal activity observed in the hippocampal network of symptomatic mecp2 mutant mice . thus , it would be interesting to determine what role mecp2 has in the maintenance of thin spines and what consequences does this have on hippocampal function . one report has already demonstrated that spine motility is slowed in mecp2 mutant mice , possibly reflecting the decrease in the proportion of thin spines that we observed . confocal microscopy of dendritic spines in organotypic hippocampal cultures has revealed that approximately 6570% of dendritic spines are juxtaposed to presynaptic terminals . for this reason , we decided to conduct unbiased stereological analyses at the electron microscopy level to determine the density and morphology of dendritic spines that were actually connected to a presynaptic terminal , for example , asymmetric spine synapses . this approach demonstrated that the density of asymmetric spine synapses in mecp2 mutant mice is comparable to that of wildtype littermates , consistent with confocal microscopy of dendritic spines . intriguingly , the areas of dendritic spines and presynaptic terminals are smaller in mecp2 mutant mice ; however , this difference was only observed for the smallest asymmetric spine synapses . considering that these spines have dimensions below the resolution of diffraction - limited light microscopy , they could not have been included in measurements of spine head width in confocal microscopy images ( figure 3(b ) ) . taken together , our observations demonstrate that while the proportion of thin spines is lower in mecp2 mutant mice , individual dendritic spines , and thus excitatory synapses , are smaller in volume . in summary , our results raise caution regarding the use of dendritic spine density in hippocampal neurons as a phenotypic endpoint for the evaluation of therapeutic interventions in symptomatic mecp2 deficient mice . however , we present data describing the importance of mecp2 on spine development in neonatal mice . future research will hopefully explain the precise molecular role of mecp2 in the establishment of the hippocampal excitatory network and how this manifest into clinical issues .

alterations in dendritic spines have been documented in numerous neurodevelopmental disorders , including rett syndrome ( rtt ) . rtt , an x chromosome - linked disorder associated with mutations in mecp2 , is the leading cause of intellectual disabilities in women . neurons in mecp2-deficient mice show lower dendritic spine density in several brain regions . to better understand the role of mecp2 on excitatory spine synapses , we analyzed dendritic spines of ca1 pyramidal neurons in the hippocampus of mecp2tm1.1jae male mutant mice by either confocal microscopy or electron microscopy ( em ) . at postnatal - day 7 ( p7 ) , well before the onset of rtt - like symptoms , ca1 pyramidal neurons from mutant mice showed lower dendritic spine density than those from wildtype littermates . on the other hand , at p15 or later showing characteristic rtt - like symptoms , dendritic spine density did not differ between mutant and wildtype neurons . consistently , stereological analyses at the em level revealed similar densities of asymmetric spine synapses in ca1 stratum radiatum of symptomatic mutant and wildtype littermates . these results raise caution regarding the use of dendritic spine density in hippocampal neurons as a phenotypic endpoint for the evaluation of therapeutic interventions in symptomatic mecp2-deficient mice . however , they underscore the potential role of mecp2 in the maintenance of excitatory spine synapses .

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

the postsynaptic sites of excitatory glutamatergic synapses in the brain , dendritic spines , are small protrusions that extend from dendrites and are associated with structural plasticity . while the role of dendritic spines in models of learning and memory and synaptic plasticity continues to gain support , alterations in dendritic spine density and morphology have been consistently documented in numerous disorders associated with intellectual disabilities [ 2 , 3 ] . one such disease associated with both intellectual disability and dendritic spine pathology is rett syndrome ( rtt ; mim 312750 ) . rtt is an x chromosome - linked disorder that affects approximately 1 : 10,00015,000 females worldwide and is the leading cause of severe intellectual disabilities in females . in addition to a reduction in the size of neuronal cell bodies , a decrease in dendritic complexity of pyramidal cells was described in several brain regions [ 57 ] . previous work in our laboratory using postmortem brain tissue from female rtt individuals demonstrated that hippocampal ca1 pyramidal neurons have lower spine density than age - matched female unaffected individuals . mutations in mecp2 , the gene encoding methyl - cpg - binding protein-2 , have been identified in ~90% of rtt individuals [ 1012 ] . using an in vitro organotypic slice culture system , we demonstrated that expression of mecp2 missense mutations commonly found in rtt individuals caused a significant reduction of dendritic spine density in hippocampal pyramidal neurons , especially of the more mature mushroom type spines . consistent with these findings , neurons generated in vitro from induced pluripotent stem cells ( ipscs ) derived from skin fibroblasts of rtt patients showed lower dendritic spine density than control neurons . while these observations shed light on the role of mecp2 in the formation and/or maintenance of excitatory synapses on dendritic spines , further characterizing the available mouse models of rtt will allow defining phenotypic endpoints to evaluate novel pharmacological interventions . quantitative confocal microscopy of lucifer yellow - labeled neurons from both of these mecp2-deficient mouse strains revealed lower dendritic spine density in several brain regions , including pyramidal neurons of the ca1 region of the hippocampus ; it should be noted that these observations were made in postnatal - day 21 mice ( before overt rtt - like symptoms ) , and that they are consistent with the lower density of excitatory synapses estimated from vglut1-psd95 puncta observed in the hippocampal ca1 region of 2-week - old mecp2 null mice . to better understand the role of mecp2 on dendritic spine maintenance , we analyzed dendritic spine density and morphology by confocal microscopy and excitatory synapses by electron microscopy in hippocampal ca1 pyramidal neurons of presymptomatic ( p7 and p15 ) and symptomatic ( p4060 ) male mecp2 mutant mice and their age - matched wildtype littermates . our observations indicate that dendritic spine density is lower only at postnatal - day 7 ( p7 ) , while it does not differ at p15 or later when symptoms are well established . consistently , stereological analyses at the em level revealed comparable density of asymmetric spine synapses between symptomatic mecp2 mutant and wildtype littermates . these results raise caution regarding the use of dendritic spine density in hippocampal neurons as a phenotypic endpoint for the evaluation of therapeutic interventions in symptomatic mecp2-deficient mice . in addition , these observations demonstrate that proper mecp2 function is required for the early development of dendritic spines in ca1 pyramidal neurons and that a secondary compensatory mechanism seems to take place in symptomatic mecp2 mutant mice . breeding pairs of mice lacking exon 3 of the x chromosome - linked mecp2 gene ( b6.cg-mecp2 , jaenisch strain ; c57bl/6 background ) were purchased from the mutant mouse regional resource center at ucdavis . hemizygous male mice of the mecp2 mutant strain are healthy until 5 - 6 weeks of age , when they begin acquiring rtt - like motor symptoms , such as hypoactivity , hind limb elevation , and reflex impairments . for the present studies , the experimental subjects were homozygous mecp2 mutant males ( called mecp2 mutants ) and wildtype male mice that were littermates of postnatal - day 7 ( p7 ) , p15 , and between p40 and p60 . mice that were postnatal - day ( p7 ) were euthanized by cervical dislocation , and the brain was immersed in 4% paraformaldehyde ( in 0.1 m phosphate buffer ) ; hippocampi were later dissected and kept in fixative for an additional 2 hours . to visualize dendritic spines by laser - scanning confocal microscopy , coronal sections containing the hippocampus were stained with the lipophilic fluorescent dye 1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate ( dii ; invitrogen , carlsbad , ca , usa ) by particle - mediated labeling ( diolistics ) [ 20 , 21 ] . high - resolution images of spiny apical secondary or tertiary dendrites showing adequate dii labeling were acquired from ca1 stratum radiatum in a fluoview fv-300 laser - scanning confocal microscope ( olympus , center valley , pa , usa ) using an oil immersion 60 x 1.45 na objective lens ( planapo , olympus ) , with additional 3x digital zoom . dendritic spines were identified as small protrusions that extended less than 3 m from the parent dendrite and counted manually in maximum - intensity projections of confocal z - stacks using imagej software ( w. s. rasband , imagej , us national institutes of health , bethesda , maryland , usa ; http://rsb.info.nih.gov/ij/ , 19972009 ) ; protrusions that were more than 3 m were classified as filopodia and rarely seen except in the p7 mice . table 1 shows the spine density and proportions of spine types in each genotype , as well as the total number of dendritic spines counted and measured and the total dendritic length analyzed . male mecp2 mutant mice and wildtype littermates ( 3 per group ) were anaesthetized with an intraperitoneal injection of chloral hydrate and transcardially perfused with ice cold 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 m phosphate buffer ( pb ; ph 7.4 ) . unbiased stereological estimation of asymmetric spine synapses ( presumptive excitatory synapses ) was performed as previously described by using the double dissector method . twenty - five pairs of nonoverlapping electron micrographs were acquired for each animal in ca1 stratum radiatum at 20,000 magnification . the cross - section area of dendritic spines and presynaptic terminals was analyzed on the same digital images using imagej as previously described . we previously showed that pyramidal neurons from rat hippocampus overexpressing human mecp2 carrying single point missense mutations commonly found in rtt individuals have lower dendritic spine density than control neurons . consistent with lower dendritic spine density in female rtt individuals , such spine loss in mutant mecp2-expressing neurons was observed after 4 days of over expression in vitro in organotypic slice cultures . in addition , 3-week - old mecp2-deficient mice have lower spine density than age - matched wildtypes , but older symptomatic mecp2 mutant mice show comparable density of vglut1-psd95 puncta to that in wildtype littermates . to address a potential developmental progression of this dendritic spine phenotype in mecp2 mutant mice ( mecp2 ) , we performed quantitative analyses of dendritic spines in dii - labeled hippocampal sections from mutant and age - matched wildtype littermates by laser - scanning confocal microscopy . figure 2(a ) shows representative examples of maximum - intensity projections of confocal stacks from segments of secondary apical dendrites of ca1 pyramidal neurons used for quantitative analyses . dendritic spine density in ca1 pyramidal neurons of p7 mecp2 mutants was lower compared to age - matched wildtype littermates ( wildtype 3.65 0.54 spines per 10 m of dendritic length , n = 5 dendrite segments from 3 mice , versus mecp2 2.23 0.40 spines/10 m , n = 10 segments/3 mice ; p = 0.030 ; figures 2(a ) and 2(d ) ) . on the other hand , dendritic spine density in p15 mecp2 mutant mice was not statistically different compared to age - matched wildtype littermates ( wildtype 5.39 1.20 spines/10 m , n = 7 segments/3 mice versus mecp2 5.87 0.43 spines/10 m , n = 9 segments/3 mice ; p = 0.359 ; figures 2(b ) and 2(d ) ) . despite differences in spine density in p7 mice , the proportions of the three main morphological types of spines did not differ between mecp2 and age - matched wildtype littermates ( stubby : wildtype 0.49 0.07 versus mecp2 0.46 0.08 ; p = 0.396 , mushroom : wildtype 0.24 0.02 versus mecp2 0.16 0.04 ; p = 0.110 , thin : wildtype 0.27 0.05 versus mecp2 0.25 0.06 ; p = 0.401 ) . dendritic spine density in ca1 pyramidal neurons of p4060 mecp2 mutant mice exhibiting characteristic rtt - like symptoms were not statistically different than in age - matched wildtype littermates ( wildtype 14.39 0.77 spines/10 m of dendritic length , n = 35 segments/5 mice versus mecp2 15.82 0.86 spines/10 m , n = 29 segments/5 mice ; p = 0.110 ; figures 2(c ) and 2(d ) ) . we next determined if the proportion of the three major morphological types of dendritic spines were affected in symptomatic mecp2 mutant mice . on the other hand , the proportion of the more motile and immature thin spines was lower in symptomatic mecp2 mutant mice ( wildtype 0.16 0.01 versus mecp2 0.11 0.01 ; p = 0.005 ) . electron microscopy of asymmetric synapses on dendritic spines ( presumptive excitatory ) within ca1 stratum radiatum of symptomatic mecp2 mutant mice was conducted to determine if mecp2 alters density of excitatory synapses . unbiased stereological analyses revealed that the density of asymmetric spine synapses in symptomatic mecp2 mutant mice ( 2.12 0.11 synapses per m ; n = 3 mice ) was not significantly different than in wildtype littermates ( 2.06 0.05 synapses/m , intriguingly , the cross - sectional area of individual spines ( figure 3(c ) ) and presynaptic terminals ( figure 3(d ) ) were smaller in mecp2 mutant mice ( kolmogorov - smirnov test ; p < 0.01 ) , but only for the smallest asymmetric spine synapses . it should be noted that these spines have dimensions below the resolution of diffraction - limited light microscopy and thus are not detectable in our dendritic spine density measurements relying on confocal microscopy ( figure 2(b ) ) . quantitative confocal microscopy of dii - labeled dendrites revealed that ca1 pyramidal neurons from p7 mecp2 mutant mice had lower spine density compared to age - matched wildtype littermates . these differences in spine density were not observed neither in slightly older but still presymptomatic mecp2 mutant mice at p15 nor in p4060 mice that express the full spectrum of rtt - like symptoms , consistent with an analysis of the density of vglut1-psd95 puncta in area ca1 . stereological analyses at the electron microscopy level revealed that the density of asymmetric spine synapses is comparable in p4060 fully symptomatic mecp2 mutant mice compared to age - matched wildtype littermates . the only significant difference between genotypes during the symptomatic stage was a lower proportion of immature thin spines and a smaller cross - sectional area of individual spines and presynaptic terminals but only for the smallest asymmetric spine synapses in mecp2 mutant mice . it should be noted that the smallest asymmetric spine synapses analyzed at the em level have dimensions below the resolution of diffraction - limited light microscopy and thus are not detectable in our measurements using confocal microscopy . these data demonstrate that proper mecp2 function is required for early development of dendritic spines in ca1 pyramidal neurons and that a compensatory mechanism that normalizes spine density seems to occur later in development . using quantitative confocal microscopy of dii - labeled hippocampal sections , we recently showed that ca1 pyramidal neurons have lower dendritic spine density in female rtt individuals than in age - matched unaffected females . our results on hippocampal dendritic spine density show that male mecp2 mutant mice at the symptomatic stage do not recapitulate the human phenotype observed in autopsy material from female rtt individuals . while many factors could contribute to these findings ( including gender differences , disease severity , seizure disorder , and x - chromosomal inactivation ratio ) , these results suggest that hippocampal neuron spine density in this particular mouse model is not a phenotype with sufficient face validity for rtt . however , these mice still yield relevant information on the role of mecp2 in the cns . further postmortem studies in more individuals with rtt as well as other mecp2-associated conditions are needed to better understand the consequences on dendritic spine density and morphology . furthermore , using a postnatal rat hippocampal slice culture preparation , we observed that the knockdown of endogenous mecp2 protein levels resulted in reduced dendritic spine density , especially of mature spines ; however , overexpression of rtt - associated human mecp2 missense mutations led to a dramatic reduction in dendritic spine density . consistently , a recent report described that neurons derived from ipscs obtained from reprogramming of skin fibroblasts of rtt patients have reduced dendritic spine density . while these exciting observations on patient - derived neurons provide additional evidence of the importance of dendritic spines in the neuropathology of rtt , it should be made clear that no causative biochemical underpinning has yet been established . however , they have also yielded varying results in terms of dendritic spine alterations . in contrast with the present observations , a quantitative study of two different mecp2 mutant lines ( mecp2 and mecp2 ) described that pyramidal neurons from hippocampal area ca1 and layers ii - iii of the motor cortex have lower dendritic spine densities than their control wildtype littermates at approximately 3 weeks of age . described significant differences in spine density between age - matched wildtype mice of the two different strains , strongly suggesting that genetic background contributes to the phenotype under study , for example , dendritic spine density and morphology . we show here that hippocampal pyramidal neurons exhibit a dendritic spine phenotype only in neonatal ( p7 ) mutant mice , well before excitatory synapse expansion , while spine density in mutants recovers to wildtype levels a week later ( p15 ) and is maintained at wildtype levels throughout the symptomatic stage ( p4060 ) . this developmental progression of the dendritic spine phenotype is also reflected in the density of vglut1-psd95 puncta , which is lower in area ca1 of 2-week - old mecp2 null mice , but comparable to wildtype levels at 5 weeks of age . together with our dendritic spine observations , those vglut1-psd95 puncta results are consistent with the present em analyses in symptomatic mecp2 mutant mice , which revealed comparable densities of asymmetric spine synapses in stratum radiatum of area ca1 of both genotypes . altogether , these data demonstrate that proper mecp2 functioning is required for dendritic spine formation during early postnatal development , and that a secondary compensatory mechanism seems to take place in symptomatic mecp2 mutant mice . one possibility is that enhanced hippocampal network activity in mecp2 mutants promotes dendritic spine formation . despite the lack of differences in dendritic spine density in fully symptomatic mecp2 mutant mice , we observed a significant , yet small , reduction in the proportion of immature thin spines in mecp2 mutants compared to age - matched wildtype animals . considering that dendritic spines are highly sensitive to the levels of neuronal activity , the shift in the proportion of morphological spine types could also reflect a response to the heightened neuronal activity observed in the hippocampal network of symptomatic mecp2 mutant mice . thus , it would be interesting to determine what role mecp2 has in the maintenance of thin spines and what consequences does this have on hippocampal function . one report has already demonstrated that spine motility is slowed in mecp2 mutant mice , possibly reflecting the decrease in the proportion of thin spines that we observed . confocal microscopy of dendritic spines in organotypic hippocampal cultures has revealed that approximately 6570% of dendritic spines are juxtaposed to presynaptic terminals . for this reason , we decided to conduct unbiased stereological analyses at the electron microscopy level to determine the density and morphology of dendritic spines that were actually connected to a presynaptic terminal , for example , asymmetric spine synapses . this approach demonstrated that the density of asymmetric spine synapses in mecp2 mutant mice is comparable to that of wildtype littermates , consistent with confocal microscopy of dendritic spines . intriguingly , the areas of dendritic spines and presynaptic terminals are smaller in mecp2 mutant mice ; however , this difference was only observed for the smallest asymmetric spine synapses . considering that these spines have dimensions below the resolution of diffraction - limited light microscopy , they could not have been included in measurements of spine head width in confocal microscopy images ( figure 3(b ) ) . taken together , our observations demonstrate that while the proportion of thin spines is lower in mecp2 mutant mice , individual dendritic spines , and thus excitatory synapses , are smaller in volume . in summary , our results raise caution regarding the use of dendritic spine density in hippocampal neurons as a phenotypic endpoint for the evaluation of therapeutic interventions in symptomatic mecp2 deficient mice . however , we present data describing the importance of mecp2 on spine development in neonatal mice . future research will hopefully explain the precise molecular role of mecp2 in the establishment of the hippocampal excitatory network and how this manifest into clinical issues .

spectrophotometric measurements of seawater ph , based on methods developed in the late 1980s , are simple , rapid , and precise . observations obtained during global surveys ( e.g. , http://cdiac.ornl.gov/oceans/ ) have demonstrated shipboard measurement precisions on the order of 0.0004 ph units . at this level of precision , ph measurements can play an important role in co2-system characterizations and quality control assessments.(6 ) spectrophotometric ph values obtained via measurements of absorbance ratios are directly grounded on indicator molecular properties : molar absorptivity ratios and protonation characteristics . these indicators have been used , for example , to monitor and evaluate the quality of aging ph standards.(7 ) ( as buffers age , they absorb atmospheric co2 and their ph declines . ) furthermore , archived spectrophotometric ph data can be quantitatively revised should improved indicator equilibrium constants and molar absorptivity ratios later become available.(3 ) however , as noted by yao et al.,(8 ) indicator impurities can introduce systematic errors in reported spectrophotometric ph even though measurement precision remains quite good . yao et al.(8 ) pointed out that indicator impurities vary with manufacturer and can also differ among batches from a single manufacturer . systematic differences in reported ph obtained using indicators from different sources were as large as 0.01 ph units . consequently , in order to fully realize the advantages of spectrophotometric ph measurements ensuring accuracy as well as precision the issue of indicator impurities must be carefully addressed . this work focused on the physical chemical characteristics of the ph indicator meta - cresol purple ( mcp ) , and endeavored to provide ( a ) an efficient procedure for indicator purification , and ( b ) a procedure for oceanic seawater ph measurements that is free of vendor - specific ph indicator impurities . purification via high - performance liquid chromatography ( hplc ) was performed , and the characteristics of purified mcp are reported for a wide range of seawater salinity and temperature . using the methods described here , independent investigators should be able to produce ph measurements that are directly comparable through time , independent of dye source . spectrophotometric ph measurements involving the use of sulfonephthalein indicators ( h2i ) are based on observations of the relative absorbance contributions of protonated ( hi ) and unprotonated ( i ) species in the sample of interest . solution ph can be calculated using the following equation : where k2 t is the dissociation constant of hi on the total hydrogen ion concentration scale,(3)and [ ] denotes concentration in mol / kg - solution . the parameter r in eq 1 is the ratio of sulfonephthalein absorbances at wavelengths 2 and 1for mcp , 2 = 578 nm and 1 = 434 nm.(3 ) the symbols e1 , e2 , and e3 are ratios of molar absorptivities of the hi and i indicator formswhere i is the molar absorptivity of i at wavelength and hi is the molar absorptivity of hi at wavelength . equation 1 can be alternatively written in a form with fewer parameters:(11)the e3/e2 term involves determinations of i molar absorptivity ratios and is directly obtained via measurements in synthetic solutions at a ph sufficiently high that species other than i are negligible ( i.e. , ph 12 ) . the e1 term involves determinations of hi molar absorptivity ratios and is obtained , as a very good approximation , through measurements at low ph ( i.e. , ph 4.5 ) , where hi is the strongly predominant form of mcp . the k2te2 term can be determined as a single parameter by measuring the absorbance ratio r in a solution of known pht ( e.g. , tris seawater buffer ) . finally , an iterative procedure is applied to refine , in an internally consistent manner , the e1 initial approximation and resulting k2te2 term . the m - cresol purple ( mcp ) indicator , in acid form or as a sodium salt , was obtained from the following vendors : acros ( batch a0182569 ) , aldrich ( batches 11517kc and 07005hh ) , alfa aesar ( batch h11n06 ) , jt baker ( batch b40626 ) , mp bio ( batch 1426k ) , ricca ( batch 4003124 ) , and tci ( batch fdp01 ) . sodium chloride ( reagentplus ) , potassium chloride ( 99.999% ) , sodium sulfate ( sigmaultra ) , magnesium chloride hexahydrate ( sigmaultra ) , calcium chloride dihydrate ( sigmaultra ) , and trifluoroacetic acid were obtained from sigma and were used without further purification . tris and all salts except mgcl2 and cacl2 were stored in a desiccator over p2o5 until the weight of each substance stabilized . solutions of mgcl2 and cacl2 , each with a concentration of 1 mol / kg solution , were prepared , and the final concentration of each was measured via icp - ms analysis . hydrochloric acid with a concentration of 1 mol / kg solution was prepared by dilution of concentrated hcl ( fisher scientific ) . the mcp used in this study was characterized and purified using a waters preplc hplc system . this system includes a prep lc controller , two hplc pumps capable of flow rates between 1 and 150 cm(3)/min , a fraction collector , and a model 2998 photodiode array detector . the primesep b2 column used in this work has a mixed - mode resin that interacts with analytes via hydrophobic and ion exchange mechanisms . an analytical column ( part b2 - 46 - 250.0510 , 4.6 250 mm , particle size 5 m ) was used to optimize separation conditions . subsequently , a preparative column ( part b2 - 220.250.0510 , 22 250 mm , particle size 5 m ) was used to purify the mcp . the primesep columns were housed in a shimadzu column oven ( model cto-10a ) at 30 c . the hplc mobile phase composition was 70% an plus 30% h2o ; 0.05% trifluoroacetic acid ( tfa ) was present as a mobile phase modifier . the pump rate was 1.5 cm / min for the analytical column and 31.26 cm / min in preparative mode . the injection loop volume was 0.020 cm in analytical mode and 7 cm in preparative mode . separations were begun by preparing a 70 mm ( 10 mol dm ) solution of mcp in the mobile phase solution . pure mcp component was collected at its characteristic retention time ( approximately 20 min ) . the solvent was separated from the mcp using a rotary evaporator ( buchi rotavapor - r ) . the evaporation flask was submerged in a 35 c water bath , with the contents of the flask under partial vacuum . evaporation of the mobile phase to dryness produced solid mcp in acid form ( h2i ) , free of salts . each injection produced approximately 0.2 g of purified mcp , resulting in a daily yield of about 2 g. thymol blue from the original batch of zhang and byrne(10 ) was also examined on the analytical column to assess its levels of impurities . batches of mcp direct from seven vendors were characterized relative to a reference ( purified ) mcp via paired ph measurements in strongly buffered solutions over a ph range of 7.2 to 8.2 . the direct - from - vendor mcps were then purified and again compared to the reference mcp . the reference mcp consisted of acros organics mcp that had been purified via the hplc method outlined above . all indicator stock solutions were adjusted to the same r ratio before use in the ph comparisons . the buffered solutions were prepared by adding 0.08 mol tris , epps , or hepes to 0.04 mol either hcl or naoh , depending on the form of the buffering agent . the solutions were brought to 0.7 m ( mol / kg solution ) ionic strength by addition of nacl . because measured ph differed slightly among different batches of the same buffer , the purified acros mcp was always used as a reference , thereby generating paired ph ( difference ) observations for each mcp comparison . for each ph measurement , the buffered solution was weighed ( 140 g ) into a custom - made quartz open - top spectrophotometric cell of 10 cm path length . after the stirred sample reached the target temperature ( 25 c ) , a blank was recorded . indicator solution ( 0.05 cm of 10 mm indicator ) was then injected into the sample and the absorbance ratio , r , was measured . addition of indicator to the strongly buffered solutions had a negligible effect on solution ph , so the wavelength accuracy of the instrument was verified using a holmium oxide standard ( nist srm 2034 ) , and the linearity of the spectrophotometer was verified with nist srm 930d glass filters . sample solutions were thermostatted in a custom - made , water - jacketed spectrophotometric cell holder that was connected via insulated tubing to a neslab refrigerating circulator . solution temperatures were monitored with a vwr digital thermometer or an ertco model 4400 thermometer . a dry n2 stream was directed to the optical windows of the spectrophotometric cell to prevent condensation of water vapor . the molar absorptivity ratio ( ei ) measurements were obtained using a 10 cm open - top quartz cell ( nsg precision cells , inc . ) . a cover for this cell had openings for insertion of a custom - made motor - driven stirring rod and a digital thermometer to ensure well - mixed solutions and accurately measured temperatures . absorbance measurements for a given solution of mcp were taken against a baseline solution that contained no mcp . values of e1 were obtained by measuring 578a and 434a in a solution that contained 0.02 m acetate buffer in 0.7 m nacl solution . the ph of the buffer solution , determined with a ross combination electrode , was adjusted to ph 4.50 by addition of naoh or hcl . the ph electrode was calibrated on the free hydrogen ion concentration scale by titrating unbuffered 0.7 m nacl solutions with 1 m ( mol dm ) standard hcl . the absorbance measurements were corrected by an iterative procedure ( described below ) to produce e1 values . ancillary experimental data showed that e1 has a negligible dependence on ionic strength and composition , indicating that this buffer solution is adequately seawater - like for the purpose of e1 determination . in contrast , e3/e2 was found to be significantly influenced by ionic strength and , to a lesser extent , ionic composition . consequently , e3/e2 values were obtained in solutions with compositions similar to seawater , over a range of temperatures ( 278.21308.37 k ) and salinities ( 2040 ) . the synthetic seawater contained 0.01 m naoh . to avoid precipitation of magnesium and sulfur salts at high ph ( ph 12 ) , titration of solutions of mcp in 0.7 m nacl with 1 m naoh demonstrated that absorbance contributions from hi were negligible in solutions containing 0.01 m naoh . titrations of acetate buffer solutions with hcl demonstrated that minima in e1 ratios were obtained near ph 4.5 . equilibrium constants ( k1 ) for dissociation of h2i ( h2i hi + h ) are required to estimate the absorbance contributions of h2i and i in the course of e1 determination . k1 values over the temperature range 279.45307.95 k were obtained from observations of mcp absorbances in aqueous hcl nacl mixtures ( 1 ph 2 ) at ionic strength 0.70 0.02 m. at each temperature , [ h ] and absorbance at = 529 nm ( the h2i absorbance maximum ) were recorded , along with isosbestic point absorbances , which were used to account for dilution due to the acid additions . hydrogen ion concentrations were calculated via the mixing ratios of the hcl and nacl solutions . to determine the log(k2te2 ) term in eq 5 , tris seawater buffer was used as a calibrating medium over a range of salinities between 20 and 40 and temperatures between 278.15 and 303.15 k. solutions of 0.04 m tris buffer were prepared at different salinities according to the recipe of delvalls and dickson.(12 ) the solutions consisted of synthetic seawater in which 0.04 mol / kg h2o of nacl was replaced with an identical molality of hcl , plus tris at a total concentration of 0.08 mol / kg h2o . the tris buffers at salinity 35 prepared in our experiments produced an r ratio identical to the measured r value of tris buffer provided by dr . the ph of each tris buffer was calculated on the total hydrogen ion concentration scale(12 ) with the following equation : to determine the log(k2te2 ) values , sulfonephthalein absorbance ratios ( r ) were obtained in the tris buffers , and eq 5 was used to calculate the log(k2te2 ) term for each t s condition . subsequent to the measurements of k2te2 , small refinements were made to the initial estimates of e1 . these quantitatively small refinements were necessary because the molar absorptivities of i and h2i at 578 nm are very large relative to that of hi . as such , even though hi is the strongly predominant form of mcp at ph 4.5 , the absorbance contributions of i and h2i are significant and must be quantitatively taken into account . the directly measured absorbances of mcp at low ph ( 578a and 434a ) are summations of the major contribution from hi and minor contributions from i and h2i : using eqs 7a and 7b , the parameter e1 can be written aswhere b is path length . the absorbances of h2i and i in eq 8 can then be expressed in terms of molar absorptivities ( i ) and path length ( b):employing eq 9 , the 578ai and 434ai absorbance terms in eq 8 can be expressed in terms of molar absorptivities and mcp species concentrations . concentrations of h2i and i can be expressed in terms of free hydrogen ion concentrations ( [ h ] ) , total concentrations of mcp ( it ) , and h2i and hi dissociation constants on the free hydrogen ion concentration scale ( k1 = [ hi][h][h2i ] and k2 = [ i][h][hi ] ) . the 578ai and 434ai absorbance terms in eq 8 can then be written asthe molar absorptivity terms 578h2i and 434h2i were calculated from measurements in 1 m hcl where h2i is a strongly predominant species ; the molar absorptivity terms 578i and 434i were calculated from experiments at ph 12 where i strongly predominates . the data used for k1 determinations consisted of coupled a and [ h ] observations at = 529 nm ( i.e. , at the h2i absorbance maximum ) . absorbance observations were fitted with the following equation , from which k1 can be obtained : k2 values used for determinations of i concentrations on the free hydrogen ion concentration scale were calculated from the k2te2 term in eq 5 . these calculations utilized e2 values obtained from paired observations of indicator absorbances for hi at = 434 nm in acetate buffer ( ph 4.5 ) and i at = 578 nm in 0.01 m naoh . the concentrations of mcp , it , in these experiments were calculated through observations of isosbestic point absorbances . k2 values on the free h scale were then calculated using the following relationship:(13)where ks = [ h][so4]/[hso4 ] , st is the total sulfate concentration at a given salinity , and ks is the dissociation constant of hso4.(14 ) using calculated values of k1 ( eq 11 ) and k2 ( eq 12 ) , eqs 8 through 10 were then used to refine e1 by accounting for small absorbance contributions from i and h2i . the refined e1 value was then used in eq 5 to obtain a refined k2 t value . iterations were repeated until log(k2te2 ) values were in agreement to 0.0001 and e1 values were in agreement to within 0.000001 . for the salinity - dependent terms in eq 5 ( k2 t and e2 ) , the salinity values are consistent with the characterizations of delvalls and dickson.(12 ) for e1 , which is not salinity - dependent , the ionic strength of the solution was maintained at 0.7 mol / kg solution . for the purposes of these calculations , it is appropriate to regard the activity coefficients of h , i , and hi as functions of ionic strength independent of medium composition . the salinity of this seawater , measured with a seabird model 59 ctd , was 35.90 ( practical salinity scale ) . total alkalinity ( at ) , total co2 ( ct ) , and pht of the seawater samples were measured to assess the internal consistency of these three carbonate - system parameters . at was measured using the method of yao and byrne.(15)ct was measured with a uic cm 5104 coulometer with the uic 5130 acidification module . sample seawater was drawn into a gastight syringe and then injected into an acidification flask . a stream of co2-free air carried evolved co2 from the acidification flask to the cathode solution for subsequent coulometric measurement . the certified reference material ( crm ) used to calibrate the coulometer was provided by dr . seawater samples were collected in 10-cm cylindrical cells ( hellma cells ) , and pht was measured using the procedures outlined in sop 6b of dickson et al.(13 ) yao et al.(8 ) demonstrated that mcp batches obtained from different vendors have variable amounts and types of impurities . figure 1a shows an hplc chromatograph of an unpurified aldrich mcp sample on a primesep b2 column . several major peaks were identified both before and after the pure mcp peak , which had a retention time of approximately 20 min . the pure mcp fraction had spectrophotometric absorption peaks at 406 and 529 nm in the 70% an mobile phase ( see inset graph ) . the component with an hplc retention time of 3.10 min had a peak absorbance at 421 nm . this peak overlaps with the mcp hi peak in aqueous solution , which is potentially problematic . components eluted at 1.68 , 8.91 , and 29.07 min all had nonzero absorbances at wavelengths greater than 400 nm , which could also affect spectrophotometric ph measurements . in contrast , the components that eluted at 2.90 and 24.21 min had no absorbances at wavelengths greater than 350 nm and so would likely not affect mcp absorbance readings and ph measurements in aqueous solutions . the small inset panels show the uv vis spectra ( 200 to 600 nm ) of indicator components eluted at different times . ( b ) purified mcp . the analytical hplc procedure , which characterizes the relative absorbance contributions of components and establishes effective separation conditions , was successfully scaled up to a preparative - scale procedure , which separates components and allows for production of purified mcp . the injection volume was increased to 7 cm at an mcp concentration of 70 mm . because salt components were eluted at the beginning of the hplc run and the rotary evaporation procedure eliminated an and tfa , pure mcp in acid form was obtained in the flask . only the mcp peak is present , indicating that the contamination components were successfully separated out . as will be shown below , the differences among purified mcps produced from various batches / vendors are insignificant . as shown in figure 1a ( see small inset panels ) , components with absorbances at wavelengths greater than 400 nm are present , which may affect spectrophotometric ph measurements in seawater . at higher ph , where concentrations of the acid form of mcp , hi , are expected to be low , the effects of the impurities that absorb at = 434 nm are likely enhanced relative to low - ph samples , where 434a is large . therefore , it is expected that the effects of mcp impurities on spectrophotometric ph measurements would be more pronounced for high - ph surface waters ( ph 8 ) than for low - ph waters ( ph 7.2 ) . using mcp from seven different vendors , spectrophotometric ph values were measured in a range of strongly buffered solutions . figure 2a compares ph values obtained using unrefined ( unpurified ) mcp to corresponding measurements obtained using purified mcp ( acros ) . using unrefined mcp yields ph deviations , relative to values obtained with purified mcp , as large as 0.010 ph units at ph 7.4 and as large as 0.018 ph units at ph 8.2 . as expected from eluate absorbances ( figure 1a ) , the effects of using unpurified mcp are greatest at higher ph values . comparison of ph values obtained using mcp from seven different vendors in strongly buffered solutions : ( a ) unpurified mcp , and ( b ) purified mcp . all ph differences are expressed relative to the ph measured with the reference mcp ( purified acros ) . the ph differences ( ph = ph(purified sample ) ph(reference ) ) are within 0.0004 ph units over a ph range between 7.4 and 8.2 . at any given ph , the maximum spread among all vendors ( ph(max ) the maximum ph deviation is approximately equal to the precision of spectrophotometric ph measurements at sea , indicating that the purification procedure described here eliminates indicator impurities to an extent that ph measurements made with purified mcp are independent of vendor and batch . the absorption spectrum for hi was obtained at ph 4.5 , where that form predominates . wavelengths of 434 nm for hi and 578 nm for i were chosen for our analyses , consistent with the choices of clayton and byrne.(3 ) the temperature dependence of the isosbestic point wavelength for hi / i can be summarized aswhere t is temperature in degrees celsius . the isosbestic point wavelength as a function of temperature for h2i / hi is summarized asisosbestic point wavelengths decrease by 0.1 nm with each 1 c increase in temperature . at ph 12 , absorbance contributions of the h2i and hi species are negligible . therefore e3/e2 can be obtained at this high ph directly from observations of the absorbance characteristics of i ( i.e. , at ph 12 , a434/a578 = e3/e2 ) . the temperature and salinity dependences of e3/e2 can be summarized as the temperature dependence of k1 in 0.7 m nacl is given by the variation of a578/a434 with temperature is small but well - defined : to determine e1 , the contributions of h2i and i to a578/a434 ( a first approximation of e1 ) were accounted for through the iterative procedure outlined in the methods section . the resulting e1 term can be summarized asvalues of e1 are approximately 0.0008 units smaller than a578/a434 values . approximately two - thirds of the difference is due to the absorbance contributions of i and one - third is attributable to h2i . the small difference between e1 and a578/a434 has very minor implications for ph calculations at 8.2 ( 0.0002 ph units ) but a larger influence at ph 7.5 ( 0.0008 ph units ) . the final equation for spectrophotometric ph measurement using purified mcp iswhere a = 246.64209 + 0.315971s + 2.8855 10s b = 7229.23864 7.098137s 0.057034s c = 44.493382 0.052711s t e3/e2 = 0.020813 + 2.60262 10 t + 1.0436 10 ( s 35 ) for 20 s 40 and 278.15 t 308.15 k. the results of ph comparisons in tris buffers over a wide range of s and t ph(tris ) from eq 6 vs. ph(mcp ) from eq 18are summarized in figure 3 . consequently , we regard the overall uncertainty of our calibration technique relative to tris buffer standards as 0.001 or better . residuals for ph ( expected ph ( eq 6 ) minus measured ph ( eq 18 ) ) determined in synthetic tris seawater buffer . although e3/e2 is significantly dependent on salinity , the dependence is sufficiently slight that an assumption of independence has only small consequences for ph calculations . for samples of s = 20 or s = 40 , assuming that e3/e2 is independent of salinity ( i.e. , that e3/e2 = 0.05626 , as at s = 30 ) yields deviations from the salinity - dependent calculation ( eq 18 ) of about 0.0002 ph units at ph 7.4 and 0.0015 units at ph 8.2 . a high - performance spectrophotometer was used in this work , but other properly calibrated spectrophotometers ( e.g. , agilent or ocean optics ) should provide ph measurements of comparable quality . to ensure that this expectation is met , we recommend ( a ) the use of nist absorbance standards to ensure spectrophotometer absorbance linearity at the wavelengths used in the ph analyses and ( b ) adherence to the measurement procedures outlined in sop 6b of dickson et al.(13 ) investigators can check their coding of the ph algorithm ( eq 18 ) by comparing their test calculations with the pht(mcp ) results given for a range of s , t , and r values in table s4 ( supporting information ) . hplc chromatographs of the kodak thymol blue used by zhang and byrne(10 ) showed no impurity peaks that extended above 400 nm . because seawater ph characterizations based on this indicator use rtb = 596a/435a , it is unlikely that impurities in the zhang and byrne(10 ) thymol blue impacted their indicator characterizations . as such , it appears that the ph measurement algorithm of zhang and byrne(10 ) is appropriate for pure thymol blue . paired ph results for gulf of mexico surface seawater ( s = 35.9 , t = 25.0 c ) , comparing purified mcp and the thymol blue used by zhang and byrne(10 ) are given below : pht(mcp ) = 8.0254 0.0002 pht(tb ) = 8.0252 0.0007 . agreement between the two indicators is better than the typical at - sea precision of ph measurements made with mcp . it should be noted , however , that assessments of thymol blue from batches other than that used by zhang and byrne(10 ) exhibited ph differences as large as 0.01 ( not shown in this work ) . therefore , indicator - purification procedures similar to those described in the methods section should be used to ensure the accuracy of ph measurements obtained with thymol blue . total dissolved inorganic carbon , ph , and total alkalinity were measured in a sample of gulf of mexico surface seawater . at was also calculated from ct and pht(mcp ) measurements using the dissociation constants of mehrbach et al.(16 ) as refitted by dickson and millero.(17 ) measured at and calculated at are in excellent agreement : ct(measured ) = 2096.6 0.2 mol / kg pht(measured ) = 8.0226 0.0002 at(measured ) = 2400.4 1.3 mol / kg at(calculated ) = 2398.6 1.0 mol / kg . these results , each based on five sets of measurements , suggest that the algorithm for calculation of pht ( eq 18 ) provides ph measurements that can be used to reliably link other measured carbon system parameters ( at , ct , and co2 fugacity ) . yao et al.(8 ) recommended that unpurified indicators used for ph measurements be archived so that comparisons and corrections could eventually be made when purified indicators come available . to correct older ph measurements obtained using unpurified indicator : use the archived unpurified indicator and purified indicator to make paired ph measurements in seawater over a range of ph values ( e.g. , 7.28.2 ) . to calculate apparent ph obtained with the unpurified indicator ( phu ) , use the ph algorithm originally employed to generate the older data set . to calculate the true ph obtained with the purified mcp ( php ) , use plot the measured ph differences ( ph = php phu ) as a function of phu ( see figure 2 of yao et al.(8 ) ) . figure 2a indicates that ph measurements made with unpurified indicator could have errors as large as 0.02 ph units relative to those made with purified indicator . fit an equation to the curve that describes the offset between the two measurements ( see eq 5 of yao et al.(8 ) ) . use this equation to correct the older ph measurements to a scale consistent with the use of purified mcp and eq 18 : php = phu + ph . equation 18 extends the single - temperature characterizations of clayton and byrne(3 ) to cover a wide range of temperature and salinity ( 278.15 k t 308.15 and 20 s 40 ) . use of purified mcp is required to facilitate direct comparisons of cross - batch ph measurements future work describing the influence of pressure on ph measurements with mcp , similar to that of hopkins et al.(18 ) for thymol blue , will enable in situ ph measurements with mcp throughout the oceanic water column .

spectrophotometric procedures allow rapid and precise measurements of the ph of natural waters . however , impurities in the acid base indicators used in these analyses can significantly affect measurement accuracy . this work describes hplc procedures for purifying one such indicator , meta - cresol purple ( mcp ) , and reports mcp physical chemical characteristics ( thermodynamic equilibrium constants and visible - light absorbances ) over a range of temperature ( t ) and salinity ( s ) . using pure mcp , seawater ph on the total hydrogen ion concentration scale ( pht ) can be expressed in terms of measured mcp absorbance ratios ( r = 2a/1a ) as follows : where log(k2te2 ) = a + ( b / t ) + c ln t dt ; a = 246.64209 + 0.315971s + 2.8855 104s2 ; b = 7229.23864 7.098137s 0.057034s2 ; c = 44.493382 0.052711s ; d = 0.0781344 ; and mcp molar absorbance ratios ( ei ) are expressed as e1 = 0.007762 + 4.5174 105 t and e3/e2 = 0.020813 + 2.60262 104 t + 1.0436 104 ( s 35 ) . the mcp absorbances , 1a and 2a , used to calculate r are measured at wavelengths ( ) of 434 and 578 nm . this characterization is appropriate for 278.15 t 308.15 and 20 s 40 .

Introduction Methods Results and Discussion

spectrophotometric measurements of seawater ph , based on methods developed in the late 1980s , are simple , rapid , and precise . , http://cdiac.ornl.gov/oceans/ ) have demonstrated shipboard measurement precisions on the order of 0.0004 ph units . (6 ) spectrophotometric ph values obtained via measurements of absorbance ratios are directly grounded on indicator molecular properties : molar absorptivity ratios and protonation characteristics . furthermore , archived spectrophotometric ph data can be quantitatively revised should improved indicator equilibrium constants and molar absorptivity ratios later become available. (3 ) however , as noted by yao et al.,(8 ) indicator impurities can introduce systematic errors in reported spectrophotometric ph even though measurement precision remains quite good . this work focused on the physical chemical characteristics of the ph indicator meta - cresol purple ( mcp ) , and endeavored to provide ( a ) an efficient procedure for indicator purification , and ( b ) a procedure for oceanic seawater ph measurements that is free of vendor - specific ph indicator impurities . purification via high - performance liquid chromatography ( hplc ) was performed , and the characteristics of purified mcp are reported for a wide range of seawater salinity and temperature . spectrophotometric ph measurements involving the use of sulfonephthalein indicators ( h2i ) are based on observations of the relative absorbance contributions of protonated ( hi ) and unprotonated ( i ) species in the sample of interest . solution ph can be calculated using the following equation : where k2 t is the dissociation constant of hi on the total hydrogen ion concentration scale,(3)and [ ] denotes concentration in mol / kg - solution . the parameter r in eq 1 is the ratio of sulfonephthalein absorbances at wavelengths 2 and 1for mcp , 2 = 578 nm and 1 = 434 nm. (3 ) the symbols e1 , e2 , and e3 are ratios of molar absorptivities of the hi and i indicator formswhere i is the molar absorptivity of i at wavelength and hi is the molar absorptivity of hi at wavelength . , ph 4.5 ) , where hi is the strongly predominant form of mcp . the m - cresol purple ( mcp ) indicator , in acid form or as a sodium salt , was obtained from the following vendors : acros ( batch a0182569 ) , aldrich ( batches 11517kc and 07005hh ) , alfa aesar ( batch h11n06 ) , jt baker ( batch b40626 ) , mp bio ( batch 1426k ) , ricca ( batch 4003124 ) , and tci ( batch fdp01 ) . sodium chloride ( reagentplus ) , potassium chloride ( 99.999% ) , sodium sulfate ( sigmaultra ) , magnesium chloride hexahydrate ( sigmaultra ) , calcium chloride dihydrate ( sigmaultra ) , and trifluoroacetic acid were obtained from sigma and were used without further purification . the mcp used in this study was characterized and purified using a waters preplc hplc system . the primesep b2 column used in this work has a mixed - mode resin that interacts with analytes via hydrophobic and ion exchange mechanisms . an analytical column ( part b2 - 46 - 250.0510 , 4.6 250 mm , particle size 5 m ) was used to optimize separation conditions . subsequently , a preparative column ( part b2 - 220.250.0510 , 22 250 mm , particle size 5 m ) was used to purify the mcp . pure mcp component was collected at its characteristic retention time ( approximately 20 min ) . the evaporation flask was submerged in a 35 c water bath , with the contents of the flask under partial vacuum . evaporation of the mobile phase to dryness produced solid mcp in acid form ( h2i ) , free of salts . each injection produced approximately 0.2 g of purified mcp , resulting in a daily yield of about 2 g. thymol blue from the original batch of zhang and byrne(10 ) was also examined on the analytical column to assess its levels of impurities . batches of mcp direct from seven vendors were characterized relative to a reference ( purified ) mcp via paired ph measurements in strongly buffered solutions over a ph range of 7.2 to 8.2 . all indicator stock solutions were adjusted to the same r ratio before use in the ph comparisons . the buffered solutions were prepared by adding 0.08 mol tris , epps , or hepes to 0.04 mol either hcl or naoh , depending on the form of the buffering agent . because measured ph differed slightly among different batches of the same buffer , the purified acros mcp was always used as a reference , thereby generating paired ph ( difference ) observations for each mcp comparison . after the stirred sample reached the target temperature ( 25 c ) , a blank was recorded . addition of indicator to the strongly buffered solutions had a negligible effect on solution ph , so the wavelength accuracy of the instrument was verified using a holmium oxide standard ( nist srm 2034 ) , and the linearity of the spectrophotometer was verified with nist srm 930d glass filters . a dry n2 stream was directed to the optical windows of the spectrophotometric cell to prevent condensation of water vapor . the molar absorptivity ratio ( ei ) measurements were obtained using a 10 cm open - top quartz cell ( nsg precision cells , inc . ) the ph of the buffer solution , determined with a ross combination electrode , was adjusted to ph 4.50 by addition of naoh or hcl . the ph electrode was calibrated on the free hydrogen ion concentration scale by titrating unbuffered 0.7 m nacl solutions with 1 m ( mol dm ) standard hcl . consequently , e3/e2 values were obtained in solutions with compositions similar to seawater , over a range of temperatures ( 278.21308.37 k ) and salinities ( 2040 ) . equilibrium constants ( k1 ) for dissociation of h2i ( h2i hi + h ) are required to estimate the absorbance contributions of h2i and i in the course of e1 determination . k1 values over the temperature range 279.45307.95 k were obtained from observations of mcp absorbances in aqueous hcl nacl mixtures ( 1 ph 2 ) at ionic strength 0.70 0.02 m. at each temperature , [ h ] and absorbance at = 529 nm ( the h2i absorbance maximum ) were recorded , along with isosbestic point absorbances , which were used to account for dilution due to the acid additions . hydrogen ion concentrations were calculated via the mixing ratios of the hcl and nacl solutions . to determine the log(k2te2 ) term in eq 5 , tris seawater buffer was used as a calibrating medium over a range of salinities between 20 and 40 and temperatures between 278.15 and 303.15 k. solutions of 0.04 m tris buffer were prepared at different salinities according to the recipe of delvalls and dickson. the ph of each tris buffer was calculated on the total hydrogen ion concentration scale(12 ) with the following equation : to determine the log(k2te2 ) values , sulfonephthalein absorbance ratios ( r ) were obtained in the tris buffers , and eq 5 was used to calculate the log(k2te2 ) term for each t s condition . subsequent to the measurements of k2te2 , small refinements were made to the initial estimates of e1 . the directly measured absorbances of mcp at low ph ( 578a and 434a ) are summations of the major contribution from hi and minor contributions from i and h2i : using eqs 7a and 7b , the parameter e1 can be written aswhere b is path length . the absorbances of h2i and i in eq 8 can then be expressed in terms of molar absorptivities ( i ) and path length ( b):employing eq 9 , the 578ai and 434ai absorbance terms in eq 8 can be expressed in terms of molar absorptivities and mcp species concentrations . concentrations of h2i and i can be expressed in terms of free hydrogen ion concentrations ( [ h ] ) , total concentrations of mcp ( it ) , and h2i and hi dissociation constants on the free hydrogen ion concentration scale ( k1 = [ hi][h][h2i ] and k2 = [ i][h][hi ] ) . absorbance observations were fitted with the following equation , from which k1 can be obtained : k2 values used for determinations of i concentrations on the free hydrogen ion concentration scale were calculated from the k2te2 term in eq 5 . these calculations utilized e2 values obtained from paired observations of indicator absorbances for hi at = 434 nm in acetate buffer ( ph 4.5 ) and i at = 578 nm in 0.01 m naoh . the concentrations of mcp , it , in these experiments were calculated through observations of isosbestic point absorbances . k2 values on the free h scale were then calculated using the following relationship:(13)where ks = [ h][so4]/[hso4 ] , st is the total sulfate concentration at a given salinity , and ks is the dissociation constant of hso4. (14 ) using calculated values of k1 ( eq 11 ) and k2 ( eq 12 ) , eqs 8 through 10 were then used to refine e1 by accounting for small absorbance contributions from i and h2i . for the salinity - dependent terms in eq 5 ( k2 t and e2 ) , the salinity values are consistent with the characterizations of delvalls and dickson. for the purposes of these calculations , it is appropriate to regard the activity coefficients of h , i , and hi as functions of ionic strength independent of medium composition . total alkalinity ( at ) , total co2 ( ct ) , and pht of the seawater samples were measured to assess the internal consistency of these three carbonate - system parameters . the certified reference material ( crm ) used to calibrate the coulometer was provided by dr . seawater samples were collected in 10-cm cylindrical cells ( hellma cells ) , and pht was measured using the procedures outlined in sop 6b of dickson et al. several major peaks were identified both before and after the pure mcp peak , which had a retention time of approximately 20 min . the pure mcp fraction had spectrophotometric absorption peaks at 406 and 529 nm in the 70% an mobile phase ( see inset graph ) . components eluted at 1.68 , 8.91 , and 29.07 min all had nonzero absorbances at wavelengths greater than 400 nm , which could also affect spectrophotometric ph measurements . in contrast , the components that eluted at 2.90 and 24.21 min had no absorbances at wavelengths greater than 350 nm and so would likely not affect mcp absorbance readings and ph measurements in aqueous solutions . the small inset panels show the uv vis spectra ( 200 to 600 nm ) of indicator components eluted at different times . because salt components were eluted at the beginning of the hplc run and the rotary evaporation procedure eliminated an and tfa , pure mcp in acid form was obtained in the flask . as shown in figure 1a ( see small inset panels ) , components with absorbances at wavelengths greater than 400 nm are present , which may affect spectrophotometric ph measurements in seawater . at higher ph , where concentrations of the acid form of mcp , hi , are expected to be low , the effects of the impurities that absorb at = 434 nm are likely enhanced relative to low - ph samples , where 434a is large . using mcp from seven different vendors , spectrophotometric ph values were measured in a range of strongly buffered solutions . using unrefined mcp yields ph deviations , relative to values obtained with purified mcp , as large as 0.010 ph units at ph 7.4 and as large as 0.018 ph units at ph 8.2 . as expected from eluate absorbances ( figure 1a ) , the effects of using unpurified mcp are greatest at higher ph values . comparison of ph values obtained using mcp from seven different vendors in strongly buffered solutions : ( a ) unpurified mcp , and ( b ) purified mcp . all ph differences are expressed relative to the ph measured with the reference mcp ( purified acros ) . the ph differences ( ph = ph(purified sample ) ph(reference ) ) are within 0.0004 ph units over a ph range between 7.4 and 8.2 . at any given ph , the maximum spread among all vendors ( ph(max ) the maximum ph deviation is approximately equal to the precision of spectrophotometric ph measurements at sea , indicating that the purification procedure described here eliminates indicator impurities to an extent that ph measurements made with purified mcp are independent of vendor and batch . wavelengths of 434 nm for hi and 578 nm for i were chosen for our analyses , consistent with the choices of clayton and byrne. (3 ) the temperature dependence of the isosbestic point wavelength for hi / i can be summarized aswhere t is temperature in degrees celsius . at ph 12 , absorbance contributions of the h2i and hi species are negligible . therefore e3/e2 can be obtained at this high ph directly from observations of the absorbance characteristics of i ( i.e. the temperature and salinity dependences of e3/e2 can be summarized as the temperature dependence of k1 in 0.7 m nacl is given by the variation of a578/a434 with temperature is small but well - defined : to determine e1 , the contributions of h2i and i to a578/a434 ( a first approximation of e1 ) were accounted for through the iterative procedure outlined in the methods section . the resulting e1 term can be summarized asvalues of e1 are approximately 0.0008 units smaller than a578/a434 values . approximately two - thirds of the difference is due to the absorbance contributions of i and one - third is attributable to h2i . the final equation for spectrophotometric ph measurement using purified mcp iswhere a = 246.64209 + 0.315971s + 2.8855 10s b = 7229.23864 7.098137s 0.057034s c = 44.493382 0.052711s t e3/e2 = 0.020813 + 2.60262 10 t + 1.0436 10 ( s 35 ) for 20 s 40 and 278.15 t 308.15 k. the results of ph comparisons in tris buffers over a wide range of s and t ph(tris ) from eq 6 vs. ph(mcp ) from eq 18are summarized in figure 3 . , that e3/e2 = 0.05626 , as at s = 30 ) yields deviations from the salinity - dependent calculation ( eq 18 ) of about 0.0002 ph units at ph 7.4 and 0.0015 units at ph 8.2 . a high - performance spectrophotometer was used in this work , but other properly calibrated spectrophotometers ( e.g. , agilent or ocean optics ) should provide ph measurements of comparable quality . to ensure that this expectation is met , we recommend ( a ) the use of nist absorbance standards to ensure spectrophotometer absorbance linearity at the wavelengths used in the ph analyses and ( b ) adherence to the measurement procedures outlined in sop 6b of dickson et al. (13 ) investigators can check their coding of the ph algorithm ( eq 18 ) by comparing their test calculations with the pht(mcp ) results given for a range of s , t , and r values in table s4 ( supporting information ) . because seawater ph characterizations based on this indicator use rtb = 596a/435a , it is unlikely that impurities in the zhang and byrne(10 ) thymol blue impacted their indicator characterizations . as such , it appears that the ph measurement algorithm of zhang and byrne(10 ) is appropriate for pure thymol blue . paired ph results for gulf of mexico surface seawater ( s = 35.9 , t = 25.0 c ) , comparing purified mcp and the thymol blue used by zhang and byrne(10 ) are given below : pht(mcp ) = 8.0254 0.0002 pht(tb ) = 8.0252 0.0007 . it should be noted , however , that assessments of thymol blue from batches other than that used by zhang and byrne(10 ) exhibited ph differences as large as 0.01 ( not shown in this work ) . therefore , indicator - purification procedures similar to those described in the methods section should be used to ensure the accuracy of ph measurements obtained with thymol blue . total dissolved inorganic carbon , ph , and total alkalinity were measured in a sample of gulf of mexico surface seawater . (16 ) as refitted by dickson and millero. (17 ) measured at and calculated at are in excellent agreement : ct(measured ) = 2096.6 0.2 mol / kg pht(measured ) = 8.0226 0.0002 at(measured ) = 2400.4 1.3 mol / kg at(calculated ) = 2398.6 1.0 mol / kg . these results , each based on five sets of measurements , suggest that the algorithm for calculation of pht ( eq 18 ) provides ph measurements that can be used to reliably link other measured carbon system parameters ( at , ct , and co2 fugacity ) . to correct older ph measurements obtained using unpurified indicator : use the archived unpurified indicator and purified indicator to make paired ph measurements in seawater over a range of ph values ( e.g. to calculate apparent ph obtained with the unpurified indicator ( phu ) , use the ph algorithm originally employed to generate the older data set . to calculate the true ph obtained with the purified mcp ( php ) , use plot the measured ph differences ( ph = php phu ) as a function of phu ( see figure 2 of yao et al. equation 18 extends the single - temperature characterizations of clayton and byrne(3 ) to cover a wide range of temperature and salinity ( 278.15 k t 308.15 and 20 s 40 ) . use of purified mcp is required to facilitate direct comparisons of cross - batch ph measurements future work describing the influence of pressure on ph measurements with mcp , similar to that of hopkins et al.

during recent decades , users of mental health services have steadily increased the strength of their voice in mental health care policies , education and practice ( who 2010 ) . patient - centered care , with its emphasis on the patient s voice and choice , has become a common good in mental health care ( dahlberg et al . 2009 ) , and service users acting as experts by experience are pivotal in their own care and treatment plans ( pilgrim 2008 ) . one can wonder what happened to knowledge derived from firsthand experience when mental health service users themselves became experts and actively began to choose care . the ensuing expertise is socially structured and constrained in terms of mental illness being a psychopathological intra - personal disturbance of the mind , in line with dominant psychiatric practice ( speed 2005 ; bracken and thomas 2005 ; coles et al . . however , alternative views on mental health are also endorsed ; mental health can be seen as a relational phenomenon which is never static , in line with so - called relation - based mental health work . in such views , mental health can not be reduced to brain activity or individual life histories , but emerges in and through processes of interaction between the person and other persons ( bracken and thomas 2005 ; crossley 2011 ; frie and coburn 2010 ; kirschner and martin 2010 ) . in our study , we take a closer look at the expertise that users as consumers bring to the mental health care field and how their views on mental health have developed vis - - vis the mainstream psychiatric view . in doing so , we will try to unravel the apparent paradox that experts by experience become part of the dominant medical paradigm they wished to move away from . in other words , in this study , we ask to what extent mental health users are experts among experts at the expense of being persons among persons. we approach these questions by generating a rational reconstruction of how the interpersonal ( person among persons ) view of mental health first gained and then lost coherence among its conceptions of mental health , the practice of mental health care , and the experience of the user . by rational reconstruction , we mean that it is only on the basis of what we know of user knowledge in mental health care today that we can reconstruct how this has come to be so . moreover , we study how past participants in mental health argued and thought in order to revive their voices in the present - day context , enabling us to create a dialogue between these and currently active players in the field . we acknowledge the risk of writing whiggish history but find support in rorty , who writes that but if they are conducted in full knowledge of their anachronism , they are unobjectable sullivan s mid - twentieth century interpersonal psychiatry , because it is a paradigmatic example of a coherent approach based on an interpersonal view of mental health . secondly , we address how sullivan s coherence was lost by those whom he inspired to revolt against psychiatry , i.e. , both anti - psychiatrists and service users . thirdly , we show how a partnership between service users and mental health professionals paved the way for a shared acceptance of the breaking up of mental health into independent parameters . we discuss the consequences of a parametric view of mental health in terms of standardized mental health and the subsequent logic of choice in mental health services ( kugelmann 2003 ; mol 2008 ) . we conclude with the suggestion that a humanistic approach to mental health can allow for the voice of the service user as a person among persons rather than an expert among experts . sullivan ( 18921949 ) has been credited for being the first to formulate an explicitly interpersonal theory of psychiatry , by psychiatrists as well as by service users in the so - called he formulated an interpersonal theory of the self , mental disorder , treatment and recovery . the theory was formulated in a series of lectures during the period 19461947 , which were published together in the book the interpersonal theory of psychiatry ( sullivan et al . sullivan lived his interpersonal theory by creating a therapeutic setting in which the psychiatrist could participate in the everyday activities of the clinic . he saw himself as a person among other persons in any patient s social world . sullivan strongly believed that the self - contained individual with a unique individuality was an illusion . he criticized society s focus on the individual and how the medical model of psychiatry sustained this view , referring to this as the very mother of illusions , the ever pregnant source of preconceptions that invalidate almost all our efforts to understand other people ( sullivan 2000/1938 , p. 114 ) . to sullivan there is a different self in each interpersonal relationship , and the person has as many understandings of him- or herself as he or she has interpersonal relationships . the self is fundamentally social in nature and personality is the relatively enduring pattern of recurrent interpersonal situations which characterize a human life ( sullivan 1953 , p. 110111 ) . the ideal of human maturity and independence leads us to believe that we are dependent on others only when we feel ill ( sullivan et al . however , in sullivan s view , health and illness are both facets of living as a person among persons ( sullivan 1953 , p. 313 ) . sullivan s view of mental disorder was consistent with his interpersonal view of the self . he was inspired by meyer s psychobiology , which endeavored to abandon a dualistic spilt between body and mind ( dowbiggin 2011 ) , seeing them as two aspects of the same process . like meyer , sullivan argued that mental illness comes from problems with living and adjustment to society . if a person is unable to adjust to or withstand the social organization in which he is embedded but experiences this organization as important , mental illness can develop ( sullivan 1953 , p. 208 ) . in this way sullivan envisioned a continuum between mental health and illness ; a person suffering from a mental illness is not fundamentally different from any other person . hence sullivan s famous one - genus postulate which states that everyone is much more simply human than otherwise sullivan emphasized the importance of taking part in the patient s everyday life as a participating observer and listening to his or her point of view , which is a crucial aspect of making a social recovery . for several years he lived and worked on a daily basis among schizophrenic patients . when he was given a patient s story by a staff member he read first - person accounts about living in a psychiatric ward , such as the self - biographical book by beers , a pioneer of the mental health movement of the first half of the 19th century , who underscored the relevance of the local community for any person s mental health . sullivan conceived of the psychiatric ward as a self - standing social world , implying psychiatric care and treatment during the other 23 h ( bloom 2002 ) . at the psychiatric hospital , he established a one - class society and did everything to minimize the impact of his own status . he hand - picked male attendants and patients in recovery to staff the ward and taught them his socio - psychiatric program in which the schizophrenic patient was to partake as a person among persons ( conci 1997 , p. 131 ; italics in the original ) . with this backdrop , we can state that sullivan had a coherent interpersonal view of the self , mental health and treatment . relational , a view in which body and mind feature as an embodied self within a situation where mental health and illness are ways of experiencing and acting in situations and where mental health care takes place in a situation between two persons who meet as persons , attempting social recovery through participant observation rather than medical treatment of an alien body . in the next two sections , we aim to show how this coherence in sullivan s approach was lost in the work of those whom he inspired : anti - psychiatry and the user movement in mental health care . sullivan inspired the psychiatrists t. szasz ( 19202012 ) and r. d. laing ( 19271989 ) to develop their own ideas about mental disorders and care in a revolt against mainstream psychiatry , the so - called anti - psychiatry of the 1960s and 1970s . crossley ( 1998 ) describes their work as a revolt from above ( p. 878 ) , implying a skeptical stance towards psychiatry as an instance of power and social control . anti - psychiatry was never a broad revolutionary movement with the intent to demolish the mental health field , but rather an attempt to promote a new paradigm to normalize madness by placing behavior in an interpersonal context rather than an allegedly narrow , normative context that sustains a controlling , medical , and objectifying gaze at patients . the anti - psychiatrists argued that symptoms of mental illness are easier to understand if context is taken into account . sullivan s approach impressed szasz ( 2010 , p. 220 ) with his portrayal of disease as problems in living . though skeptical of the anti - psychiatry label , szasz famously held that mental illness is a myth . mind is not brain , he argued ; behavior can not be a disease and is not detectable as a physical defect within the individual . phenomena studied by psychiatrists , such as racism , suicide , and murder , can not be revealed through studies of the brain . in his view , the psychiatric establishment exercised negative power over psychiatric patients by depriving them of the rights to self - determination and freedom and the right to be regarded as citizens who can take the consequences of their own actions . seen in this light , psychiatry is an ideology and a technology for the radical remaking of man ( szasz 1973 , p. 11 ) . szasz maintained that the medical model was not suitable to help people with their personal , social and ethical problems with living ( szasz 2010 , p. 262 ) . hardship for modern man derives from stresses and strains inherent in the social intercourse of complex human personalities ( szasz 1973 , p. 14 ) . difficulties in human relations must be analyzed and given meaning merely within specific social and ethical contexts . problems of living , as szasz ( 1973 , p. 22 ) contended , are due to man s awareness of himself in an expanding world and the increasing burden of understanding ; however , modernity and man s increasing knowledge do not take away the individual s responsibility for his or her actions by hiding it in the notion of mental illness . the therapist is not responsible for the patient s actions , only the patient him- or herself , and any preoccupation with the therapist is a way of not attending to one s own life . szasz , as the libertine he was , encouraged his patients to assume responsibility and develop a self that was independent of the therapist as a way out of their problems with living . he concurs with sullivan s well - known assertion that the psychotic person is primarily simply human ( laing 2010 , p. 34 ) . human beings may exist in isolation but are first and foremost intimately related to other persons and the world . problems of living arise when the person is placed in a situation of conflicting expectations from family members ( laing and esterson 1970 ) . for laing , mental illness was a normal response to a mad world and thereby socially intelligible ( op cit . actions are embedded in meaningful contexts , not controlled settings , and therefore , laing argued , existential problems can arise that are beyond the person s control . when lacking a solid foundation of existence , the person can experience an ontological insecurity and become preoccupied with preserving rather than gratifying himself ( laing 2010 , p. 42 ) . an ontologically insecure individual dreads engulfment by the other , implosion into complete emptiness and transformation into a dead thing ( laing 2010 , p. 43 ) . when failing to come up with a unique voice , the person may experience a lack of autonomy and become intertwined with the other . to laing , people with so - called mental illness are trying to find their way back ; psychotic episodes are understandable as an attempt to communicate worries and concerns , often in situations where this was not possible or not permitted . they should thus be seen as self - acting agents , responsible and capable of choice ( laing 2010 , p. 22 ) . in laing s view , the person needs help to cultivate such independence ; accordingly he set up therapeutic communities the most famous being kinsley hall with the aim to support the person through his or her own voyage . szasz and laing both see the psychiatric patient as a person more human than anything else having problems with living . however , where sullivan followed through with a relational view of the self and mental health care , szasz and laing emphasize an independent self as the driving force to break out of unhealthy social relationships ( bracken and thomas 2005 ) . more often than not , human relationships were the problem , and becoming a self - standing individual was the solution . szasz proclaimed individual liberty and independence ; persons can make their own choices , solve their own problems and are responsible for their actions . laing suggested ways that the person might cultivate a more independent self and urged for freedom and subjectivity . hence , the anti - psychiatrists , through the voices of szasz and laing , did not maintain the coherent relational view of self , mental disorder , and mental health care that had typified sullivan . while anti - psychiatry revolted from above , at the delivering end of mental health care , those at the receiving end of psychiatry also let their voices be heard , initiating a revolt from below ( crossley 1998 , p. 878 ) . judi chamberlin ( 19442010 ) , an active leader in the user movement , notes that users of mental health services considered anti - psychiatry largely an intellectual exercise of academics with little willingness to reach out to struggling ex - patients and their perspectives ( chamberlin 1990 ) . initially , user movements may have been nurtured by the anti - psychiatry movement s critique of psychiatry s standard medical model and the everyday life it generated in the psychiatric ward , providing a joint platform for individuals who had expressed similar criticism , such as beers ( 1908 ) and packard ( cf . dowbiggin 2011 ) . however , user groups were also eager to create an identity formulated in positive terms , separate from anti - psychiatry . the user movements merged with a larger class struggle , the counter - culture of the 1960s , which provided impetus and legitimacy ( reaume 2002 ) . the user movement mushroomed into many different user organizations with different agendas and ideologies , and even controversy among themselves , but with a shared belief in the rights to interpret their own experiences of mental disorder , to self - determination and to help on their own terms ( barnes and cotterell 2012 ) . the first - person singular voice of a few patients in the late nineteenth and early twentieth centuries had thereby gradually developed into a first - person plural identity as patients / users , service users went from formalized ascribed roles to a growing self - consciousness and self - confidence that was anchored in their unique understanding of their own illness . they did not subscribe to the anti - psychiatric claim that mental illness is not a disease but rather confirmed its status by becoming the new experts on mental illness . as experience experts side by side with medical experts , the majority of user organizations have now turned from an anti - establishment movement into a consumer coalition ( rissmiller and rissmiller 2006 ) . as consumers , mental health service users have acquired much of the equality they aspired to through grass - roots lobbying rather than by radical revolt and have made peace with the mental health establishment [ with ] no sympathy for szasz s theory that mental health is a myth the improvement in equality between service user and provider is based on a shared understanding of mental illness as a medical disease , i.e. , as an individual s that is to say that while the user movement strengthened the user voice , a sullivan - like interpersonal conception of mental health was weakened in favor of a intrapersonal conception of mental health ; it is only such a conception that allows one to acquire and express experiential expertise . why are the voices of experts by experience unable to give us a coherent perspective on care and health ? to explore this question we use kugelmann s ( 2003 ) distinction between standardized health and being healthy . according to kugelmann ( 2003 ) , standardized health appears when norms and risk factors are determined by biomedicine , and biomedicine intersects with an economic definition of wellbeing . parameters of health are turned into medical and economic parameters . lived experiences are turned into third - person objective categories of sickness and are thus not the health of any particular person ; given are statistical averages in the intersection of biomedicine and economy . science and health care are increasingly organized like businesses ( gadamer 1996 ) . transposed to mental health , standardized mental health can be compared with a procrustean bed that reduces and deforms its object . the first - person narratives of relational experience of distress , suffering and healing are made into parts of value - free parameters by health care providers and patients . these parameters influence service users behavior in such a way that they identify themselves with constructed labels even though all they may have in common is the wide range of parameters used to categorize them . they become exposed to the anonymity of the clinical apparatus ( gadamer 1996 , p. 20 ) . they become dependent on the classifications of diseases to attain support because the socially significant outcome that determines different forms of interventions is what constitutes sickness ( young 1982 ) . to put it differently , the sickness does not define the intervention as much as the intervention defines the sickness . mental health work materializes as a series of direct relational interventions to standardize these parameters . the immediate grasp of the life world disappears the moment the service user raises a question from an expert perspective or third person perspective about his or her disease . the life world is no longer experienced but considered as if in the third - person plural . in other words , they become part of the problem of the reification of mental health . in the emerging partnership , service users become part of the family of professionals who , by their expertise of subjectivity , can classify , measure , diagnose and prescribe remedies for psychologic illnesses ( rose 1989 ) . users as experience experts are trapped in a race to keep up with professional experts , as they do their own research , write their stories , campaign politically , etc . accordingly , the more users invest in becoming experts among experts , the farther they become removed from the relational upwards to become experts , they are captured in what hacking ( 1999 ) termed the looping effect , where conceptual labels of mental health evoke new behaviors and where original behaviors may give rise to new labels . it implies that help - seekers descriptions of their experience of mental health interact with the professional knowledge that categorizes them in the first place ( dehue 2009 ) . this profoundly illuminates the idea that when service user expertise stands on equal footing with research results and professional judgment , user experience is a moot contribution to evidence - based treatment and social support . to sustain an alternative to the intrapersonal conception of mental health , it has been argued that user groups need to return to their roots ( lakeman et al . they need an approach where the person is portrayed in terms of lived experience ; one can not portray a person purely from outside as a the person and the world belong together and are inseparable ; the interpersonal aspect of illness can not be separated from the person s lived experience ( van den berg 1972 ) . an alternative is required where standardized mental health is replaced by perspectives that capture the experience of being mentally healthy . according to kugelmann ( 2003 ) , being healthy is a relational experience ; one is not separated from lived experience but rather effortlessly engaged with others in situations in the world . he adopts gadamer s ( 1996 ) view that health is not something that can simply be made or produced ( p. vii ) it is a condition of being involved being together with one s fellow human beings in this perspective , being mentally healthy involves becoming engaged in something caring about something meaningful , for oneself , for family and friends . such a perspective on mental health rests on a relational view of the person , and should cohere with a relational view of care . the preoccupation with user voice and choice is grounded in a logic that enforces a split between the person and the world . to make this point we use mol s ( 2008 ) distinction between the logic of choice and the logic of care . in the logic of choice , professionals are preoccupied with what patients want and opt for . the role of professionals is then to provide users with care options to choose from . in the logic of care , by contrast , professionals and patients are not concerned with choice but with what one actually does . in this logic , one s ability to act depends on others in a continuing process that goes on and on until the day you die ( mol 2008 , p. 62 ) . in the pas de deux between service users and professionals , good treatment is realized when the service user s voice is heard and when mental health workers emphasize the service user s choices . more choice is regarded as the road to better mental health , and it is logical to think that if some choice is good , more is better . schwartz ( 2004 ) argues , however , that in a psychological sense this assumption may be wrong . although some choice is undoubtedly better than none , more is not always better than less in the caring industry . too many choices can raise your expectations to an unrealistic level , and it can make you blame yourself for any failures . you can blame yourself for not making a choice that is more or less per impossible . a good swimmer and a person who can not swim well do not have the same experience of autonomy when choosing whether to save a drowning man ( merleau - ponty 2002 , p. 508 ) . in the logic of choice the world materializes as a conglomerate of neutral objects or facts that are detached from the individual . the individual engaged in the world is replaced by an individual looking at the world as sheer facts from a third - person perspective , and in this way gives up living ( merleau - ponty 1964 , p. 159 ) in it , failing to see that we live in it from the inside however , in the lived world of care , we can not simply separate subject from object or values from facts as these are internally related . mol ( 2008 ) argues that care has little to do with separate individuality and user choice because care is attuned to people with respect to their basic relationship to one another . mol puts forward an alternative logic of care where the self emerges in interpersonal situations in everyday living . in the logic of care , care is always situational , and it does not make an unbridgeable distinction between fact and value . facts are not just neutral facts ; in the logic of care , one attends to facts and values jointly ( mol 2008 ) . in the context of care , knowledge is not information to provide a better map of reality to gradually increase certainty before risk but to gather knowledge is a way of crafting more bearable ways of living with , or in reality ( p. 46 ; italics in the original ) . care can not be separated from experience , and therefore care aims to improve the situation that meddles with every detail of our daily lives ( mol 2008 , p. 37 ) . care appears as tinkering , dealing with the messiness and quirkiness of everyday life , in our relationships with the world , others , and self , over time . in the relational perspective , care is not receiving care , or choosing care , but engaging in care with professionals as much as oneself in an environment which may facilitate or hamper it . in mol s analysis of the logic of choice , the collective user voice is formed by individuals who are facing similar problems in living , share a diagnosis , or get the same type of treatment . the users influence the market as consumers , and as citizens the sum of their votes may bring about democratic decisions . however , in the logic of care , the collective aspect forms the starting point of reasoning . the person is embedded in the family tradition , or is immersed in the work climate , with all of the habits and customs involved . in this view , the community is not one of added equals , but of entangled roles in a relational network , where not everybody is characterized by the same health- or illness - related variable . yet , within the network , mental health can become the focal point at which the actions of persons in the network are directed . the person and his or her mental health are not characterized by open choices , but rather by living as making a plethora of small adjustments in a network of relationships to improve one s existence . the current demand by mental health service users for a stronger voice and more choice is a positive development because users then take center stage in the practice of care . service users are driven to seek care and treatment within the system of knowledge they once denied but that defines and negotiates their life world by standardizing values . those service users who strive for equality between users and providers of care have the opportunity to contribute to care and treatment on a par with providers , insofar as this is practically possible . however , this implies that service users who become experts among experts maintain and uphold the pivotal contribution of care providers . while users have acquired an influential presence as experts among experts , they also have gradually distanced themselves from being persons among persons . upwards to become experts , research should reach downwards and focus more on mental health as a relational persons among persons phenomenon . mental health emerges as a lived relational phenomenon between i - oriented persons among persons , whereas it emerges as a reified object when discussed among they - oriented experts . these two different views on mental health are endorsed by different treatment and care practices but also by different systems of knowledge . those who take a relational perspective on mental health argue that mental health emerges continually in time , in the world , in the body and in relationships with significant others and can not be objectified to standardized parameters within an autonomous self ( crossley 2011 ; frie and coburn 2010 ; martin et al . such a relational understanding of mental health requires a human scientific approach that is in tune with the essence of human beings ( giorgi 1985 , p. 20 ) , a science that is close to practice and anchored in life worlds ( todres et al . the choice of a human scientific approach to mental health implies criticism of powerful governmental demands for evidence - based mental health care in which biomedical access to the patient s experiences of illness makes the management of human beings become all the more powerful and coercive ( taussig 1980 ) . however , it is also a critique of those service users who strive to become experts among experts and start to see themselves in a medical and perhaps even economic light . mcknight ( 1995 ) argues in his book the careless society that revolutions begin when people who are defined as problems achieve the power to redefine the problem ( p. 16 ) . the spark of a revolution from above in the anti - psychiatric critique of the legitimacy of experts power to define others disappeared when the revolt from below ebbed out into a consumer movement with service users who were eager to collaborate . what we need is a new service user movement grounded outside reductionist standardized health in a logic that encourages a new understanding of what experience of life is as a person among persons .

since the revolutionary mood of the 1960s , patient - centered mental health care and a research emphasis on service users as experts by experience have emerged hand in hand with a view of service users as consumers . what happens to knowledge derived from firsthand experience when mental health users become experts and actively choose care ? what kind of perspective do service users pursue on psychological distress ? these are important questions in a field where psychiatric expertise on mental illness is socially structured and constrained as an intra - personal disturbance of the mind . we argue that experience experts have lost a coherent perspective on care and health . we illustrate this by rationally reconstructing how the interpersonal view of mental health first gained and then lost coherence between the conception of mental health , the practice of mental health care , and the user experience . harry stack sullivan s interpersonal theory was a paradigm case for such coherence . the inclusion of mental health consumers as experts by experience in the mental health field took place at the cost of sullivan s coherent interpersonal theory . service users who interact side by side with medical experts as experience experts are constrained by the evidence - based imperative and consumerism . service users are caught up in a race among experts to gain knowledge about mental problems from a third - person perspective instead of from first - person experience . to make a contribution service users have more to gain from a research approach that appreciates that they are persons among persons rather than experts among experts .

Introduction Sullivans coherent interpersonal approach Anti-psychiatry: the revolt from above The mental health service user movement: the revolt from below Standardized versus relational health Logic of choice versus logic of care Concluding remarks

during recent decades , users of mental health services have steadily increased the strength of their voice in mental health care policies , education and practice ( who 2010 ) . patient - centered care , with its emphasis on the patient s voice and choice , has become a common good in mental health care ( dahlberg et al . 2009 ) , and service users acting as experts by experience are pivotal in their own care and treatment plans ( pilgrim 2008 ) . one can wonder what happened to knowledge derived from firsthand experience when mental health service users themselves became experts and actively began to choose care . the ensuing expertise is socially structured and constrained in terms of mental illness being a psychopathological intra - personal disturbance of the mind , in line with dominant psychiatric practice ( speed 2005 ; bracken and thomas 2005 ; coles et al . however , alternative views on mental health are also endorsed ; mental health can be seen as a relational phenomenon which is never static , in line with so - called relation - based mental health work . in such views , mental health can not be reduced to brain activity or individual life histories , but emerges in and through processes of interaction between the person and other persons ( bracken and thomas 2005 ; crossley 2011 ; frie and coburn 2010 ; kirschner and martin 2010 ) . in our study , we take a closer look at the expertise that users as consumers bring to the mental health care field and how their views on mental health have developed vis - - vis the mainstream psychiatric view . in doing so , we will try to unravel the apparent paradox that experts by experience become part of the dominant medical paradigm they wished to move away from . in other words , in this study , we ask to what extent mental health users are experts among experts at the expense of being persons among persons. we approach these questions by generating a rational reconstruction of how the interpersonal ( person among persons ) view of mental health first gained and then lost coherence among its conceptions of mental health , the practice of mental health care , and the experience of the user . by rational reconstruction , we mean that it is only on the basis of what we know of user knowledge in mental health care today that we can reconstruct how this has come to be so . moreover , we study how past participants in mental health argued and thought in order to revive their voices in the present - day context , enabling us to create a dialogue between these and currently active players in the field . we acknowledge the risk of writing whiggish history but find support in rorty , who writes that but if they are conducted in full knowledge of their anachronism , they are unobjectable sullivan s mid - twentieth century interpersonal psychiatry , because it is a paradigmatic example of a coherent approach based on an interpersonal view of mental health . thirdly , we show how a partnership between service users and mental health professionals paved the way for a shared acceptance of the breaking up of mental health into independent parameters . we discuss the consequences of a parametric view of mental health in terms of standardized mental health and the subsequent logic of choice in mental health services ( kugelmann 2003 ; mol 2008 ) . we conclude with the suggestion that a humanistic approach to mental health can allow for the voice of the service user as a person among persons rather than an expert among experts . sullivan ( 18921949 ) has been credited for being the first to formulate an explicitly interpersonal theory of psychiatry , by psychiatrists as well as by service users in the so - called he formulated an interpersonal theory of the self , mental disorder , treatment and recovery . the theory was formulated in a series of lectures during the period 19461947 , which were published together in the book the interpersonal theory of psychiatry ( sullivan et al . sullivan lived his interpersonal theory by creating a therapeutic setting in which the psychiatrist could participate in the everyday activities of the clinic . he criticized society s focus on the individual and how the medical model of psychiatry sustained this view , referring to this as the very mother of illusions , the ever pregnant source of preconceptions that invalidate almost all our efforts to understand other people ( sullivan 2000/1938 , p. 114 ) . to sullivan there is a different self in each interpersonal relationship , and the person has as many understandings of him- or herself as he or she has interpersonal relationships . however , in sullivan s view , health and illness are both facets of living as a person among persons ( sullivan 1953 , p. 313 ) . sullivan s view of mental disorder was consistent with his interpersonal view of the self . he was inspired by meyer s psychobiology , which endeavored to abandon a dualistic spilt between body and mind ( dowbiggin 2011 ) , seeing them as two aspects of the same process . in this way sullivan envisioned a continuum between mental health and illness ; a person suffering from a mental illness is not fundamentally different from any other person . hence sullivan s famous one - genus postulate which states that everyone is much more simply human than otherwise sullivan emphasized the importance of taking part in the patient s everyday life as a participating observer and listening to his or her point of view , which is a crucial aspect of making a social recovery . when he was given a patient s story by a staff member he read first - person accounts about living in a psychiatric ward , such as the self - biographical book by beers , a pioneer of the mental health movement of the first half of the 19th century , who underscored the relevance of the local community for any person s mental health . sullivan conceived of the psychiatric ward as a self - standing social world , implying psychiatric care and treatment during the other 23 h ( bloom 2002 ) . he hand - picked male attendants and patients in recovery to staff the ward and taught them his socio - psychiatric program in which the schizophrenic patient was to partake as a person among persons ( conci 1997 , p. 131 ; italics in the original ) . with this backdrop , we can state that sullivan had a coherent interpersonal view of the self , mental health and treatment . relational , a view in which body and mind feature as an embodied self within a situation where mental health and illness are ways of experiencing and acting in situations and where mental health care takes place in a situation between two persons who meet as persons , attempting social recovery through participant observation rather than medical treatment of an alien body . in the next two sections , we aim to show how this coherence in sullivan s approach was lost in the work of those whom he inspired : anti - psychiatry and the user movement in mental health care . sullivan inspired the psychiatrists t. szasz ( 19202012 ) and r. d. laing ( 19271989 ) to develop their own ideas about mental disorders and care in a revolt against mainstream psychiatry , the so - called anti - psychiatry of the 1960s and 1970s . anti - psychiatry was never a broad revolutionary movement with the intent to demolish the mental health field , but rather an attempt to promote a new paradigm to normalize madness by placing behavior in an interpersonal context rather than an allegedly narrow , normative context that sustains a controlling , medical , and objectifying gaze at patients . the anti - psychiatrists argued that symptoms of mental illness are easier to understand if context is taken into account . though skeptical of the anti - psychiatry label , szasz famously held that mental illness is a myth . phenomena studied by psychiatrists , such as racism , suicide , and murder , can not be revealed through studies of the brain . in his view , the psychiatric establishment exercised negative power over psychiatric patients by depriving them of the rights to self - determination and freedom and the right to be regarded as citizens who can take the consequences of their own actions . problems of living , as szasz ( 1973 , p. 22 ) contended , are due to man s awareness of himself in an expanding world and the increasing burden of understanding ; however , modernity and man s increasing knowledge do not take away the individual s responsibility for his or her actions by hiding it in the notion of mental illness . for laing , mental illness was a normal response to a mad world and thereby socially intelligible ( op cit . when lacking a solid foundation of existence , the person can experience an ontological insecurity and become preoccupied with preserving rather than gratifying himself ( laing 2010 , p. 42 ) . when failing to come up with a unique voice , the person may experience a lack of autonomy and become intertwined with the other . to laing , people with so - called mental illness are trying to find their way back ; psychotic episodes are understandable as an attempt to communicate worries and concerns , often in situations where this was not possible or not permitted . however , where sullivan followed through with a relational view of the self and mental health care , szasz and laing emphasize an independent self as the driving force to break out of unhealthy social relationships ( bracken and thomas 2005 ) . hence , the anti - psychiatrists , through the voices of szasz and laing , did not maintain the coherent relational view of self , mental disorder , and mental health care that had typified sullivan . while anti - psychiatry revolted from above , at the delivering end of mental health care , those at the receiving end of psychiatry also let their voices be heard , initiating a revolt from below ( crossley 1998 , p. 878 ) . judi chamberlin ( 19442010 ) , an active leader in the user movement , notes that users of mental health services considered anti - psychiatry largely an intellectual exercise of academics with little willingness to reach out to struggling ex - patients and their perspectives ( chamberlin 1990 ) . initially , user movements may have been nurtured by the anti - psychiatry movement s critique of psychiatry s standard medical model and the everyday life it generated in the psychiatric ward , providing a joint platform for individuals who had expressed similar criticism , such as beers ( 1908 ) and packard ( cf . the user movements merged with a larger class struggle , the counter - culture of the 1960s , which provided impetus and legitimacy ( reaume 2002 ) . the user movement mushroomed into many different user organizations with different agendas and ideologies , and even controversy among themselves , but with a shared belief in the rights to interpret their own experiences of mental disorder , to self - determination and to help on their own terms ( barnes and cotterell 2012 ) . the first - person singular voice of a few patients in the late nineteenth and early twentieth centuries had thereby gradually developed into a first - person plural identity as patients / users , service users went from formalized ascribed roles to a growing self - consciousness and self - confidence that was anchored in their unique understanding of their own illness . they did not subscribe to the anti - psychiatric claim that mental illness is not a disease but rather confirmed its status by becoming the new experts on mental illness . as experience experts side by side with medical experts , the majority of user organizations have now turned from an anti - establishment movement into a consumer coalition ( rissmiller and rissmiller 2006 ) . as consumers , mental health service users have acquired much of the equality they aspired to through grass - roots lobbying rather than by radical revolt and have made peace with the mental health establishment [ with ] no sympathy for szasz s theory that mental health is a myth the improvement in equality between service user and provider is based on a shared understanding of mental illness as a medical disease , i.e. , as an individual s that is to say that while the user movement strengthened the user voice , a sullivan - like interpersonal conception of mental health was weakened in favor of a intrapersonal conception of mental health ; it is only such a conception that allows one to acquire and express experiential expertise . why are the voices of experts by experience unable to give us a coherent perspective on care and health ? lived experiences are turned into third - person objective categories of sickness and are thus not the health of any particular person ; given are statistical averages in the intersection of biomedicine and economy . science and health care are increasingly organized like businesses ( gadamer 1996 ) . transposed to mental health , standardized mental health can be compared with a procrustean bed that reduces and deforms its object . the first - person narratives of relational experience of distress , suffering and healing are made into parts of value - free parameters by health care providers and patients . these parameters influence service users behavior in such a way that they identify themselves with constructed labels even though all they may have in common is the wide range of parameters used to categorize them . the immediate grasp of the life world disappears the moment the service user raises a question from an expert perspective or third person perspective about his or her disease . the life world is no longer experienced but considered as if in the third - person plural . in other words , they become part of the problem of the reification of mental health . in the emerging partnership , service users become part of the family of professionals who , by their expertise of subjectivity , can classify , measure , diagnose and prescribe remedies for psychologic illnesses ( rose 1989 ) . users as experience experts are trapped in a race to keep up with professional experts , as they do their own research , write their stories , campaign politically , etc . accordingly , the more users invest in becoming experts among experts , the farther they become removed from the relational upwards to become experts , they are captured in what hacking ( 1999 ) termed the looping effect , where conceptual labels of mental health evoke new behaviors and where original behaviors may give rise to new labels . it implies that help - seekers descriptions of their experience of mental health interact with the professional knowledge that categorizes them in the first place ( dehue 2009 ) . this profoundly illuminates the idea that when service user expertise stands on equal footing with research results and professional judgment , user experience is a moot contribution to evidence - based treatment and social support . to sustain an alternative to the intrapersonal conception of mental health , it has been argued that user groups need to return to their roots ( lakeman et al . they need an approach where the person is portrayed in terms of lived experience ; one can not portray a person purely from outside as a the person and the world belong together and are inseparable ; the interpersonal aspect of illness can not be separated from the person s lived experience ( van den berg 1972 ) . such a perspective on mental health rests on a relational view of the person , and should cohere with a relational view of care . the preoccupation with user voice and choice is grounded in a logic that enforces a split between the person and the world . to make this point we use mol s ( 2008 ) distinction between the logic of choice and the logic of care . in the logic of care , by contrast , professionals and patients are not concerned with choice but with what one actually does . in the pas de deux between service users and professionals , good treatment is realized when the service user s voice is heard and when mental health workers emphasize the service user s choices . more choice is regarded as the road to better mental health , and it is logical to think that if some choice is good , more is better . the individual engaged in the world is replaced by an individual looking at the world as sheer facts from a third - person perspective , and in this way gives up living ( merleau - ponty 1964 , p. 159 ) in it , failing to see that we live in it from the inside however , in the lived world of care , we can not simply separate subject from object or values from facts as these are internally related . in the logic of care , care is always situational , and it does not make an unbridgeable distinction between fact and value . facts are not just neutral facts ; in the logic of care , one attends to facts and values jointly ( mol 2008 ) . in the context of care , knowledge is not information to provide a better map of reality to gradually increase certainty before risk but to gather knowledge is a way of crafting more bearable ways of living with , or in reality ( p. 46 ; italics in the original ) . in the relational perspective , care is not receiving care , or choosing care , but engaging in care with professionals as much as oneself in an environment which may facilitate or hamper it . in mol s analysis of the logic of choice , the collective user voice is formed by individuals who are facing similar problems in living , share a diagnosis , or get the same type of treatment . the users influence the market as consumers , and as citizens the sum of their votes may bring about democratic decisions . however , in the logic of care , the collective aspect forms the starting point of reasoning . the person is embedded in the family tradition , or is immersed in the work climate , with all of the habits and customs involved . in this view , the community is not one of added equals , but of entangled roles in a relational network , where not everybody is characterized by the same health- or illness - related variable . yet , within the network , mental health can become the focal point at which the actions of persons in the network are directed . the person and his or her mental health are not characterized by open choices , but rather by living as making a plethora of small adjustments in a network of relationships to improve one s existence . the current demand by mental health service users for a stronger voice and more choice is a positive development because users then take center stage in the practice of care . service users are driven to seek care and treatment within the system of knowledge they once denied but that defines and negotiates their life world by standardizing values . those service users who strive for equality between users and providers of care have the opportunity to contribute to care and treatment on a par with providers , insofar as this is practically possible . however , this implies that service users who become experts among experts maintain and uphold the pivotal contribution of care providers . while users have acquired an influential presence as experts among experts , they also have gradually distanced themselves from being persons among persons . upwards to become experts , research should reach downwards and focus more on mental health as a relational persons among persons phenomenon . mental health emerges as a lived relational phenomenon between i - oriented persons among persons , whereas it emerges as a reified object when discussed among they - oriented experts . these two different views on mental health are endorsed by different treatment and care practices but also by different systems of knowledge . those who take a relational perspective on mental health argue that mental health emerges continually in time , in the world , in the body and in relationships with significant others and can not be objectified to standardized parameters within an autonomous self ( crossley 2011 ; frie and coburn 2010 ; martin et al . such a relational understanding of mental health requires a human scientific approach that is in tune with the essence of human beings ( giorgi 1985 , p. 20 ) , a science that is close to practice and anchored in life worlds ( todres et al . the choice of a human scientific approach to mental health implies criticism of powerful governmental demands for evidence - based mental health care in which biomedical access to the patient s experiences of illness makes the management of human beings become all the more powerful and coercive ( taussig 1980 ) . however , it is also a critique of those service users who strive to become experts among experts and start to see themselves in a medical and perhaps even economic light . the spark of a revolution from above in the anti - psychiatric critique of the legitimacy of experts power to define others disappeared when the revolt from below ebbed out into a consumer movement with service users who were eager to collaborate . what we need is a new service user movement grounded outside reductionist standardized health in a logic that encourages a new understanding of what experience of life is as a person among persons .

degenerative flat back ( dfb ) is frequent in asian countries , which suggests that typical life style or working posture is associated with occurrence of dfb . they stand up or sit on the floor with waist flexion and frequently work at home or farm with stooped posture , which contributes to decreased lumbar lordosis ( ll ) and weakness of paraspinal extensor muscles ( pses ) . decreased ll causes anterior displacement of the center of gravity , which leads to spinopelvic angular changes and causes various disabilities in daily life including gait difficulty.12 although the exact pathophysiology of dfb has not yet been confirmed , extensive degeneration , and weakness of lumbar extensor muscles are thought to be the most important cause.34 the quantitative evaluations of lumbar extensor muscle have been developed using magnetic resonance image ( mri).456 the typical signs of muscle degeneration detected on mri are muscle cross - sectional area ( csa ) and fatty replacement of muscle , expressed as increased signal intensity ( si).67 previous study using mri histogram reported that dfb patients showed higher mean si of back extensor muscles suggesting fat infiltration compared to normal control.5 although radiographic examination can show characteristic sagittal deviation of spinopelvic alignment,8 it has the limitation only to reveal the static posture , and not to identify dynamic status such as ambulation . however , daily activities or functional aspects of patients are more related to dynamic status of spinopelvic segment than static posture . treatment outcomes or patient satisfaction can be influenced more by dynamical parameters than by static parameters . thus , it is assumed that assessment of dynamic parameters of dfb by three - dimensional motion analysis can provide clinically useful data about patients functional status and treatment outcomes.9101112 it can provide specific characteristics of spinopelvic and lower limb joints motion in patients with dfb and also reveal which improvement occurred following surgery in quantitative and objective way . the purposes of this study were ( 1 ) to evaluate correlation between preoperative lumbar extensor muscle condition and angular severity of dfb and ( 2 ) to evaluate correlation between preoperative lumbar extensor muscle condition and degree of improvement of dfb obtained by corrective fusion surgery , in terms of static parameters by simple radiography and dynamic parameters by three - dimensional motion analysis . 45 patients who underwent corrective fusion surgery for dfb between 2010 and 2012 , ( 4 men and 41 women , mean age 69.9 6.01 years ) were included in this retrospective study . lumbar mri , whole spine x - ray , and three dimensional motion analysis were done before and 6 months after surgery in all the patients . the patients showed characteristic clinical features , including stooped posture while walking , inability to lift heavy objects , difficulty in climbing , and the need to use a support ( such as an elbow ) when standing in the kitchen . the patients who had history of previous lumbar surgery and prominent lower extremity pain or weakness affecting gait function were excluded from the study . fusion levels were as follows ; t10-s1 in two patients , t11-s1 in four , l1-s1 in seven , l2l5 in one , l2-s1 in 22 , l2-s2 in two , and l3-s1 in seven . as for spinal sagittal parameters , thoracic kyphosis ( tk ) , thoracolumbar junction ( tlj ) , and ll were measured in whole spine lateral view . the tk was measured from the t5 superior end plate to t12 inferior end plate . the tlj was measured from the t10 superior end plate to l2 inferior end plate . the ll was measured from the t12 inferior end plate to s1 superior end plate by the cobb method [ figure 1 ] . as for the pelvic parameters , pelvic incidence ( pi ) , the ss was the angle between the s1 superior end plate and a horizontal line . the pt was defined as the angle between a vertical line originating at the center of the bicoxofemoral axis and a line drawn between the same point and the middle of the superior end plate of s1 . the pi was defined as the angle between the line perpendicular to the sacral plate and the line connecting the midpoint of the sacral plate to the bicoxofemoral axis [ figure 2].813 x - ray thoracolumbosacral spine lateral view showing spinal parameters such as thoracic kyphosis , thoracolumbar junction , and lumbar lordosis . ( a ) the thoracic kyphosis was measured from the t5 superior end plate to t12 inferior end plate . ( b ) the thoracolumbar junction was measured from the t10 superior end plate to l2 inferior end plate . ( c ) the lumbar lordosis was measured from the t12 inferior end plate to s1 superior end plate by the cobb method x - ray lumbosacral spine lateral view showing pelvic parameters including pelvic tilt , pelvic incidence , and sacral slope . ( a ) the pelvic tilt is defined as the angle between a vertical line originating at the center of the bicoxofemoral axis and a line drawn between the same point and the middle of the superior end plate of s1 . ( b ) the pelvic incidence is defined as the angle between the line perpendicular to the sacral plate and the line connecting the midpoint of the sacral plate to the bicoxofemoral axis . ( c ) the sacral slope is the angle between the s1 superior end plate and a horizontal line the conditions of lumbar pses were analyzed at the l1l2 , l2l3 , l3l4 , and l4l5 levels . the l5-s1 level was not included because the axial cutting gantry was obstructed by iliac crest and the muscular anatomy was quite different from the upper levels . the images were displayed and analyzed using piview ( infinitt , seoul , korea ) digital image viewing software . the regions of interest ( roi ) were outlined with a graphic cursor around the lumbar pse including multifidi and erector spinae and on the intervertebral disc at each level.14 the degree of muscle atrophy was determined by measuring the csa of the pse compartment . in order to decrease the bias caused by differences in individual body size , the area of the pse compartment was divided by intervertebral disc area of the same level ( the ratio between the csa of the pse and the csa of the intervertebral disc [ pse / disc csa ] ) to represent the relative muscle compartment volume in each individual . the si of the muscle within the roi was additionally measured using a histogram , and the mean value of the si in the patient 's group was obtained [ figure 3 ] . mean value of the signal intensity was measured within paraspinal extensor muscle , using histogram of magnetic resonance image gait analysis was conducted using three - dimensional motion analyzer ( motion analysis ; motion analysis company , santa rosa , ca , usa ) . markers of diameter 2.5 cm were attached bilaterally to the bony landmarks of the pelvis and lower extremities , including the l5-s1 intervertebral space , anterior superior iliac spine , anterior side of the midthigh , midpoint of the lateral knee , anterior side of the mid - tibia , lateral malleolus of the fibula , dorsal side between the second and third metatarsal heads , and the calcaneal area on the same line as the metatarsal marker to assessing lower limb kinematics during ambulation . in addition , markers were attached to bony landmarks of the c7 , t6 , t12 , l2 , and l5 spinous processes for assessing kinematics of thoracic and lumbar vertebrae segments during ambulation [ figure 4 ] . the participants went through a 10 m walkway for dynamic test at a range of self - selected . the data were sent to simm ( software for interactive musculoskeletal modeling ) program(motionanalysis company , santa rosa , ca , usa ) program and joint motions were calculated and analyzed . maximal and minimal angle of posterior pt , hip , knee and ankle joints flexion angles in sagittal plane was measured during ambulation . in addition , maximal and minimal sagittal angle of thoracic vertebrae and lumbar vertebrae segment was obtained . positive values of spinal columns and lower limbs meant kyphotic angle and flexion ( ankle joint dorsiflexion ) . clinical photographs ( a and b ) showing location of markers during three - dimensional motion analysis all of these measurements were performed by single physician ( jhl ) who was expert in spine radiology and three - dimensional motion analysis . the correlation between preoperative pse measurements and static / dynamic parameters and the correlation between preoperative pse measurements and degree of improvement of static / dynamic parameters obtained by corrective fusion surgery were determined using the pearson correlation coefficient . as for spinal sagittal parameters , thoracic kyphosis ( tk ) , thoracolumbar junction ( tlj ) , and ll were measured in whole spine lateral view . the tk was measured from the t5 superior end plate to t12 inferior end plate . the tlj was measured from the t10 superior end plate to l2 inferior end plate . the ll was measured from the t12 inferior end plate to s1 superior end plate by the cobb method [ figure 1 ] . as for the pelvic parameters , pelvic incidence ( pi ) , the ss was the angle between the s1 superior end plate and a horizontal line . the pt was defined as the angle between a vertical line originating at the center of the bicoxofemoral axis and a line drawn between the same point and the middle of the superior end plate of s1 . the pi was defined as the angle between the line perpendicular to the sacral plate and the line connecting the midpoint of the sacral plate to the bicoxofemoral axis [ figure 2].813 x - ray thoracolumbosacral spine lateral view showing spinal parameters such as thoracic kyphosis , thoracolumbar junction , and lumbar lordosis . ( a ) the thoracic kyphosis was measured from the t5 superior end plate to t12 inferior end plate . ( b ) the thoracolumbar junction was measured from the t10 superior end plate to l2 inferior end plate . ( c ) the lumbar lordosis was measured from the t12 inferior end plate to s1 superior end plate by the cobb method x - ray lumbosacral spine lateral view showing pelvic parameters including pelvic tilt , pelvic incidence , and sacral slope . ( a ) the pelvic tilt is defined as the angle between a vertical line originating at the center of the bicoxofemoral axis and a line drawn between the same point and the middle of the superior end plate of s1 . ( b ) the pelvic incidence is defined as the angle between the line perpendicular to the sacral plate and the line connecting the midpoint of the sacral plate to the bicoxofemoral axis . ( c ) the sacral slope is the angle between the s1 superior end plate and a horizontal line the conditions of lumbar pses were analyzed at the l1l2 , l2l3 , l3l4 , and l4l5 levels . the l5-s1 level was not included because the axial cutting gantry was obstructed by iliac crest and the muscular anatomy was quite different from the upper levels . the images were displayed and analyzed using piview ( infinitt , seoul , korea ) digital image viewing software . the regions of interest ( roi ) were outlined with a graphic cursor around the lumbar pse including multifidi and erector spinae and on the intervertebral disc at each level.14 the degree of muscle atrophy was determined by measuring the csa of the pse compartment . in order to decrease the bias caused by differences in individual body size , the area of the pse compartment was divided by intervertebral disc area of the same level ( the ratio between the csa of the pse and the csa of the intervertebral disc [ pse / disc csa ] ) to represent the relative muscle compartment volume in each individual . the si of the muscle within the roi was additionally measured using a histogram , and the mean value of the si in the patient 's group was obtained [ figure 3 ] . mean value of the signal intensity was measured within paraspinal extensor muscle , using histogram of magnetic resonance image gait analysis was conducted using three - dimensional motion analyzer ( motion analysis ; motion analysis company , santa rosa , ca , usa ) . markers of diameter 2.5 cm were attached bilaterally to the bony landmarks of the pelvis and lower extremities , including the l5-s1 intervertebral space , anterior superior iliac spine , anterior side of the midthigh , midpoint of the lateral knee , anterior side of the mid - tibia , lateral malleolus of the fibula , dorsal side between the second and third metatarsal heads , and the calcaneal area on the same line as the metatarsal marker to assessing lower limb kinematics during ambulation . in addition , markers were attached to bony landmarks of the c7 , t6 , t12 , l2 , and l5 spinous processes for assessing kinematics of thoracic and lumbar vertebrae segments during ambulation [ figure 4 ] . the participants went through a 10 m walkway for dynamic test at a range of self - selected . the data were sent to simm ( software for interactive musculoskeletal modeling ) program(motionanalysis company , santa rosa , ca , usa ) program and joint motions were calculated and analyzed . maximal and minimal angle of posterior pt , hip , knee and ankle joints flexion angles in sagittal plane was measured during ambulation . in addition , maximal and minimal sagittal angle of thoracic vertebrae and lumbar vertebrae segment was obtained . positive values of spinal columns and lower limbs meant kyphotic angle and flexion ( ankle joint dorsiflexion ) . clinical photographs ( a and b ) showing location of markers during three - dimensional motion analysis all of these measurements were performed by single physician ( jhl ) who was expert in spine radiology and three - dimensional motion analysis . chicago , il , usa ) . a p < 0.05 was considered statistically significant . the correlation between preoperative pse measurements and static / dynamic parameters and the correlation between preoperative pse measurements and degree of improvement of static / dynamic parameters obtained by corrective fusion surgery were determined using the pearson correlation coefficient . tlj was correlated significantly with l1l2 pse / disc csa and l1l2 si . tk was correlated with l1l2 si , l3l4 pse / disc csa , and l4l5 si [ table 1 ] . correlation between paraspinal extensor muscle measurement and static parameters maximal thoracic angle was correlated with l1l2 pse / disc csa , l1l2 si , and l2l3 pse / disc csa . maximal lumbar angle was correlated with l3l4 pse / disc csa and l4l5 pse / disc csa . minimal thoracic angle was correlated with l1l2 pse / disc csa . minimal lumbar angle was correlated with l2l3 si , l3l4 pse / disc csa , and l3l4 si , and l4l5 pse / disc csa [ table 2 ] . correlation between paraspinal extensor muscle measurement and dynamic parameters maximal right hip and knee flexion angle were correlated with l4l5 pse / disc csa . minimal posterior pt angle was correlated with l2l3 si . minimal right hip flexion angle was correlated with l3l4 pse / disc csa and l4l5 pse / disc csa . minimal left ankle dorsiflexion angle was correlated with l1l2 , l2l3 , l3l4 , and l4l5 si . the change ( ) of tk was significantly correlated with l1l2 and l2l3 pse / disc csa [ table 3 ] . correlation between preoperative paraspinal extensor muscle measurement and improvement of static parameters obtained by surgery the maximal thoracic angle showed significant correlation with l1l2 and l2l3 pse / disc csa . the maximal lumbar angle showed significant correlation with l1l2 , l2l3 , l3l4 , and l4l5 pse / disc csa . the minimal thoracic angle showed significant correlation with l1l2 and l2l3 pse / disc csa . the minimal lumbar angle showed significant correlation with l1l2 , l2l3 , l3l4 , and l4l5 pse / disc csa [ table 4 ] . correlation between preoperative paraspinal extensor measurement and improvement of dynamic parameters obtained surgery the maximal pelvic posterior tilt was also correlated with l4l5 pse / disc csa . the maximal right and left hip flexion angle was correlated with l2l3 , l3l4 , and l4l5 pse / disc csa . the minimal right ankle flexion angle was correlated with l1l2 pse / disc csa . tlj was correlated significantly with l1l2 pse / disc csa and l1l2 si . tk was correlated with l1l2 si , l3l4 pse / disc csa , and l4l5 si [ table 1 ] . maximal thoracic angle was correlated with l1l2 pse / disc csa , l1l2 si , and l2l3 pse / disc csa . maximal lumbar angle was correlated with l3l4 pse / disc csa and l4l5 pse / disc csa . minimal thoracic angle was correlated with l1l2 pse / disc csa . minimal lumbar angle was correlated with l2l3 si , l3l4 pse / disc csa , and l3l4 si , and l4l5 pse / disc csa [ table 2 ] . correlation between paraspinal extensor muscle measurement and dynamic parameters maximal right hip and knee flexion angle were correlated with l4l5 pse / disc csa . minimal posterior pt angle was correlated with l2l3 si . minimal right hip flexion angle was correlated with l3l4 pse / disc csa and l4l5 pse / disc csa . minimal left ankle dorsiflexion angle was correlated with l1l2 , l2l3 , l3l4 , and l4l5 si . the change ( ) of tk was significantly correlated with l1l2 and l2l3 pse / disc csa [ table 3 ] . the maximal thoracic angle showed significant correlation with l1l2 and l2l3 pse / disc csa . the maximal lumbar angle showed significant correlation with l1l2 , l2l3 , l3l4 , and l4l5 pse / disc csa . the minimal thoracic angle showed significant correlation with l1l2 and l2l3 pse / disc csa . the minimal lumbar angle showed significant correlation with l1l2 , l2l3 , l3l4 , and l4l5 pse / disc csa [ table 4 ] . correlation between preoperative paraspinal extensor measurement and improvement of dynamic parameters obtained surgery the maximal pelvic posterior tilt was also correlated with l4l5 pse / disc csa . the maximal right and left hip flexion angle was correlated with l2l3 , l3l4 , and l4l5 pse / disc csa . the minimal posterior pt was correlated with l2l3 si . the minimal right ankle flexion angle was correlated with l1l2 pse / disc csa . the degeneration and weakness of pse were thought to be important causes of dfb because the pse played a role in maintaining spinal curvatures and their degeneration or atrophy resulted in a progressive kyphosis and sagittal imbalance.135 degeneration of pse was significantly associated with facet arthropathy , which additionally contributed to progression of lumbar kyphosis.15 facet arthropathy could elicit a reflex mechanism that inhibited muscle activity and further caused muscle atrophy.16 as well , kyphotic deformity , in turn , aggravated degeneration and weakness of pse in more severe degree . over lengthening of pse caused by increased lumbar kyphotic angle lost the lever arm effects enough for appropriate contraction , which further aggravated muscle degeneration and atrophy.17 low back pain caused by dfb could contribute to limited back movement and consequently , muscle atrophy . vascular compression between the convexed lumbar spine and the superficial fascia promoted atrophy of pse by disturbing vascular supply.4 in spite of debates about causal relationship , extensor muscle degeneration and development of dfb were closely related with each other . histographic analysis using mri has been reported to be an effective method to assess the degree of muscle degeneration . high si in histography means severe degree of replacement of muscle by fat tissues , and this also suggests muscle degeneration or weakness as small csa did.57 several literatures demonstrated that the patients with dfb had significantly smaller csa and higher si of the pse compared with normal control or patients suffering from chronic low back pain without dfb.345 current study demonstrated that pse degeneration , expressed by reduced csa and higher si , was related to more kyphotic lumbar segment and more flat / lordotic thoracic segment as well as was also associated with less improvement after corrective surgery . in static parameters , as pse condition was more severe , thoracic angle was more lordotic . it meant that more severe pse condition lead to more severe lumbar kyphosis ( less ll ) , consequently resulted in flat / lordotic thoracic angle . after surgery , as upper pse condition was more severe preoperatively , less improvement of tk was accomplished by corrective surgery . in dynamic parameters , decreased thoracic kyphotic angle showed correlation with severity of upper pse condition , whereas increased lumbar kyphotic angle had correlation with severity of middle to lower pse condition . similar results were found in terms of improvement after surgery . while improvement of tk after surgery was associated with upper pse condition , improvement of ll was related to more extensive pse condition , from l1l2 to l4l5 levels . dynamic parameters showed more prominent correlation with preoperative pse conditions than static parameters and also identify segmental specificity in the relationship between pse condition and angular deformity . while upper pse condition was related to angular change of thoracic segment , middle to lower pse condition was related to angular change of lumbar segment . this study showed that pse conditions had more remarkable relationship with dynamic parameters than static parameters . considering muscle activity influenced functional aspect , it was expected that pse condition was more closely associated with dynamic parameters , which were relevant to functionality in daily activities . it has been reported that pse condition was more relevant to patients with lower muscle strength , poorer functionality , or more severe pain . among the patients with low back pain , those with smaller csa of pse revealed lower muscle strength.18 the patients with better functional performance among those diagnosed as lumbar stenosis exhibited a significantly larger csa and a lower fat infiltration in pse.15 in lumbar discectomy patients , patients with persistent pain showed significantly smaller csa of multifidus and erector spinae than those with pain - free patients.19 because functional impairments regarding daily activity were critical problems to patients quality of life or satisfaction with treatment , assessment of dynamic parameters was more important . three - dimensional motion analysis fulfilled more objective and quantitative evaluation of functional aspects than that of clinical assessments . in lower limb dynamic parameters , even if no typical correlation was found , posterior pt angle , hip and knee flexion angle or ankle dorsiflexion angle was increased as pse atrophy and fat infiltration was more severe . the less improvement of hip flexion angle was acquired after surgery , as pse atrophy was more severe . it has been reported that dfb also leads to change of lower limb kinematics in addition to spinopelvic kinematics such as increased knee flexion and ankle extension ( dorsiflexion).20 the change of low limb kinematics was assumed to be compensatory mechanism to prevent excessive anterior translation of body center related to stooped posture in dfb , including increased hip and knee flexion , ankle dorsiflexion , which further led to posterior pt.22021 the poorer pse conditions led to more severe lumbar kyphosis , which , further , caused the more prominent compensatory mechanism of lower limbs . all of these surgeries were conducted by posterior approach , which could affect pse conditions . however , it was assumed that because all of surgeries were conducted by posterior approach , all patients were in same conditions . although all subjects underwent the corrective surgery by posterior approach , we could obtain the results that more severe preoperative pse resulted in less improvement after corrective surgery and segmental specificity was found between pse condition and angular deformity . in addition , fusion levels were variable and more wide excisions could produce more severe muscles atrophy . but most of patients underwent corrective surgery within lumbosacral area and even the corrective surgeries including thoracic area were conducted at lower levels of thoracic spine . the number of subjects undergoing surgeries of thoracic segments was relatively small , so that we could not stratify the results according to surgery level . however , we obtained the segmental specificity that thoracic angle showed correlation with preoperative upper pse conditions , whereas lumbar angle had correlation with preoperative middle to lower pse conditions , which was mainly found in dynamic parameters , not in static parameters . this suggested that surgical outcomes were influenced by preoperative pse conditions rather than surgical level . first , this study was retrospective study , so we recruited only the patients who conducted three - dimensional gait analysis before and 6 months after corrective surgeries . the tests about other daily activities regarding sit - to - stand movement , trunk flexion , or picking up object could provide more useful information related to patients functional aspects . followup study such as 1 or 2 years could assess the change of patients functional status in terms of long term surgical outcomes . the severe atrophy or fat infiltration of pse was related to severe angular deformity in patients with dfb and to less improvement after corrective surgery in terms of static as well as dynamic parameters . dynamic parameters were more prominently correlated with pse conditions than static parameters and showed segmental specificity such that upper pse condition was more correlated with thoracic curve and middle to lower pse condition was more correlated with lumbar curve .

background : degenerative flat back ( dfb ) is characterized by sagittal imbalance resulting from the loss of lumbar lordosis ( ll ) . extensive degeneration and weakness of lumbar paraspinal extensor muscle ( pse ) are thought to be the main cause of dfb . this study is to evaluate correlation between preoperative pse conditions and angular severity of dfb and to evaluate correlation between preoperative pse conditions and degree of improvement of dfb obtained by corrective surgery.materials and methods : forty five patients with dfb who took magnetic resonance image ( mri ) preoperatively and conducted simple radiography and three - dimensional gait analysis before and 6 months after corrective surgery were included . to determine the severity of pse atrophy , the ratio between cross - sectional area of pse and disc was calculated from l1l2 to l4l5 on mri . to assess the degree of fat infiltration , the signal intensity of pse was measured . static parameters of spinopelvic segment were measured by simple radiography . dynamic parameters of spinopelvic and lower limb joints were obtained by three - dimensional gait analysis.results:in static parameters , thoracic angle was correlated with atrophy and fat infiltration of upper pse . thoracic angle was less improved after surgery , as atrophy of upper pse was more severe . in dynamic parameters , thoracic angle showed correlation with upper pse conditions , whereas lumbar angle had correlation with middle to lower pse conditions . while thoracic kyphosis was less improved after surgery , as atrophy of upper pse was more severe , ll was less improved , as atrophy and fat infiltration of pse from l1l2 to l4l5 were more severe.conclusions:severity of atrophy or fat infiltration of pse showed correlation with degree of angular deformity in patients with dfb and with less improvement after corrective surgery . dynamic parameters showed more prominent correlation with pse conditions than static parameters and also showed segmental specificity between pse and angular deformity .

I M Radiological evaluation (static parameters) Lumbar paraspinal extensor muscle measurement on magnetic resonance image Three-dimensional gait analysis (dynamic parameters) Statistical analysis R Correlation between paraspinal extensor muscle measurements and static parameters before surgery Correlation between paraspinal extensor muscle measurements and dynamic parameters before surgery Correlation between paraspinal extensor muscle measurement and improvement of static parameters obtained by surgery Correlation between paraspinal extensor muscle measurement and improvement of dynamic parameters obtained surgery D C Financial support and sponsorship Conflicts of interest

degenerative flat back ( dfb ) is frequent in asian countries , which suggests that typical life style or working posture is associated with occurrence of dfb . they stand up or sit on the floor with waist flexion and frequently work at home or farm with stooped posture , which contributes to decreased lumbar lordosis ( ll ) and weakness of paraspinal extensor muscles ( pses ) . decreased ll causes anterior displacement of the center of gravity , which leads to spinopelvic angular changes and causes various disabilities in daily life including gait difficulty.12 although the exact pathophysiology of dfb has not yet been confirmed , extensive degeneration , and weakness of lumbar extensor muscles are thought to be the most important cause.34 the quantitative evaluations of lumbar extensor muscle have been developed using magnetic resonance image ( mri).456 the typical signs of muscle degeneration detected on mri are muscle cross - sectional area ( csa ) and fatty replacement of muscle , expressed as increased signal intensity ( si).67 previous study using mri histogram reported that dfb patients showed higher mean si of back extensor muscles suggesting fat infiltration compared to normal control.5 although radiographic examination can show characteristic sagittal deviation of spinopelvic alignment,8 it has the limitation only to reveal the static posture , and not to identify dynamic status such as ambulation . however , daily activities or functional aspects of patients are more related to dynamic status of spinopelvic segment than static posture . thus , it is assumed that assessment of dynamic parameters of dfb by three - dimensional motion analysis can provide clinically useful data about patients functional status and treatment outcomes.9101112 it can provide specific characteristics of spinopelvic and lower limb joints motion in patients with dfb and also reveal which improvement occurred following surgery in quantitative and objective way . the purposes of this study were ( 1 ) to evaluate correlation between preoperative lumbar extensor muscle condition and angular severity of dfb and ( 2 ) to evaluate correlation between preoperative lumbar extensor muscle condition and degree of improvement of dfb obtained by corrective fusion surgery , in terms of static parameters by simple radiography and dynamic parameters by three - dimensional motion analysis . lumbar mri , whole spine x - ray , and three dimensional motion analysis were done before and 6 months after surgery in all the patients . as for spinal sagittal parameters , thoracic kyphosis ( tk ) , thoracolumbar junction ( tlj ) , and ll were measured in whole spine lateral view . the tk was measured from the t5 superior end plate to t12 inferior end plate . the ll was measured from the t12 inferior end plate to s1 superior end plate by the cobb method [ figure 1 ] . as for the pelvic parameters , pelvic incidence ( pi ) , the ss was the angle between the s1 superior end plate and a horizontal line . the pi was defined as the angle between the line perpendicular to the sacral plate and the line connecting the midpoint of the sacral plate to the bicoxofemoral axis [ figure 2].813 x - ray thoracolumbosacral spine lateral view showing spinal parameters such as thoracic kyphosis , thoracolumbar junction , and lumbar lordosis . ( a ) the thoracic kyphosis was measured from the t5 superior end plate to t12 inferior end plate . ( b ) the thoracolumbar junction was measured from the t10 superior end plate to l2 inferior end plate . ( c ) the lumbar lordosis was measured from the t12 inferior end plate to s1 superior end plate by the cobb method x - ray lumbosacral spine lateral view showing pelvic parameters including pelvic tilt , pelvic incidence , and sacral slope . the regions of interest ( roi ) were outlined with a graphic cursor around the lumbar pse including multifidi and erector spinae and on the intervertebral disc at each level.14 the degree of muscle atrophy was determined by measuring the csa of the pse compartment . in order to decrease the bias caused by differences in individual body size , the area of the pse compartment was divided by intervertebral disc area of the same level ( the ratio between the csa of the pse and the csa of the intervertebral disc [ pse / disc csa ] ) to represent the relative muscle compartment volume in each individual . mean value of the signal intensity was measured within paraspinal extensor muscle , using histogram of magnetic resonance image gait analysis was conducted using three - dimensional motion analyzer ( motion analysis ; motion analysis company , santa rosa , ca , usa ) . markers of diameter 2.5 cm were attached bilaterally to the bony landmarks of the pelvis and lower extremities , including the l5-s1 intervertebral space , anterior superior iliac spine , anterior side of the midthigh , midpoint of the lateral knee , anterior side of the mid - tibia , lateral malleolus of the fibula , dorsal side between the second and third metatarsal heads , and the calcaneal area on the same line as the metatarsal marker to assessing lower limb kinematics during ambulation . clinical photographs ( a and b ) showing location of markers during three - dimensional motion analysis all of these measurements were performed by single physician ( jhl ) who was expert in spine radiology and three - dimensional motion analysis . the correlation between preoperative pse measurements and static / dynamic parameters and the correlation between preoperative pse measurements and degree of improvement of static / dynamic parameters obtained by corrective fusion surgery were determined using the pearson correlation coefficient . as for spinal sagittal parameters , thoracic kyphosis ( tk ) , thoracolumbar junction ( tlj ) , and ll were measured in whole spine lateral view . the ll was measured from the t12 inferior end plate to s1 superior end plate by the cobb method [ figure 1 ] . the pi was defined as the angle between the line perpendicular to the sacral plate and the line connecting the midpoint of the sacral plate to the bicoxofemoral axis [ figure 2].813 x - ray thoracolumbosacral spine lateral view showing spinal parameters such as thoracic kyphosis , thoracolumbar junction , and lumbar lordosis . ( a ) the thoracic kyphosis was measured from the t5 superior end plate to t12 inferior end plate . ( b ) the thoracolumbar junction was measured from the t10 superior end plate to l2 inferior end plate . ( c ) the lumbar lordosis was measured from the t12 inferior end plate to s1 superior end plate by the cobb method x - ray lumbosacral spine lateral view showing pelvic parameters including pelvic tilt , pelvic incidence , and sacral slope . the regions of interest ( roi ) were outlined with a graphic cursor around the lumbar pse including multifidi and erector spinae and on the intervertebral disc at each level.14 the degree of muscle atrophy was determined by measuring the csa of the pse compartment . in order to decrease the bias caused by differences in individual body size , the area of the pse compartment was divided by intervertebral disc area of the same level ( the ratio between the csa of the pse and the csa of the intervertebral disc [ pse / disc csa ] ) to represent the relative muscle compartment volume in each individual . mean value of the signal intensity was measured within paraspinal extensor muscle , using histogram of magnetic resonance image gait analysis was conducted using three - dimensional motion analyzer ( motion analysis ; motion analysis company , santa rosa , ca , usa ) . markers of diameter 2.5 cm were attached bilaterally to the bony landmarks of the pelvis and lower extremities , including the l5-s1 intervertebral space , anterior superior iliac spine , anterior side of the midthigh , midpoint of the lateral knee , anterior side of the mid - tibia , lateral malleolus of the fibula , dorsal side between the second and third metatarsal heads , and the calcaneal area on the same line as the metatarsal marker to assessing lower limb kinematics during ambulation . clinical photographs ( a and b ) showing location of markers during three - dimensional motion analysis all of these measurements were performed by single physician ( jhl ) who was expert in spine radiology and three - dimensional motion analysis . the correlation between preoperative pse measurements and static / dynamic parameters and the correlation between preoperative pse measurements and degree of improvement of static / dynamic parameters obtained by corrective fusion surgery were determined using the pearson correlation coefficient . tk was correlated with l1l2 si , l3l4 pse / disc csa , and l4l5 si [ table 1 ] . correlation between paraspinal extensor muscle measurement and static parameters maximal thoracic angle was correlated with l1l2 pse / disc csa , l1l2 si , and l2l3 pse / disc csa . maximal lumbar angle was correlated with l3l4 pse / disc csa and l4l5 pse / disc csa . minimal thoracic angle was correlated with l1l2 pse / disc csa . minimal lumbar angle was correlated with l2l3 si , l3l4 pse / disc csa , and l3l4 si , and l4l5 pse / disc csa [ table 2 ] . correlation between paraspinal extensor muscle measurement and dynamic parameters maximal right hip and knee flexion angle were correlated with l4l5 pse / disc csa . minimal posterior pt angle was correlated with l2l3 si . minimal right hip flexion angle was correlated with l3l4 pse / disc csa and l4l5 pse / disc csa . minimal left ankle dorsiflexion angle was correlated with l1l2 , l2l3 , l3l4 , and l4l5 si . correlation between preoperative paraspinal extensor muscle measurement and improvement of static parameters obtained by surgery the maximal thoracic angle showed significant correlation with l1l2 and l2l3 pse / disc csa . the maximal lumbar angle showed significant correlation with l1l2 , l2l3 , l3l4 , and l4l5 pse / disc csa . the minimal thoracic angle showed significant correlation with l1l2 and l2l3 pse / disc csa . the minimal lumbar angle showed significant correlation with l1l2 , l2l3 , l3l4 , and l4l5 pse / disc csa [ table 4 ] . correlation between preoperative paraspinal extensor measurement and improvement of dynamic parameters obtained surgery the maximal pelvic posterior tilt was also correlated with l4l5 pse / disc csa . the maximal right and left hip flexion angle was correlated with l2l3 , l3l4 , and l4l5 pse / disc csa . the minimal right ankle flexion angle was correlated with l1l2 pse / disc csa . tk was correlated with l1l2 si , l3l4 pse / disc csa , and l4l5 si [ table 1 ] . maximal thoracic angle was correlated with l1l2 pse / disc csa , l1l2 si , and l2l3 pse / disc csa . maximal lumbar angle was correlated with l3l4 pse / disc csa and l4l5 pse / disc csa . minimal thoracic angle was correlated with l1l2 pse / disc csa . minimal lumbar angle was correlated with l2l3 si , l3l4 pse / disc csa , and l3l4 si , and l4l5 pse / disc csa [ table 2 ] . correlation between paraspinal extensor muscle measurement and dynamic parameters maximal right hip and knee flexion angle were correlated with l4l5 pse / disc csa . minimal posterior pt angle was correlated with l2l3 si . minimal right hip flexion angle was correlated with l3l4 pse / disc csa and l4l5 pse / disc csa . minimal left ankle dorsiflexion angle was correlated with l1l2 , l2l3 , l3l4 , and l4l5 si . the maximal thoracic angle showed significant correlation with l1l2 and l2l3 pse / disc csa . the maximal lumbar angle showed significant correlation with l1l2 , l2l3 , l3l4 , and l4l5 pse / disc csa . the minimal thoracic angle showed significant correlation with l1l2 and l2l3 pse / disc csa . the minimal lumbar angle showed significant correlation with l1l2 , l2l3 , l3l4 , and l4l5 pse / disc csa [ table 4 ] . correlation between preoperative paraspinal extensor measurement and improvement of dynamic parameters obtained surgery the maximal pelvic posterior tilt was also correlated with l4l5 pse / disc csa . the maximal right and left hip flexion angle was correlated with l2l3 , l3l4 , and l4l5 pse / disc csa . the minimal posterior pt was correlated with l2l3 si . the minimal right ankle flexion angle was correlated with l1l2 pse / disc csa . the degeneration and weakness of pse were thought to be important causes of dfb because the pse played a role in maintaining spinal curvatures and their degeneration or atrophy resulted in a progressive kyphosis and sagittal imbalance.135 degeneration of pse was significantly associated with facet arthropathy , which additionally contributed to progression of lumbar kyphosis.15 facet arthropathy could elicit a reflex mechanism that inhibited muscle activity and further caused muscle atrophy.16 as well , kyphotic deformity , in turn , aggravated degeneration and weakness of pse in more severe degree . over lengthening of pse caused by increased lumbar kyphotic angle lost the lever arm effects enough for appropriate contraction , which further aggravated muscle degeneration and atrophy.17 low back pain caused by dfb could contribute to limited back movement and consequently , muscle atrophy . vascular compression between the convexed lumbar spine and the superficial fascia promoted atrophy of pse by disturbing vascular supply.4 in spite of debates about causal relationship , extensor muscle degeneration and development of dfb were closely related with each other . histographic analysis using mri has been reported to be an effective method to assess the degree of muscle degeneration . high si in histography means severe degree of replacement of muscle by fat tissues , and this also suggests muscle degeneration or weakness as small csa did.57 several literatures demonstrated that the patients with dfb had significantly smaller csa and higher si of the pse compared with normal control or patients suffering from chronic low back pain without dfb.345 current study demonstrated that pse degeneration , expressed by reduced csa and higher si , was related to more kyphotic lumbar segment and more flat / lordotic thoracic segment as well as was also associated with less improvement after corrective surgery . in static parameters , as pse condition was more severe , thoracic angle was more lordotic . it meant that more severe pse condition lead to more severe lumbar kyphosis ( less ll ) , consequently resulted in flat / lordotic thoracic angle . after surgery , as upper pse condition was more severe preoperatively , less improvement of tk was accomplished by corrective surgery . in dynamic parameters , decreased thoracic kyphotic angle showed correlation with severity of upper pse condition , whereas increased lumbar kyphotic angle had correlation with severity of middle to lower pse condition . similar results were found in terms of improvement after surgery . while improvement of tk after surgery was associated with upper pse condition , improvement of ll was related to more extensive pse condition , from l1l2 to l4l5 levels . dynamic parameters showed more prominent correlation with preoperative pse conditions than static parameters and also identify segmental specificity in the relationship between pse condition and angular deformity . while upper pse condition was related to angular change of thoracic segment , middle to lower pse condition was related to angular change of lumbar segment . this study showed that pse conditions had more remarkable relationship with dynamic parameters than static parameters . considering muscle activity influenced functional aspect , it was expected that pse condition was more closely associated with dynamic parameters , which were relevant to functionality in daily activities . it has been reported that pse condition was more relevant to patients with lower muscle strength , poorer functionality , or more severe pain . among the patients with low back pain , those with smaller csa of pse revealed lower muscle strength.18 the patients with better functional performance among those diagnosed as lumbar stenosis exhibited a significantly larger csa and a lower fat infiltration in pse.15 in lumbar discectomy patients , patients with persistent pain showed significantly smaller csa of multifidus and erector spinae than those with pain - free patients.19 because functional impairments regarding daily activity were critical problems to patients quality of life or satisfaction with treatment , assessment of dynamic parameters was more important . in lower limb dynamic parameters , even if no typical correlation was found , posterior pt angle , hip and knee flexion angle or ankle dorsiflexion angle was increased as pse atrophy and fat infiltration was more severe . the less improvement of hip flexion angle was acquired after surgery , as pse atrophy was more severe . it has been reported that dfb also leads to change of lower limb kinematics in addition to spinopelvic kinematics such as increased knee flexion and ankle extension ( dorsiflexion).20 the change of low limb kinematics was assumed to be compensatory mechanism to prevent excessive anterior translation of body center related to stooped posture in dfb , including increased hip and knee flexion , ankle dorsiflexion , which further led to posterior pt.22021 the poorer pse conditions led to more severe lumbar kyphosis , which , further , caused the more prominent compensatory mechanism of lower limbs . although all subjects underwent the corrective surgery by posterior approach , we could obtain the results that more severe preoperative pse resulted in less improvement after corrective surgery and segmental specificity was found between pse condition and angular deformity . however , we obtained the segmental specificity that thoracic angle showed correlation with preoperative upper pse conditions , whereas lumbar angle had correlation with preoperative middle to lower pse conditions , which was mainly found in dynamic parameters , not in static parameters . this suggested that surgical outcomes were influenced by preoperative pse conditions rather than surgical level . first , this study was retrospective study , so we recruited only the patients who conducted three - dimensional gait analysis before and 6 months after corrective surgeries . the severe atrophy or fat infiltration of pse was related to severe angular deformity in patients with dfb and to less improvement after corrective surgery in terms of static as well as dynamic parameters . dynamic parameters were more prominently correlated with pse conditions than static parameters and showed segmental specificity such that upper pse condition was more correlated with thoracic curve and middle to lower pse condition was more correlated with lumbar curve .

spasticity is defined as a velocity - dependent increase in the tonic stretch response with excessive tendon jerk reflexes and is caused by the reduction of inhibitory impulses on lower motor neurons . spasticity can cause pain , muscle shortening , and orthopedic malformations , and it can severely interfere with functional abilities and gait pattern . cerebral palsy is a group of disorders of movement and posture due to a nonprogressive lesion of the developing brain . as a consequence of this definition , its causes are diverse . selective dorsal rhizotomy ( sdr ) is a neurosurgical treatment that is mainly performed at lumbar level in patients with bilateral spastic paresis . by incomplete transection of the ( sensory ) posterior lumbosacral rootlets , sdr reduces the excitatory input from the lower limbs that enters the spinal cord . in children with spastic cp , sdr leads to a more normal gait pattern [ 36 ] , better performance of the activities of daily life [ 7 , 8 ] , and improvement in gross motor function [ 613 ] . data on functional outcome after sdr in patients with other conditions than spastic cp are limited , though promising . detailed information about the clinical characteristics of the selected children , other than spasticity alone , is often lacking . medical history and clinical examination does not always provide sufficient evidence to establish the diagnosis of the underlying disorder in patients with cp . the quality standards subcommittee of the american academy of neurology and the practice committee of the child neurology society both advise magnetic resonance imaging ( mri ) of the brain for all cp patients . in patients with bilateral spastic cp , the most common mri abnormality is periventricular leucomalacia ( pvl ) , which is characterized by a damage of the periventricular white matter [ 1619 ] . gray matter lesions , early developmental abnormalities of the central nervous system , and abnormalities of the cerebrospinal fluid space , such as hydrocephalus , are less commonly found [ 1719 ] , and in some children with bilateral spastic cp no abnormalities are found with mri [ 1619 ] . in patients with normal mri findings and bilateral spastic paresis , genetic causes or spinal cord involvement should be considered . the goal of this study was to identify possible relationships between the mri findings with the level of gross motor function of the patient before sdr and the change in functioning after sdr . for this purpose , we retrospectively analyzed the mris of the patients who underwent sdr in our clinic . this study was part of the project : investigation of long - term effects of sdr in children with spastic diplegia and was approved by the medical ethical committee of the vu university medical center , amsterdam , the netherlands ( project number 2006/105 ) . the study population consisted of all the patients who underwent sdr in our clinic between january 1998 and december 2007 . the patients were selected for sdr according to the criteria shown in table 1 . in our previous study , we only included patients with bilateral spastic cp and documented pvl . in the present study , , we included data on patients who underwent pre- and postoperative gross motor function measure ( gmfm-66 ) analysis with a follow - up period of at least 12 months and of whom mri was available for review . table 1selection criteria for selective dorsal rhizotomy in the vu university medical centercriteria for selective dorsal rhizotomy1.)bilateral spasticity of the lower extremities interfering with walking performance2.)presence of spasticity ( defined as velocity - dependent resistance to passive stretch ) in at least six muscle groups of the lower limbs3.)sufficient force in the quadriceps femoris muscle ( squatting at least seven times ) and the hip extensors ( kneel with extended hips , support for balance allowed)4.)absence of structural orthopedic deformities or contractures at hip , knee , or ankle5.)presence of moderate to good selective motor control in the lower limbs6.)gross motor function classification system ( gmfcs ) level i , ii , or iii7.)good support from parents and rehabilitation setting selection criteria for selective dorsal rhizotomy in the vu university medical center selective dorsal rhizotomy was performed by the same neurosurgeon ( wvo ) in all children . dorsal roots l2-s1 were exposed and separated into different rootlets after laminotomy l2-l5 and opening of the dura . transection of the rootlets was performed after electro - stimulation , according to palpable muscle contraction and emg response . at most , 50% of the rootlets to prevent sexual and bladder disturbances , rootlets / fascicles showing electrical response after stimulation of the penis / the clitoris were spared . the mris were assessed at two different points in time by two investigators one neuropediatrician ( 8 years of experience in the assessment of mr neuroimaging ; rjv ) and one pediatrician with specialization in pediatric neurorehabilitation ( 2 years of experience in the assessment of mr neuroimaging ; sg ) . imaging was classified into three diagnostic groups according to the following criteria : periventricular leucomalacia ( pvl ; increased signal intensity of the periventricular white matter in t2 weighted imaging and/or flair ; other abnormalities optional ) ; hydrocephalus ( signs of ventricular dilatation and increased intracranial pressure ) ; normal mri ( see fig . 1a i ) . in one patient , the mr abnormalities could not be classified into one of the three diagnostic groups and he was excluded from further data analysis ( patient number 19 , table 2 ; his mri showed delayed myelinisation and slightly enlarged ventricular size but no other white matter abnormalities and there were no signs of increased intracranial pressure ) . in the patients with pvl , we graded the severity of their mri abnormalities based on the work of cioni et al . . the following items were assessed : ventricular size , evidence and extension of white matter signal intensity , evidence and extension of white matter loss , thinning of the corpus callosum , dimension ( size ) of subarachnoidal space , evidence and size of cysts , and presence of gray matter abnormalities . the items were scored on a three - point scale , with a score of three indicating the most severe mri abnormalities . the scores were summed to obtain a total score for each child ( minimal score , 7 and maximal score , 21 ) . if the two investigators did not agree , the classification and/or scoring were determined by a consensus . 1a i mr imaging of three patients with bilateral spastic paresis undergoing sdr . a , d , g midsagittal t1 weighted images . b , e , h transversal t2 weighted images at the level of the centrum semiovale . g , h , i transversal t2 weighted imaging at the level of the basal ganglia . d f mr images classified as periventricular leucomalacia showing thinning of the corpus callosum involving the total body ( fig . 1e ) , a slight ventricular enlargement , and a loss of the occipital white matter ( fig . 1i ) are enlargedtable 2summary of the characteristics of the patients included in the studycasegenderga ( weeks)bw ( g)age sdr years ( mts)follow - up years ( mts)mri classificationdiagnosisgmfm-66 before sdrgmfm-66 ( mean ) after sdr1f302,0005 ( 7)8 ( 5)pvlpvl50.8553.292f402,5005(7)9 ( 0)pvlpvl47.6849.663f403,4505 ( 8)8 ( 11)pvlpvl54.3855.714m301,6506 ( 8)8 ( 11)pvlpvl46.9148.325f281,2854 ( 11)7 ( 10)pvlpvl50.3254.756m261,0005 ( 3)7 ( 10)pvlpvl64.9867.827m322,5105 ( 1)6 ( 7)pvlpvl54.1560.388m331,2658 ( 9)5 ( 1)pvlpvl47.0955.929m268706 ( 11)3 ( 11)pvlpvl50.0954.3210m267805 ( 4)3 ( 0)pvlpvl52.3262.9811f413,0608 ( 4)5 ( 2)hydrocephaluscongenital hydrocephalus55.6256.3512m271,02010 ( 1)4 ( 5)hydrocephaluscongenital hydrocephalus68.8668.6913m403,3005 ( 9)7 ( 2)normalunknown65.3367.3114m343,1556 ( 11)3 ( 0)normalspinal process76.7585.1515m384,2808 ( 0)2 ( 7)normalunknown82.9989.7016f403,8758 ( 10)1 ( 7)normalhiv - encephalo(myelo)pathy73.6380.4617m394,0406 ( 7)2 ( 0)normalunknown65.9878.3818m403,4606 ( 5)1 ( 1)normalunknown73.6381.9319m373,4003 ( 11)7 ( 1)not classifiedlaurence moon syndrome47.2683.01f female , m male , ga gestational age , bw birth weight , sdr selective dorsal rhizotomy , gmfcs gross motor function classification system , gmfm gross motor function measure , pvl periventricular leucomalacia , g gram , mts months a b , e , h transversal t2 weighted images at the level of the centrum semiovale . g , h , i transversal t2 weighted imaging at the level of the basal ganglia . d f mr images classified as periventricular leucomalacia showing thinning of the corpus callosum involving the total body ( fig . 1e ) , a slight ventricular enlargement , and a loss of the occipital white matter ( fig . 1i ) are enlarged summary of the characteristics of the patients included in the study f female , m male , ga gestational age , bw birth weight , sdr selective dorsal rhizotomy , gmfcs gross motor function classification system , gmfm gross motor function measure , pvl periventricular leucomalacia , g gram , mts months all patients had a detailed pre- and postoperative clinical evaluation , including spasticity assessment , range of motion of single joints , gait analysis , and the gmfm . in this study the gmfm is a criterion - referenced observational measure that was developed to assess children with cp . the validity , reliability , and responsiveness of the gmfm have been demonstrated in a population of patients similar to the participants in the present study . for our data analysis , we used the gmfm-66 version , which assesses 66 items covering five gross motor dimensions ( lying and rolling / crawling and kneeling / standing and walking / running / jumping ) and is elaborated to a numerical scale ranging from 0 to 100 . as outcome parameters we used the mean of all postoperative gmfm-66 scores from 1 year after surgery . the changes after sdr were expressed as the difference between gmfm-66 score and the preoperative gmfm-66 score ( delta - gmfm-66 ) . agreement strengths for the kappa values were classified according to landis and koch . for the comparison of the outcome in patients with different mri characteristics , we used nonparametric tests ( kruskal walis test for group comparison ; post hoc analysis was performed using the mann this study was part of the project : investigation of long - term effects of sdr in children with spastic diplegia and was approved by the medical ethical committee of the vu university medical center , amsterdam , the netherlands ( project number 2006/105 ) . the study population consisted of all the patients who underwent sdr in our clinic between january 1998 and december 2007 . the patients were selected for sdr according to the criteria shown in table 1 . in our previous study , we only included patients with bilateral spastic cp and documented pvl . in the present study , , we included data on patients who underwent pre- and postoperative gross motor function measure ( gmfm-66 ) analysis with a follow - up period of at least 12 months and of whom mri was available for review . table 1selection criteria for selective dorsal rhizotomy in the vu university medical centercriteria for selective dorsal rhizotomy1.)bilateral spasticity of the lower extremities interfering with walking performance2.)presence of spasticity ( defined as velocity - dependent resistance to passive stretch ) in at least six muscle groups of the lower limbs3.)sufficient force in the quadriceps femoris muscle ( squatting at least seven times ) and the hip extensors ( kneel with extended hips , support for balance allowed)4.)absence of structural orthopedic deformities or contractures at hip , knee , or ankle5.)presence of moderate to good selective motor control in the lower limbs6.)gross motor function classification system ( gmfcs ) level i , ii , or iii7.)good support from parents and rehabilitation setting selection criteria for selective dorsal rhizotomy in the vu university medical center selective dorsal rhizotomy was performed by the same neurosurgeon ( wvo ) in all children . dorsal roots l2-s1 were exposed and separated into different rootlets after laminotomy l2-l5 and opening of the dura . transection of the rootlets was performed after electro - stimulation , according to palpable muscle contraction and emg response . at most , 50% of the rootlets rootlets / fascicles showing electrical response after stimulation of the penis / the clitoris were spared . the mris were assessed at two different points in time by two investigators one neuropediatrician ( 8 years of experience in the assessment of mr neuroimaging ; rjv ) and one pediatrician with specialization in pediatric neurorehabilitation ( 2 years of experience in the assessment of mr neuroimaging ; sg ) . imaging was classified into three diagnostic groups according to the following criteria : periventricular leucomalacia ( pvl ; increased signal intensity of the periventricular white matter in t2 weighted imaging and/or flair ; other abnormalities optional ) ; hydrocephalus ( signs of ventricular dilatation and increased intracranial pressure ) ; normal mri ( see fig . 1a i ) . in one patient , the mr abnormalities could not be classified into one of the three diagnostic groups and he was excluded from further data analysis ( patient number 19 , table 2 ; his mri showed delayed myelinisation and slightly enlarged ventricular size but no other white matter abnormalities and there were no signs of increased intracranial pressure ) . in the patients with pvl , we graded the severity of their mri abnormalities based on the work of cioni et al . . the following items were assessed : ventricular size , evidence and extension of white matter signal intensity , evidence and extension of white matter loss , thinning of the corpus callosum , dimension ( size ) of subarachnoidal space , evidence and size of cysts , and presence of gray matter abnormalities . the items were scored on a three - point scale , with a score of three indicating the most severe mri abnormalities . the scores were summed to obtain a total score for each child ( minimal score , 7 and maximal score , 21 ) . if the two investigators did not agree , the classification and/or scoring were determined by a consensus . 1a i mr imaging of three patients with bilateral spastic paresis undergoing sdr . a , d , g midsagittal t1 weighted images . b , e , h transversal t2 weighted images at the level of the centrum semiovale . g , h , i transversal t2 weighted imaging at the level of the basal ganglia . d f mr images classified as periventricular leucomalacia showing thinning of the corpus callosum involving the total body ( fig . 1e ) , a slight ventricular enlargement , and a loss of the occipital white matter ( fig . 1i ) are enlargedtable 2summary of the characteristics of the patients included in the studycasegenderga ( weeks)bw ( g)age sdr years ( mts)follow - up years ( mts)mri classificationdiagnosisgmfm-66 before sdrgmfm-66 ( mean ) after sdr1f302,0005 ( 7)8 ( 5)pvlpvl50.8553.292f402,5005(7)9 ( 0)pvlpvl47.6849.663f403,4505 ( 8)8 ( 11)pvlpvl54.3855.714m301,6506 ( 8)8 ( 11)pvlpvl46.9148.325f281,2854 ( 11)7 ( 10)pvlpvl50.3254.756m261,0005 ( 3)7 ( 10)pvlpvl64.9867.827m322,5105 ( 1)6 ( 7)pvlpvl54.1560.388m331,2658 ( 9)5 ( 1)pvlpvl47.0955.929m268706 ( 11)3 ( 11)pvlpvl50.0954.3210m267805 ( 4)3 ( 0)pvlpvl52.3262.9811f413,0608 ( 4)5 ( 2)hydrocephaluscongenital hydrocephalus55.6256.3512m271,02010 ( 1)4 ( 5)hydrocephaluscongenital hydrocephalus68.8668.6913m403,3005 ( 9)7 ( 2)normalunknown65.3367.3114m343,1556 ( 11)3 ( 0)normalspinal process76.7585.1515m384,2808 ( 0)2 ( 7)normalunknown82.9989.7016f403,8758 ( 10)1 ( 7)normalhiv - encephalo(myelo)pathy73.6380.4617m394,0406 ( 7)2 ( 0)normalunknown65.9878.3818m403,4606 ( 5)1 ( 1)normalunknown73.6381.9319m373,4003 ( 11)7 ( 1)not classifiedlaurence moon syndrome47.2683.01f female , m male , ga gestational age , bw birth weight , sdr selective dorsal rhizotomy , gmfcs gross motor function classification system , gmfm gross motor function measure , pvl periventricular leucomalacia , g gram , mts months a i mr imaging of three patients with bilateral spastic paresis undergoing sdr . a , d , g midsagittal t1 weighted images . b , e , h transversal t2 weighted images at the level of the centrum semiovale . g , h , i transversal t2 weighted imaging at the level of the basal ganglia . d f mr images classified as periventricular leucomalacia showing thinning of the corpus callosum involving the total body ( fig . 1e ) , a slight ventricular enlargement , and a loss of the occipital white matter ( fig . 1i ) are enlarged summary of the characteristics of the patients included in the study f female , m male , ga gestational age , bw birth weight , sdr selective dorsal rhizotomy , gmfcs gross motor function classification system , gmfm gross motor function measure , pvl periventricular leucomalacia , g gram , mts months all patients had a detailed pre- and postoperative clinical evaluation , including spasticity assessment , range of motion of single joints , gait analysis , and the gmfm . in this study , we only report the results of the gmfm . the gmfm is a criterion - referenced observational measure that was developed to assess children with cp . the validity , reliability , and responsiveness of the gmfm have been demonstrated in a population of patients similar to the participants in the present study . for our data analysis , we used the gmfm-66 version , which assesses 66 items covering five gross motor dimensions ( lying and rolling / crawling and kneeling / standing and walking / running / jumping ) and is elaborated to a numerical scale ranging from 0 to 100 . as outcome parameters we used the mean of all postoperative gmfm-66 scores from 1 year after surgery . the changes after sdr were expressed as the difference between gmfm-66 score and the preoperative gmfm-66 score ( delta - gmfm-66 ) . agreement strengths for the kappa values were classified according to landis and koch . for the comparison of the outcome in patients with different mri characteristics , we used nonparametric tests ( kruskal walis test for group comparison ; post hoc analysis was performed using the mann thirty - six patients underwent sdr of whom 32 patients had a brain mri performed before sdr , and 26 mris were available for review . of the patients with available mri , four did not have any preoperative gmfm-66 assessments . three patients had a gmfm-66 assessment before sdr but with a follow - up period of less than 12 months . the detailed patient characteristics of the remaining 19 patients are summarized in table 2 . the mean number of gmfm assessments per patient was 4.9 ( standard deviation ( sd ) 3.5 , range 1 to 12 ) , and the mean follow - up period was 5 years and 4 months ( sd 2 years and 9 months , range 1 year and 1 month to 9 years ) . the mean age at the time of the operation was 6 years and 10 months ( sd 1 year and 6 months , range 5 years and 9 months to 10 years and1 month ) . the age at the time of sdr neither correlate with the preoperative gmfm-66 ( rho , 0.328 ; p = 0.184 ) nor with the postoperative gmfm-66 scores ( rho , 0.336 ; p = 0.173 ) and the improvement after sdr ( rho , 0.049 ; p = 0.847 ) . we diagnosed pvl in ten patients , hydrocephalus in two patients , and no mri abnormalities in six patients . with respect to the grading of the mr abnormalities , the intrarater agreement was perfect for the frontal and occipital white matter loss , cysts , thinning of the corpus callosum , subarachnoidal space , and gray matter abnormalities ( kappa , 1.0 ) and almost perfect for the ventricular size ( kappa , 0.90 ) and the white matter signal intensities ( kappa , 0.87 ) . it was perfect for the subarachnoidal space and the gray matter abnormalities ( kappa , 1.0 ) , almost perfect for the ventricular size ( kappa , 0.90 ) , substantial for the occipital white matter loss ( kappa , 0.74 ) , and moderate for the frontal white matter loss ( kappa , 0.45 ) . kappa statistics could not be calculated for the white matter signal intensities , cysts , and thinning of the corpus callosum as only one of the two observers graded severe abnormalities ( according grade three on the point scale ) . with respect to the white matter signal intensities and the thinning of the corpus callosum , the observers disagreed in 25% of the scorings , with respect to the cysts there was a disagreement in 36% . the outcome parameters in patients with different mri classification are summarized in table 3 and in fig . the preoperative gmfm-66 scores were significantly higher in patients with normal mri than in patients with pvl ( p = 0.001 ) . patients with hydrocephalus had intermediate scores and did not differ significantly from the other groups . in the follow - up measurements , the mean gmfm-66 was the highest in patients with a normal mri ( group difference , p = 0.003 ; difference between pvl and normal , p = 0.002 ) . almost no improvement was observed in the patients with hydrocephalus ( group difference p = 0.030 ; difference between pvl and hydrocephalus , p = 0.032 ; difference between hydrocephalus and normal mri , p = 0.046 ) . there was a significant group difference in age at the time of the operation ( p = 0.028 ) . patients with normal mri and patients with hydrocephalus were older at the time of the operation than patients with pvl for the patients with normal mri this difference was significant ( p = 0.044 ) . table 3differences of gross motor outcome in with patients with different mri classificationnormal mri ( n = 6)pvl ( n = 10)hydrocephalus ( n = 2)total ( n = 18)p valuemeansdrangemeansdrangemeansdrangemeansdrangegmfm-66 before sdr73.16.765.383.051.95.346.965.062.29.455.668.960.111.546.983.00.002mean gmfm-66 after sdr80.57.667.389.756.35.948.367.862.58.756.368.765.113.048.389.70.003delta - gmfm-667.43.42.012.44.43.21.310.70.30.60.20.75.03.70.212.40.030mri magnetic resonance imaging , pvl periventricular leucomalacia , sd standard deviation , gmfm gross motor function measuresignificant difference with p < 0.05 ( mann whitney test ) between normal mri and pvlsignificant difference with p < 0.05 ( mann whitney test ) between normal mri and hydrocephalussignificant difference with p < 0.05 ( mann whitney test ) between hydrocephalus and pvlfig . 2box plots of the preoperative gmfm-66 ( white boxes ) and the mean values of all postoperative gmfm-66 measurements ( dashed boxes ) in patients with different mri classification . note that the largest improvements after sdr were observed in patients with normal mri and that the patients with hydrocephalus did not improve after sdr differences of gross motor outcome in with patients with different mri classification mri magnetic resonance imaging , pvl periventricular leucomalacia , sd standard deviation , gmfm gross motor function measure significant difference with p < 0.05 ( mann whitney test ) between normal mri and pvl significant difference with p < 0.05 ( mann whitney test ) between normal mri and hydrocephalus significant difference with p < 0.05 ( mann whitney test ) between hydrocephalus and pvl box plots of the preoperative gmfm-66 ( white boxes ) and the mean values of all postoperative gmfm-66 measurements ( dashed boxes ) in patients with different mri classification . note that the largest improvements after sdr were observed in patients with normal mri and that the patients with hydrocephalus did not improve after sdr the scoring of the mri abnormalities in the patients with pvl is summarized in table 4 . two patients with pvl did not show ventricular enlargement , and eight patients had moderate ventricular enlargement . nine patients had moderate occipital white matter loss , and one patient had moderate frontal white matter loss . small cysts were found in one patient , and large cysts were found in two . four patients had no thinning of the corpus callosum , and six patients had moderate thinning of the corpus callosum . none of the patients had gray matter abnormalities or an enlargement of the subarachnoidal space . the mean total score was 10.8 ( sd , 1.1 ; range , 8 to 12 ) . the ventricular size showed a significant correlation with the preoperative gmfm-66 score ( rho , 0.696 ; p = 0.025 ) . however , none of the items in the scoring correlated with the postoperative changes of the gmfm-66 . table 4scoring of the mr abnormalities in patients with periventricular leucomalacia ( n = 10)normalmoderatesevereventricular size2 ( 20%)8 ( 80%)0 ( 0%)white matter signal intensity0 ( 0%)10 ( 100%)0 ( 0%)white matter loss1 ( 10%)9 ( 90%)0 ( 0%)thinning of the corpus callosum4 ( 40%)6 ( 60%)0 ( 0%)cysts7 ( 70%)1 ( 10%)2 ( 20%)gray matter abnormality10 ( 100%)0 ( 0%)0 ( 0%)enlargement of the subarachnoidal space10 ( 100%)0 ( 0%)0 ( 0%)meansdrangetotal score10.81.1812 scoring of the mr abnormalities in patients with periventricular leucomalacia thirty - six patients underwent sdr of whom 32 patients had a brain mri performed before sdr , and 26 mris were available for review . of the patients with available mri , four did not have any preoperative gmfm-66 assessments . three patients had a gmfm-66 assessment before sdr but with a follow - up period of less than 12 months . the detailed patient characteristics of the remaining 19 patients are summarized in table 2 . the mean number of gmfm assessments per patient was 4.9 ( standard deviation ( sd ) 3.5 , range 1 to 12 ) , and the mean follow - up period was 5 years and 4 months ( sd 2 years and 9 months , range 1 year and 1 month to 9 years ) . the mean age at the time of the operation was 6 years and 10 months ( sd 1 year and 6 months , range 5 years and 9 months to 10 years and1 month ) . the age at the time of sdr neither correlate with the preoperative gmfm-66 ( rho , 0.328 ; p = 0.184 ) nor with the postoperative gmfm-66 scores ( rho , 0.336 ; p = 0.173 ) and the improvement after sdr ( rho , 0.049 ; p = 0.847 ) . we diagnosed pvl in ten patients , hydrocephalus in two patients , and no mri abnormalities in six patients . with respect to the grading of the mr abnormalities , the intrarater agreement was perfect for the frontal and occipital white matter loss , cysts , thinning of the corpus callosum , subarachnoidal space , and gray matter abnormalities ( kappa , 1.0 ) and almost perfect for the ventricular size ( kappa , 0.90 ) and the white matter signal intensities ( kappa , 0.87 ) . it was perfect for the subarachnoidal space and the gray matter abnormalities ( kappa , 1.0 ) , almost perfect for the ventricular size ( kappa , 0.90 ) , substantial for the occipital white matter loss ( kappa , 0.74 ) , and moderate for the frontal white matter loss ( kappa , 0.45 ) . kappa statistics could not be calculated for the white matter signal intensities , cysts , and thinning of the corpus callosum as only one of the two observers graded severe abnormalities ( according grade three on the point scale ) . with respect to the white matter signal intensities and the thinning of the corpus callosum , the observers disagreed in 25% of the scorings , with respect to the cysts there was a disagreement in 36% . the outcome parameters in patients with different mri classification are summarized in table 3 and in fig . 2 . the preoperative gmfm-66 scores were significantly higher in patients with normal mri than in patients with pvl ( p = 0.001 ) . patients with hydrocephalus had intermediate scores and did not differ significantly from the other groups . in the follow - up measurements , the mean gmfm-66 was the highest in patients with a normal mri ( group difference , p = 0.003 ; difference between pvl and normal , p = 0.002 ) . almost no improvement was observed in the patients with hydrocephalus ( group difference p = 0.030 ; difference between pvl and hydrocephalus , p = 0.032 ; difference between hydrocephalus and normal mri , p = 0.046 ) . there was a significant group difference in age at the time of the operation ( p = 0.028 ) . patients with normal mri and patients with hydrocephalus were older at the time of the operation than patients with pvl for the patients with normal mri this difference was significant ( p = 0.044 ) . table 3differences of gross motor outcome in with patients with different mri classificationnormal mri ( n = 6)pvl ( n = 10)hydrocephalus ( n = 2)total ( n = 18)p valuemeansdrangemeansdrangemeansdrangemeansdrangegmfm-66 before sdr73.16.765.383.051.95.346.965.062.29.455.668.960.111.546.983.00.002mean gmfm-66 after sdr80.57.667.389.756.35.948.367.862.58.756.368.765.113.048.389.70.003delta - gmfm-667.43.42.012.44.43.21.310.70.30.60.20.75.03.70.212.40.030mri magnetic resonance imaging , pvl periventricular leucomalacia , sd standard deviation , gmfm gross motor function measuresignificant difference with p < 0.05 ( mann whitney test ) between normal mri and pvlsignificant difference with p < 0.05 ( mann whitney test ) between normal mri and hydrocephalussignificant difference with p < 0.05 ( mann whitney test ) between hydrocephalus and pvlfig . 2box plots of the preoperative gmfm-66 ( white boxes ) and the mean values of all postoperative gmfm-66 measurements ( dashed boxes ) in patients with different mri classification . note that the largest improvements after sdr were observed in patients with normal mri and that the patients with hydrocephalus did not improve after sdr differences of gross motor outcome in with patients with different mri classification mri magnetic resonance imaging , pvl periventricular leucomalacia , sd standard deviation , gmfm gross motor function measure significant difference with p < 0.05 ( mann whitney test ) between normal mri and pvl significant difference with p < 0.05 ( mann whitney test ) between normal mri and hydrocephalus significant difference with p < 0.05 ( mann whitney test ) between hydrocephalus and pvl box plots of the preoperative gmfm-66 ( white boxes ) and the mean values of all postoperative gmfm-66 measurements ( dashed boxes ) in patients with different mri classification . note that the largest improvements after sdr were observed in patients with normal mri and that the patients with hydrocephalus did not improve after sdr the scoring of the mri abnormalities in the patients with pvl is summarized in table 4 . two patients with pvl did not show ventricular enlargement , and eight patients had moderate ventricular enlargement . nine patients had moderate occipital white matter loss , and one patient had moderate frontal white matter loss . small cysts were found in one patient , and large cysts were found in two . four patients had no thinning of the corpus callosum , and six patients had moderate thinning of the corpus callosum . none of the patients had gray matter abnormalities or an enlargement of the subarachnoidal space . the mean total score was 10.8 ( sd , 1.1 ; range , 8 to 12 ) . the ventricular size showed a significant correlation with the preoperative gmfm-66 score ( rho , 0.696 ; p = 0.025 ) . however , none of the items in the scoring correlated with the postoperative changes of the gmfm-66 . table 4scoring of the mr abnormalities in patients with periventricular leucomalacia ( n = 10)normalmoderatesevereventricular size2 ( 20%)8 ( 80%)0 ( 0%)white matter signal intensity0 ( 0%)10 ( 100%)0 ( 0%)white matter loss1 ( 10%)9 ( 90%)0 ( 0%)thinning of the corpus callosum4 ( 40%)6 ( 60%)0 ( 0%)cysts7 ( 70%)1 ( 10%)2 ( 20%)gray matter abnormality10 ( 100%)0 ( 0%)0 ( 0%)enlargement of the subarachnoidal space10 ( 100%)0 ( 0%)0 ( 0%)meansdrangetotal score10.81.1812 scoring of the mr abnormalities in patients with periventricular leucomalacia ( n = 10 ) the aim of this study was to identify possible relationships between mr characteristics and the level of functioning before and after sdr in patients with bilateral spastic paresis . we classified the mris in three mri categories : pvl , hydrocephalus , and normal mri and compared the postoperative improvement in the gmfm in the three groups . the best results were found in the group with normal mri , and the poorest results were found in patients with hydrocephalus . the poor outcome of patients with hydrocephalus could be a consequence of persistently elevated intracranial pressure , which can be observed in the absence of characteristic symptoms in children with cp . in the present study , both patients with hydrocephalus ( patient 11 and patient 12 ) underwent surgical correction . patient 12 was diagnosed with hydrocephalus a few months before sdr , and he underwent third ventriculostomy . unfortunately , there was no follow - up imaging available for either of these patients , and although neither of them showed any clinical evidence of elevated intracranial pressure , it can not be ruled out definitively . previous studies found greater postoperative improvements after sdr in children with less motor impairment [ 7 , 13 ] . in contrast , in the three randomized controlled trials comparing functional outcome after sdr , the poorest results were found in the study which included children with the best preoperative gross motor skills [ 9 , 10 ] . in the present study , the patients with normal mri had much better preoperative gross motor skills than the patients with pvl and hydrocephalus . mclaughlin et al . found an inverse correlation between the age at the time of the operation and the postoperative changes in the gmfm . this could be explained as a consequence of faster spontaneous motor development in early childhood . however , in our study , not only the children with hydrocephalus ( who showed the poorest outcome ) , but also the children with normal mri ( who showed the best outcome ) were older than the patients with pvl when sdr was performed . there was no correlation between the age at the time of sdr and outcome . in the patients with pvl , we graded the severity of the mr abnormalities and correlated them with the gross motor abilities . for this purpose , we used a grading scale that has previously been used to describe mr abnormalities in patients with spastic cp [ 20 , 24 ] . several explanations should be considered : it was difficult to detect cysts in an area with extensive gliosis , there is a difference between the level of experience with brain mri in the two investigators ( 2 years vs 8 years ) . correlations between ventricular size and gross motor abilities have been described previously in patients with spastic cp . in contrast to the findings of previous studies [ 20 , 24 , 26 ] , we did not find a correlation between gross motor skills and the total mri score / the thinning of the corpus callosum . however , the present study was limited to ambulatory patients with gmfcs levels i to iii , and patients with more severe motor handicaps where more severe brain abnormalities could be expected on mri have not been studied . no correlations were found between the severity of the brain anomalies and the outcome after sdr . the outcome assessment in the present study consisted in the gmfm-66 which according to the international classification of functioning , disability , and healths ( icf ) assesses the domain of activity . however , the other two domains of the icf body structure and participation have not been evaluated . unfortunately , we did not have access to all mri scans and not all patients had a preoperative gmfm assessment . we could only include data on a small number of patients in our analysis . notably , the group with hydrocephalus was small and comprised only two patients . therefore , the association between the type of brain lesion and the outcome after sdr needs to be proved in future studies with larger study samples , including the other icf domains body structure and participation . we found significant differences in the post - operative changes in the gmfm-66 in patients with different brain mri abnormalities . the largest postoperative improvement was observed in patients with normal mri , and the poorest outcome was observed in patients with hydrocephalus . in patients with pvl , we could not detect any relation between mri abnormalities and the postoperative improvement in of gross motor function after sdr . we conclude that mri of the brain can provide additional information for the selection of patients for sdr . however , the degree of pvl does not provide information about the degree of improvement in gross motor function after sdr .

purposeto identify mri characteristics that may predict the functional effect of selective dorsal rhizotomy ( sdr ) in children with bilateral spastic paresis.methodswe performed sdr in a group of 36 patients . the gross motor functioning measure-66 ( gmfm-66 ) was applied before and after sdr . available cerebral mris were retrospectively classified into three diagnostic groups : periventricular leucomalacia ( pvl ; n = 10 ) , hydrocephalus ( n = 2 ) , and normal ( n = 6 ) . in patients with pvl , we scored the severity of the mr abnormalities . we compared the changes in the gmfm-66 after sdr in the diagnostic groups . in patients with pvl , we correlated the severity of the mr abnormalities with the changes in the gmfm-66.resultsthe mean follow - up period was 5 years and 4 months ( range , 1 year and 1 month to 9 years ) . the best improvement in gross motor function was observed in patients with normal mri , and the slightest improvement was observed in patients with hydrocephalus . the severity of the pvl did correlate with the gmfm-66 score before sdr but not with the functional effect of sdr.conclusionwe conclude that with respect to gross motor skills , the improvements after sdr are good in patients with no mri abnormalities . in the patients with hydrocephalus , the improvements after sdr were insignificant . in patients with pvl , the improvements were intermediate and did not correlate with the degree of pvl .

Introduction Methods and materials Subjects Procedure Neuroimaging Outcome measure Statistical analysis Results Clinical characteristics and level of functioning Neuroimaging Discussion Conclusion

selective dorsal rhizotomy ( sdr ) is a neurosurgical treatment that is mainly performed at lumbar level in patients with bilateral spastic paresis . in children with spastic cp , sdr leads to a more normal gait pattern [ 36 ] , better performance of the activities of daily life [ 7 , 8 ] , and improvement in gross motor function [ 613 ] . in patients with bilateral spastic cp , the most common mri abnormality is periventricular leucomalacia ( pvl ) , which is characterized by a damage of the periventricular white matter [ 1619 ] . in the present study , , we included data on patients who underwent pre- and postoperative gross motor function measure ( gmfm-66 ) analysis with a follow - up period of at least 12 months and of whom mri was available for review . imaging was classified into three diagnostic groups according to the following criteria : periventricular leucomalacia ( pvl ; increased signal intensity of the periventricular white matter in t2 weighted imaging and/or flair ; other abnormalities optional ) ; hydrocephalus ( signs of ventricular dilatation and increased intracranial pressure ) ; normal mri ( see fig . in the patients with pvl , we graded the severity of their mri abnormalities based on the work of cioni et al . 1i ) are enlargedtable 2summary of the characteristics of the patients included in the studycasegenderga ( weeks)bw ( g)age sdr years ( mts)follow - up years ( mts)mri classificationdiagnosisgmfm-66 before sdrgmfm-66 ( mean ) after sdr1f302,0005 ( 7)8 ( 5)pvlpvl50.8553.292f402,5005(7)9 ( 0)pvlpvl47.6849.663f403,4505 ( 8)8 ( 11)pvlpvl54.3855.714m301,6506 ( 8)8 ( 11)pvlpvl46.9148.325f281,2854 ( 11)7 ( 10)pvlpvl50.3254.756m261,0005 ( 3)7 ( 10)pvlpvl64.9867.827m322,5105 ( 1)6 ( 7)pvlpvl54.1560.388m331,2658 ( 9)5 ( 1)pvlpvl47.0955.929m268706 ( 11)3 ( 11)pvlpvl50.0954.3210m267805 ( 4)3 ( 0)pvlpvl52.3262.9811f413,0608 ( 4)5 ( 2)hydrocephaluscongenital hydrocephalus55.6256.3512m271,02010 ( 1)4 ( 5)hydrocephaluscongenital hydrocephalus68.8668.6913m403,3005 ( 9)7 ( 2)normalunknown65.3367.3114m343,1556 ( 11)3 ( 0)normalspinal process76.7585.1515m384,2808 ( 0)2 ( 7)normalunknown82.9989.7016f403,8758 ( 10)1 ( 7)normalhiv - encephalo(myelo)pathy73.6380.4617m394,0406 ( 7)2 ( 0)normalunknown65.9878.3818m403,4606 ( 5)1 ( 1)normalunknown73.6381.9319m373,4003 ( 11)7 ( 1)not classifiedlaurence moon syndrome47.2683.01f female , m male , ga gestational age , bw birth weight , sdr selective dorsal rhizotomy , gmfcs gross motor function classification system , gmfm gross motor function measure , pvl periventricular leucomalacia , g gram , mts months a b , e , h transversal t2 weighted images at the level of the centrum semiovale . 1i ) are enlarged summary of the characteristics of the patients included in the study f female , m male , ga gestational age , bw birth weight , sdr selective dorsal rhizotomy , gmfcs gross motor function classification system , gmfm gross motor function measure , pvl periventricular leucomalacia , g gram , mts months all patients had a detailed pre- and postoperative clinical evaluation , including spasticity assessment , range of motion of single joints , gait analysis , and the gmfm . for the comparison of the outcome in patients with different mri characteristics , we used nonparametric tests ( kruskal walis test for group comparison ; post hoc analysis was performed using the mann this study was part of the project : investigation of long - term effects of sdr in children with spastic diplegia and was approved by the medical ethical committee of the vu university medical center , amsterdam , the netherlands ( project number 2006/105 ) . in the present study , , we included data on patients who underwent pre- and postoperative gross motor function measure ( gmfm-66 ) analysis with a follow - up period of at least 12 months and of whom mri was available for review . imaging was classified into three diagnostic groups according to the following criteria : periventricular leucomalacia ( pvl ; increased signal intensity of the periventricular white matter in t2 weighted imaging and/or flair ; other abnormalities optional ) ; hydrocephalus ( signs of ventricular dilatation and increased intracranial pressure ) ; normal mri ( see fig . in the patients with pvl , we graded the severity of their mri abnormalities based on the work of cioni et al . 1i ) are enlargedtable 2summary of the characteristics of the patients included in the studycasegenderga ( weeks)bw ( g)age sdr years ( mts)follow - up years ( mts)mri classificationdiagnosisgmfm-66 before sdrgmfm-66 ( mean ) after sdr1f302,0005 ( 7)8 ( 5)pvlpvl50.8553.292f402,5005(7)9 ( 0)pvlpvl47.6849.663f403,4505 ( 8)8 ( 11)pvlpvl54.3855.714m301,6506 ( 8)8 ( 11)pvlpvl46.9148.325f281,2854 ( 11)7 ( 10)pvlpvl50.3254.756m261,0005 ( 3)7 ( 10)pvlpvl64.9867.827m322,5105 ( 1)6 ( 7)pvlpvl54.1560.388m331,2658 ( 9)5 ( 1)pvlpvl47.0955.929m268706 ( 11)3 ( 11)pvlpvl50.0954.3210m267805 ( 4)3 ( 0)pvlpvl52.3262.9811f413,0608 ( 4)5 ( 2)hydrocephaluscongenital hydrocephalus55.6256.3512m271,02010 ( 1)4 ( 5)hydrocephaluscongenital hydrocephalus68.8668.6913m403,3005 ( 9)7 ( 2)normalunknown65.3367.3114m343,1556 ( 11)3 ( 0)normalspinal process76.7585.1515m384,2808 ( 0)2 ( 7)normalunknown82.9989.7016f403,8758 ( 10)1 ( 7)normalhiv - encephalo(myelo)pathy73.6380.4617m394,0406 ( 7)2 ( 0)normalunknown65.9878.3818m403,4606 ( 5)1 ( 1)normalunknown73.6381.9319m373,4003 ( 11)7 ( 1)not classifiedlaurence moon syndrome47.2683.01f female , m male , ga gestational age , bw birth weight , sdr selective dorsal rhizotomy , gmfcs gross motor function classification system , gmfm gross motor function measure , pvl periventricular leucomalacia , g gram , mts months a i mr imaging of three patients with bilateral spastic paresis undergoing sdr . 1i ) are enlarged summary of the characteristics of the patients included in the study f female , m male , ga gestational age , bw birth weight , sdr selective dorsal rhizotomy , gmfcs gross motor function classification system , gmfm gross motor function measure , pvl periventricular leucomalacia , g gram , mts months all patients had a detailed pre- and postoperative clinical evaluation , including spasticity assessment , range of motion of single joints , gait analysis , and the gmfm . for the comparison of the outcome in patients with different mri characteristics , we used nonparametric tests ( kruskal walis test for group comparison ; post hoc analysis was performed using the mann thirty - six patients underwent sdr of whom 32 patients had a brain mri performed before sdr , and 26 mris were available for review . the mean number of gmfm assessments per patient was 4.9 ( standard deviation ( sd ) 3.5 , range 1 to 12 ) , and the mean follow - up period was 5 years and 4 months ( sd 2 years and 9 months , range 1 year and 1 month to 9 years ) . with respect to the grading of the mr abnormalities , the intrarater agreement was perfect for the frontal and occipital white matter loss , cysts , thinning of the corpus callosum , subarachnoidal space , and gray matter abnormalities ( kappa , 1.0 ) and almost perfect for the ventricular size ( kappa , 0.90 ) and the white matter signal intensities ( kappa , 0.87 ) . in the follow - up measurements , the mean gmfm-66 was the highest in patients with a normal mri ( group difference , p = 0.003 ; difference between pvl and normal , p = 0.002 ) . almost no improvement was observed in the patients with hydrocephalus ( group difference p = 0.030 ; difference between pvl and hydrocephalus , p = 0.032 ; difference between hydrocephalus and normal mri , p = 0.046 ) . note that the largest improvements after sdr were observed in patients with normal mri and that the patients with hydrocephalus did not improve after sdr differences of gross motor outcome in with patients with different mri classification mri magnetic resonance imaging , pvl periventricular leucomalacia , sd standard deviation , gmfm gross motor function measure significant difference with p < 0.05 ( mann whitney test ) between normal mri and pvl significant difference with p < 0.05 ( mann whitney test ) between normal mri and hydrocephalus significant difference with p < 0.05 ( mann whitney test ) between hydrocephalus and pvl box plots of the preoperative gmfm-66 ( white boxes ) and the mean values of all postoperative gmfm-66 measurements ( dashed boxes ) in patients with different mri classification . note that the largest improvements after sdr were observed in patients with normal mri and that the patients with hydrocephalus did not improve after sdr the scoring of the mri abnormalities in the patients with pvl is summarized in table 4 . table 4scoring of the mr abnormalities in patients with periventricular leucomalacia ( n = 10)normalmoderatesevereventricular size2 ( 20%)8 ( 80%)0 ( 0%)white matter signal intensity0 ( 0%)10 ( 100%)0 ( 0%)white matter loss1 ( 10%)9 ( 90%)0 ( 0%)thinning of the corpus callosum4 ( 40%)6 ( 60%)0 ( 0%)cysts7 ( 70%)1 ( 10%)2 ( 20%)gray matter abnormality10 ( 100%)0 ( 0%)0 ( 0%)enlargement of the subarachnoidal space10 ( 100%)0 ( 0%)0 ( 0%)meansdrangetotal score10.81.1812 scoring of the mr abnormalities in patients with periventricular leucomalacia thirty - six patients underwent sdr of whom 32 patients had a brain mri performed before sdr , and 26 mris were available for review . the mean number of gmfm assessments per patient was 4.9 ( standard deviation ( sd ) 3.5 , range 1 to 12 ) , and the mean follow - up period was 5 years and 4 months ( sd 2 years and 9 months , range 1 year and 1 month to 9 years ) . with respect to the grading of the mr abnormalities , the intrarater agreement was perfect for the frontal and occipital white matter loss , cysts , thinning of the corpus callosum , subarachnoidal space , and gray matter abnormalities ( kappa , 1.0 ) and almost perfect for the ventricular size ( kappa , 0.90 ) and the white matter signal intensities ( kappa , 0.87 ) . in the follow - up measurements , the mean gmfm-66 was the highest in patients with a normal mri ( group difference , p = 0.003 ; difference between pvl and normal , p = 0.002 ) . almost no improvement was observed in the patients with hydrocephalus ( group difference p = 0.030 ; difference between pvl and hydrocephalus , p = 0.032 ; difference between hydrocephalus and normal mri , p = 0.046 ) . table 3differences of gross motor outcome in with patients with different mri classificationnormal mri ( n = 6)pvl ( n = 10)hydrocephalus ( n = 2)total ( n = 18)p valuemeansdrangemeansdrangemeansdrangemeansdrangegmfm-66 before sdr73.16.765.383.051.95.346.965.062.29.455.668.960.111.546.983.00.002mean gmfm-66 after sdr80.57.667.389.756.35.948.367.862.58.756.368.765.113.048.389.70.003delta - gmfm-667.43.42.012.44.43.21.310.70.30.60.20.75.03.70.212.40.030mri magnetic resonance imaging , pvl periventricular leucomalacia , sd standard deviation , gmfm gross motor function measuresignificant difference with p < 0.05 ( mann whitney test ) between normal mri and pvlsignificant difference with p < 0.05 ( mann whitney test ) between normal mri and hydrocephalussignificant difference with p < 0.05 ( mann whitney test ) between hydrocephalus and pvlfig . note that the largest improvements after sdr were observed in patients with normal mri and that the patients with hydrocephalus did not improve after sdr differences of gross motor outcome in with patients with different mri classification mri magnetic resonance imaging , pvl periventricular leucomalacia , sd standard deviation , gmfm gross motor function measure significant difference with p < 0.05 ( mann whitney test ) between normal mri and pvl significant difference with p < 0.05 ( mann whitney test ) between normal mri and hydrocephalus significant difference with p < 0.05 ( mann whitney test ) between hydrocephalus and pvl box plots of the preoperative gmfm-66 ( white boxes ) and the mean values of all postoperative gmfm-66 measurements ( dashed boxes ) in patients with different mri classification . note that the largest improvements after sdr were observed in patients with normal mri and that the patients with hydrocephalus did not improve after sdr the scoring of the mri abnormalities in the patients with pvl is summarized in table 4 . table 4scoring of the mr abnormalities in patients with periventricular leucomalacia ( n = 10)normalmoderatesevereventricular size2 ( 20%)8 ( 80%)0 ( 0%)white matter signal intensity0 ( 0%)10 ( 100%)0 ( 0%)white matter loss1 ( 10%)9 ( 90%)0 ( 0%)thinning of the corpus callosum4 ( 40%)6 ( 60%)0 ( 0%)cysts7 ( 70%)1 ( 10%)2 ( 20%)gray matter abnormality10 ( 100%)0 ( 0%)0 ( 0%)enlargement of the subarachnoidal space10 ( 100%)0 ( 0%)0 ( 0%)meansdrangetotal score10.81.1812 scoring of the mr abnormalities in patients with periventricular leucomalacia ( n = 10 ) the aim of this study was to identify possible relationships between mr characteristics and the level of functioning before and after sdr in patients with bilateral spastic paresis . we classified the mris in three mri categories : pvl , hydrocephalus , and normal mri and compared the postoperative improvement in the gmfm in the three groups . the best results were found in the group with normal mri , and the poorest results were found in patients with hydrocephalus . in contrast , in the three randomized controlled trials comparing functional outcome after sdr , the poorest results were found in the study which included children with the best preoperative gross motor skills [ 9 , 10 ] . in the present study , the patients with normal mri had much better preoperative gross motor skills than the patients with pvl and hydrocephalus . however , in our study , not only the children with hydrocephalus ( who showed the poorest outcome ) , but also the children with normal mri ( who showed the best outcome ) were older than the patients with pvl when sdr was performed . in the patients with pvl , we graded the severity of the mr abnormalities and correlated them with the gross motor abilities . the largest postoperative improvement was observed in patients with normal mri , and the poorest outcome was observed in patients with hydrocephalus . in patients with pvl , we could not detect any relation between mri abnormalities and the postoperative improvement in of gross motor function after sdr . however , the degree of pvl does not provide information about the degree of improvement in gross motor function after sdr .

in 2007 , 246 million people had diabetes worldwide , and its prevalence is expected to continue increasing . a close relationship between type 2 diabetes and the development of atherosclerosis exists , and type 2 diabetes is associated with a two to fourfold increase in coronary artery disease ( cad ) . in fact , cardiovascular disease is the leading cause of death in this patient population . myocardial ischemia in patients with diabetes is often asymptomatic and frequently in an advanced stage when it becomes clinically manifest . once cad is symptomatic in patients with diabetes , morbidity and mortality are high and significantly worse than those in patients without diabetes . noninvasive tests , including electrocardiogram ( ecg ) , echocardiography and myocardial perfusion scintigraphy , have been used to detect cad in diabetic patients . nonetheless , after normal findings of the tests , elevated event rates are still observed in diabetic patients compared with nondiabetic individuals . furthermore , direct visualization of the coronary arteries is preferred because patients with diabetes frequently have diffuse , multivessel cad . although conventional angiography is carried out to evaluate the presence and extent of cad , this is an invasive approach associated with a minimal but definitive risk of complications . therefore , noninvasive techniques , which are capable of directly visualizing the coronary arteries , are required for further refinement of prognostication in diabetes patients . recently , contrastenhanced multidetector computed tomography ( mdct ) coronary angiography has been shown to be a tool for noninvasive visualization of the coronary arteries . mdct permits the detection of coronary lesions with high sensitivity and specificity , which could be valuable for preventing risk during examination . the purpose of the present study was to evaluate the indication of mdct for the screening of cad , especially asymptomatic cad , and to identify the predictors of cad in japanese patients with type 2 diabetes . between june 2006 and december 2007 , 56 patients with type 2 diabetes were enrolled for the present study . the inclusion criteria were : ( i ) diabetes was diagnosed according to the american diabetes association criteria ; ( ii ) absence of a history of myocardial infarction ; ( iii ) absence of renal failure ( serum creatinine > 1.5 mg / dl [ 114 mol / l ] ) ; and ( iv ) absence of an allergic history to iodinated contrast media . four patients were excluded because they manifested arrhythmia or tachycardia of more than 75 b.p.m . ( n = 3 ) , and severe calcification of coronary arteries that caused blooming artifacts and obscured over 75% of the entire vessel lumen in the proximal segment of the coronary artery ( n = 1 ) . consequently , 52 patients were available for the assessment , they were 37 men and 15 women ranging in age from 34 to 81 years ( mean 66.2 11.8 years ) . the present study was approved by the ethical committee of kumamoto university school of medicine . physical examinations were carried , and blood and urine samples were obtained from patients for laboratory testing . a resting 12lead ecg was recorded , and we determined qwave myocardial infarction , ischemic stsegment change ( horizontal and downsloping stsegment depression of over 0.3 mv or stsegment elevations of more than 0.1 mv ) or twave change were determined to be ischemic change positive. anklebrachial blood pressure index ( abi ) and pulse wave velocity ( pwv ) were measured using an automatic waveform analyzer ( bp203rpeii , colin , komaki , japan ) . ultrasonographic scanning of the carotid arteries was carried out using an echotomographic system ( sdu2200 , shimadzu , kyoto , japan ) and carotid intimamedia thickness ( imt ) was measured as described previously . echocardiography ( vivid7 , gevingmed , milwaukee , wi , usa and ssa770a , toshiba medical systems , tokyo , japan ) was also carried out . wall motion was analyzed using the 17segment model and evaluated by a fourpoint scale according to the guideline . each patient received an additional oral betablocker ( metoprolol , 20 mg , single dose ) 23 h before examination and 0.3 mg of nitroglycerin ( nitropen , nippon kayaku , tokyo , japan ) sublingually 5 min before scanning . all patients were scanned in the supine position during a single breathhold with inspiration during scanning on a 64detector computed tomography ( ct ) scanner ( brilliance64 , philips medical systems , cleveland , oh , usa ) . in all patients , iohexol350 ( omnipaque350 , daiichisankyo , tokyo , japan ) was delivered through a 20gauge catheter inserted into an antecubital vein and a power injector ( autoenhance a250 , nemoto kyorindo , tokyo , japan ) . we used the testbolus technique to synchronize the arrival of contrast media ( cm ) . this technique is based on the intravenous injection of a small amount ( 10 ml ) of contrast material during the acquisition of a series of dynamic lowdose ( 120 kv , 20 mas ) monitoring scans at the level of the ascending aorta . the time interval between each monitoring scan acquisition was 1 s. acquisition of the dynamic monitoring scans started 5 s after the beginning of the injection of intravenous contrast material ( 10 ml of cm injected at 5 ml / s ) . a region of interest ( roi ) was drawn inside the ascending aorta to generate an enhancement curve ( generated by test injection bolus timing application , philips medical systems ) , which showed the time needed to reach the peak of maximum enhancement for the testbolus . we selected the delay applied for angiographic scanning as 3 s after the time of peak enhancement at the testbolus in the ascending aorta . our contrast injection protocol was a patient bodyweight ( bw)tailored small contrast dose protocol , 0.7 ml / kg bw of cm at a fixed injection duration of 9 s. the scan parameters were as follows : detector collimation 64 0.625 mm , 11.9 mm / s table feed , 0.20 helical pitch ( beam pitch ) , 420 msec tube rotation time , 120 kv tube voltage , 900 mas tube current timeproduct . depending on the cardiac dimensions , the scanning time varied from 6 to 8 s. image reconstruction was in a 16.520.0cm display fieldofview depending on the patient s physique . all scans started at the upper end of the coronary sinus in a craniocaudal direction . we reconstructed axial images with a section thickness of 0.67 mm , a section interval of 0.33 mm , and a 16.520.0cm display fieldofview depending on the patient s physique using a medium cardiac kernel ( xcb ) with ecg gating . initially , a single data set was reconstructed during the middiastolic phase ( 75% of the rwave to rwave interval ) . in cases with unsatisfactory image quality , image reconstruction of the raw data was carried out at 0 , 10 , 20 , 30 , 40 , 50 , 60 , 70 , 80 and 90% of the rwave to rwave interval to improve the image quality of all available coronary segments . together , two boardcertified radiologists ( tn and ka ) with 5 and 7 years of experience interpreting cardiac ct analyzed the generated images on the same workstation ( zaio . coronary arteries were divided into 17 segments according to the modified american heart association classification . the presence of coronary lesions was evaluated visually using a volume rendering view , angiographic view , curved multiplanar reconstructions and a crosssectional image . plaques were classified as stenosis and no stenosis using a 75% threshold of luminal narrowing , and one coronary plaque was assigned per coronary segment . values of p < 0.05 were considered to show statistically relevant differences . a forward stepwise logisticregression procedure was then carried out to adjust cad risk factors with the use of covariates that were found to be significant predictors of mdct detecting stenosis . furthermore , to identify the threshold value of each significant predictor , twotailed test was used for each variable and the odds ratios were calculated by crosstabulation . we carried out further receiver operating characteristics ( roc ) curve analyses to evaluate the sensitivity and specificity of imt and the duration of diabetes on mdct detecting stenosis . statistical analyses were carried out using computer software ( sigmastat for windows version 3.5 , systat software , chicago , il , usa ) . between june 2006 and december 2007 , 56 patients with type 2 diabetes were enrolled for the present study . the inclusion criteria were : ( i ) diabetes was diagnosed according to the american diabetes association criteria ; ( ii ) absence of a history of myocardial infarction ; ( iii ) absence of renal failure ( serum creatinine > 1.5 mg / dl [ 114 mol / l ] ) ; and ( iv ) absence of an allergic history to iodinated contrast media . four patients were excluded because they manifested arrhythmia or tachycardia of more than 75 b.p.m . ( n = 3 ) , and severe calcification of coronary arteries that caused blooming artifacts and obscured over 75% of the entire vessel lumen in the proximal segment of the coronary artery ( n = 1 ) . consequently , 52 patients were available for the assessment , they were 37 men and 15 women ranging in age from 34 to 81 years ( mean 66.2 11.8 years ) . the present study was approved by the ethical committee of kumamoto university school of medicine . physical examinations were carried , and blood and urine samples were obtained from patients for laboratory testing . a resting 12lead ecg was recorded , and we determined qwave myocardial infarction , ischemic stsegment change ( horizontal and downsloping stsegment depression of over 0.3 mv or stsegment elevations of more than 0.1 mv ) or twave change were determined to be ischemic change positive. anklebrachial blood pressure index ( abi ) and pulse wave velocity ( pwv ) were measured using an automatic waveform analyzer ( bp203rpeii , colin , komaki , japan ) . ultrasonographic scanning of the carotid arteries was carried out using an echotomographic system ( sdu2200 , shimadzu , kyoto , japan ) and carotid intimamedia thickness ( imt ) was measured as described previously . echocardiography ( vivid7 , gevingmed , milwaukee , wi , usa and ssa770a , toshiba medical systems , tokyo , japan ) was also carried out . wall motion was analyzed using the 17segment model and evaluated by a fourpoint scale according to the guideline . each patient received an additional oral betablocker ( metoprolol , 20 mg , single dose ) 23 h before examination and 0.3 mg of nitroglycerin ( nitropen , nippon kayaku , tokyo , japan ) sublingually 5 min before scanning . all patients were scanned in the supine position during a single breathhold with inspiration during scanning on a 64detector computed tomography ( ct ) scanner ( brilliance64 , philips medical systems , cleveland , oh , usa ) . in all patients , iohexol350 ( omnipaque350 , daiichisankyo , tokyo , japan ) was delivered through a 20gauge catheter inserted into an antecubital vein and a power injector ( autoenhance a250 , nemoto kyorindo , tokyo , japan ) . we used the testbolus technique to synchronize the arrival of contrast media ( cm ) . this technique is based on the intravenous injection of a small amount ( 10 ml ) of contrast material during the acquisition of a series of dynamic lowdose ( 120 kv , 20 mas ) monitoring scans at the level of the ascending aorta . the time interval between each monitoring scan acquisition was 1 s. acquisition of the dynamic monitoring scans started 5 s after the beginning of the injection of intravenous contrast material ( 10 ml of cm injected at 5 ml / s ) . a region of interest ( roi ) was drawn inside the ascending aorta to generate an enhancement curve ( generated by test injection bolus timing application , philips medical systems ) , which showed the time needed to reach the peak of maximum enhancement for the testbolus . we selected the delay applied for angiographic scanning as 3 s after the time of peak enhancement at the testbolus in the ascending aorta . our contrast injection protocol was a patient bodyweight ( bw)tailored small contrast dose protocol , 0.7 ml / kg bw of cm at a fixed injection duration of 9 s. the scan parameters were as follows : detector collimation 64 0.625 mm , 11.9 mm / s table feed , 0.20 helical pitch ( beam pitch ) , 420 msec tube rotation time , 120 kv tube voltage , 900 mas tube current timeproduct . depending on the cardiac dimensions , the scanning time varied from 6 to 8 s. image reconstruction was in a 16.520.0cm display fieldofview depending on the patient s physique . all scans started at the upper end of the coronary sinus in a craniocaudal direction . we reconstructed axial images with a section thickness of 0.67 mm , a section interval of 0.33 mm , and a 16.520.0cm display fieldofview depending on the patient s physique using a medium cardiac kernel ( xcb ) with ecg gating . initially , a single data set was reconstructed during the middiastolic phase ( 75% of the rwave to rwave interval ) . in cases with unsatisfactory image quality , image reconstruction of the raw data was carried out at 0 , 10 , 20 , 30 , 40 , 50 , 60 , 70 , 80 and 90% of the rwave to rwave interval to improve the image quality of all available coronary segments . together , two boardcertified radiologists ( tn and ka ) with 5 and 7 years of experience interpreting cardiac ct analyzed the generated images on the same workstation ( zaio . coronary arteries were divided into 17 segments according to the modified american heart association classification . the presence of coronary lesions was evaluated visually using a volume rendering view , angiographic view , curved multiplanar reconstructions and a crosssectional image . plaques were classified as stenosis and no stenosis using a 75% threshold of luminal narrowing , and one coronary plaque was assigned per coronary segment . a forward stepwise logisticregression procedure was then carried out to adjust cad risk factors with the use of covariates that were found to be significant predictors of mdct detecting stenosis . furthermore , to identify the threshold value of each significant predictor , twotailed test was used for each variable and the odds ratios were calculated by crosstabulation . we carried out further receiver operating characteristics ( roc ) curve analyses to evaluate the sensitivity and specificity of imt and the duration of diabetes on mdct detecting stenosis . statistical analyses were carried out using computer software ( sigmastat for windows version 3.5 , systat software , chicago , il , usa ) . the study group consisted of 52 patients with type 2 diabetes ( age 66.2 11.8 years ; 37 men ; bmi 24.7 4.2 ; glycated hemoglobin [ hba1c ] 7.9 1.7% ) . the average duration of diabetes was 15.9 10.8 years at the time of mdct . data are mean se or n. hdl , high density lipoprotein ; ldl , low density lipoprotein . in mdct , of the 19 patients , onevessel disease was identified in 12 patients , twovessel disease in four patients , and threevessel disease in three patients . accidents during mdct or sideeffects as a result of contrast material were not detected in the present study . the correlation between mdct and symptoms , ecg , and echocardiography are provided in tables 2 and 3 . of the 19 patients who had stenosis detected by mdct , eight patients ( 42% ) were identified with neither a positive ischemic change in ecg nor a wall motion abnormality in echocardiography ( table 2 ) . thirteen patients ( 25% of 52 patients ) who had no symptoms of ischemic heart disease ( ihd ) had coronary artery stenosis detected by mdct , indicating the presence of silent myocardial ischemia . more importantly , of the 19 patients with stenosis , seven patients ( 37% ) had no symptoms , including chest pain , nor ischemic changes in ecg ( table 3 ) . these data indicate the high prevalence of asymptomatic ischemia in diabetes patients and the usefulness of mdct for the screening of silent ischemic heart disease in diabetes patients . ecg , electrocardiogram ; lv , left ventricular ; mdct , multidetector computed tomography results . ecg , electrocardiogram ; mdct , multidetector computed tomography . the comparison of the patient characteristics and clinical variables between mdct stenosis detection and no mdct stenosis detection are presented in table 4 . interestingly , there were no significant differences in age ( 69 8 years in stenosis vs 64 13 years in no stenosis , p = 0.061 ) , blood pressure ( 131 22/72 13 mmhg vs 133 20/72 11 mmhg ) , serum low density lipoprotein ( ldl)cholesterol ( 123 44 mg / dl vs 126 37 mg / dl , p = 0.388 ) , serum high density lipoprotein ( hdl)cholesterol ( 55 14 mg / dl vs 51 15 mg / dl , p = 0.195 ) , urinary albumin excretion ( 80.7 149.6 mg / g creatinin vs 83.1 167.2 mg / g creatinin , p = 0.481 ) , estimated glomerular filtration rate ( egfr ) ( 64.9 25.3 ml / min/1.73mvs 74.8 20.6 ml / min/1.73 m , p = 0.066 ) and pwv ( 1917 323 cm / s vs 1899 433 cm / s , p = 0.411 ) between the patients with stenosis and those with no stenosis . significant differences were detected in mean imt ( 1.21 0.44 mm in stenosis vs 0.95 0.26 mm in no stenosis , p = 0.007 ) and duration of diabetes ( 20 11 years vs 13 10 years , p = 0.008 ) . data are mean se and n. * p < 0.05 by unpaired ttest and mann abi , ankle brachial index ; egfr , estimated glomerular filtration rate ; hdl , high density lipoprotein ; imt , intimamedia thickness ; ldl , low density lipoprotein ; mdct , multidetector computed tomography ; pwv , pulse wave velocity . to identify the correlation between the presence of stenosis of coronary arteries on mdct and cad risk factors although a significant correlation was not found between mdct detecting stenosis and age , bmi , hba1c , egfr , diastolic blood pressure , serum ldl and hdl cholesterol concentrations , triglyceride , urinemicroalbumine , abi or pwv , the correlation of mdct detecting stenosis and duration of diabetes , medication of statin and meanimt of carotid artery remained statistically significant after correction for baseline characteristics . the dependent variable of mdct detecting stenosis can be predicted from a linear combination of the independent variables as follows : ( i ) duration of diabetes ( p = 0.004 ) , ( ii ) treatment with statin ( p = 0.029 ) ; and ( iii ) mean imt ( p = 0.023 ) . p = 0.011 ) in duration of diabetes ( per year ) , 9.867 ( 95% ci 1.65558.882 , p = 0.012 ) in treatment with statin , 1.359 ( 95% ci 1.0181.814 , p = 0.038 ) in mean imt ( per 0.1 mm ) by multiple logistic regression analysis . in the present study , it was shown that symptomatic or asymptomatic cad in type 2 diabetes patients can be diagnosed noninvasively by the use of mdct . the population of the present study group was considered to be representative of japanese diabetic patients , although the bmi had a tendency to be lower compared with other studies of caucasian patients . the patients in the present study were receiving contemporary medical treatment and were under almost reasonable metabolic control ( blood pressure 132 21/72 12 mmhg , ldlcholesterol 124 39 mg / dl , hdlcholesterol 52 15 mg / dl , triglyceride 133 94 mg / dl ) , although hba1c ( 7.9 1.7% ) was considered to be higher . previous studies reported that both sensitivity and specificity of mdct were high enough for the diagnosis of coronary artery stenosis to be made . continuous modification of hardware , scan protocol and renewing scanner generation has led to a significant stabilization and improvement of image quality . recently , studies using 64slice scanners have been reported , showing a more accurate assessment for the diagnosis of cad and characteristics of plaque . however , several general limitations of mdct , including the administration of an iodinated contrast agent and elevated radiation dose , should be mentioned . in the present study , we generated a protocol to carry out mdct with less iodinated agent and lower radiation exposure . in the present study group , 13 patients ( 25.0% of 52 patients ) who had no symptoms of ihd had coronary artery stenosis detected by mdct . wackers et al . indicated that 133/522 patients with diabetes ( 25.5% ) were diagnosed with silent myocardial ischemia using adenosine technetium99 m sestamibi single photon emissioncomputed tomography myocardial perfusion imaging . the prevalence of mdct detecting stenosis detection without ihd symptoms in the present study was similar to those of previous reports , suggesting that mdct is useful in screening for silent ischemia in patients with diabetes . furthermore , of 19 patients who had coronary artery stenosis detected by mdct , seven patients had neither positive ischemic change in rest ecg nor symptoms of ihd . however , dewey et al . reported that both sensitivity and specificity of mdct were significantly higher than those of exerciseecg . it was also reported that exercise ecg had a certain risk , the most relevant being myocardial infarction or death which have been confirmed in multiple surveys to occur in approximately 10/10 000 tests . taken together , it is suggested that the mdct is effective for the screening of cad , especially silent myocardial ischemia . it was reported that the incidences of coronary heart disease ( chd ) , per 1000 patients per year , among japanese diabetes patients were 9.8 in men and 5.5 in women , although that of chd among caucasian diabetes patients was 17.4 . the prevalence of cad in type 2 diabetes in the caucasian population has been reported to be 3040% . in the present study , even the incidence of chd in japanese type 2 diabetes patients was much lower than that of caucasian patients , as 19/52 patients ( 36.5% ) had coronary artery stenosis detected by mdct . therefore , it is thought that mdct might detect more cad in type 2 diabetes patients in the caucasian population . another important finding of the present study is the assessment of the predictors of mdct detecting stenosis in diabetes patients . in the guidelines for early detection of chd in asymptomatic patients with diabetes from the american diabetes association ( ada ) , the presence of multiple cardiovascular risk factors including ldlcholesterol , hdlcholesterol , blood pressure , micro/macroalbuminuria is mentioned . in the analysis of risk factors that contribute to chd risk in diabetic patients in the united kingdom prospective diabetes study , ldlcholesterol , hdlcholesterol , blood pressure and hba1c were reported to be important . however , in the present study , significant differences were not detected in blood pressure , ldlcholesterol , hdlcholesterol , triglyceride , blood glucose , hba1c , microalbuminuria , abi or pwv between the mdct detected stenosis and mdct detecting no stenosis groups . the reason why there is no significant difference in these markers between mdct detecting stenosis and mdct detecting no stenosis groups might be a result of the limited number of subjects in our study group . with regard to blood pressure , it was also possible that aggressive blood pressure control using calcium channel blockers , angiotensin ii receptor inhibitors , etc . in both mdct detecting stenosis and mdct detecting no stenosis groups led to no significant difference . we could not find a significant correlation between oral agents for hypertension and mdct detecting stenosis . in addition , there was no significant difference in the presence / absence of diabetic retinopathy between the mdct detecting stenosis and mdct detecting no stenosis groups ( data not shown ) . in contrast , significant differences were detected in mean imt and duration of diabetes between mdct detecting stenosis and mdct no stenosis groups in the present study . the data of multiple logistic regression analysis indicated that the predictors of mdct detecting stenosis were mean imt , treatment with statin and duration of diabetes . this multiple regression analysis showed that the administration of statin is a predictor of mdct detecting stenosis although the ldlcholesterol is not . these results indicate that the subjects with diabetes and dyslipidemia , who were given statin and had relatively lower ldlcholesterol levels , still were at risk of having coronary artery stenosis . furthermore , to determine the threshold value of the duration of diabetes and mean imt at the carotid artery for the prediction of mdct detecting stenosis , twotailed test was used for each variable and the odds ratios were calculated by crosstabulation , with a 95% ci . more than 20 years of duration of diabetes significantly correlated with the detection of stenosis of coronary arteries by mdct ( odds ratio 6.222 [ 95% ci 1.67923.064 , p = 0.011 ] , sensitivity 0.474 , specificity 0.805 ) , and more than 1.1 mm of mean imt in carotid arteries significantly correlated with mdct detecting stenosis ( odds ratio 4.600 [ 95% ci 1.20717.525 , p = 0.047 ] , sensitivity 0.500 , specificity 0.833 ) . recently , the american heart association reported that routine surveillance with mdct in asymptomatic patients at low risk for ihd was not recommended . the results of this study indicated that the type 2 diabetic patients with longer duration of diabetes or increased thickness of mean imt in carotid arteries have a high risk of ihd . thus , it is recommended that diabetic patients with more than 1.1 mm mean imt in the carotid arteries and/or more than 20 years duration of diabetes should receive mdct for screening of cad even though they are in good control of blood pressure and lipid metabolism . 52 patients were included , and examinations were carried out at a single timepoint and were not repeated over time . prospective studies with larger patient cohorts are required . in summary , it was shown that mdct detects coronary artery stenosis in diabetic patients without symptoms of ihd or ecg abnormality . from the data of the present study , the predictors of cad in japanese type 2 diabetes patients were mean imt and duration of diabetes . thus , mdct is a noninvasive , effective method to detect or rule out cad , especially silent myocardial ischemia in patients with diabetes , and it is recommended that patients with more than 1.1 mm mean imt at the carotid artery and/or more than 20 years duration of diabetes should be screened for cad . we thank dr yoko horibata , department of diagnostic medicine , graduate school of medical sciences , kumamoto university , kumamoto , japan for suggestions .

abstractaims / introduction : multidetector computed tomography ( mdct ) coronary angiography has been applied as a tool for noninvasive evaluation of the coronary arteries . the purpose of the present study was to evaluate the effectiveness of mdct in screening for coronary artery disease ( cad ) , and to identify the indications for screening in diabetes patients with cad.materials and methods : the study population consisted of 52 japanese type 2 diabetes patients who underwent examination with a 64slice mdct scanner , electrocardiogram ( ecg ) , echocardiography and ultrasonographic scanning of the carotid arteries . regression analysis was carried out to assess the correlation between mdct results and cad risk factors.results : stenosis of the coronary artery was detected in 19/52 patients . of the 19 patients , 7 patients had no symptoms , including chest pain , and no ischemic changes in ecg . significant differences between patients with stenosis and those without stenosis were detected by mean imt ( 1.21 vs 0.95 mm ) , and duration of diabetes ( 20 vs 13 years ) . twotailed 2test showed that a duration of diabetes of more than 20 years ( odds ratio 6.222 ) and more than 1.1 mm of meanimt ( odds ratio 4.600 ) significantly correlated with the stenosis.conclusions : it was shown that mdct is useful in detecting coronary artery stenosis in diabetic patients without symptoms of cad or ecg abnormality , and the predictors of cad are mean imt and duration of diabetes . it is recommended that patients with more than 1.1 mm mean imt at the carotid artery and/or more than 20 years duration of diabetes should be screened for cad by carrying out mdct .

Introduction Materials and methods Subjects MDCT Data Acquisition and Analysis Statistical Analysis Results Discussion Acknowledgements

a close relationship between type 2 diabetes and the development of atherosclerosis exists , and type 2 diabetes is associated with a two to fourfold increase in coronary artery disease ( cad ) . noninvasive tests , including electrocardiogram ( ecg ) , echocardiography and myocardial perfusion scintigraphy , have been used to detect cad in diabetic patients . nonetheless , after normal findings of the tests , elevated event rates are still observed in diabetic patients compared with nondiabetic individuals . furthermore , direct visualization of the coronary arteries is preferred because patients with diabetes frequently have diffuse , multivessel cad . although conventional angiography is carried out to evaluate the presence and extent of cad , this is an invasive approach associated with a minimal but definitive risk of complications . therefore , noninvasive techniques , which are capable of directly visualizing the coronary arteries , are required for further refinement of prognostication in diabetes patients . recently , contrastenhanced multidetector computed tomography ( mdct ) coronary angiography has been shown to be a tool for noninvasive visualization of the coronary arteries . the purpose of the present study was to evaluate the indication of mdct for the screening of cad , especially asymptomatic cad , and to identify the predictors of cad in japanese patients with type 2 diabetes . between june 2006 and december 2007 , 56 patients with type 2 diabetes were enrolled for the present study . four patients were excluded because they manifested arrhythmia or tachycardia of more than 75 b.p.m . ( n = 3 ) , and severe calcification of coronary arteries that caused blooming artifacts and obscured over 75% of the entire vessel lumen in the proximal segment of the coronary artery ( n = 1 ) . the present study was approved by the ethical committee of kumamoto university school of medicine . a resting 12lead ecg was recorded , and we determined qwave myocardial infarction , ischemic stsegment change ( horizontal and downsloping stsegment depression of over 0.3 mv or stsegment elevations of more than 0.1 mv ) or twave change were determined to be ischemic change positive. ultrasonographic scanning of the carotid arteries was carried out using an echotomographic system ( sdu2200 , shimadzu , kyoto , japan ) and carotid intimamedia thickness ( imt ) was measured as described previously . all patients were scanned in the supine position during a single breathhold with inspiration during scanning on a 64detector computed tomography ( ct ) scanner ( brilliance64 , philips medical systems , cleveland , oh , usa ) . our contrast injection protocol was a patient bodyweight ( bw)tailored small contrast dose protocol , 0.7 ml / kg bw of cm at a fixed injection duration of 9 s. the scan parameters were as follows : detector collimation 64 0.625 mm , 11.9 mm / s table feed , 0.20 helical pitch ( beam pitch ) , 420 msec tube rotation time , 120 kv tube voltage , 900 mas tube current timeproduct . all scans started at the upper end of the coronary sinus in a craniocaudal direction . in cases with unsatisfactory image quality , image reconstruction of the raw data was carried out at 0 , 10 , 20 , 30 , 40 , 50 , 60 , 70 , 80 and 90% of the rwave to rwave interval to improve the image quality of all available coronary segments . plaques were classified as stenosis and no stenosis using a 75% threshold of luminal narrowing , and one coronary plaque was assigned per coronary segment . a forward stepwise logisticregression procedure was then carried out to adjust cad risk factors with the use of covariates that were found to be significant predictors of mdct detecting stenosis . furthermore , to identify the threshold value of each significant predictor , twotailed test was used for each variable and the odds ratios were calculated by crosstabulation . we carried out further receiver operating characteristics ( roc ) curve analyses to evaluate the sensitivity and specificity of imt and the duration of diabetes on mdct detecting stenosis . between june 2006 and december 2007 , 56 patients with type 2 diabetes were enrolled for the present study . ( n = 3 ) , and severe calcification of coronary arteries that caused blooming artifacts and obscured over 75% of the entire vessel lumen in the proximal segment of the coronary artery ( n = 1 ) . consequently , 52 patients were available for the assessment , they were 37 men and 15 women ranging in age from 34 to 81 years ( mean 66.2 11.8 years ) . the present study was approved by the ethical committee of kumamoto university school of medicine . a resting 12lead ecg was recorded , and we determined qwave myocardial infarction , ischemic stsegment change ( horizontal and downsloping stsegment depression of over 0.3 mv or stsegment elevations of more than 0.1 mv ) or twave change were determined to be ischemic change positive. ultrasonographic scanning of the carotid arteries was carried out using an echotomographic system ( sdu2200 , shimadzu , kyoto , japan ) and carotid intimamedia thickness ( imt ) was measured as described previously . all patients were scanned in the supine position during a single breathhold with inspiration during scanning on a 64detector computed tomography ( ct ) scanner ( brilliance64 , philips medical systems , cleveland , oh , usa ) . our contrast injection protocol was a patient bodyweight ( bw)tailored small contrast dose protocol , 0.7 ml / kg bw of cm at a fixed injection duration of 9 s. the scan parameters were as follows : detector collimation 64 0.625 mm , 11.9 mm / s table feed , 0.20 helical pitch ( beam pitch ) , 420 msec tube rotation time , 120 kv tube voltage , 900 mas tube current timeproduct . all scans started at the upper end of the coronary sinus in a craniocaudal direction . we reconstructed axial images with a section thickness of 0.67 mm , a section interval of 0.33 mm , and a 16.520.0cm display fieldofview depending on the patient s physique using a medium cardiac kernel ( xcb ) with ecg gating . in cases with unsatisfactory image quality , image reconstruction of the raw data was carried out at 0 , 10 , 20 , 30 , 40 , 50 , 60 , 70 , 80 and 90% of the rwave to rwave interval to improve the image quality of all available coronary segments . plaques were classified as stenosis and no stenosis using a 75% threshold of luminal narrowing , and one coronary plaque was assigned per coronary segment . a forward stepwise logisticregression procedure was then carried out to adjust cad risk factors with the use of covariates that were found to be significant predictors of mdct detecting stenosis . furthermore , to identify the threshold value of each significant predictor , twotailed test was used for each variable and the odds ratios were calculated by crosstabulation . we carried out further receiver operating characteristics ( roc ) curve analyses to evaluate the sensitivity and specificity of imt and the duration of diabetes on mdct detecting stenosis . the study group consisted of 52 patients with type 2 diabetes ( age 66.2 11.8 years ; 37 men ; bmi 24.7 4.2 ; glycated hemoglobin [ hba1c ] 7.9 1.7% ) . the average duration of diabetes was 15.9 10.8 years at the time of mdct . in mdct , of the 19 patients , onevessel disease was identified in 12 patients , twovessel disease in four patients , and threevessel disease in three patients . accidents during mdct or sideeffects as a result of contrast material were not detected in the present study . the correlation between mdct and symptoms , ecg , and echocardiography are provided in tables 2 and 3 . of the 19 patients who had stenosis detected by mdct , eight patients ( 42% ) were identified with neither a positive ischemic change in ecg nor a wall motion abnormality in echocardiography ( table 2 ) . thirteen patients ( 25% of 52 patients ) who had no symptoms of ischemic heart disease ( ihd ) had coronary artery stenosis detected by mdct , indicating the presence of silent myocardial ischemia . more importantly , of the 19 patients with stenosis , seven patients ( 37% ) had no symptoms , including chest pain , nor ischemic changes in ecg ( table 3 ) . these data indicate the high prevalence of asymptomatic ischemia in diabetes patients and the usefulness of mdct for the screening of silent ischemic heart disease in diabetes patients . ecg , electrocardiogram ; lv , left ventricular ; mdct , multidetector computed tomography results . ecg , electrocardiogram ; mdct , multidetector computed tomography . the comparison of the patient characteristics and clinical variables between mdct stenosis detection and no mdct stenosis detection are presented in table 4 . interestingly , there were no significant differences in age ( 69 8 years in stenosis vs 64 13 years in no stenosis , p = 0.061 ) , blood pressure ( 131 22/72 13 mmhg vs 133 20/72 11 mmhg ) , serum low density lipoprotein ( ldl)cholesterol ( 123 44 mg / dl vs 126 37 mg / dl , p = 0.388 ) , serum high density lipoprotein ( hdl)cholesterol ( 55 14 mg / dl vs 51 15 mg / dl , p = 0.195 ) , urinary albumin excretion ( 80.7 149.6 mg / g creatinin vs 83.1 167.2 mg / g creatinin , p = 0.481 ) , estimated glomerular filtration rate ( egfr ) ( 64.9 25.3 ml / min/1.73mvs 74.8 20.6 ml / min/1.73 m , p = 0.066 ) and pwv ( 1917 323 cm / s vs 1899 433 cm / s , p = 0.411 ) between the patients with stenosis and those with no stenosis . significant differences were detected in mean imt ( 1.21 0.44 mm in stenosis vs 0.95 0.26 mm in no stenosis , p = 0.007 ) and duration of diabetes ( 20 11 years vs 13 10 years , p = 0.008 ) . data are mean se and n. * p < 0.05 by unpaired ttest and mann abi , ankle brachial index ; egfr , estimated glomerular filtration rate ; hdl , high density lipoprotein ; imt , intimamedia thickness ; ldl , low density lipoprotein ; mdct , multidetector computed tomography ; pwv , pulse wave velocity . to identify the correlation between the presence of stenosis of coronary arteries on mdct and cad risk factors although a significant correlation was not found between mdct detecting stenosis and age , bmi , hba1c , egfr , diastolic blood pressure , serum ldl and hdl cholesterol concentrations , triglyceride , urinemicroalbumine , abi or pwv , the correlation of mdct detecting stenosis and duration of diabetes , medication of statin and meanimt of carotid artery remained statistically significant after correction for baseline characteristics . the dependent variable of mdct detecting stenosis can be predicted from a linear combination of the independent variables as follows : ( i ) duration of diabetes ( p = 0.004 ) , ( ii ) treatment with statin ( p = 0.029 ) ; and ( iii ) mean imt ( p = 0.023 ) . p = 0.011 ) in duration of diabetes ( per year ) , 9.867 ( 95% ci 1.65558.882 , p = 0.012 ) in treatment with statin , 1.359 ( 95% ci 1.0181.814 , p = 0.038 ) in mean imt ( per 0.1 mm ) by multiple logistic regression analysis . in the present study , it was shown that symptomatic or asymptomatic cad in type 2 diabetes patients can be diagnosed noninvasively by the use of mdct . the population of the present study group was considered to be representative of japanese diabetic patients , although the bmi had a tendency to be lower compared with other studies of caucasian patients . the patients in the present study were receiving contemporary medical treatment and were under almost reasonable metabolic control ( blood pressure 132 21/72 12 mmhg , ldlcholesterol 124 39 mg / dl , hdlcholesterol 52 15 mg / dl , triglyceride 133 94 mg / dl ) , although hba1c ( 7.9 1.7% ) was considered to be higher . previous studies reported that both sensitivity and specificity of mdct were high enough for the diagnosis of coronary artery stenosis to be made . however , several general limitations of mdct , including the administration of an iodinated contrast agent and elevated radiation dose , should be mentioned . in the present study , we generated a protocol to carry out mdct with less iodinated agent and lower radiation exposure . in the present study group , 13 patients ( 25.0% of 52 patients ) who had no symptoms of ihd had coronary artery stenosis detected by mdct . the prevalence of mdct detecting stenosis detection without ihd symptoms in the present study was similar to those of previous reports , suggesting that mdct is useful in screening for silent ischemia in patients with diabetes . furthermore , of 19 patients who had coronary artery stenosis detected by mdct , seven patients had neither positive ischemic change in rest ecg nor symptoms of ihd . taken together , it is suggested that the mdct is effective for the screening of cad , especially silent myocardial ischemia . it was reported that the incidences of coronary heart disease ( chd ) , per 1000 patients per year , among japanese diabetes patients were 9.8 in men and 5.5 in women , although that of chd among caucasian diabetes patients was 17.4 . the prevalence of cad in type 2 diabetes in the caucasian population has been reported to be 3040% . in the present study , even the incidence of chd in japanese type 2 diabetes patients was much lower than that of caucasian patients , as 19/52 patients ( 36.5% ) had coronary artery stenosis detected by mdct . therefore , it is thought that mdct might detect more cad in type 2 diabetes patients in the caucasian population . another important finding of the present study is the assessment of the predictors of mdct detecting stenosis in diabetes patients . in the guidelines for early detection of chd in asymptomatic patients with diabetes from the american diabetes association ( ada ) , the presence of multiple cardiovascular risk factors including ldlcholesterol , hdlcholesterol , blood pressure , micro/macroalbuminuria is mentioned . in the analysis of risk factors that contribute to chd risk in diabetic patients in the united kingdom prospective diabetes study , ldlcholesterol , hdlcholesterol , blood pressure and hba1c were reported to be important . however , in the present study , significant differences were not detected in blood pressure , ldlcholesterol , hdlcholesterol , triglyceride , blood glucose , hba1c , microalbuminuria , abi or pwv between the mdct detected stenosis and mdct detecting no stenosis groups . the reason why there is no significant difference in these markers between mdct detecting stenosis and mdct detecting no stenosis groups might be a result of the limited number of subjects in our study group . in contrast , significant differences were detected in mean imt and duration of diabetes between mdct detecting stenosis and mdct no stenosis groups in the present study . the data of multiple logistic regression analysis indicated that the predictors of mdct detecting stenosis were mean imt , treatment with statin and duration of diabetes . this multiple regression analysis showed that the administration of statin is a predictor of mdct detecting stenosis although the ldlcholesterol is not . furthermore , to determine the threshold value of the duration of diabetes and mean imt at the carotid artery for the prediction of mdct detecting stenosis , twotailed test was used for each variable and the odds ratios were calculated by crosstabulation , with a 95% ci . more than 20 years of duration of diabetes significantly correlated with the detection of stenosis of coronary arteries by mdct ( odds ratio 6.222 [ 95% ci 1.67923.064 , p = 0.011 ] , sensitivity 0.474 , specificity 0.805 ) , and more than 1.1 mm of mean imt in carotid arteries significantly correlated with mdct detecting stenosis ( odds ratio 4.600 [ 95% ci 1.20717.525 , p = 0.047 ] , sensitivity 0.500 , specificity 0.833 ) . the results of this study indicated that the type 2 diabetic patients with longer duration of diabetes or increased thickness of mean imt in carotid arteries have a high risk of ihd . thus , it is recommended that diabetic patients with more than 1.1 mm mean imt in the carotid arteries and/or more than 20 years duration of diabetes should receive mdct for screening of cad even though they are in good control of blood pressure and lipid metabolism . 52 patients were included , and examinations were carried out at a single timepoint and were not repeated over time . in summary , it was shown that mdct detects coronary artery stenosis in diabetic patients without symptoms of ihd or ecg abnormality . from the data of the present study , the predictors of cad in japanese type 2 diabetes patients were mean imt and duration of diabetes . thus , mdct is a noninvasive , effective method to detect or rule out cad , especially silent myocardial ischemia in patients with diabetes , and it is recommended that patients with more than 1.1 mm mean imt at the carotid artery and/or more than 20 years duration of diabetes should be screened for cad .

lung remodeling is a common end - stage sequela of idiopathic pulmonary fibrosis ( ipf ) and systemic sclerosis ( ssc ) , resulting in disruption of lung architecture , leading to progressive respiratory failure.(- ) histologically , the remodeling process is characterized by diffuse chronic interstitial inflammation and increased fibroblast proliferation , as well as by increased extracellular matrix synthesis and collagen deposition . ( , , ) therefore , modulation of inflammation , fibroblast proliferation , and collagen synthesis by effector mediators in ipf and ssc is very important . despite the characterization of a variety of key participants , the mediators and mechanisms involved in the pathogenesis of ipf and ssc have yet to be fully defined , which might explain the limited number of therapeutic approaches , with little impact on long - term survival . ( , ) it is known that cox is the key enzyme in the conversion of arachidonic acid to prostaglandin e2 ( pge2 ) , the precursor of a diverse family of bioactive lipid mediators including prostaglandins , thromboxane , and prostacyclin . the former is constitutively expressed in most tissues and acts as a housekeeping enzyme regulating vascular homeostasis , protecting the gastric mucosa , and maintaining renal integrity , ( , ) whereas the latter has lower levels of expression in most tissues but is inducible in response to growth factors , cytokines , and other proinflammatory molecules.(- ) regarding the proinflammatory and anti - inflammatory roles of cox-1 and cox-2 , immunohistochemistry can be a useful tool to detect cox-1 and cox-2 in the remodeled lung in patients with ssc and ipf . data on the assessment of cox-1 and cox-2 in the remodeled lung have previously been reported in serum ( , ) and bronchoalveolar lavage fluid ( ) from patients with ssc , as well as in fibroblast cultures ( ) and biopsies ( , ) from patients with ipf . however , the roles of cox-1 and cox-2 in the mechanisms involved in the remodeled lung in ipf and ssc patients are still unclear , and there has been uncertainty regarding the best way to detect cox-2 . the aim of the present study was to study the expression of cox-1 and cox-2 in lung biopsy specimens ( cox-1 and cox-2 expression being separately evaluated in alveolar septa , bronchioles , and vessels ) and correlate it with patient survival . between january of 2002 and july of 2008 , 24 consecutive patients with ssc and interstitial lung disease and 30 patients suspected of having ipf on the basis of hrct findings were submitted to open lung biopsy at the university of so paulo school of medicine hospital das clnicas , located in the city of so paulo , brazil . all patients fulfilled the diagnostic criteria for ssc ( ) and ipf ( ) open lung biopsy was performed by formal thoracotomy , areas of honeycombing being avoided . all patients gave written informed consent , and the study was approved by the local research ethics committee ( protocol no . disease duration was determined on the basis of the onset of the first symptom . pulmonary function testing and hrct were performed within up to 3 months before the biopsy . pulmonary function testing included vc , fev1 , fvc , fev1/fvc , tlc , rv , and dlco . physiological assessment was performed before open lung biopsy and before the initiation of treatment . all pulmonary function tests , including spirometry , determination of lung volumes , and measurement of dlco , were performed on the same day . all spirometric tests were performed with a calibrated pneumotachograph ( medical graphics co. , st . paul , mn , usa ) , all values being expressed as a percentage of their respective predicted value , the reference values having been established by pereira et al . ( ) lung volumes were measured with a whole - body plethysmograph ( medical graphics co. ) , all values being expressed as a percentage of the predicted values . ( ) all patients were followed regularly after treatment until death , blood and lung function tests being regularly performed . the primary endpoint was to evaluate the impact of cox-1 and cox-2 changes on survival and analyze differences between ssc and ipf . , normal lung tissue was obtained from 10 individuals ( 6 males and 4 females ) whose median age was 46.6 5.8 years and who had died suddenly of nonpulmonary causes . table 1 clinical data of the patients with systemic sclerosis and of those with idiopathic pulmonary fibrosis.a regarding open lung biopsy findings , usual interstitial pneumonia ( uip ) , the histological pattern of ipf , was characterized by patchy subpleural and paraseptal distribution of parenchymal injury . temporal heterogeneity was seen at low magnification , areas of normal lung parenchyma alternating with alveolar collapse , interstitial mononuclear infiltrates , septal fibromyxoid tissue ( fibroblastic foci ) , and honeycomb lung . ( ) all of the patients with ssc had histological patterns consistent with fibrotic nonspecific interstitial pneumonia ( nsip ) , as defined by temporally homogeneous septal thickening and interstitial fibrosis . ( ) for immunohistochemistry analysis , a standard peroxidase technique was used ( harris 's hematoxylin being used as the counterstain ) in order to identify cox-1 and cox-2 expression in alveolar septa , bronchiolar walls , and vascular walls in normal lung tissue ( the control tissue ) , in lung tissue showing the uip pattern ( the uip tissue ) , and in lung tissue showing the nsip pattern ( the nsip tissue ) . anti - cox-1 and anti - cox-2 antibodies ( santa cruz biotechnology , inc . , santa cruz , ca , usa ) were incubated with tissue sections at dilutions of 1:50 and 1:100 , respectively . the novolink max polymer amplification kit ( leica biosystems newcastle ltd , newcastle upon tyne , uk ) was used for signal amplification , and 3,3'-diaminobenzidine tetrahydrochloride ( 0.25 mg dissolved in 1 ml of 0.02% hydrogen peroxide ) was used as a precipitating substrate for signal detection . the specificity of primary antibodies was confirmed by appropriate reagent controls ( the primary antibody being omitted or nonimmune serum being substituted for the primary antibody in the staining protocol ) , which revealed no staining . regarding histomorphometry , cox-1 expression and cox-2 expression were assessed by a point - counting technique in 50 and 30 fields in alveolar septa , bronchiolar walls , and vascular walls in the control tissue , in the uip tissue , and in the nsip tissue . the technique was performed with a 100-point grid ( area , 187,500 m ( ) ; magnification , 400 ) attached to the microscope eyepiece . ( ) at a magnification of 400 , the septal , bronchiolar , and vascular areas in each field were calculated on the basis of the number of points overlying connective tissue , as a proportion of the total grid area . subsequently , the number of immunostained cells within the septal , bronchiolar , and vascular areas was counted . the areal fraction of immunostained cells represents the percentage ratio of the area of labeled cells in relationship to the total area covered by the grid in the eyepiece . in order to assess interobserver variability , we compared the results obtained by two observers in 20% of the slides . the coefficient of variation for the interobserver error of the cell count was 5% . the student 's t - test for independent samples was used in order to test the relationship between continuous variables , and the residuals were examined to ensure that they were approximately normally distributed . the relationship between cellularity ( as determined by immunostaining ) and pulmonary function test results was evaluated by pearson 's correlation coefficient . for all cases , measured variable values were arranged in ascending order and divided into two groups on the basis of the median value of each variable . for each variable , the groups were designated low degree and high degree , as follows : alveolar septal cox-1 ( low degree , < 2.35% ; high degree , 2.35% ) ; vascular cox-1 ( low degree , < 2.91% ; high degree , 2.91% ) ; bronchiolar cox-1 ( low degree , < 2.88% ; high degree , 2.88% ) ; total cox-1 ( low degree , < 2.77% ; high degree , 2.77% ) ; alveolar septal cox-2 ( low degree , < 2.04% ; high degree , 2.04% ) ; vascular cox-2 ( low degree , < 2.34% ; high degree , 2.34% ) ; bronchiolar cox-2 ( low degree , < 2.34% ; high degree , 2.34% ) ; and total cox-2 ( low degree , < 2.16% ; high degree , 2.16% ) . first , we performed a univariate analysis relating overall follow - up to each of the measured variables by means of the kaplan - meier method and then analyzed survival using the log - rank test . the variables that were found to be significant in the univariate analysis were included in the multivariate analysis based on the cox proportional hazards regression model . deaths from causes other than ipf or ssc and living patients were included in the models as censored cases . all statistical procedures were performed with the statistical package for the social sciences , version 18.0 ( spss inc . table 1 summarizes the clinical features of the patients with ssc ( n = 24 ) and those of those with ipf ( n = 30 ) . six of 17 ssc patients ( 35.29% ) and 13 of 19 ipf patients ( 68.42% ) had restrictive lung disease . respiratory function test results were as follows : fvc < 80% in 18 ( 75% ) of the 24 ssc patients and in 19 of 22 ipf patients ( 86.36% ) ; tlc < 80% in 6 of 17 ssc patients ( 35.9% ) and in 13 of 19 ipf patients ( 68.42% ) ; dlco < 80% in 12 of 15 ssc patients ( 80% ) and in 8 of 9 ipf patients ( 88.88% ) ; and dlco / alveolar volume < 80% in 11 of 18 ssc patients ( 61.11% ) and in 11 of 14 ipf patients ( 78.57% ) . a significant negative correlation was found between cox-2 expression in vessels and fvc ( r= 0.28 ; p = 0.05 ) , as well as between cox-2 expression in alveolar septa and dlco ( r = 0.80 ; p = 0.009 ) . figure 1 shows alveolar septa , vessels , and bronchioles in the control tissue , in the nsip tissue , and in the uip tissue immunostained for cox-1 ( in a , c , e , g , i , k , m , o , and q ) and cox-2 ( in b , d , f , h , j , l , n , p , and r ) . the nsip and uip tissues differed from the control tissue in terms of the immunostaining intensity of epithelial cells , endothelial cells , myofibroblasts , and smooth muscle cells in the alveolar septa , vessels , and bronchioles . figure 1 cellular expression of cox-1 and cox-2 in alveolar septa , intrapulmonary vessels , and bronchioles in normal lung tissue ( control tissue ) ; in lung tissue obtained from patients with systemic sclerosis ( ssc ) and showing fibrotic nonspecific interstitial pneumonia ( nsip ) ; and in lung tissue obtained from patients with idiopathic pulmonary fibrosis ( ipf ) and showing usual interstitial pneumonia ( uip ) . the intensity of cox-1 immunostaining of epithelial cells , endothelial cells , myofibroblasts , and smooth muscle cells in ssc - nsip and ipf - uip tissue alveolar septa ( g and m , respectively ) , vessels ( i and o , respectively ) , and bronchioles ( k and q , respectively ) was higher than was that of cox-1 immunostaining of those cells in control tissue alveolar septa ( a ) , vessels ( c ) , and bronchioles ( e ) . likewise , the intensity of cox-2 immunostaining of those cells in ssc - nsip and ipf - uip tissue alveolar septa ( h and n , respectively ) , vessels ( j and p , respectively ) , and bronchioles ( l and r , respectively ) was higher than was that of cox-2 immunostaining of those cells in control tissue alveolar septa ( b ) , vessels ( d ) , and bronchioles ( f ) . the bar plots show the quantification of cox-1 and cox-2 immunostaining of cells in alveolar septa ( s ) , total lung parenchyma ( t ) , and bronchioles ( w ) in the control tissue , in the ssc - nsip tissue , and in the ipf - uip tissue ( immunohistochemical staining ; magnification , 400 ) . the proportion of alveolar septal cells immunostained for cox-1 and cox-2 was significantly higher in the uip and nsip tissues than in the control tissue . in other words , high proportions of alveolar septal cells staining for cox-1 and cox-2 were associated with the uip and nsip patterns . as can be seen in the bar plots in figure 1 ( s and t ) the relationship of cox-1 and cox-2 with ipf ( the uip pattern ) was stronger than was that of cox-1 and cox-2 with ssc ( the nsip pattern ) . although the proportion of bronchiolar cells immunostained for cox-2 was lower in the nsip and uip tissues than in the control tissue ( figure 1w ) , the difference was not statistically significant . in addition , although the proportion of bronchiolar cells immunostained for cox-1 was higher in the uip and nsip tissues than in the control tissue ( figure 1w ) , the difference was not significant . no differences were found among the tissues in terms of the cox-1 or cox-2 immunostaining , for vessels or for the total parenchyma ( table 2 ) . table 2 morphometric results in normal lung tissue ( control tissue ) , in lung tissue showing the usual interstitial pneumonia pattern ( from patients with idiopathic pulmonary fibrosis ) , and in lung tissue showing the nonspecific interstitial pneumonia pattern ( from patients with systemic sclerosis).a a preliminary analysis of the kaplan - meier survival curves showed that survival was better in the patients with ssc ( the fibrotic nsip pattern ) and cox-2 expression > 2.25% ( median survival , 70.75 months ) than in those with ipf ( the uip pattern ) and cox-2 expression < 2.25% ( median survival , 46.32 months ; figure 2 ) . therefore , we coded the fibrotic nsip pattern as a single dummy variable with a value of 1 and the uip pattern with a value of 2 . the results of the multivariate analysis based on the cox proportional hazards regression model are shown in table 3 . after controlling for age , pulmonary function test results , the uip pattern , and the fibrotic nsip pattern , we found that only two variables were significantly associated with survival time : the fibrotic nsip pattern and alveolar septal cox-2 ( p = 0.02 ) . once these two variables were accounted for , none of the others were related to survival . the multivariate analysis showed a low risk of death for young patients with low fev1/fvc , fibrotic nsip pattern , and high - degree alveolar septal cox-2 . figure 2 cox regression plots for risk of death risk versus duration of follow - up ( in months ) in young patients with low dlco / alveolar volume , systemic sclerosis ( and a histological pattern of cellular nonspecific interstitial pneumonia ) , high - degree total cox-1 , and low - degree alveolar septal cox-2 . the top curve represents the group of patients with systemic sclerosis and cellular nonspecific interstitial pneumonia . the bottom curve represents two groups of patients : those with systemic sclerosis and fibrotic nonspecific interstitial pneumonia ; and those with idiopathic pulmonary fibrosis and the usual interstitial pneumonia pattern . table 3 cox proportional hazards regression to ascertain the individual contribution of the histological pattern and morphological factors associated with survival and to compare adjusted survival between the two groups . the limited number of therapeutic approaches that have any impact on long - term survival in patients with ipf - uip and in those with ssc and fibrotic nsip is due to the lack of definition regarding the mediators and mechanisms involved in the pathogenesis of ipf and ssc . therefore , the question of interest is whether additional mediators can provide a better understanding of the pathogenesis of these diseases . the repair process involves two distinct stages : a regenerative , inflammatory phase , in which the microenvironment attempts to replace injured cells ; and a fibrotic phase , in which connective tissue replaces normal parenchymal tissue.(- ) in the repair process , pge2 production by fibroblasts is increased , ( , ) which constitutes further evidence of the antiproliferative , anti - inflammatory and antifibrotic properties of cox-2/pge2 . ( ) therefore , our finding that immunohistochemistry staining for cox provides important information on the repair processes in pulmonary fibrosis is not surprising , and our results confirm that the expression of cox-2 is increased in ipf and ssc , with improved outcome in a group of patients . we found that the proportion of alveolar septal cells immunostained for cox-1 and cox-2 was significantly higher in lung tissue showing the uip pattern and the fibrotic nsip pattern than in normal lung tissue . increased cox-1 expression was expected because cox-1 is constitutively expressed in most cells and tissues , whereas cox-2 is induced by inflammatory or mitogenic stimuli . ( , , ) those studies showed reduced cox-2 expression in pulmonary fibroblasts secondary to decreased cox-2 production . however , in those studies , cox-2 expression was measured only in fibroblasts , whereas in our study it was measured in the alveolar septa , including epithelial cells and fibroblasts in normal areas , collapsed areas , and fibroblast foci . ( ) found increased expression of cox-2 in metaplastic epithelium and fibroblasts from fibrotic areas in ipf - uip . first , reduced cox-2 expression in normal areas , collapsed areas , and fibroblastic foci suggests an anti - inflammatory role for cox-2 in early - stage ipf - uip . second , the presence of progressive fibrosis even in the presence of increased cox-2 expression suggests that fibroblasts are unable to respond to stimulation by cox-2 and its main product ( pge2 ) so as to inhibit fibroblast proliferation , myofibroblastic transformation , and increased production of collagen and other extracellular matrix molecules . in the present study , the proportion of alveolar septal cells immunostained for cox-1 and cox-2 was found to be lower in fibrotic nsip tissue ( from ssc patients ) than in uip tissue ( from ipf patients ) . this finding contrasts with those of previous studies showing that cox-2 levels are higher in ssc patients.(- ) in addition , cox-2 production has been shown to be much greater in the inflammatory resolution phase than in the early phase . ( ) these contradictory findings suggest that cox-2 has a dual role in a normal inflammatory process , playing a proinflammatory role in the early phase and an anti - inflammatory role in the resolution phase . ( ) therefore , in view of the abovementioned evidence and of the latent inflammation in patients with ssc and lung involvement , our results emphasize the idea that cox-2 does not exert its anti - inflammatory effect properly , because there is inflammation even when cox-2 expression is increased in patients with ssc and fibrotic nsip . however , further studies are needed in order to clarify the real reason why the cox-2 mechanism is deficient . we hypothesize that this is due to an inability of cox-2 to stimulate the production of pge2 or other anti - inflammatory mediators in opposition to its own proinflammatory effects or an inability of the cells to respond appropriately to cox-2 . we sought to establish a correlation between cox-2 and patient survival controlled for age , pulmonary function test results , the uip pattern ( in patients with ipf ) and the nsip pattern ( in patients with ssc ) . our multivariate analysis showed a low risk of death for younger patients with low dlco / alveolar volume , ssc ( and the nsip histological pattern ) , high - degree total cox-2 , and high - degree alveolar septal cox-1 . in conclusion , the expression of cox-1 and cox-2 in the lung parenchyma offers us the potential to control repair processes involved in the progression of ssc - nsip and ipf - uip , suggesting that strategies aimed at preventing low cox-1 synthesis will have a greater impact on ssc , whereas those aimed at preventing high cox-2 synthesis will have a greater impact on ipf . estudar a expresso de cox-1 e cox-2 em reas pulmonares remodeladas em pacientes com esclerose sistmica ( es ) ou fibrose pulmonar idioptica ( fpi ) e correlacion - la com a sobrevida desses pacientes . examinamos espcimes de bipsia pulmonar a cu aberto de 24 pacientes com es e de 30 pacientes com fpi , utilizando - se tecido pulmonar normal como controle . os padres histolgicos incluram pneumonia intersticial no especfica ( pine ) fibrtica em pacientes com es e pneumonia intersticial usual ( piu ) nos pacientes com fpi . imuno - histoqumica e histomorfometria foram usadas para avaliar a expresso celular de cox-1 e cox-2 em septos alveolares , vasos e bronquolos , sua correlao com provas de funo pulmonar e seu impacto na sobrevida . a expresso de cox-1 e cox-2 em septos alveolares foi significativamente maior em fpi - piu e es - pine do que no tecido controle . no houve diferena entre fpi - piu e es - pine quanto expresso de cox-1 e cox-2 . a anlise multivariada baseada no modelo de regresso de cox mostrou que os fatores associados a baixo risco de morte foram ter idade menor , valores elevados de dlco / volume alveolar , fpi , e alta expresso de cox-1 em septos alveolares , ao passo que os fatores associados a alto risco de morte foram ter idade maior , valores baixos de dlco / volume alveolar , es ( com pine ) e baixa expresso de cox-1 em septos alveolares nossos resultados sugerem que estratgias de preveno de baixa sntese de cox-1 tero maior impacto sobre a es , ao passo que as de preveno de alta sntese de cox-2 tero maior impacto sobre a fpi . o remodelamento pulmonar uma sequela terminal comum da fibrose pulmonar idioptica ( fpi ) e da esclerose sistmica ( es ) ; resulta em desorganizao da arquitetura pulmonar e , consequentemente , insuficincia respiratria progressiva.(- ) histologicamente , o processo de remodelamento caracterizado por inflamao intersticial crnica difusa e aumento da proliferao de fibroblastos , da sntese de matriz extracelular e da deposio de colgeno . ( , , ) portanto , a modulao da inflamao , da proliferao de fibroblastos e da sntese de colgeno por mediadores efetores na fpi e na es muito importante . no obstante a caracterizao de vrios participantes cruciais , os mediadores e mecanismos envolvidos na patognese da fpi e da es ainda no foram totalmente definidos , o que pode explicar o nmero limitado de opes teraputicas , com pouco impacto na sobrevida em longo prazo . ( , ) sabe - se que a cox a enzima essencial para a converso de cido araquidnico em prostaglandina e2 ( pge2 ) , que o precursor de uma famlia de mediadores lipdicos bioativos diversos , como as prostaglandinas , o tromboxano e a prostaciclina . a cox existe em duas isoformas : cox-1 e cox-2 . a cox-1 expressa constitutivamente na maioria dos tecidos e atua como uma enzima " que mantm a casa em ordem " , isto , que regula a homeostase vascular , protege a mucosa gstrica e mantm a integridade renal , ( , ) ao passo que a cox-2 tem nveis mais baixos de expresso na maioria dos tecidos , embora possa ser induzida em resposta a fatores de crescimento , citocinas e outras molculas pr - inflamatrias.(- ) no tocante ao papel pr - inflamatrio e anti - inflamatrio da cox-1 e da cox-2 , a imuno - histoqumica pode ser til para detectar essas enzimas no pulmo remodelado de pacientes com es e fpi . dados relativos avaliao de cox-1 e cox-2 no pulmo remodelado foram anteriormente relatados no soro ( , ) e lavado broncoalveolar ( ) de pacientes com es , bem como em culturas de fibroblastos ( ) e bipsias ( , ) de pacientes com fpi . no entanto , o papel da cox-1 e da cox-2 nos mecanismos envolvidos no remodelamento pulmonar em pacientes com fpi e es ainda no est claro , e h incerteza sobre a melhor maneira de detectar a cox-2 . o objetivo do presente estudo foi estudar a expresso de cox-1 e cox-2 ( avaliada separadamente em septos alveolares , bronquolos e vasos ) em espcimes obtidos por meio de bipsia pulmonar e correlacion - la com a sobrevida dos pacientes . entre janeiro de 2002 e julho de 2008 , 24 pacientes consecutivos com es e doena pulmonar intersticial e 30 pacientes com suspeita de fpi baseada em achados de tcar foram submetidos a bipsia pulmonar a cu aberto no hospital das clnicas da faculdade de medicina da universidade de so paulo , na cidade de so paulo ( sp ) . todos os pacientes preencheram os critrios para o diagnstico de es ( ) e fpi . ( ) a bipsia pulmonar a cu aberto foi realizada por meio de toracotomia formal , evitando - se reas de faveolamento . todos os pacientes assinaram um termo de consentimento livre e esclarecido , e o estudo foi aprovado pelo comit de tica em pesquisa da instituio ( protocolo n 0960/08 ) . foram analisados os pronturios clnicos de todos os pacientes . a durao da doena foi determinada com base no incio do primeiro sintoma . os testes de funo pulmonar e a tcar foram realizados em um prazo de at 3 meses antes da bipsia . os testes de funo pulmonar incluram cv , vef1 , cvf , vef1/cvf , cpt , vr e dlco . a avaliao fisiolgica foi realizada antes da bipsia pulmonar a cu aberto e antes do incio do tratamento . todos os testes de funo pulmonar , incluindo espirometria , determinao dos volumes pulmonares e medio da dlco , foram realizados no mesmo dia . todos os testes espiromtricos foram realizados com um pneumotacgrafo calibrado ( medical graphics co. , st . paul , mn , eua ) ; todos os valores foram expressos em porcentagem de seus respectivos valores previstos , os valores de referncia tendo sido estabelecidos por pereira et al . ( ) os volumes pulmonares foram medidos com um pletismgrafo de corpo inteiro ( medical graphics co. ) ; todos os valores foram expressos em porcentagem dos valores previstos . ( ) todos os pacientes foram acompanhados regularmente aps o tratamento at o bito ; exames de sangue e testes de funo pulmonar foram realizados regularmente . o desfecho primrio foi avaliar o impacto de alteraes em cox-1 e cox-2 na sobrevida e analisar as diferenas entre es e fpi . a tabela 1 mostra os dados demogrficos . como controle , obtivemos tecido pulmonar normal de 10 indivduos ( 6 homens e 4 mulheres ) cuja mediana de idade foi de 46,6 5,8 anos e que morreram subitamente de causas no pulmonares . tabela 1 dados clnicos dos pacientes com esclerose sistmica e daqueles com fibrose pulmonar idioptica.a quanto aos achados da bipsia pulmonar a cu aberto , a pneumonia intersticial usual ( piu ) , o padro histolgico da fpi , foi caracterizada por distribuio subpleural e parasseptal segmentada de leses parenquimatosas . com um aumento pequeno , observou - se heterogeneidade temporal , com reas de parnquima pulmonar normal se alternando com colapso alveolar , infiltrado mononuclear intersticial , tecido fibromixoide septal ( focos fibroblsticos ) e faveolamento . ( ) todos os pacientes com es apresentavam padres histolgicos consistentes com pneumonia intersticial no especfica ( pine ) fibrtica , definida por espessamento septal temporalmente homogneo e fibrose intersticial . ( ) para a anlise imuno - histoqumica , foi usada uma tcnica - padro de peroxidase - com a hematoxilina de harris como contracorante - a fim de identificar a expresso de cox-1 e cox-2 em septos alveolares , paredes bronquiolares e paredes vasculares em tecido pulmonar normal ( o tecido controle ) , em tecido pulmonar com padro histolgico de piu ( o tecido piu ) e em tecido pulmonar com padro histolgico de pine ( o tecido pine ) . anticorpos anti - cox-1 e anti - cox-2 ( santa cruz biotechnology , inc . , santa cruz , ca , eua ) foram incubados com cortes histolgicos em diluies de 1:50 e 1:100 , respectivamente . o kit de amplificao novolink max polymer ( leica biosystems newcastle ltd , newcastle upon tyne , reino unido ) foi usado para a amplificao do sinal , e 3,3'-diaminobenzidina tetra - hidrocloreto ( 0,25 mg dissolvidos em 1 ml de perxido de hidrognio a 0,02% ) foi usado como substrato precipitante para a deteco do sinal . a especificidade dos anticorpos primrios foi confirmada por controles de reagentes apropriados - o anticorpo primrio sendo omitido ou soro no imune sendo usado em vez do anticorpo primrio no protocolo de colorao - os quais no revelaram nenhuma colorao . quanto histomorfometria , avaliamos a expresso de cox-1 e cox-2 por meio de uma tcnica de contagem de pontos em 50 e 30 campos em septos alveolares , paredes bronquiolares , e paredes vasculares no tecido controle , no tecido piu e no tecido pine . a tcnica foi executada com um retculo de 100 pontos ( rea : 187.500 m ( ) ; aumento : 400 ) ligado ocular do microscpio . ( ) com um aumento de 400 , as reas septais , bronquiolares e vasculares em cada campo foram calculadas com base no nmero de pontos sobrepostos ao tecido conjuntivo , na forma de porcentagem da rea total do retculo . posteriormente , contou - se o nmero de clulas imunomarcadas nas reas septais , bronquiolares e vasculares . a frao de rea de clulas imunomarcadas representa a relao percentual entre a rea de clulas marcadas e a rea total coberta pelo retculo na ocular . para avaliar a variabilidade interobservador , comparamos os resultados obtidos por dois observadores em 20% das lminas . o coeficiente de variao para o erro interobservador da contagem de clulas foi de 5% . os dados so apresentados na forma de mdia dp e ic95% . o teste t de student para amostras independentes foi usado a fim de testar a relao entre variveis contnuas , e os resduos foram examinados para certificar que sua distribuio era aproximadamente normal . a relao entre celularidade ( determinada pela imunomarcao ) e os resultados dos testes de funo pulmonar foi avaliada pelo coeficiente de correlao de pearson . para todos os casos , os valores das variveis medidas foram dispostos em ordem crescente e divididos em dois grupos com base na mediana de cada varivel . para cada varivel , os grupos foram denominados baixo grau e alto grau , a saber : cox-1 em septos alveolares ( baixo grau : < 2,35% ; alto grau : 2,35% ) ; cox-1 em vasos ( baixo grau : < 2,91% ; alto grau : 2,91% ) ; cox-1 em bronquolos ( baixo grau : < 2,88% ; alto grau : 2,88% ) ; cox-1 total ( baixo grau : < 2,77% ; alto grau : 2,77% ) ; cox-2 em septos alveolares ( baixo grau : < 2,04% ; alto grau : 2,04% ) ; cox-2 em vasos ( baixo grau : < 2,34% ; alto grau : 2,34% ) ; cox-2 em bronquolos ( baixo grau : < 2,34% ; alto grau : 2,34% ) e cox-2 total ( baixo grau : < 2,16% ; alto grau : 2,16% ) . primeiro , realizamos uma anlise univariada relacionando o acompanhamento global com cada uma das variveis medidas por meio do mtodo de kaplan - meier e ento analisamos a sobrevida por meio do teste de log - rank . as variveis que se mostraram significantes na anlise univariada foram includas na anlise multivariada baseada no modelo de regresso de riscos proporcionais de cox . mortes cujas causas foram outras que no fpi ou es e pacientes vivos foram includos nos modelos como casos censurados . todos os procedimentos estatsticos foram realizados com o programa statistical package for the social sciences , verso 18.0 ( spss inc . , a tabela 1 resume as caractersticas clnicas dos pacientes com es ( n = 24 ) e dos pacientes com fpi ( n = 30 ) . seis de 17 pacientes com es ( 35,29% ) e 13 de 19 pacientes com fpi ( 68,42% ) apresentaram distrbio ventilatrio restritivo . os resultados dos testes de funo respiratria foram os seguintes : cvf < 80% em 18 ( 75% ) dos 24 pacientes com es e em 19 de 22 pacientes com fpi ( 86,36% ) ; cpt < 80% em 6 de 17 pacientes com es ( 35,9% ) e em 13 de 19 pacientes com fpi ( 68,42% ) ; dlco < 80% em 12 de 15 pacientes com es ( 80% ) e em 8 de 9 pacientes com fpi ( 88,88% ) ; dlco / volume alveolar < 80% em 11 de 18 pacientes com es ( 61,11% ) e em 11 de 14 pacientes com fpi ( 78,57% ) . houve correlao negativa significativa entre a expresso de cox-2 em vasos e a cvf ( r = 0,28 ; p = 0,05 ) e entre a expresso de cox-2 em septos alveolares e a dlco ( r = 0,80 ; p = 0,009 ) . a figura 1 mostra septos alveolares , vasos e bronquolos no tecido controle , no tecido pine e no tecido piu com imunomarcao positiva para cox-1 ( em a , c , e , g , i , k , m , o e q ) e cox-2 ( em b , d , f , h , j , l , n , p e r ) . os tecidos pine e piu diferiram do tecido controle quanto intensidade da imunomarcao de clulas epiteliais , clulas endoteliais , miofibroblastos e clulas musculares lisas nos septos alveolares , vasos , e bronquolos . figura 1 expresso celular de cox-1 e cox-2 em septos alveolares , vasos intrapulmonares e bronquolos em tecido pulmonar normal ( tecido controle ) ; em tecido pulmonar de pacientes com esclerose sistmica ( es ) e pneumonia intersticial no especfica ( pine ) fibrtica ; e em tecido pulmonar de pacientes com fibrose pulmonar idioptica ( fpi ) e pneumonia intersticial usual ( piu ) . a intensidade da imunomarcao positiva para cox-1 em clulas epiteliais , clulas endoteliais , miofibroblastos e clulas musculares lisas em septos alveolares de tecido es - pine e fpi - piu ( g e m , respectivamente ) , em vasos de tecido es - pine e fpi - piu ( i e o , respectivamente ) e em bronquolos de tecido es - pine e fpi - piu ( k e q , respectivamente ) foi maior que a da imunomarcao positiva para cox-1 nessas clulas em septos alveolares do tecido controle ( a ) , em vasos do tecido controle ( c ) e em bronquolos do tecido controle ( e ) . do mesmo modo , a intensidade da imunomarcao positiva para cox-2 nessas clulas em septos alveolares de tecido es - pine e fpi - piu ( h e n , respectivamente ) , em vasos de tecido es - pine e fpi - piu ( j e p , respectivamente ) e em bronquolos de tecido es - pine e fpi - piu ( l e r , respectivamente ) foi maior que a da imunomarcao positiva para cox-2 nessas clulas em septos alveolares do tecido controle ( b ) , em vasos do tecido controle ( d ) e em bronquolos do tecido controle ( f ) . os grficos de barras mostram a quantificao da imunomarcao positiva para cox-1 e cox-2 em clulas em septos alveolares ( s ) , parnquima pulmonar total ( t ) e bronquolos ( w ) no tecido controle , no tecido es - pine e no tecido fpi - piu ( marcao imuno - histoqumica ; aumento : 400 ) . a proporo de clulas em septos alveolares com imunomarcao positiva para cox-1 e cox-2 foi significativamente maior nos tecidos piu e pine do que no tecido controle . de clulas em septos alveolares marcadas positivamente para cox-1 e cox-2 associaram - se aos padres histolgicos de piu e pine . como se pode observar nos grficos de barras na figura 1 ( s e t ) , a relao de cox-1 e cox-2 com a fpi ( o padro histolgico de piu ) foi mais forte do que a de cox-1 e cox-2 com a es ( o padro histolgico de pine ) . embora a proporo de clulas bronquiolares com imunomarcao positiva para cox-2 tenha sido menor nos tecidos pine e piu do que no tecido controle ( figura 1w ) , a diferena no foi estatisticamente significante . alm disso , embora a proporo de clulas bronquiolares com imunomarcao positiva para cox-1 tenha sido maior nos tecidos piu e pine do que no tecido controle ( figura 1w ) , a diferena no foi significante . no foram encontradas diferenas entre os tecidos no tocante imunomarcao de cox-1 e cox-2 em vasos e no parnquima total ( tabela 2 ) . tabela 2 resultados morfomtricos em tecido pulmonar normal ( tecido controle ) , em tecido pulmonar com o padro histolgico de pneumonia intersticial usual ( proveniente de pacientes com fibrose pulmonar idioptica ) e em tecido pulmonar com o padro histolgico de pneumonia intersticial no especfica ( proveniente de pacientes com esclerose sistmica).a uma anlise preliminar das curvas de sobrevida de kaplan - meier mostrou que a sobrevida foi melhor nos pacientes com es ( o padro histolgico de pine fibrtica ) e expresso de cox-2 > 2,25% ( mediana da sobrevida : 70,75 meses ) do que naqueles com fpi ( o padro histolgico de piu ) e expresso de cox-2 < 2,25% ( mediana da sobrevida : 46,32 meses ; figura 2 ) . nica varivel dummy com valor = 1 e o padro histolgico de piu com valor = 2 . os resultados da anlise multivariada baseada no modelo de regresso de riscos proporcionais de cox so apresentados na tabela 3 . aps termos controlado a idade , os resultados dos testes de funo pulmonar , o padro histolgico de piu e o padro histolgico de pine fibrtica , constatamos que apenas duas variveis se associaram de maneira significativa ao tempo de sobrevida : o padro histolgico de pine fibrtica e cox-2 em septos alveolares ( p = 0,02 ) . uma vez que essas duas variveis foram contabilizadas , nenhuma das demais se relacionou sobrevida . a anlise multivariada revelou baixo risco de morte para pacientes jovens com vef1/cvf baixa , padro histolgico de pine fibrtica e cox-2 de alto grau em septos alveolares . figura 2 curvas de regresso de cox para risco de bito versus tempo de acompanhamento ( em meses ) em pacientes jovens com dlco / volume alveolar baixa , esclerose sistmica ( e padro histolgico de pneumonia intersticial no especfica celular ) , cox-1 total de alto grau e cox-2 de baixo grau em septos alveolares . a curva superior representa o grupo de pacientes com esclerose sistmica e pneumonia intersticial no especfica celular . a curva inferior representa dois grupos de pacientes : aqueles com esclerose sistmica e pneumonia intersticial no especfica fibrtica e aqueles com fibrose pulmonar idioptica e padro histolgico de pneumonia intersticial usual . tabela 3 regresso de riscos proporcionais de cox para verificar a contribuio individual do padro histolgico e fatores morfolgicos associados sobrevida e comparar os dois grupos quanto sobrevida ajustada . o nmero limitado de abordagens teraputicas que tenham qualquer impacto na sobrevida em longo prazo em pacientes com fpi - piu e naqueles com es e pine fibrtica deve - se falta de definio a respeito dos mediadores e mecanismos envolvidos na patognese da fpi e da es . portanto , a questo de interesse se mediadores adicionais podem ajudar a compreender melhor a patognese dessas doenas . o processo de reparo envolve duas fases distintas : uma fase inflamatria regenerativa , em que o microambiente tenta substituir as clulas lesadas , e uma fase fibrtica , em que o tecido conjuntivo substitui o tecido parenquimatoso normal.(- ) no processo de reparo , aumenta a produo de pge2 por fibroblastos , ( , ) o que constitui evidncia adicional das propriedades antiproliferativas , anti - inflamatrias e antifibrticas da cox-2/pge2 . ( ) portanto , nosso achado de que a marcao imuno - histoqumica para cox fornece informaes importantes sobre os processos de reparo na fibrose pulmonar no surpreendente , e nossos resultados confirmam que a expresso de cox-2 maior na fpi e na es , com desfecho melhor em um grupo de pacientes . constatamos que a proporo de clulas em septos alveolares com imunomarcao positiva para cox-1 e cox-2 foi significativamente maior em tecido pulmonar com padro histolgico de piu e pine fibrtica do que em tecido pulmonar normal . a maior expresso de cox-1 era esperada , pois a cox-1 expressa constitutivamente na maioria das clulas e tecidos , ao passo que a cox-2 induzida por estmulos inflamatrios ou mitognicos . ( ) esses resultados contrastam com os de estudos anteriores nos quais se investigou a fpi . ( , , ) esses estudos mostraram uma reduo da expresso de cox-2 em fibroblastos pulmonares em consequncia de uma diminuio da produo de cox-2 . no entanto , nesses estudos , a expresso de cox-2 foi medida apenas em fibroblastos , ao passo que em nosso estudo ela foi medida nos septos alveolares , incluindo clulas epiteliais e fibroblastos em reas normais , reas em colapso , e focos de fibroblastos . outros estudos , inclusive um estudo realizado por lappi - blanco et al . , ( ) encontraram um aumento da expresso de cox-2 em epitlio metaplsico e fibroblastos provenientes de reas fibrticas em fpi - piu . esses resultados divergentes sugerem que a cox-2 desempenha um papel duplo na fpi - piu . primeiro , a reduo da expresso da cox-2 em reas normais , reas em colapso e focos fibroblsticos sugere que a cox-2 desempenha um papel anti - inflamatrio na fpi - piu em fase inicial . segundo , a presena de fibrose progressiva mesmo na presena de aumento da expresso de cox-2 sugere que os fibroblastos so incapazes de responder ao estmulo da cox-2 e seu produto principal ( pge2 ) de modo a inibir a proliferao de fibroblastos , a transformao miofibroblstica e o aumento da produo de colgeno e outras molculas da matriz extracelular . no presente estudo , a proporo de clulas em septos alveolares com imunomarcao positiva para cox-1 e cox-2 foi menor em tecido fibrtico pine ( proveniente de pacientes com es ) do que em tecido piu ( de pacientes com fpi ) . esse achado contrasta com os de estudos anteriores em que se mostrou que os nveis de cox-2 so mais elevados em pacientes com es.(- ) alm disso , relatou - se que a produo de cox-2 muito maior na fase de resoluo inflamatria do que na fase inicial . ( ) esses achados divergentes sugerem que a cox-2 tem um papel duplo em um processo inflamatrio normal , desempenhando um papel pr - inflamatrio na fase inicial e um papel anti - inflamatrio na fase de resoluo . ( ) portanto , diante das evidncias mencionadas acima e da inflamao latente em pacientes com es e envolvimento pulmonar , nossos resultados reforam a ideia de que a cox-2 no exerce seu efeito anti - inflamatrio de maneira apropriada , pois h inflamao mesmo quando a expresso de cox-2 est aumentada em pacientes com es e pine fibrtica . no entanto , mais estudos so necessrios para esclarecer o verdadeiro motivo pelo qual o mecanismo da cox-2 deficiente . supomos que isso se deva incapacidade da cox-2 de estimular a produo de pge2 ou de outros mediadores anti - inflamatrios em oposio a seus prprios efeitos pr - inflamatrios ou incapacidade das clulas procuramos estabelecer uma correlao entre a cox-2 e a sobrevida dos pacientes controlada quanto idade , aos resultados dos testes de funo pulmonar , ao padro histolgico de piu ( em pacientes com fpi ) e ao padro histolgico de pine ( em pacientes com es ) . nossa anlise multivariada revelou baixo risco de morte para pacientes mais jovens com dlco / volume alveolar baixa , es ( e o padro histolgico de pine ) cox-2 total de alto grau e cox-1 de alto grau em septos alveolares . em suma , a expresso de cox-1 e cox-2 no parnquima pulmonar nos oferece o potencial de controlar processos de reparo envolvidos na progresso da es - pine e da fpi - piu , sugerindo que estratgias destinadas a prevenir a baixa sntese de cox-1 tero maior impacto na es , ao passo que as destinadas a prevenir a elevada sntese de cox-2 tero maior impacto na fpi . ensaios randomizados prospectivos so necessrios para confirmar isso .

objective : to study the expression of cox-1 and cox-2 in the remodeled lung in systemic sclerosis ( ssc ) and idiopathic pulmonary fibrosis ( ipf ) patients , correlating that expression with patient survival . methods : we examined open lung biopsy specimens from 24 ssc patients and 30 ipf patients , using normal lung tissue as a control . the histological patterns included fibrotic nonspecific interstitial pneumonia ( nsip ) in ssc patients and usual interstitial pneumonia ( uip ) in ipf patients . we used immunohistochemistry and histomorphometry to evaluate the expression of cox-1 and cox-2 in alveolar septa , vessels , and bronchioles . we then correlated that expression with pulmonary function test results and evaluated its impact on patient survival . results : the expression of cox-1 and cox-2 in alveolar septa was significantly higher in ipf - uip and ssc - nsip lung tissue than in the control tissue . no difference was found between ipf - uip and ssc - nsip tissue regarding cox-1 and cox-2 expression . multivariate analysis based on the cox regression model showed that the factors associated with a low risk of death were younger age , high dlco / alveolar volume , ipf , and high cox-1 expression in alveolar septa , whereas those associated with a high risk of death were advanced age , low dlco / alveolar volume , ssc ( with nsip ) , and low cox-1 expression in alveolar septa . conclusions : our findings suggest that strategies aimed at preventing low cox-1 synthesis will have a greater impact on ssc , whereas those aimed at preventing high cox-2 synthesis will have a greater impact on ipf . however , prospective randomized clinical trials are needed in order to confirm that .

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

lung remodeling is a common end - stage sequela of idiopathic pulmonary fibrosis ( ipf ) and systemic sclerosis ( ssc ) , resulting in disruption of lung architecture , leading to progressive respiratory failure. ( , , ) therefore , modulation of inflammation , fibroblast proliferation , and collagen synthesis by effector mediators in ipf and ssc is very important . despite the characterization of a variety of key participants , the mediators and mechanisms involved in the pathogenesis of ipf and ssc have yet to be fully defined , which might explain the limited number of therapeutic approaches , with little impact on long - term survival . ( , ) it is known that cox is the key enzyme in the conversion of arachidonic acid to prostaglandin e2 ( pge2 ) , the precursor of a diverse family of bioactive lipid mediators including prostaglandins , thromboxane , and prostacyclin . the former is constitutively expressed in most tissues and acts as a housekeeping enzyme regulating vascular homeostasis , protecting the gastric mucosa , and maintaining renal integrity , ( , ) whereas the latter has lower levels of expression in most tissues but is inducible in response to growth factors , cytokines , and other proinflammatory molecules. (- ) regarding the proinflammatory and anti - inflammatory roles of cox-1 and cox-2 , immunohistochemistry can be a useful tool to detect cox-1 and cox-2 in the remodeled lung in patients with ssc and ipf . data on the assessment of cox-1 and cox-2 in the remodeled lung have previously been reported in serum ( , ) and bronchoalveolar lavage fluid ( ) from patients with ssc , as well as in fibroblast cultures ( ) and biopsies ( , ) from patients with ipf . however , the roles of cox-1 and cox-2 in the mechanisms involved in the remodeled lung in ipf and ssc patients are still unclear , and there has been uncertainty regarding the best way to detect cox-2 . the aim of the present study was to study the expression of cox-1 and cox-2 in lung biopsy specimens ( cox-1 and cox-2 expression being separately evaluated in alveolar septa , bronchioles , and vessels ) and correlate it with patient survival . between january of 2002 and july of 2008 , 24 consecutive patients with ssc and interstitial lung disease and 30 patients suspected of having ipf on the basis of hrct findings were submitted to open lung biopsy at the university of so paulo school of medicine hospital das clnicas , located in the city of so paulo , brazil . all patients fulfilled the diagnostic criteria for ssc ( ) and ipf ( ) open lung biopsy was performed by formal thoracotomy , areas of honeycombing being avoided . pulmonary function testing included vc , fev1 , fvc , fev1/fvc , tlc , rv , and dlco . physiological assessment was performed before open lung biopsy and before the initiation of treatment . all pulmonary function tests , including spirometry , determination of lung volumes , and measurement of dlco , were performed on the same day . ( ) lung volumes were measured with a whole - body plethysmograph ( medical graphics co. ) , all values being expressed as a percentage of the predicted values . the primary endpoint was to evaluate the impact of cox-1 and cox-2 changes on survival and analyze differences between ssc and ipf . table 1 clinical data of the patients with systemic sclerosis and of those with idiopathic pulmonary fibrosis.a regarding open lung biopsy findings , usual interstitial pneumonia ( uip ) , the histological pattern of ipf , was characterized by patchy subpleural and paraseptal distribution of parenchymal injury . temporal heterogeneity was seen at low magnification , areas of normal lung parenchyma alternating with alveolar collapse , interstitial mononuclear infiltrates , septal fibromyxoid tissue ( fibroblastic foci ) , and honeycomb lung . ( ) all of the patients with ssc had histological patterns consistent with fibrotic nonspecific interstitial pneumonia ( nsip ) , as defined by temporally homogeneous septal thickening and interstitial fibrosis . ( ) for immunohistochemistry analysis , a standard peroxidase technique was used ( harris 's hematoxylin being used as the counterstain ) in order to identify cox-1 and cox-2 expression in alveolar septa , bronchiolar walls , and vascular walls in normal lung tissue ( the control tissue ) , in lung tissue showing the uip pattern ( the uip tissue ) , and in lung tissue showing the nsip pattern ( the nsip tissue ) . the specificity of primary antibodies was confirmed by appropriate reagent controls ( the primary antibody being omitted or nonimmune serum being substituted for the primary antibody in the staining protocol ) , which revealed no staining . regarding histomorphometry , cox-1 expression and cox-2 expression were assessed by a point - counting technique in 50 and 30 fields in alveolar septa , bronchiolar walls , and vascular walls in the control tissue , in the uip tissue , and in the nsip tissue . ( ) at a magnification of 400 , the septal , bronchiolar , and vascular areas in each field were calculated on the basis of the number of points overlying connective tissue , as a proportion of the total grid area . the student 's t - test for independent samples was used in order to test the relationship between continuous variables , and the residuals were examined to ensure that they were approximately normally distributed . the relationship between cellularity ( as determined by immunostaining ) and pulmonary function test results was evaluated by pearson 's correlation coefficient . the variables that were found to be significant in the univariate analysis were included in the multivariate analysis based on the cox proportional hazards regression model . table 1 summarizes the clinical features of the patients with ssc ( n = 24 ) and those of those with ipf ( n = 30 ) . six of 17 ssc patients ( 35.29% ) and 13 of 19 ipf patients ( 68.42% ) had restrictive lung disease . respiratory function test results were as follows : fvc < 80% in 18 ( 75% ) of the 24 ssc patients and in 19 of 22 ipf patients ( 86.36% ) ; tlc < 80% in 6 of 17 ssc patients ( 35.9% ) and in 13 of 19 ipf patients ( 68.42% ) ; dlco < 80% in 12 of 15 ssc patients ( 80% ) and in 8 of 9 ipf patients ( 88.88% ) ; and dlco / alveolar volume < 80% in 11 of 18 ssc patients ( 61.11% ) and in 11 of 14 ipf patients ( 78.57% ) . a significant negative correlation was found between cox-2 expression in vessels and fvc ( r= 0.28 ; p = 0.05 ) , as well as between cox-2 expression in alveolar septa and dlco ( r = 0.80 ; p = 0.009 ) . figure 1 shows alveolar septa , vessels , and bronchioles in the control tissue , in the nsip tissue , and in the uip tissue immunostained for cox-1 ( in a , c , e , g , i , k , m , o , and q ) and cox-2 ( in b , d , f , h , j , l , n , p , and r ) . the nsip and uip tissues differed from the control tissue in terms of the immunostaining intensity of epithelial cells , endothelial cells , myofibroblasts , and smooth muscle cells in the alveolar septa , vessels , and bronchioles . figure 1 cellular expression of cox-1 and cox-2 in alveolar septa , intrapulmonary vessels , and bronchioles in normal lung tissue ( control tissue ) ; in lung tissue obtained from patients with systemic sclerosis ( ssc ) and showing fibrotic nonspecific interstitial pneumonia ( nsip ) ; and in lung tissue obtained from patients with idiopathic pulmonary fibrosis ( ipf ) and showing usual interstitial pneumonia ( uip ) . the intensity of cox-1 immunostaining of epithelial cells , endothelial cells , myofibroblasts , and smooth muscle cells in ssc - nsip and ipf - uip tissue alveolar septa ( g and m , respectively ) , vessels ( i and o , respectively ) , and bronchioles ( k and q , respectively ) was higher than was that of cox-1 immunostaining of those cells in control tissue alveolar septa ( a ) , vessels ( c ) , and bronchioles ( e ) . likewise , the intensity of cox-2 immunostaining of those cells in ssc - nsip and ipf - uip tissue alveolar septa ( h and n , respectively ) , vessels ( j and p , respectively ) , and bronchioles ( l and r , respectively ) was higher than was that of cox-2 immunostaining of those cells in control tissue alveolar septa ( b ) , vessels ( d ) , and bronchioles ( f ) . the bar plots show the quantification of cox-1 and cox-2 immunostaining of cells in alveolar septa ( s ) , total lung parenchyma ( t ) , and bronchioles ( w ) in the control tissue , in the ssc - nsip tissue , and in the ipf - uip tissue ( immunohistochemical staining ; magnification , 400 ) . the proportion of alveolar septal cells immunostained for cox-1 and cox-2 was significantly higher in the uip and nsip tissues than in the control tissue . in other words , high proportions of alveolar septal cells staining for cox-1 and cox-2 were associated with the uip and nsip patterns . as can be seen in the bar plots in figure 1 ( s and t ) the relationship of cox-1 and cox-2 with ipf ( the uip pattern ) was stronger than was that of cox-1 and cox-2 with ssc ( the nsip pattern ) . although the proportion of bronchiolar cells immunostained for cox-2 was lower in the nsip and uip tissues than in the control tissue ( figure 1w ) , the difference was not statistically significant . in addition , although the proportion of bronchiolar cells immunostained for cox-1 was higher in the uip and nsip tissues than in the control tissue ( figure 1w ) , the difference was not significant . table 2 morphometric results in normal lung tissue ( control tissue ) , in lung tissue showing the usual interstitial pneumonia pattern ( from patients with idiopathic pulmonary fibrosis ) , and in lung tissue showing the nonspecific interstitial pneumonia pattern ( from patients with systemic sclerosis).a a preliminary analysis of the kaplan - meier survival curves showed that survival was better in the patients with ssc ( the fibrotic nsip pattern ) and cox-2 expression > 2.25% ( median survival , 70.75 months ) than in those with ipf ( the uip pattern ) and cox-2 expression < 2.25% ( median survival , 46.32 months ; figure 2 ) . therefore , we coded the fibrotic nsip pattern as a single dummy variable with a value of 1 and the uip pattern with a value of 2 . the results of the multivariate analysis based on the cox proportional hazards regression model are shown in table 3 . after controlling for age , pulmonary function test results , the uip pattern , and the fibrotic nsip pattern , we found that only two variables were significantly associated with survival time : the fibrotic nsip pattern and alveolar septal cox-2 ( p = 0.02 ) . the multivariate analysis showed a low risk of death for young patients with low fev1/fvc , fibrotic nsip pattern , and high - degree alveolar septal cox-2 . figure 2 cox regression plots for risk of death risk versus duration of follow - up ( in months ) in young patients with low dlco / alveolar volume , systemic sclerosis ( and a histological pattern of cellular nonspecific interstitial pneumonia ) , high - degree total cox-1 , and low - degree alveolar septal cox-2 . the top curve represents the group of patients with systemic sclerosis and cellular nonspecific interstitial pneumonia . the bottom curve represents two groups of patients : those with systemic sclerosis and fibrotic nonspecific interstitial pneumonia ; and those with idiopathic pulmonary fibrosis and the usual interstitial pneumonia pattern . table 3 cox proportional hazards regression to ascertain the individual contribution of the histological pattern and morphological factors associated with survival and to compare adjusted survival between the two groups . the limited number of therapeutic approaches that have any impact on long - term survival in patients with ipf - uip and in those with ssc and fibrotic nsip is due to the lack of definition regarding the mediators and mechanisms involved in the pathogenesis of ipf and ssc . (- ) in the repair process , pge2 production by fibroblasts is increased , ( , ) which constitutes further evidence of the antiproliferative , anti - inflammatory and antifibrotic properties of cox-2/pge2 . ( ) therefore , our finding that immunohistochemistry staining for cox provides important information on the repair processes in pulmonary fibrosis is not surprising , and our results confirm that the expression of cox-2 is increased in ipf and ssc , with improved outcome in a group of patients . we found that the proportion of alveolar septal cells immunostained for cox-1 and cox-2 was significantly higher in lung tissue showing the uip pattern and the fibrotic nsip pattern than in normal lung tissue . increased cox-1 expression was expected because cox-1 is constitutively expressed in most cells and tissues , whereas cox-2 is induced by inflammatory or mitogenic stimuli . ( , , ) those studies showed reduced cox-2 expression in pulmonary fibroblasts secondary to decreased cox-2 production . however , in those studies , cox-2 expression was measured only in fibroblasts , whereas in our study it was measured in the alveolar septa , including epithelial cells and fibroblasts in normal areas , collapsed areas , and fibroblast foci . ( ) found increased expression of cox-2 in metaplastic epithelium and fibroblasts from fibrotic areas in ipf - uip . first , reduced cox-2 expression in normal areas , collapsed areas , and fibroblastic foci suggests an anti - inflammatory role for cox-2 in early - stage ipf - uip . second , the presence of progressive fibrosis even in the presence of increased cox-2 expression suggests that fibroblasts are unable to respond to stimulation by cox-2 and its main product ( pge2 ) so as to inhibit fibroblast proliferation , myofibroblastic transformation , and increased production of collagen and other extracellular matrix molecules . in the present study , the proportion of alveolar septal cells immunostained for cox-1 and cox-2 was found to be lower in fibrotic nsip tissue ( from ssc patients ) than in uip tissue ( from ipf patients ) . this finding contrasts with those of previous studies showing that cox-2 levels are higher in ssc patients. (- ) in addition , cox-2 production has been shown to be much greater in the inflammatory resolution phase than in the early phase . ( ) these contradictory findings suggest that cox-2 has a dual role in a normal inflammatory process , playing a proinflammatory role in the early phase and an anti - inflammatory role in the resolution phase . however , further studies are needed in order to clarify the real reason why the cox-2 mechanism is deficient . we sought to establish a correlation between cox-2 and patient survival controlled for age , pulmonary function test results , the uip pattern ( in patients with ipf ) and the nsip pattern ( in patients with ssc ) . our multivariate analysis showed a low risk of death for younger patients with low dlco / alveolar volume , ssc ( and the nsip histological pattern ) , high - degree total cox-2 , and high - degree alveolar septal cox-1 . in conclusion , the expression of cox-1 and cox-2 in the lung parenchyma offers us the potential to control repair processes involved in the progression of ssc - nsip and ipf - uip , suggesting that strategies aimed at preventing low cox-1 synthesis will have a greater impact on ssc , whereas those aimed at preventing high cox-2 synthesis will have a greater impact on ipf . figura 2 curvas de regresso de cox para risco de bito versus tempo de acompanhamento ( em meses ) em pacientes jovens com dlco / volume alveolar baixa , esclerose sistmica ( e padro histolgico de pneumonia intersticial no especfica celular ) , cox-1 total de alto grau e cox-2 de baixo grau em septos alveolares .

conformer generation has been a topic of considerable interest to the modeling community for a number of years , and a large number of methods for conformer generation have been presented . these methods have utilized a wide variety of approaches to the problem , including systematic enumeration , knowledge - based rule sets , random coordinate embedding , and energy - based or energy - biased sampling . all these approaches attempt to generate a set of conformations designated to be different from one another by some measure , usually geometric , and thereby aim to sample some subset of the conformational space available to a molecule . the method used by omega is based on random coordinate embedding and refinement , followed by torsion driving using a set of rules derived from experimental structures and energy profile calculations , and then subsequent sampling of the resulting conformer ensemble by geometric and energetic criteria . publications that demonstrate the utility of conformer generators have approached the problem of validation of such tools in a number of different ways . conformer generation is not an end in itself but a means to obtain input for other applications , most frequently shape comparison tools ( or other 3d ligand matching approaches ) and docking engines . indeed conformer generation is often subsumed as a portion of a docking algorithm , which prevents independent evaluation of the conformer generation within such tools . one approach to validation has , therefore , been to generate conformer ensembles for use in a downstream tool ( e.g. , pharmacophore perception).(9 ) the quality of the conformer ensembles is then judged by the quality of the output from the downstream tool . while it might be argued that the real - life use of conformer generators is well reflected in this approach , judging the quality of a conformer generator based on the performance of a separate tool is fraught with problems . if this tool has been optimized to use conformers from a certain engine , then it would be expected to produce better results from this engine over all others . other approaches to validating conformer generators have emphasized estimation of conformational coverage based on both pharmacophoric and geometric measures.(10 ) in studies of this kind , coverage of conformational space is held to be an absolute good and the tools under study are assessed based on their ability to cover the greatest amount of conformational space . however , there are large areas of accessible conformational space for a flexible molecule that are populated by conformers very different in shape and geometry from any experimentally determined conformer ( be it in the solid state , in solution , or in the gas phase ) . as such , simply maximizing coverage of conformational space may result in generation of a conformer set containing a high proportion of conformers irrelevant to any measurable experimental property of the molecule . by far , the most common , though not always the most well - executed , method for validation of conformer generators , and the method used in this paper , is the reproduction of experimental crystal structures(11 ) ( often those retrieved from the protein databank ( pdb)(12 ) ) . the implicit argument proffered for this approach is that coverage of conformational space near an experimental conformation of a molecule is more important than coverage in other regions of conformational space . since experimental structures , while frequently not at a local minimum for a force field,(13 ) are generally considered to exhibit low levels of strain,(14 ) the regions of conformational space near an experimental structure can be considered as basins in the molecule s energy profile . accordingly , assessing sampling in these regions of the energy hypersurface can be a good guide to the performance of a method in generating low - energy conformations . to perform this sort of validation , the connection tables of these ligands are used as input for the conformer generator to provide a set of conformers for each ligand , unbiased by any structural information of the original experimental conformation . a metric of quality is then calculated that provides a measure of the three - dimensional similarity between the experimental conformation for the ligand and the conformers in the set . there are , therefore , two critical choices to this method of validation : selection of the appropriate metric of quality to perform the comparison and selection of suitable cocrystal structures to provide the ligands . while many metrics for comparing computed and experimental conformations are available , such as rms error in cartesian space ( rmsd ) , rms error in torsion space ( rmst ) , and relative displacement error ( rde),(15 ) choosing the metric of quality presents some subtle problems . atom - based metrics like rmsd , while very popular , have significant problems in their interpretation and necessarily have a dependence for their significance on the experimental error / precision of the crystal structure ( a particularly difficult issue for pdb ligands ) . in addition , rde and rmsd have no easily calculable upper bound ( unlike rmst ) , and all these measures have a size dependence that is not easily compensated for . in this paper , two different metrics are used to compare computed and experimental conformations of druglike molecules , one atom based ( rmsd ) and one shape based ( tanimoto combo ) . , it is hoped to provide a more complete picture of the ability of omega ( or any other conformer generator ) to match experimental conformations . identifying suitable cocomplex structures from the pdb to provide the experimental conformations of small molecules unfortunately , in most publications in this area , insufficient care is taken in the selection of the structures , resulting in unwarranted or questionable conclusions being drawn based on unreliable data . some of the issues that should be considered in the selection of structures from the pdb include the following : are the deposited models of good quality at both the global and local levels ? there is little sense in trying to reproduce a deposited structure that is a poor model of the experimental density or a model that is not completely defined by the density . when using atom - based metrics like rmsd , paying attention to the precision in the atomic coordinates is critical : an rmsd or any measure of conformer reproduction more precise than the inherent inaccuracy / experimental error in the atomic coordinates is meaningless . significant atomic clashes , either between the ligand and the protein or between atoms of the ligand , must be artifacts of the process of fitting the ligand to the experimental electron density and not an inherent property of the structure . the binding energy available upon proteinligand complexation is unlikely to be sufficient to drive atoms into close contact with one another . a set of ligands with a large degree of structural redundancy does not provide as rigorous a test of conformer generators as a structurally nonredundant set does . also , in order for the results of the validation to be useful in a predictive or prospective manner , they should be readily generalizable to any set of applicable molecules . this can only be done with validation sets that cover chemical space in a reasonable way . consideration of these questions has allowed the development of quantitative criteria for structural quality in the pdb . these criteria were applied as successive filters to three previously published large data sets of cocrystal structures , pdbbind,(16 ) the sadowski set(13 ) and the kirchmair set,(17 ) to provide a high quality group of cocrystal structures to establish a gold standard for the validation of approaches to conformer sampling . a set of small molecule structures from the cambridge structural database ( csd)(18 ) was also collected to provide complementary data to that obtained with the ligands from the pdb . these two data sets are applied to the evaluation of the omega conformer generation application using the metrics mentioned above . here , the omega algorithm is introduced in some detail ; the metrics for comparison of the generated conformers to the experimental structures are outlined , and the criteria for the identification of suitable proteinligand cocomplexes and small molecule structures with which to validate the algorithm are discussed . the design goal for omega is to provide a thorough , though not exhaustive , sampling of conformational space for druglike molecules at as high a speed as possible ( typically 22.5 s per molecule on a workstation running sled 10 with a 2.4 ghz processor and 4 gb of ram ) . the process used by omega for conformer generation can be considered in several parts , two of which are generic and are precalculated and the rest of which are molecule specific and occur at run time . the first two steps are construction of a database of fragments from which the molecule will be assembled and derivation of a torsion library ; the remaining steps generate a large ensemble of conformations and sample from this ensemble to deliver the final set of conformers . the omega fragment database contains one or more 3d conformations for every entry ( flexible rings have multiple conformations , rigid rings and acyclic groups have one conformation ) . the database is generated by fragmenting a very large collection of commercially available compounds into contiguous ring systems and small linear linkers . one or more 3d conformations for each fragment are generated by the following procedure : a distance bounds matrix is generated based on the connection table of the fragment ; the distance bounds are augmented by volume constraints for chirality and planarity ; the coordinates of each atom are randomly embedded in a cartesian space and optimized to fulfill the bounds and constraints ; the fragment is further refined against a modified version of the merck molecular force field ( mmff94),(19 ) in which the electrostatic and attractive van der waals terms are removed . the procedure is repeated hundreds of times for each fragment , with cycles being sampled more heavily than acyclics , and the lowest energy conformers that are unique in rmsd space are retained . a hierarchy of torsions defined by smarts patterns is preorganized so that every rotatable bond in a small molecule matches at least one torsion rule . every torsion definition is associated with a list of angles at which the torsion should be sampled . very common or structurally unique torsions may be represented by very specific rules , while unusual torsions are only covered by generic torsion rules , often with relatively heavy sampling . specific rules are employed when the barrier to rotation is high and the minima in the torsion profile are well - known . the torsion angles specified by the torsion library were derived by analysis of a set of experimental crystal structures ( mostly from the pdb ) and from energy scans of certain torsions against mmff94 . each graph is fragmented in the same manner as the fragment database and fragment conformations are drawn directly from the fragment database described in step 1 . if a molecular fragment is not found in the fragment database , the procedure described in step 1 is carried out on - the - fly to generate coordinates for the missing fragment . the fragments are assembled into the parent molecule by overlapping fragments using geometric and chemical rules , thereby providing one or a small number of initial conformations for the molecule . this set of conformers includes all necessary ring conformer sampling . every rotatable bond in the conformers generated in step 3 is compared to the torsion library from step 2 , and the appropriate torsion angles are noted . a torsion buildup procedure is applied to all the torsions in the molecule to generate a large ensemble of conformations that does not contain severe internal clashes or duplicates due to common symmetries . the design goal of omega is to produce up to a few hundred low energy , structurally diverse conformers , and as such , the much larger collection of conformers produced by step 4 must be sampled . this process is begun by ordering the conformers using a simple scoring function that eliminates conformers with internal clashes . while many functions are suitable for this process , omega uses a modified form of mmff94 ( vide supra ) . next , beginning with the lowest scoring conformer , all higher scoring conformers that are less than a user - defined rmsd ( including heavy atom automorphisms ) to the lower scoring conformer are eliminated ( the default rmsd cutoff is 0.5 ) . this process is continued with each successive conformer in the ordered list until ( a ) all conformers have a score less than 10 units higher than the lowest energy conformer produced or ( b ) 200 mutually unique conformers are identified . at no stage in this process is minimization performed because this tends to produce highly compact ( folded ) conformations not reflective of the conformation(s ) found in solution or bound to a protein . this is mostly due to the differences between the gas phase and the solution phase or complexed potential energy surfaces for small molecules . extensive experimentation in - house has shown that minimization of ensembles of conformers either before or after deduplication improves neither solid - state structure reproduction nor virtual screening performance with tools like rocs or fred . two metrics for comparison of conformer ensembles to an experimental conformation were chosen , rmsd and the tanimoto combo , calculated using the shape toolkit from openeye scientific software . the tanimoto combo ( or tc ) score represents a combination of shape matching ( shape tanimoto ) and functional group matching ( color tanimoto ) . tc offers a complementary approach to the atom - based rmsd measure for a number of reasons treated at greater length in the discussion section . an important difference between tc and rmsd is that tc is bounded ( by 0 and 2 ) . it should be noted that the processes used to overlay and score conformers using rmsd or tc are very different , and therefore , the conformer giving the minimum rmsd to the experimental structure will almost always be different than the conformer giving the maximum tc to the experimental structure . two sources of small molecule structures , the pdb and the csd , were utilized . to find small molecules from the pdb , first , suitable cocrystal structures were identified from larger sets using global criteria ; then , the ligands were extracted and inappropriate ligands were removed using local , ligand - centric criteria . small molecule structures from the csd were obtained from a previously published data set,(18 ) which was subjected to some of the ligand - centric criteria used to identify suitable pdb ligands . there are two kinds of criteria that must be considered here : global criteria for the structure as a whole and local criteria pertaining to the fit of the ligand to its local density and the properties of the ligand itself . a number of possible criteria of quality for crystal structures were enumerated in a paper by hartshorn et al . on the selection of a set of structures suitable for validating docking programs for were that all structures must have good nominal resolution and have deposited structure factors , while the local criteria were that the ligand must be well fit to its local density and can not be covalently bound to the protein . this list has been extended in this work , both at the global level and the local level . in addition to the hartshorn criteria at the global level ( good nominal resolution , deposited structure factors in the pdb ) , the model must have good overall metrics of model quality and reasonable coordinate precision . at the local level , it is demanded that a ligand be well fit ( using different criteria to hartshorn et al . it is further required that the ligands show no intra- or intermolecular clashes and that they be diverse at the graph level . nominal resolution , while very commonly used as a measure of structure quality , is in fact a measure of the quantity of data gathered , not quality of the structure , and as such can only be used as a very rough guide to selecting a good structure.(21 ) in many studies in this area , the nominal resolution has been used as a surrogate for experimental accuracy , which is inappropriate ( see ref ( 22 ) and the discussion ) . however , since nominal resolution indicates the quantity of data gathered , at a resolution of 2.7 or better , the ratio of experimental data points to parameters in the model is greater than 1 . as such , structures with a resolution of 2.7 or better offer the possibility ( but only the possibility ) of a well determined model . the quality of a deposited model can be independently checked only if structure factors ( electron density maps ) are available in the pdb , so all structures must have deposited structure factors . in order to ensure that the models used are globally well fit to the data ( and not overfit ) , we use the difference between the r - factor and the rfree , as introduced by brunger.(23 ) in this work , we demand that a structure must have a rfree within 0.05 of the r - factor.(24 ) in comparing computed and experimental poses for ligands , it is almost uniformly assumed that the experimental pose is infinitely precise and without error , which is untrue for any piece of experimental data . the average positional errors for atoms in a crystal structure can be assessed using the diffraction - component precision index ( dpi).(25 ) we calculate dpi according to the approximation published by blow(26 ) ( using the blow rearrangement allows us to calculate an estimate for dpi based solely upon information commonly contained in the header of a pdb file ) . the comparison of a computed conformer to the experimental conformer by rmsd obviously requires comparison of atom positions between the two conformers . accordingly , the positional uncertainty in the atoms in the experimental structures should be less than the rmsd for the comparison to be meaningful . in this work , we estimate positional uncertainty in atomic positions as sqrt(2 ) dpi , following goto et al.(27 ) the maximum for the average positional uncertainty in a structure was set at 0.6 , which sets the maximum for the dpi to be 0.42 . in this way , any rmsd result from this set greater than 0.6 is guaranteed to be a prediction greater than the error in the experimental data , and therefore , such a prediction will be significant . whatever the magnitude of the rmsd , a correction to it will have to be made to account for positional uncertainty in the ligand atoms ( see the results section ) . the assumption used in this paper is that the ligand atoms have average positional uncertainty . while the real uncertainty for the ligand atoms may well be different , it is not reliably calculable at the present time ; therefore , we use dpi as our best estimate . when considering whether or not a ligand is suitable for conformer generation studies , the local quality of fit of the ligand to its density , as well as global metrics of quality , should be considered . in this study , three local metrics , the real - space correlation coefficient ( rscc),(28 ) the real - space r - value ( rsr),(29 ) and the occupancy - weighted b - factor ( owab ) were used to ensure that the ligand has been sensibly fitted to the electron density . the use of the occupancy weighted b - factor as a criterion was based not on the idea that this number necessarily measures thermal mobility in the ligand , as is often assumed . rather , we used the owab to indicate either structures where the ligand is genuinely disordered in the active site , and therefore , the idea of reproducing the bioactive conformation is questionable or structures where there is some pathology of the model that led , in the process of fitting the ligand to its density , to very high or very low b - factors . these models are inherently of dubious value and should not be included in the set . the criteria used in this work were that rscc > 0.9 , rsr < 0.2 , and 1 < owab < 50 . these data were obtained by download from the electron density server ( eds).(30 ) when applied sequentially , these global and local filters should ensure that a ligand structure arises from a good quality overall model and has been well fit to its local density . the filters were applied to a large set of pdb structures ( see results ) , and the cocomplexes that survived were then processed to separate the ligand and the protein . these pairs were checked to ensure that they do not show any intermolecular clashes , and those ligands that showed no clashes were designated as well - solved structures , to be used in the final , ligand - based filters . in these final steps , we focus on identifying suitable molecules using simple physicochemical and graph - based properties . in the last filtering step , we removed ligands that are not representative of the population of molecules for which this validation is intended . as a first step , we removed all well - known cofactors , as these were not as relevant to the goals of this study ( validation of omega s performance on druglike molecules ) . as mentioned above , omega depends upon mmff94 for its energy calculations , so molecules that contain elements unknown to mmff94 ( such as boron ) must be removed . to ensure that we used a collection of ligands that provides as broad a set of tests for the omega algorithm as possible , we attempted to select a structurally diverse set of ligands . at the simplest level , the molecules can be neither too rigid ( so that the conformer sampling problem is trivial ) nor too flexible ( so that a close reproduction of the experimental conformation is more a matter of good luck than a good algorithm because of the high dimensional nature of the search space ) and neither too small nor too large . we , therefore , applied the following criteria : 3 maximum number of rotatable bonds 16 ; 8 heavy atom count 50 . preliminary analysis revealed that molecules designed to bind to thrombin were over - represented in the set of well - solved ligands identified by the criteria used thus far . these ligands tended to share a high degree of substructural similarity , so a method to introduce some level of structural diversity at the graph level was sought . we applied a simple measure of molecular similarity using the lingos method;(31 ) any pair of molecules that had a lingos tanimoto > 0.9 was flagged for removal , and the molecule with the better set of local fitting metrics for the pair ( rscc , rsr and owab ) was retained . the use of graph - based metrics like lingos for structural diversity is a surrogate for the more relevant but more difficult to assess diversity in the torsions and flexible rings present in the molecule . to further reduce the influence of experimental error on this data set , we demand that the ligand does not show any nonbonded intramolecular atomic clashes , as these are a mark of a poorly solved model . as further discussed in the results section , we turned to the csd for precise distributions of interatomic distances with which to identify clashing atoms . a final visual check was performed to remove molecules that are structurally analogous but were not identified as such by the lingos comparison ( those with large common substructures and/or a large fraction of their functional groups in common ) . the attrition rates for the different data sets and an analysis of omega s ability to reproduce these 197 surviving structures is given in the results section . a parallel analysis was performed on high quality small molecule structures from the csd , as outlined in the following paragraphs . an important part of the selection criteria for structures from the pdb focused on removing structures that were not reliably and accurately fit to their density , and a large majority of the candidate structures failed these criteria ( see the results section ) . a further problem with molecules from the pdb is the high level of inherent experimental uncertainty in the atomic positions . both of these problems can be avoided entirely if small molecule structures from the cambridge structural database ( csd ) are used . a high quality set of 492 druglike structures has been extracted from the csd as part of a previously published study,(18 ) and these were used as the basis for generating a complementary set of structures to those we obtained from the pdb . no filtering of these structures based on model fit ( global or local ) was necessary as all atoms are accurately located in these models . applying the same physicochemical and diversity criteria as were used for the ligands from the pdb gave a set of 480 small molecule structures upon which we could perform the same validation experiments . comparison of these results to those from the ligands from the pdb can be found in the results section . all code was written in python ( version 2.4 ) , and statistical calculations were performed with rpy version 1.03 . cheminformatics functions ( including rmsd and lingos calculations ) were performed using openeye s oechem ( version 1.6.1 ) , and shape calculations were performed using the openeye shape toolkit ( version 1.6 ) . proteinligand cocrystal structures were downloaded from the pdb and filtered according to the criteria laid out in the methods section . rscc , rsr , and owab data for each pdb ligand were obtained from the eds . molecules from the csd were obtained from one of the authors of the original study ( personal communication , k. brameld ) . molecules were converted into isomeric smiles format using the oechem toolkit before being used for conformer generation . conformer databases were generated using omega version 2.3 , with an energy window for acceptable conformers of 10 kcal / mol above the ground state using a modified version of the mmff94 force field , a maximum number of conformations per molecule of 200 , and an rmsd cutoff of 0.5 ( the default settings in omega2.3 ) . in the methods section , we presented a set of criteria for selecting suitable proteinligand cocomplexes from the pdb and then identifying suitable ligands from those complexes . we applied these criteria to three large data sets ( two of which have previously been used in conformer validation studies ) that together consist of over 4500 cocrystal structures : 778 structures that kirchmair et al . used in their comparison of catalyst and omega,(17 ) the set used by sadowski and bostrom in their study on the omega 1.8.1 torsion library(13 ) ( 1267 structures ) and a subset of the pdbbind database(16 ) ( 2516 structures ) . the percentages of structures remaining after each filter was applied are illustrated in figure 1 . in all three databases , we found very large levels of loss when we applied our filtering criteria ( over 90% of the structures in every database were removed ) . it can easily be seen from figure 1 that there are three criteria that , when applied in the given order , remove the highest percentages of the structures : nominal resolution greater than 2.7 ( criterion a ) , poor fits of the ligand to its density ( criterion e ) , and inappropriate physicochemical properties ( criterion f ) . the filters used are ( a ) nominal resolution 2.7 ; ( b ) structure factors must be present ; ( c ) r rfree must be < 0.05 ; ( d ) dpi < 0.42 ; ( e ) rscc > 0.9 , rsr < 0.2 , owab < 50 , no intermolecular clashes ; ( f ) physicochemical properties ; ( g ) pairwise lingos similarity < 0.9 , no intramolecular clashes . the problem of identifying atomatom clashes ( filters e and g ) based on analysis of interatomic distances in structures from the pdb is nontrivial , due to the inherent uncertainty in those atom positions . therefore , we chose to perform an analysis of nonbonded contacts for commonly occurring atoms ( c , n , o , s , p , f , cl ) using high quality structures ( r - factor < 0.05 , no disorder ) of organic molecules from the csd , where the atoms are located with very high precision . the cutoffs identifying clashes were set at an interatomic distance above which 95% of the distances for that ( nonbonded ) atom pair in the csd occurred . results are shown as mean / median for each column heading ; u = the coordinate uncertainty ( sqrt(2 ) dpi ) . the only well fit ligands that can show intermolecular clashes are those that are covalently bound to their target protein , and these are explicitly excluded from this set . therefore , noncovalent ligands that have clashes , by these criteria , with their cognate protein are incorrectly solved . none of the ligands that passed the previous filters ( a to e in figure 1 ) showed any intermolecular clashes , indicating that the criteria used in these filters ( especially the rsr and rscc filters ) can be useful in identifying misfitted ligands . similarly , no ligands were removed based on the intramolecular clash filter , further reinforcing the idea that rscc and rsr can provide useful information on the quality of a ligand s fit to its density . however , it is noteworthy that several structures from such well - known data sets as the perola and charifson set(33 ) show intramolecular clashes , as these authors selected structures using only global criteria of fit , omitting local measures . as such , these structures should not be used in conformer generation studies , as has been done by some authors,(34 ) because the deposited coordinates for the ligand are incorrect ; atomatom clashes can not be enforced by protein binding and simply reflect an error in the solution of the structure . after the filters had been applied , the surviving ligands from each of these three sets were combined and deduplicated and close analogues were removed as discussed in the methods section . the mean pairwise similarity of these molecules using the lingos tanimoto measure is 0.18 , so the structures are relatively diverse from one another at a graph level . conformers were generated for these molecules using omega with the default settings ( see the methods section ) . the average cpu time per molecule in this set was 2.05 s. in figure 2 , we show the reproduction of the experimental conformation using the lowest rmsd and highest tanimoto combo metrics . approximately 83% of the structures are reproduced within a 1 rmsd and 66% are reproduced with a tanimoto combo of 1.5 or better . distribution of rmsd and tanimoto combo between the closest conformer in a set and the experimental conformation for 197 ligands from the pdb . the relationship between best ( lowest ) rmsd and best ( highest ) tanimoto combo for this data set is shown in figure 3 , where the lowest rmsd for any conformer to the experimental conformation in a set is plotted against the highest tanimoto combo for any conformer to the same experimental conformation . as expected , the cases where the rmsd is low ( < 0.25 ) all show very high tanimoto combos . as rmsd increases , the relationship to tanimoto combo becomes increasingly less linear , in that there are several examples where constant rmsd gives a widely varying tanimoto combo and vice versa . quantitatively , we can assess the correlation between the two measures using either the spearman rank correlation coefficient or the kendall tau . for this data , the spearman coefficient is 0.925 and the kendall tau is 0.775 . as such , the two measures of conformer reproduction are correlated ( as would be hoped for two measures attempting to assess the same thing ) but are by no means identical . best rmsd v. best 3d shape and chemical similarity ( tanimoto combo ) for reproduction of 197 ligands from the pdb . that these two measures are assessing conformer reproduction differently is to be expected , since they are generated in rather different ways and , therefore , provide complementary means to compare conformations . a striking difference in the two overlay methods is illustrated in figure 4 , where two different overlays of conformers of the ligand from the pdb structure 1 v2n are shown . the experimental conformation is in green ; the best overlay based on lowest rmsd is in red , and the best overlay based on highest tanimoto combo is in blue . the best rmsd overlay here is 0.89 , while the best tanimoto combo is only 1.06 ( tanimoto combo s range is 02 , see methods section ) . the origin of this difference in the assessment of the quality of reproduction when using these two different metrics is most clearly seen by inspecting the alignments of the benzamidine functional groups . comparison of the experimental conformation of the ligand 1 v2n ( green ) with the best overlay as provided by rmsd ( red ) and the best overlay as provided by tanimoto combo ( blue ) . as can easily be seen in figure 4 , the rmsd overlay in red symmetrically , but poorly , matches both benzamidine functional groups , while the tanimoto combo overlay matches one benzamidine group very well ( the one on the left in figure 4 ) while the position of the other benzamidine group is much less well reproduced , giving the surprisingly low tanimoto combo . this illustrates a consistent difference between the two methods of comparison ; rmsd tends to produce overlays that are equally good ( or bad ) everywhere across the molecule , while tanimoto combo tends to produce overlays that are very close in some portions but can be much more divergent in others . while all the structures in this validation set have high coordinate precision , there is still some level of inherent uncertainty in the atomic coordinates . therefore , when comparing a computed pose to an experimental pose using atom - based metrics like rmsd , this experimental uncertainty , u , in the atom positions must be taken into account . there are two possible approaches to this , both of which are shown in table 1 . the first is to use as the figure of merit the maximum of the rmsd and the coordinate uncertainty , u ( max(rmsd , u ) in table 1 ) , since the prediction can not be more accurate than the error in the experimental data ( the coordinate uncertainty ) . the second is to use the difference between the rmsd and the coordinate uncertainty ( rmsd - u in table 1 ) , which reflects the additional error in the model introduced by conformer generation . this number is set to zero if rmsd < u. as can be seen from column 3 ( rmsd - u ) in table 1 , the average amount of noise introduced by omega in generating a conformer close to the experimental is small . also , due to carefully choosing structures that have low levels of uncertainty , the mean and median for the maximum of the rmsd and experimental uncertainty ( column 4 ) are very close to the mean and median for the raw rmsd ( column 2 ) . therefore , the level of accuracy in our predictions is only minimally affected by the experimental uncertainty in the structures that make up the data set . an issue that is most often ignored in studies of this kind is the prospective utility of the performance metrics presented , or how robust are the performance metrics to changes in the composition of the data set and , therefore , how predictive are the results of future performance ? this question is addressed here by bootstrapping : we omit a randomly selected 10% of the results ( allowing duplicates ) , recalculate the mean rmsd and repeat 10 000 times , and then find the 5% and 95% quantiles . the 5% quantile is 0.647 , and the 95% quantile is 0.688 , thereby giving us the 90% confidence interval . therefore , we can say that for a future experiment on a set of molecules with similar properties to those of this pdb - derived set 90% of the time the mean rmsd will lie in the range 0.6470.688 . this small interval implies that , even though the data set is not as large as we had hoped , the mean found here is a reliable indicator of omega s average performance on as yet untested collections of ligands . the 90% confidence interval for the mean tanimoto combo can be calculated in the same fashion and is 1.54 to 1.64 . we may also use these confidence intervals as a method of comparing two sets of results to determine if they are significantly different from one another ( see discussion ) . so far , we have dealt with the properties of the single conformer closest by a given metric to the experimental . while this measure has significant utility , in another view of the problem , the proportion of conformers within a certain distance of the experimental structure is important . this method of measuring success is very rarely used(35 ) but provides a useful complement to focusing solely on the matching of only the best single conformer from a possibly large set , as we have done to this point . a common upper bound for successful reproduction of an experimental structure in docking is an rmsd of 2 , this number being thought of as providing a sufficiently close match of the important interaction points on the ligand ( hydrogen bond partners , etc . ) . accordingly , a close reproduction of an experimental pose could be considered to have an rmsd of less than 2 . in this work , since we calculate each metric after aligning the conformers to the experimental structure , a smaller cutoff is more relevant , so we have selected an rmsd of 1.25 as our definition of close . figure 5 shows the cumulative frequency of cases where conformers are close to the experimental . distribution of percentages of conformers that are close ( rmsd < 1.25 ) to the experimental conformation of 197 structures from the pdb . it can be seen from the figure that , for around half of the cases ( 47% ) , less than 25% of the conformers generated for a given molecule are close to the experimental conformation , while for around 8% of the cases 100% of the conformers generated are close . in total , 19% of the conformers generated for this set are within 1.25 rmsd of the protein - bound conformation . having a reasonable proportion of the conformers in the set close to the protein - bound structure is helpful for downstream protein - based tasks such as docking . however , as is seen in the next section , the same molecule may have multiple solid - state structures and , therefore , have too high a fraction of the conformational ensemble , for that molecule close to only one of them would not be desirable . as mentioned previously , 492 molecules from the csd were obtained from a previous publication , of which 480 survived the ligand filtering process ( physicochemical and graph diversity filters ) . the mean pairwise similarity of this set using the lingos tanimoto is 0.14 , again indicating that the molecules are diverse at a graph level . it was hoped that these molecules from the csd would represent a different set of challenges in conformer generation ; the pdb structures are obtained in an aqueous environment , and the majority of csd structures are obtained in nonpolar environments . accordingly , using sets of high - quality structures from both sources will further challenge omega s abilities to reproduce solid - state structures . while there are large numbers of small molecule structures in the pdb and the csd , the two databases show only a low level of overlap ; a comparison of over 4000 druglike ligands from the pdb with over 57 000 druglike molecules in the csd finds only 224 molecules in common ( see supporting information for pdb and csd codes ) . of these 224 molecules , only 32 meet the quality criteria for pdb structures laid out previously ( see methods ) . the differences in the conformations of these 32 molecules ( as measured by rmsd ) found both in the pdb and the csd sets are shown in figure 6 . the mean rmsd for this set is 1.02 , a figure close to that found in similar analyses . rmsd distribution for the same molecule found in both the csd and the pdb ( n = 32 ) . clearly , the same molecule in the csd and the pdb can sometimes adopt rather different conformations ( over 7% have rmsd > 2 ) , while other protein - bound and uncomplexed conformations can be quite similar ( 16% have rmsd 0.5 ) . it is likely that at least some of these differences in conformation are due to the crystal packing forces often found in csd structures . these can have a profound effect on the conformation of a molecule , sometimes enforcing a conformation not found in protein - bound structures . the larger differences in conformation for the same molecule indicate that there could be several basins in a molecule s energy hyper - surface in which solid - state structures are found , so sampling across a range of the hyper - surface could be useful . while the molecules from the csd were selected to be druglike , they have somewhat different physicochemical properties than those from the pdb . table 2 illustrates the differences between the csd molecules and the pdb molecules for two simple properties , heavy atom count and rotatable bond count , and figure 7 compares the distributions for these two properties . it can easily be seen that for both properties the molecules from the csd occupy a smaller range than those from the pdb . most importantly for this study , while the molecules from both sets are approximately equal in size ( as judged by their heavy atom counts ) , the csd - derived molecules are somewhat less flexible than the pdb derived set , having , on average , one fewer rotatable bond . therefore , the csd derived set is expected to be an easier test of conformer sampling and reproduction than the pdb set . given as mean / median . property distributions for molecules from the csd and the pdb . given the difference in rotatable bond count , the numbers of conformers produced by omega2 for this set and for the pdb set are rather different ; the median number of confomers per molecule was 47 for the csd set and 123 for the pdb set . in figure 8 is shown omega s performance in reproducing these structures , as judged by rmsd and tanimoto combo . distribution of rmsds and tanimoto combos between the closest conformer in a set and the experimental conformation for 480 molecules from the csd . as can be seen in figure 8 , 441/480 ( 84.5% ) of the csd structures are reproduced within 1 rmsd , while 400/480 ( 83.3% ) are reproduced within 1.5 tanimoto combo . note that in the case of these structures from the csd the positional uncertainty for the atoms is sufficiently small to be disregarded when calculating rmsds . the spearman rank correlation for the two metrics in table 3 is 0.89 , and the kendall tau is 0.72 , in both cases slightly smaller than those found for the pdb set , again indicating that these two measures are assessing different aspects of omega s ability to reproduce the experimental conformation . as with the results from the pdb ligands , these small intervals imply that our results are not strongly dependent upon the exact composition of this data set and as such are reliable indicators of future performance on data sets of similar property distributions to the one used in this study . it can also be said with greater than 90% confidence that the csd results , either for rmsd or for tc , are better than the pdb results since the 90% confidence intervals for either metric do not overlap . the results for close reproduction ( rmsd < 1.25 ) of the experimental conformations are much better than for the pdb ; overall , 39% of the conformations generated for the csd set are close to the experimental conformation . this is entirely consistent with the results from the csd set relative to the pdb set for best pose reproduction ( as judged by either tanimoto combo or rmsd ) . it is possible that the superior performance of omega on the csd set is not due to the lower average flexibility of the molecules but due to some consistent and unexpected difference in the nature of the molecules between the two sets . therefore , a subset of the pdb ligands was derived to closely match relevant physical properties of the csd set ( heavy atom count and rotor count ) . the aggregate performance metrics on this subset are much closer , though not identical , to those seen from the csd set , see table 4 . reproduction of three data sets by omega , using both tanimoto combo ( tc ) and rmsd . the 90% confidence intervals of the entire pdb set and the csd - matched subset of the pdb set do not quite overlap , though they are very close ( the difference could be entirely due to the physical property distributions of the two sets not being perfectly matched ) . therefore , we can not with complete assurance attribute the entire change in omega s performance to differences in the rotatable bond count between the two sets . however , we conclude that the superior performance of omega on the csd set derives to some large degree from the lower average flexibility of the csd set compared to the pdb set and not from bias in the omega knowledge base . we used both the pdb- and csd - derived test sets to investigate another possible source of bias in the results ; the torsion library , which was assembled mostly by examination of ligand structures from the pdb . if there is significant overlap between the molecules used to arrive at specific angles in the torsion library and the molecules in the test sets used in this study , then the results will be artificially good ( or artificially better for one test set over another ) . we investigated possible training set bias by eliminating all training set knowledge from the torsion library . in these experiments , the simplest possible torsion settings were used : every rotatable bond was sampled at 30 degree increments , and new conformer sets were produced using defaults for all other settings . interestingly , omega s ability to reproduce the solid - state structures from the pdb or the csd sets was not significantly changed ( whether measured by rmsd or tanimoto combo ) when using this naive torsion library . results for the pdb set using rmsd are shown in table 5 , where it can be seen that the 90% confidence intervals overlap substantially . therefore , with 90% confidence , we can assert that omega s accuracy is not affected by the use of the knowledge - based torsion library . however , some other effects on performance were noted . for the pdb set , when the naive torsion library was used , the average number of conformations produced per molecule increased by almost 15% over using the default library ( from 123.3 to 139.6 conformers / molecule ) , while the total run time increased by nearly 40% . it was also seen that the worst failures ( highest rmsd , lowest tc ) were less poor when the default , knowledge - based , torsion library was used than when the 30 degree library was used . qualitatively similar results were obtained for the csd data set ( data not shown ) . the object of this study was 2-fold : to arrive at reliable and challenging sets of solid - state structures to validate omega and to examine its default parameters for their effectiveness on these well chosen solid - state structures ( not to exhaustively evaluate a large number of combinations of parameters in omega for their efficacy ) . the omega algorithm presented here combines knowledge - based and first principles approaches to conformer generation , so it can be described as systematic and rule based . the knowledge - based part is the torsion library , while the fragment library , ensemble buildup , and sampling are all performed on a first principles basis . we have developed a gold standard set of pdb ligand structures by paying particular attention to identifying structures that are good models for their electron density , an approach rarely taken in the literature . many of the properties of a model that are used here are ignored or misinterpreted in other publications in this area . for example , based on the published literature , it is commonly believed that selecting a cocrystal structure with a resolution below some cutoff value ( for example 2 ) ensures a good quality ligand structure . that this is clearly mistaken can easily be seen by inspecting the ligand models for two structures , both of 2 resolution , 1nhu and 1iy7 ( see figure 10 ) . figure 10electron density for two ligands both solved at 2 resolution ; 1nhu on the left , 1iy7 on the right . electron density for two ligands both solved at 2 resolution ; 1nhu on the left , 1iy7 on the right . the 1iy7 ligand model is clearly a good model of complete density , while the 1nhu model is an interpretation of partial density that is obviously poor ( the deposited ligand coordinates show severe atomic clashes ) . in two other cases at 2 resolution or better , 1atl and 1eta , there is no significant density for the ligand at all ( even when viewed at 1 ) making the deposited coordinates at best highly speculative educated guesses . therefore , resolution of the parent structure alone is no guide to the quality of a ligand s conformation but should only be used as one criterion among many . cases like 1nhu , 1atl , and 1eta also caution against using b - factors as a representation of thermal mobility in a structure . if there is no density for a set of atoms , what physical meaning is there in the b - factors for those atoms ? models built from data collected to at least 2.7 resolution have a parameter to data point ratio of at least 1 , allowing the model to be well constructed . the confidence in the fit of a model at the global level is increased if the difference in r and rfree is low ( < 0.05 herein ) , as a large difference in r and rfree is indicative of an overfit , though not necessarily poor , model . a low level of difference also means that the local measures of fit , rscc and rsr , are meaningful , which is not the case for overfit models ( good values of rscc and rsr can be obtained for poor fits when r rfree is large ) . by ensuring that the structures in the set all have low experimental error in their atomic coordinates ( dpi ) , we can use atom - based metrics like rmsd , take appropriate account of this experimental error , and still generate meaningful measures of performance . the rscc and rsr metrics often delineate ligands that show good fits to their local density from those that do not , and we thereby avoid many of the problems with structures from other publications . cases like 1nhu and 1atl are easily identified by these fit criteria as unsuitable for inclusion in conformation generator validation sets ( or any other kind of validation set ) , as the coordinates are not supported by the experimental data . for example in 1nhu , the rsccs for the two versions of the ligand in the unit cell are 0.768 and 0.744 , and the rsrs are 0.27 and 0.25 ; while in 1atl , the ligand rsccs are 0.709 and 0.722 , and the rsrs are 0.35 and 0.35 , greatly exceeding the cutoffs used in this work . sets,(33 ) and their presence in these sets only weakens the conclusions that can be drawn therefrom . any ligand conformation from the pbd that shows intramolecular atomic clashes is an error on the part of the crystallographer , and such structures are easily avoided by the use of the rscc , rsr , and owab criteria used here . further , by paying attention to the physicochemical properties and graph diversity of the ligands , we ensure a reasonable level of independence among the molecules in our data set . that this is a factor often ignored is easily seen by the example of the kirchmair set,(17 ) which contains no less than 50 duplicate molecules . such a level of duplication is likely to bias the results obtained from that data set . while individually all the criteria we deploy seem reasonable and even relatively benign , when combined , they present a significant hurdle for a structure to surmount . even though we began to assemble our validation set using three large data sets , two of which had already been selected for validation of conformer generators , we found it impossible to assemble a substantial set of well - solved structures . by applying relatively loose criteria for the quality of the crystallographic models at a global level overall , more than 90% of all the input structures failed our filtering criteria , a surprisingly high level . figure 1 illustrates the attrition rates for each of the three databases used and shows that they are quite similar , which was unexpected . given that the kirchmair and sadowski sets were selected with the explicit goal of testing conformer generators , we expected lower attrition rates for these two sets than for pdbbind , which is simply a collation of cocrystal structures for which there exists a published binding affinity . however , the percentages of surviving structures are quite similar : 5.3% for the kirchmair set , 6.4% for pdbbind , and 8.1% for the sadowski set . these very low levels of survival emphasize that the number of structures suitable for this sort of study in the pdb as a whole is very small , as has been seen in a study using pdb structures for docking validation.(37 ) however , given that recently the pdb has made deposition of structure factors along with coordinates a requirement , we hope that that this situation will improve quickly in the future . the most commonly used metrics in conformer generation studies are based on comparing each conformer in the set to the experimental conformation , using some atom - based geometric measure such as rmsd . metrics like rmsd are used almost without exception in conformer reproduction studies , probably because they are relatively easy to understand and require no specialized applications to calculate . there are , however , a number of objections to the use of rmsd as a metric of quality ; it has no upper bound , it scales with molecular size ( so that an rmsd of 2 for a molecule of 6 heavy atoms is much different than an rmsd of 2 for a molecule with 60 heavy atoms ) , and it can give an inaccurate picture of the overall quality of a prediction.(38 ) the most serious of the problems with rmsd may be that it does not directly compare a prediction with an experimental value but rather compares a prediction or model ( the conformer set ) with another model ( the atomic positions in the crystal structure ) . this problem has been eloquently discussed in a paper by yusuf et al.(39 ) in which the authors advocate comparison between the experimental data ( electron density ) with calculated density derived from a docking pose ( or computed conformation ) using the rsr metric ( which is bounded by 0 and 1 ) . these objections notwithstanding the large amount of published literature using rmsd militates against not using it as one metric of quality in a validation study , but it should not be the only one . a complementary approach , pace yusuf et al . , is to use a bounded metric that is not derived directly from the atom positions in the two conformers being compared . one recent approach in this vein has been the comparison of the overall shape of the experimental conformation to the shape of a docking pose using the shape tanimoto metric employed by warren et al.(40 ) we have extended their shape - based comparison to include an additional term ( the color tanimoto ) that compares the alignment of functional groups between the conformers . the score representing this combination of shape matching ( shape tanimoto ) and functional group matching ( color tanimoto ) is known as the tanimoto combo ( tc ) . since it represents the match of both shape and functional groups in space , tc allows for a greater discrimination between poses than using shape alone . use of a metric like tc avoids some major problems with rmsd : while rmsd has no defined range , the range of tc is , by definition , 02 ; since tc is bounded by 0 and 2 comparisons using tc is independent of molecular size ; large rmsds can arise from differences in the conformations of only small parts of the molecule , while tc is not as sensitive to these divergences ; tc provides an extra weight for matching chemical functionality ( the color tanimoto term represents the matching of the chemical features only ) while rmsd weights the matching of all atoms equally ; the gaussian representations of molecular properties used in the calculation of tc are soft so that the significance of results are not as affected by experimental uncertainties in atomic positions ( though it is more difficult to quantitatively correct for their effect on tc ) . while any use of a cutoff value for good reproduction is difficult , we find that if tc is below 1.0 the reproduction of the experimental pose is always bad and if tc is above 1.5 the reproduction is almost always satiusfactory . another major issue with the common use of atom - based measures like rmsd , rde , etc . is that no account is taken of experimental coordinate error in the structures being reproduced . to our knowledge , this is the first work in which reported rmsds are corrected for the atomic coordinate precision of the structure being reproduced . we have chosen to allow for coordinate error or uncertainty by the use of , as a metric of quality , either the maximum of the rmsd and the uncertainty or the difference between them . the second of these can be considered as an estimate of the level of computational noise introduced by the conformer generation process atop the existing experimental noise . while careful selection of our data set resulted in the corrected and uncorrected results not being significantly different , the correction of rmsds by coordinate uncertainty as outlined herein allows the future use of interesting structures with poorer coordinate precision than used in this study . there are clearly more sophisticated approaches that can be taken to this problem of experimental noise in pdb ligand structures , among which is to calculate a set of conformers that all fit the electron density equally well within some limit and compare these with the set computed by omega . this more realistically reflects the fact that a crystal structure is an average over time and space , and so , a small molecule is likely to be found in a number of slightly different conformations in a solid - state structure . the pdb - derived data set used here , while of good quality , is relatively small ( 197 structures ) , and so , a possible concern is that the results generated are not robust indicators of future performance . we find that in both cases the 5% and 95% quantiles are close and that the standard deviations of the bootstrap means are small . we infer that our results are quite stable to changes in the composition of the data sets used and , therefore , can be considered reliable indicators of future performance on molecule sets of similar physicochemical properties . the usual practice in this area has been to compare an aggregate statistic such as mean or median results , from a number of different tools or parameter sets and to declare one superior , without any account of the errors in these terms . however , by the use of confidence intervals , we can quantitatively assign a probability that one tool or parameter set actually is superior ; for example based on data in this paper , it is over 90% likely that omega with default parameters is better at reproducing small molecule structures from the csd than from the pdb . the torsion library in omega is based upon analysis of a number of crystal structures from the pdb , coupled with analysis of energy profiles for certain torsions in the mmff94 force field . therefore , the problem of overtraining the torsion library arises if many of the structures used to derive the torsion library entries are also in the test sets used in this study . this problem was addressed by the use of a naive torsion library containing no torsion specific information at all . comparison of the results from this naive library with the default one showed that the main impact of the torsion library is not to improve omega s ability to closely reproduce experimental structures but rather to reduce the size of the conformer ensemble required for good reproduction and the run time required . this result reduces any possible concern about the effect of over - training the torsion library so that it contains matches for many known structures . as above , the use of bootstrapping is key in interpreting the results ; it is over 90% certain that the use of the default torsion library produces no improvement in reproduction of the pdb structures compared to the use of a naive torsion library . while the purpose of this study was not to extensively compare parameter sets and versions of omega , a comparison of previous versions of omega with the current version ( on the pdb ligand set ) is provided in the supporting information . we have presented omega , an algorithm for rapid generation of conformers using a prebuilt library of fragments and a knowledge base of torsion angles and test sets of solid - state structures for its evaluation . the test sets of structures were obtained from both the pdb and the csd . identifying a set of small molecules from the pdb suitable for validation proved difficult , with the vast majority of ligand structures used in some previous validation sets for conformer generators proving unsuitable for this purpose . however , by applying a set of sequential quality filters to over 4500 structures from the pdb , a set of 197 ligands was found that were accurate and well fit to their electron density . most of the structures failed due to insufficient nominal resolution , poor fits of the ligand to its density , and inappropriate physicochemical properties . it is hoped that the pdb data set used herein will be of general use to the community since it contains only highly reliable structures with well fit ligands , so conclusions based on this data set will be highly reliable . a set of 492 druglike molecules from the csd was also subjected to a subset of these filters , and the surviving 480 molecules were tested against omega . in contrast to most studies in this area , we have used two different metrics for success in reproducing these experimental structures , rmsd and the tanimoto combo ( which estimates overall similarity in three dimensions ) , as well as a metric of conformational coverage close to the experimental structure . we , thereby , obtained complementary information on omega s ability to satisfactorily sample the conformational space of molecules around their solid - state structures . omega s performance on both of the data sets was good when judged by any of the metrics used ( performance was particularly good against the csd structures , due to their lower flexibility ) . the use of bootstrapping has allowed us to determine confidence intervals for our results and to make quantitative discrimination between the performance of omega on different data sets and with different amounts of information . in sum , we found that omega was able to satisfactorily sample the conformational space around solid - state structures of druglike molecules , which is omega s design goal .

here , we present the algorithm and validation for omega , a systematic , knowledge - based conformer generator . the algorithm consists of three phases : assembly of an initial 3d structure from a library of fragments ; exhaustive enumeration of all rotatable torsions using values drawn from a knowledge - based list of angles , thereby generating a large set of conformations ; and sampling of this set by geometric and energy criteria . validation of conformer generators like omega has often been undertaken by comparing computed conformer sets to experimental molecular conformations from crystallography , usually from the protein databank ( pdb ) . such an approach is fraught with difficulty due to the systematic problems with small molecule structures in the pdb . methods are presented to identify a diverse set of small molecule structures from cocomplexes in the pdb that has maximal reliability . a challenging set of 197 high quality , carefully selected ligand structures from well - solved models was obtained using these methods . this set will provide a sound basis for comparison and validation of conformer generators in the future . validation results from this set are compared to the results using structures of a set of druglike molecules extracted from the cambridge structural database ( csd ) . omega is found to perform very well in reproducing the crystallographic conformations from both these data sets using two complementary metrics of success .

Introduction Methods Results Discussion Conclusion

these methods have utilized a wide variety of approaches to the problem , including systematic enumeration , knowledge - based rule sets , random coordinate embedding , and energy - based or energy - biased sampling . all these approaches attempt to generate a set of conformations designated to be different from one another by some measure , usually geometric , and thereby aim to sample some subset of the conformational space available to a molecule . the method used by omega is based on random coordinate embedding and refinement , followed by torsion driving using a set of rules derived from experimental structures and energy profile calculations , and then subsequent sampling of the resulting conformer ensemble by geometric and energetic criteria . publications that demonstrate the utility of conformer generators have approached the problem of validation of such tools in a number of different ways . while it might be argued that the real - life use of conformer generators is well reflected in this approach , judging the quality of a conformer generator based on the performance of a separate tool is fraught with problems . if this tool has been optimized to use conformers from a certain engine , then it would be expected to produce better results from this engine over all others . by far , the most common , though not always the most well - executed , method for validation of conformer generators , and the method used in this paper , is the reproduction of experimental crystal structures(11 ) ( often those retrieved from the protein databank ( pdb)(12 ) ) . to perform this sort of validation , the connection tables of these ligands are used as input for the conformer generator to provide a set of conformers for each ligand , unbiased by any structural information of the original experimental conformation . identifying suitable cocomplex structures from the pdb to provide the experimental conformations of small molecules unfortunately , in most publications in this area , insufficient care is taken in the selection of the structures , resulting in unwarranted or questionable conclusions being drawn based on unreliable data . some of the issues that should be considered in the selection of structures from the pdb include the following : are the deposited models of good quality at both the global and local levels ? when using atom - based metrics like rmsd , paying attention to the precision in the atomic coordinates is critical : an rmsd or any measure of conformer reproduction more precise than the inherent inaccuracy / experimental error in the atomic coordinates is meaningless . a set of ligands with a large degree of structural redundancy does not provide as rigorous a test of conformer generators as a structurally nonredundant set does . these criteria were applied as successive filters to three previously published large data sets of cocrystal structures , pdbbind,(16 ) the sadowski set(13 ) and the kirchmair set,(17 ) to provide a high quality group of cocrystal structures to establish a gold standard for the validation of approaches to conformer sampling . a set of small molecule structures from the cambridge structural database ( csd)(18 ) was also collected to provide complementary data to that obtained with the ligands from the pdb . here , the omega algorithm is introduced in some detail ; the metrics for comparison of the generated conformers to the experimental structures are outlined , and the criteria for the identification of suitable proteinligand cocomplexes and small molecule structures with which to validate the algorithm are discussed . the design goal for omega is to provide a thorough , though not exhaustive , sampling of conformational space for druglike molecules at as high a speed as possible ( typically 22.5 s per molecule on a workstation running sled 10 with a 2.4 ghz processor and 4 gb of ram ) . the first two steps are construction of a database of fragments from which the molecule will be assembled and derivation of a torsion library ; the remaining steps generate a large ensemble of conformations and sample from this ensemble to deliver the final set of conformers . the torsion angles specified by the torsion library were derived by analysis of a set of experimental crystal structures ( mostly from the pdb ) and from energy scans of certain torsions against mmff94 . every rotatable bond in the conformers generated in step 3 is compared to the torsion library from step 2 , and the appropriate torsion angles are noted . a torsion buildup procedure is applied to all the torsions in the molecule to generate a large ensemble of conformations that does not contain severe internal clashes or duplicates due to common symmetries . tc offers a complementary approach to the atom - based rmsd measure for a number of reasons treated at greater length in the discussion section . two sources of small molecule structures , the pdb and the csd , were utilized . to find small molecules from the pdb , first , suitable cocrystal structures were identified from larger sets using global criteria ; then , the ligands were extracted and inappropriate ligands were removed using local , ligand - centric criteria . small molecule structures from the csd were obtained from a previously published data set,(18 ) which was subjected to some of the ligand - centric criteria used to identify suitable pdb ligands . on the selection of a set of structures suitable for validating docking programs for were that all structures must have good nominal resolution and have deposited structure factors , while the local criteria were that the ligand must be well fit to its local density and can not be covalently bound to the protein . in addition to the hartshorn criteria at the global level ( good nominal resolution , deposited structure factors in the pdb ) , the model must have good overall metrics of model quality and reasonable coordinate precision . the quality of a deposited model can be independently checked only if structure factors ( electron density maps ) are available in the pdb , so all structures must have deposited structure factors . (25 ) we calculate dpi according to the approximation published by blow(26 ) ( using the blow rearrangement allows us to calculate an estimate for dpi based solely upon information commonly contained in the header of a pdb file ) . the comparison of a computed conformer to the experimental conformer by rmsd obviously requires comparison of atom positions between the two conformers . whatever the magnitude of the rmsd , a correction to it will have to be made to account for positional uncertainty in the ligand atoms ( see the results section ) . the filters were applied to a large set of pdb structures ( see results ) , and the cocomplexes that survived were then processed to separate the ligand and the protein . these pairs were checked to ensure that they do not show any intermolecular clashes , and those ligands that showed no clashes were designated as well - solved structures , to be used in the final , ligand - based filters . as a first step , we removed all well - known cofactors , as these were not as relevant to the goals of this study ( validation of omega s performance on druglike molecules ) . to ensure that we used a collection of ligands that provides as broad a set of tests for the omega algorithm as possible , we attempted to select a structurally diverse set of ligands . preliminary analysis revealed that molecules designed to bind to thrombin were over - represented in the set of well - solved ligands identified by the criteria used thus far . as further discussed in the results section , we turned to the csd for precise distributions of interatomic distances with which to identify clashing atoms . the attrition rates for the different data sets and an analysis of omega s ability to reproduce these 197 surviving structures is given in the results section . a parallel analysis was performed on high quality small molecule structures from the csd , as outlined in the following paragraphs . an important part of the selection criteria for structures from the pdb focused on removing structures that were not reliably and accurately fit to their density , and a large majority of the candidate structures failed these criteria ( see the results section ) . a further problem with molecules from the pdb is the high level of inherent experimental uncertainty in the atomic positions . both of these problems can be avoided entirely if small molecule structures from the cambridge structural database ( csd ) are used . a high quality set of 492 druglike structures has been extracted from the csd as part of a previously published study,(18 ) and these were used as the basis for generating a complementary set of structures to those we obtained from the pdb . applying the same physicochemical and diversity criteria as were used for the ligands from the pdb gave a set of 480 small molecule structures upon which we could perform the same validation experiments . comparison of these results to those from the ligands from the pdb can be found in the results section . proteinligand cocrystal structures were downloaded from the pdb and filtered according to the criteria laid out in the methods section . in the methods section , we presented a set of criteria for selecting suitable proteinligand cocomplexes from the pdb and then identifying suitable ligands from those complexes . the problem of identifying atomatom clashes ( filters e and g ) based on analysis of interatomic distances in structures from the pdb is nontrivial , due to the inherent uncertainty in those atom positions . therefore , we chose to perform an analysis of nonbonded contacts for commonly occurring atoms ( c , n , o , s , p , f , cl ) using high quality structures ( r - factor < 0.05 , no disorder ) of organic molecules from the csd , where the atoms are located with very high precision . however , it is noteworthy that several structures from such well - known data sets as the perola and charifson set(33 ) show intramolecular clashes , as these authors selected structures using only global criteria of fit , omitting local measures . best rmsd v. best 3d shape and chemical similarity ( tanimoto combo ) for reproduction of 197 ligands from the pdb . a striking difference in the two overlay methods is illustrated in figure 4 , where two different overlays of conformers of the ligand from the pdb structure 1 v2n are shown . the origin of this difference in the assessment of the quality of reproduction when using these two different metrics is most clearly seen by inspecting the alignments of the benzamidine functional groups . an issue that is most often ignored in studies of this kind is the prospective utility of the performance metrics presented , or how robust are the performance metrics to changes in the composition of the data set and , therefore , how predictive are the results of future performance ? therefore , we can say that for a future experiment on a set of molecules with similar properties to those of this pdb - derived set 90% of the time the mean rmsd will lie in the range 0.6470.688 . distribution of percentages of conformers that are close ( rmsd < 1.25 ) to the experimental conformation of 197 structures from the pdb . having a reasonable proportion of the conformers in the set close to the protein - bound structure is helpful for downstream protein - based tasks such as docking . it was hoped that these molecules from the csd would represent a different set of challenges in conformer generation ; the pdb structures are obtained in an aqueous environment , and the majority of csd structures are obtained in nonpolar environments . while there are large numbers of small molecule structures in the pdb and the csd , the two databases show only a low level of overlap ; a comparison of over 4000 druglike ligands from the pdb with over 57 000 druglike molecules in the csd finds only 224 molecules in common ( see supporting information for pdb and csd codes ) . given the difference in rotatable bond count , the numbers of conformers produced by omega2 for this set and for the pdb set are rather different ; the median number of confomers per molecule was 47 for the csd set and 123 for the pdb set . note that in the case of these structures from the csd the positional uncertainty for the atoms is sufficiently small to be disregarded when calculating rmsds . as with the results from the pdb ligands , these small intervals imply that our results are not strongly dependent upon the exact composition of this data set and as such are reliable indicators of future performance on data sets of similar property distributions to the one used in this study . the results for close reproduction ( rmsd < 1.25 ) of the experimental conformations are much better than for the pdb ; overall , 39% of the conformations generated for the csd set are close to the experimental conformation . this is entirely consistent with the results from the csd set relative to the pdb set for best pose reproduction ( as judged by either tanimoto combo or rmsd ) . it is possible that the superior performance of omega on the csd set is not due to the lower average flexibility of the molecules but due to some consistent and unexpected difference in the nature of the molecules between the two sets . reproduction of three data sets by omega , using both tanimoto combo ( tc ) and rmsd . the 90% confidence intervals of the entire pdb set and the csd - matched subset of the pdb set do not quite overlap , though they are very close ( the difference could be entirely due to the physical property distributions of the two sets not being perfectly matched ) . however , we conclude that the superior performance of omega on the csd set derives to some large degree from the lower average flexibility of the csd set compared to the pdb set and not from bias in the omega knowledge base . we used both the pdb- and csd - derived test sets to investigate another possible source of bias in the results ; the torsion library , which was assembled mostly by examination of ligand structures from the pdb . interestingly , omega s ability to reproduce the solid - state structures from the pdb or the csd sets was not significantly changed ( whether measured by rmsd or tanimoto combo ) when using this naive torsion library . it was also seen that the worst failures ( highest rmsd , lowest tc ) were less poor when the default , knowledge - based , torsion library was used than when the 30 degree library was used . the knowledge - based part is the torsion library , while the fragment library , ensemble buildup , and sampling are all performed on a first principles basis . we have developed a gold standard set of pdb ligand structures by paying particular attention to identifying structures that are good models for their electron density , an approach rarely taken in the literature . the confidence in the fit of a model at the global level is increased if the difference in r and rfree is low ( < 0.05 herein ) , as a large difference in r and rfree is indicative of an overfit , though not necessarily poor , model . by ensuring that the structures in the set all have low experimental error in their atomic coordinates ( dpi ) , we can use atom - based metrics like rmsd , take appropriate account of this experimental error , and still generate meaningful measures of performance . even though we began to assemble our validation set using three large data sets , two of which had already been selected for validation of conformer generators , we found it impossible to assemble a substantial set of well - solved structures . by applying relatively loose criteria for the quality of the crystallographic models at a global level overall , more than 90% of all the input structures failed our filtering criteria , a surprisingly high level . (37 ) however , given that recently the pdb has made deposition of structure factors along with coordinates a requirement , we hope that that this situation will improve quickly in the future . the most commonly used metrics in conformer generation studies are based on comparing each conformer in the set to the experimental conformation , using some atom - based geometric measure such as rmsd . there are , however , a number of objections to the use of rmsd as a metric of quality ; it has no upper bound , it scales with molecular size ( so that an rmsd of 2 for a molecule of 6 heavy atoms is much different than an rmsd of 2 for a molecule with 60 heavy atoms ) , and it can give an inaccurate picture of the overall quality of a prediction. use of a metric like tc avoids some major problems with rmsd : while rmsd has no defined range , the range of tc is , by definition , 02 ; since tc is bounded by 0 and 2 comparisons using tc is independent of molecular size ; large rmsds can arise from differences in the conformations of only small parts of the molecule , while tc is not as sensitive to these divergences ; tc provides an extra weight for matching chemical functionality ( the color tanimoto term represents the matching of the chemical features only ) while rmsd weights the matching of all atoms equally ; the gaussian representations of molecular properties used in the calculation of tc are soft so that the significance of results are not as affected by experimental uncertainties in atomic positions ( though it is more difficult to quantitatively correct for their effect on tc ) . there are clearly more sophisticated approaches that can be taken to this problem of experimental noise in pdb ligand structures , among which is to calculate a set of conformers that all fit the electron density equally well within some limit and compare these with the set computed by omega . the pdb - derived data set used here , while of good quality , is relatively small ( 197 structures ) , and so , a possible concern is that the results generated are not robust indicators of future performance . however , by the use of confidence intervals , we can quantitatively assign a probability that one tool or parameter set actually is superior ; for example based on data in this paper , it is over 90% likely that omega with default parameters is better at reproducing small molecule structures from the csd than from the pdb . the torsion library in omega is based upon analysis of a number of crystal structures from the pdb , coupled with analysis of energy profiles for certain torsions in the mmff94 force field . comparison of the results from this naive library with the default one showed that the main impact of the torsion library is not to improve omega s ability to closely reproduce experimental structures but rather to reduce the size of the conformer ensemble required for good reproduction and the run time required . as above , the use of bootstrapping is key in interpreting the results ; it is over 90% certain that the use of the default torsion library produces no improvement in reproduction of the pdb structures compared to the use of a naive torsion library . while the purpose of this study was not to extensively compare parameter sets and versions of omega , a comparison of previous versions of omega with the current version ( on the pdb ligand set ) is provided in the supporting information . we have presented omega , an algorithm for rapid generation of conformers using a prebuilt library of fragments and a knowledge base of torsion angles and test sets of solid - state structures for its evaluation . identifying a set of small molecules from the pdb suitable for validation proved difficult , with the vast majority of ligand structures used in some previous validation sets for conformer generators proving unsuitable for this purpose . however , by applying a set of sequential quality filters to over 4500 structures from the pdb , a set of 197 ligands was found that were accurate and well fit to their electron density . a set of 492 druglike molecules from the csd was also subjected to a subset of these filters , and the surviving 480 molecules were tested against omega . in contrast to most studies in this area , we have used two different metrics for success in reproducing these experimental structures , rmsd and the tanimoto combo ( which estimates overall similarity in three dimensions ) , as well as a metric of conformational coverage close to the experimental structure . in sum , we found that omega was able to satisfactorily sample the conformational space around solid - state structures of druglike molecules , which is omega s design goal .

development & in vitro testing : designed and fabricated at south india textile research association ( sitra ) , coimbatore , the main testing and clinical work of woven grafts was carried out at sree chitra tirunal institute for medical sciences and technology ( sctimst ) , thiruvananthapuram , kerala , india . the prosthesis was essentially made of polyester polyethylene terephthalate ( pet ) . the design and developmental process included yarn preparation , weaving of the tubular fabric , crimping and sterilization . the fabric was a seamless woven tube of 75 denier texturised polyester yarn which was suitably crimped ( fig . 1 ) to provide large diameter vascular graft with a porosity of 20050 ml / min / cm of normal saline at 120 mmhg . the physical characteristics of the yarn , in terms of the yarn count , yarn twist and tensile strength to weave the grafts were determined by sitra . the chemical composition of the material was analysed by infrared spectroscopy using spectrophotometer ( shimadzu , japan ) , and differential thermal analysis at the laboratory for technical evaluation of biomaterials at the biomedical division of sctimst . toxicity / biocompatibility studies such as acute systemic toxicity , intracutaneous irritation , in vitro haemolysis and implantation in muscle were studied as per international standards3 for the vascular graft material . acute systemic toxicity was studied in mice using physiological saline and cotton seed oil extracts of the material . intracutaneous irritation was done by injecting the extracts of material and control intradermally into rabbits under ketamine anaesthesia . grading of erythema and oedema of material in experimental and control rabbits were recorded at 24 , 48 and 72 h. in vitro haemolysis was carried out with material and extract of the material using centrifugation ( remi r-8c dx , india ) with rabbit blood . the implanted animals were sacrificed at the end of one , four and 12 wk , the tissue with the implanted materials were collected , sectioned using rm2255 microtome ( leica , biosystems , india ) and subjected to histopathological analysis ( axio imager z1 microscope , carl zeiss ) . the test vascular polyester polyethylene terephthalate ( pet ) graft with internal diameter ranging from 8 - 30 mm . pre - clinical animal testing : during 1982 - 1983 , as part of the pilot study4 , 30 pigs had received the test graft . further , in vivo experiments in 1992 - 1994 , reported herein , involved 15 pigs as per ethics committee requirements in accordance with draft document of association for the advancement of medical instrumentation5 . protocol was drawn to evaluate 11 mm test graft and 12 mm usci debakey graft ( usa ) as concurrent controls ( fig . 2 ) . protocol of pre - clinical testing in yorkshire pigs using test and control grafts and the explantation data . four to six months old large white yorkshire pigs ( source : government pig farm , parassala , thiruvananthapuram ) weighing 40 - 45 kg , were used for each experiment after due conditioning as per animal testing guidelines6 . following overnight fasting and premedication , general anaesthesia was employed using thiopentone sodium ( 10 mg / kg ) and muscle relaxants and maintained with endotracheal intubation and inhalation anaesthetic agents . open cut down was employed to obtain central venous access through saphenous vein and arterial access through femoral artery . continuous monitoring of heart rate , blood pressure , electrocardiogram ( ecg ) and arterial blood gases ( abg ) at regular intervals formed the standard protocol . left postero - lateral thoracotomy was performed and pleural cavity entered by excising 4 or 5 rib . lung was gently retracted and mediastinal pleura over descending thoracic aorta incised and aorta dissected and looped . dissection was continued as far as feasible towards suprahiatal aorta whenever long grafts had to be implanted . heparin ( 1 mg / kg ) was administered three minutes prior to clamping in some cases except where blood retrieval strategy was employed in which case 3 mg / kg was required7 . replacement of segment of descending thoracic aorta in pig with the test graft using standard protocol via left postero - lateral thoracotomy ( a ) . gross healing characterstics of test vascular graft at explant post-6 month in vivo in pig in 8 , 4 and 12 cm lengths respectively ( b , c , d ) . neointima was found to be smooth in short replacements while it was less regular and even , nonetheless thrombus - free , in long grafts . all animals except one could be extubated at the end of procedure and walked back to cage 4 - 6 h after completion of the procedure . grafts were explanted to study the healing characteristics , blood / tissue interactions and patency rates . explantation was performed at three months or six months following graft insertion under general anaesthesia ; 1 mg / kg heparin was administered to avoid misinterpretation of pre- or postmortem thrombus . phase i clinical trial : ( i ) design - prospective randomized study was designed to prove non - inferiority of the test prosthesis with concurrent controls using commercially available prosthetic grafts . objective patency assessment was made by non - invasive haemodynamic indices , duplex ultrasound and catheter angiography / ct angiography . ( ii ) parameters evaluated were ( a ) ease of pre - clotting ; ( b ) pliability / conformability ; ( c ) ease of suturing / suture retention ; and ( d ) short - term patency at one year , long - term patency at five years and beyond . ( iii ) institutional ethics committee ( iec ) gave permission to use large diameter prosthesis of length less than 10 cm for a total of 10 patients during phase i trial as a matter of abundant caution . this restricted its use only for repair of abdominal aortic aneurysms ( aaa ) and coarctation of thoracic aorta ( coa ) . ( iv ) methods - between september 1998 and november 1999 , 10 patients were included in phase i clinical trial using the test graft at sctimst , six of whom underwent repair for aaa and four for coa ( table ii ) . aaa involved the infra - renal segment of aorta which was repaired through xipho - pubic midline laparotomy and transperitoneal approach to the aorta . standard inclusion graft technique was performed using appropriate ( 14 - 20 mm internal diameter ) test graft using 3/0 monofilament polypropylene suture for proximal anastomosis and 4/0 suture for distal . inferior mesenteric artery was re - implantated into the prosthesis using carrel 's patch technique8 in selected cases upon indication . excision of coarct segment and interposition graft placement using 14 - 16 mm i d test graft was done in one patient while left subclavian artery ( lsa ) to descending thoracic aorta bypass ( jump graft ) using 12 - 14 mm i d graft was done in the other three . the latter involved end - to - side anastomosis to the dilated lsa proximally and post - coarct aorta distally using 4/0 polypropylene suture . in the control group , eight patients with aaa and two with coa underwent elective implantation of the control dacron graft . clinical details and results of phase i ( controlled ) clinical trial phase ii clinical trial : twenty two patients who underwent indexed vascular reconstructions at four designated hospitals viz . medwin hospital , hyderabad ; g. kuppuswamy naidu memorial hospital , coimbatore ; medical college hospital , and sctimst , thiruvananthapuram ; formed the basis of this part of the study conducted from august 2005 to september 2008 . patients chosen were all symptomatic and investigated , operated upon and followed up using an already established and common protocol . age ranged from 19 to 74 yr with mean age of 55 yr ; four patients were female ( > 4:1 male : female ratio ) . clinical indications for graft implantations included coa in three , middle aortic syndrome in one , thoraco - abdominal aortic aneurysm ( taaa ) in two , aaa in 10 , iliac artery occlusion in five and iliac artery aneurysm in one patient . parameters were carefully noted regarding handling qualities of prosthesis and procedure related complications or mortality . patients were followed up for a maximum period of five years using clinical and imaging documentation of graft patency and integrity . clinical details and results of phase ii ( multicentric ) clinical trial long - term follow up till date : patients recruited in the clinical trials that involved implantation of the test graft at the institute as well as the control group were followed up by clinical examination and duplex ultrasound on yearly basis . ct scan / magnetic resonance angiography ( mra ) were performed yearly for first two years and thereafter once in 2 - 3 years of follow up . parameters evaluated were ( i ) dilatation and elongation - no progressive dilatation > 15 per cent / that of control grafts ( as measured in systole ) . ( iv ) infection - < 1 per cent in intracavity grafts , < 2 per cent for grafts crossing inguinal ligament and <3 per cent for extremity grafts . in vitro testing : the results of the study did not show any significant irritation or systemic toxicity with physiological saline and cotton seed oil extracts of the material . results of the histopathological evaluation suggested that the material did not produce any histopathological changes . hence the toxicity study concluded that the material was non toxic , non irritant , non haemolytic and biocompatible . ( i ) immediate - all animals , except one ( 15 ) , were extubated at the end of procedure and walked back to their cage 6 - 8 h thereafter . the 15 animal which underwent interposition graft using cardio - pulmonary bypass to support circulation succumbed to air embolism at the end of an otherwise satisfactory procedure . another animal died on 2 post - operative day following hyperpyrexia of 104f and at autopsy graft was noted to be patent . the third animal developed delayed paraplegia 24 h after operation that involved 45 min of aortic cross - clamping , had to be sacrificed and at autopsy the prosthetic graft was found to be patent and void of any thrombus . hence the remaining 12 animals were available for explantation to study the healing characteristics . ( ii ) explantation data - explantation was performed at three and six months after graft implantation under general anaesthesia . animals weighed 55 and 65 kg , respectively ; 1 mg / kg heparin was administered to avoid misinterpretation of pre- and post - mortem thrombus . gross examination showed that the grafts were well incorporated in a 1 - 4 mm thick perigraft capsule . cut surface showed that suture lines were tidy and clean with no evidence of thrombus formation . however , neoimtima inside long grafts appeared irregular and uneven but well organized in contradistinction to smooth glistening thin lining in short replacements ( fig . 3b - d ) histologically , the inner lining was thin and measured 0.6 - 1.0 mm , and showed uniform regular neointima of fibro - collagenous tissue , a few islands of calcification along with cellular infiltration of lymphocytes and a few foreign body giant cells ( fig . low power microphotographs using hematoxylin & eosin ( h&e ) staining showing lack of thrombosis at anastomotic site ( black arrow ) with tissue ingrowth between the graft ( a ) , and endothelial - lined neointima ( green arrow ) on the inner aspect of the graft ( b ) 6 months after implantation in porcine model . phase i clinical trial : first patient following repair of aaa was discharged from hospital on 10 day . at discharge , her blood pressure was controlled with 50 mg atenolol ( beta - aderenergic blocker ) and her ankle brachial index was 1.1 . ct scan prior to discharge showed patent graft , intact suture lines , smooth luminal outline and patent inferior mesenteric artery . all patients survived major surgical procedure and made satisfactory early recovery except one patient ( 4 ) who underwent surgical repair for complex post - subclavian coarctation of aorta with large heavily calcified post - coarct aneurysm , who developed paraplegia . prolonged aortic cross - clamp time of > 60 min along with significant bleeding requiring blood transfusions led to spinal cord dysfunction . minor sequelae in others included prolonged paralytic ileus in two and respiratory infection in one ( table ii ) . all patients were discharged from hospital on or before 10 postoperative day except the patient who developed paraplegia ( 20 days ) . all patients underwent duplex ultrasound and ct scan before discharge from hospital which showed satisfactory repair , patent graft and smooth regular luminal surface of prosthesis . complete follow up was available for all 10 patients at three , six months , one year and yearly thereafter . all patients were in good health and active except the 4 patient who did not recover from paraplegia inspite of aggressive physiotherapy . ct / conventional aortogram was done at discharge and on yearly follow up ( fig . 5 ) . the inner diameter of the graft as measured in ct / aortogram was 0.5 - 0.7 mm less than the internal diameter of the appropriate graft size . contrast - enhanced abdominal ct scan axial section of a symptomatic patient in phase i clinical trial showing an infra - renal abdominal aortic aneurysm ( a ) . abdominal contrast enhanced ct scan of the same patient taken 8 years after repair showing intact and patent test vascular graft ( yellow arrow ) ( b ) . follow - up imaging of an adult patient with coarctation of aorta treated by interposition grafting with test vascular graft as part of phase i trial . digital subtraction angiogram ( dsa ) at 1 year ( c ) and ct angiogram at 6 years ( d ) showing preserved and patent graft ( red arrows ) . all patients who received the test implant survived the procedures and recovered well except the patient who developed paraplegia ; however , digital substraction angiography ( dsa ) at one year and ct aortogram at five years showed intact repair with patent graft . host - graft interaction was reflected at three sites namely , aorta to graft interphase , outside the graft and its inner luminal surface . no untoward sequelae of excessive intimal hyperplasia or pseudoaneurysm formation were noted in and around the proximal and distal suture lines in these patients . the luminal side of the grafts were clean and regular with no evidence of thrombus formation inside the test or control grafts . phase ii clinical trial : prosthesis was found to be surgeon friendly , with excellent suturing and suture retention qualities . all centres reported good graft handling qualities with pliability , lack of fraying , ease of pre - clotting and suturing and absence of excessive weeping following implantation similar to control dacron grafts . patients were electively ventilated for 4 to 24 h with mean of 8 h as dictated by their clinical condition . all 22 patients at 4 centres of study made satisfactory early recovery from appropriate procedures . two patients ( both taaa ) developed transient renal dysfunction ( increase in serum creatinine > 1 mg / dl higher than pre - operative level ) eventually normalising at discharge . follow up visits were planned at three and six months , one year and yearly thereafter . apart from regular clinical assessment , duplex scan evaluation was performed in patients with graft implantation in abdomen . check mr / ct angiogram was performed on follow up showed preserved patency with no evidence of graft - related complications ( fig . follow up ct / mr angiograms of patients in the multicentric trials who underwent vascular reconstruction using test graft ( white arrows ) , showing good results following open repair of abdominal aortic aneurysm at 5 years ( a ) , aorto - femoral bypass for iliac occlusion at 4 years ( b ) , and open repair of thoraco - abdominal aortic aneurysm at 6 years ( c ) . one patient succumbed to stroke 18 months after aorto - femoral graft procedure ; and another to road traffic accident leading to irreversible brain damage three years after aaa repair . post - operative abi at 1 - 4 years were 0.9 or more in 15 , 0.8 in 4 , and 0.7 in 3 ( table iii ) . long - term follow up till date : follow up was complete in eight patients at > 10 yr after implantation of test vascular prosthesis in phase i trial and > 5 yr in 14 patients in phase ii trial . three patients who were around 70 yr of age in 1998 - 1999 died of old age and cardiac events beyond five years after the operation . present evaluation has confirmed excellent clinical status of the survivors and satisfactory graft function with no incidence of pseudoaneurysm , aneurysm , infection , thrombosis , dilatation or any other untoward sequelae with reference to implanted vascular prosthesis ( fig . 7)9 , except one 32 yr old patient with thoraco - abdominal aortic aneurysm due to takayasu 's disease was noted to have pseudoaneurysm at distal aortic anastomosis five years following surgery requiring endovascular aortic stent grafting . ct angiogram , volume - rendered image at 14 years following jump graft bypass ( white arrow ) from left subclavian artery to descending thoracic aorta for coarctation of aorta ( green arrow ) using 14 mm test graft . ( i ) immediate - all animals , except one ( 15 ) , were extubated at the end of procedure and walked back to their cage 6 - 8 h thereafter . the 15 animal which underwent interposition graft using cardio - pulmonary bypass to support circulation succumbed to air embolism at the end of an otherwise satisfactory procedure . another animal died on 2 post - operative day following hyperpyrexia of 104f and at autopsy graft was noted to be patent . the third animal developed delayed paraplegia 24 h after operation that involved 45 min of aortic cross - clamping , had to be sacrificed and at autopsy the prosthetic graft was found to be patent and void of any thrombus . hence the remaining 12 animals were available for explantation to study the healing characteristics . ( ii ) explantation data - explantation was performed at three and six months after graft implantation under general anaesthesia . animals weighed 55 and 65 kg , respectively ; 1 mg / kg heparin was administered to avoid misinterpretation of pre- and post - mortem thrombus . gross examination showed that the grafts were well incorporated in a 1 - 4 mm thick perigraft capsule . cut surface showed that suture lines were tidy and clean with no evidence of thrombus formation . however , neoimtima inside long grafts appeared irregular and uneven but well organized in contradistinction to smooth glistening thin lining in short replacements ( fig . 3b - d ) histologically , the inner lining was thin and measured 0.6 - 1.0 mm , and showed uniform regular neointima of fibro - collagenous tissue , a few islands of calcification along with cellular infiltration of lymphocytes and a few foreign body giant cells ( fig . low power microphotographs using hematoxylin & eosin ( h&e ) staining showing lack of thrombosis at anastomotic site ( black arrow ) with tissue ingrowth between the graft ( a ) , and endothelial - lined neointima ( green arrow ) on the inner aspect of the graft ( b ) 6 months after implantation in porcine model . phase i clinical trial : first patient following repair of aaa was discharged from hospital on 10 day . at discharge , her blood pressure was controlled with 50 mg atenolol ( beta - aderenergic blocker ) and her ankle brachial index was 1.1 . ct scan prior to discharge showed patent graft , intact suture lines , smooth luminal outline and patent inferior mesenteric artery . all patients survived major surgical procedure and made satisfactory early recovery except one patient ( 4 ) who underwent surgical repair for complex post - subclavian coarctation of aorta with large heavily calcified post - coarct aneurysm , who developed paraplegia . prolonged aortic cross - clamp time of > 60 min along with significant bleeding requiring blood transfusions led to spinal cord dysfunction . minor sequelae in others included prolonged paralytic ileus in two and respiratory infection in one ( table ii ) . all patients were discharged from hospital on or before 10 postoperative day except the patient who developed paraplegia ( 20 days ) . all patients underwent duplex ultrasound and ct scan before discharge from hospital which showed satisfactory repair , patent graft and smooth regular luminal surface of prosthesis . complete follow up was available for all 10 patients at three , six months , one year and yearly thereafter . all patients were in good health and active except the 4 patient who did not recover from paraplegia inspite of aggressive physiotherapy . ct / conventional aortogram was done at discharge and on yearly follow up ( fig . the inner diameter of the graft as measured in ct / aortogram was 0.5 - 0.7 mm less than the internal diameter of the appropriate graft size . contrast - enhanced abdominal ct scan axial section of a symptomatic patient in phase i clinical trial showing an infra - renal abdominal aortic aneurysm ( a ) . abdominal contrast enhanced ct scan of the same patient taken 8 years after repair showing intact and patent test vascular graft ( yellow arrow ) ( b ) . follow - up imaging of an adult patient with coarctation of aorta treated by interposition grafting with test vascular graft as part of phase i trial . digital subtraction angiogram ( dsa ) at 1 year ( c ) and ct angiogram at 6 years ( d ) showing preserved and patent graft ( red arrows ) . all patients who received the test implant survived the procedures and recovered well except the patient who developed paraplegia ; however , digital substraction angiography ( dsa ) at one year and ct aortogram at five years showed intact repair with patent graft . host - graft interaction was reflected at three sites namely , aorta to graft interphase , outside the graft and its inner luminal surface . no untoward sequelae of excessive intimal hyperplasia or pseudoaneurysm formation were noted in and around the proximal and distal suture lines in these patients . the luminal side of the grafts were clean and regular with no evidence of thrombus formation inside the test or control grafts . phase ii clinical trial : prosthesis was found to be surgeon friendly , with excellent suturing and suture retention qualities . all centres reported good graft handling qualities with pliability , lack of fraying , ease of pre - clotting and suturing and absence of excessive weeping following implantation similar to control dacron grafts . patients were electively ventilated for 4 to 24 h with mean of 8 h as dictated by their clinical condition . all 22 patients at 4 centres of study made satisfactory early recovery from appropriate procedures . two patients ( both taaa ) developed transient renal dysfunction ( increase in serum creatinine > 1 mg / dl higher than pre - operative level ) eventually normalising at discharge . follow up visits were planned at three and six months , one year and yearly thereafter . apart from regular clinical assessment , duplex scan evaluation was performed in patients with graft implantation in abdomen . check mr / ct angiogram was performed on follow up showed preserved patency with no evidence of graft - related complications ( fig . follow up ct / mr angiograms of patients in the multicentric trials who underwent vascular reconstruction using test graft ( white arrows ) , showing good results following open repair of abdominal aortic aneurysm at 5 years ( a ) , aorto - femoral bypass for iliac occlusion at 4 years ( b ) , and open repair of thoraco - abdominal aortic aneurysm at 6 years ( c ) . one patient succumbed to stroke 18 months after aorto - femoral graft procedure ; and another to road traffic accident leading to irreversible brain damage three years after aaa repair . post - operative abi at 1 - 4 years were 0.9 or more in 15 , 0.8 in 4 , and 0.7 in 3 ( table iii ) . long - term follow up till date : follow up was complete in eight patients at > 10 yr after implantation of test vascular prosthesis in phase i trial and > 5 yr in 14 patients in phase ii trial . three patients who were around 70 yr of age in 1998 - 1999 died of old age and cardiac events beyond five years after the operation . present evaluation has confirmed excellent clinical status of the survivors and satisfactory graft function with no incidence of pseudoaneurysm , aneurysm , infection , thrombosis , dilatation or any other untoward sequelae with reference to implanted vascular prosthesis ( fig . 7)9 , except one 32 yr old patient with thoraco - abdominal aortic aneurysm due to takayasu 's disease was noted to have pseudoaneurysm at distal aortic anastomosis five years following surgery requiring endovascular aortic stent grafting . ct angiogram , volume - rendered image at 14 years following jump graft bypass ( white arrow ) from left subclavian artery to descending thoracic aorta for coarctation of aorta ( green arrow ) using 14 mm test graft . ( i ) immediate - all animals , except one ( 15 ) , were extubated at the end of procedure and walked back to their cage 6 - 8 h thereafter . the 15 animal which underwent interposition graft using cardio - pulmonary bypass to support circulation succumbed to air embolism at the end of an otherwise satisfactory procedure . another animal died on 2 post - operative day following hyperpyrexia of 104f and at autopsy graft was noted to be patent . the third animal developed delayed paraplegia 24 h after operation that involved 45 min of aortic cross - clamping , had to be sacrificed and at autopsy the prosthetic graft was found to be patent and void of any thrombus . hence the remaining 12 animals were available for explantation to study the healing characteristics . ( ii ) explantation data - explantation was performed at three and six months after graft implantation under general anaesthesia . animals weighed 55 and 65 kg , respectively ; 1 mg / kg heparin was administered to avoid misinterpretation of pre- and post - mortem thrombus . gross examination showed that the grafts were well incorporated in a 1 - 4 mm thick perigraft capsule . cut surface showed that suture lines were tidy and clean with no evidence of thrombus formation . however , neoimtima inside long grafts appeared irregular and uneven but well organized in contradistinction to smooth glistening thin lining in short replacements ( fig . 3b - d ) histologically , the inner lining was thin and measured 0.6 - 1.0 mm , and showed uniform regular neointima of fibro - collagenous tissue , a few islands of calcification along with cellular infiltration of lymphocytes and a few foreign body giant cells ( fig . low power microphotographs using hematoxylin & eosin ( h&e ) staining showing lack of thrombosis at anastomotic site ( black arrow ) with tissue ingrowth between the graft ( a ) , and endothelial - lined neointima ( green arrow ) on the inner aspect of the graft ( b ) 6 months after implantation in porcine model . phase i clinical trial : first patient following repair of aaa was discharged from hospital on 10 day . at discharge , her blood pressure was controlled with 50 mg atenolol ( beta - aderenergic blocker ) and her ankle brachial index was 1.1 . ct scan prior to discharge showed patent graft , intact suture lines , smooth luminal outline and patent inferior mesenteric artery . all patients survived major surgical procedure and made satisfactory early recovery except one patient ( 4 ) who underwent surgical repair for complex post - subclavian coarctation of aorta with large heavily calcified post - coarct aneurysm , who developed paraplegia . prolonged aortic cross - clamp time of > 60 min along with significant bleeding requiring blood transfusions led to spinal cord dysfunction . minor sequelae in others included prolonged paralytic ileus in two and respiratory infection in one ( table ii ) . all patients were discharged from hospital on or before 10 postoperative day except the patient who developed paraplegia ( 20 days ) . all patients underwent duplex ultrasound and ct scan before discharge from hospital which showed satisfactory repair , patent graft and smooth regular luminal surface of prosthesis . complete follow up was available for all 10 patients at three , six months , one year and yearly thereafter . all patients were in good health and active except the 4 patient who did not recover from paraplegia inspite of aggressive physiotherapy . ct / conventional aortogram was done at discharge and on yearly follow up ( fig . the inner diameter of the graft as measured in ct / aortogram was 0.5 - 0.7 mm less than the internal diameter of the appropriate graft size . contrast - enhanced abdominal ct scan axial section of a symptomatic patient in phase i clinical trial showing an infra - renal abdominal aortic aneurysm ( a ) . abdominal contrast enhanced ct scan of the same patient taken 8 years after repair showing intact and patent test vascular graft ( yellow arrow ) ( b ) . follow - up imaging of an adult patient with coarctation of aorta treated by interposition grafting with test vascular graft as part of phase i trial . digital subtraction angiogram ( dsa ) at 1 year ( c ) and ct angiogram at 6 years ( d ) showing preserved and patent graft ( red arrows ) . all patients who received the test implant survived the procedures and recovered well except the patient who developed paraplegia ; however , digital substraction angiography ( dsa ) at one year and ct aortogram at five years showed intact repair with patent graft . host - graft interaction was reflected at three sites namely , aorta to graft interphase , outside the graft and its inner luminal surface . no untoward sequelae of excessive intimal hyperplasia or pseudoaneurysm formation were noted in and around the proximal and distal suture lines in these patients . the luminal side of the grafts were clean and regular with no evidence of thrombus formation inside the test or control grafts . phase ii clinical trial : prosthesis was found to be surgeon friendly , with excellent suturing and suture retention qualities . all centres reported good graft handling qualities with pliability , lack of fraying , ease of pre - clotting and suturing and absence of excessive weeping following implantation similar to control dacron grafts . patients were electively ventilated for 4 to 24 h with mean of 8 h as dictated by their clinical condition . all 22 patients at 4 centres of study made satisfactory early recovery from appropriate procedures . two patients ( both taaa ) developed transient renal dysfunction ( increase in serum creatinine > 1 mg / dl higher than pre - operative level ) eventually normalising at discharge . follow up visits were planned at three and six months , one year and yearly thereafter . apart from regular clinical assessment , duplex scan evaluation was performed in patients with graft implantation in abdomen . check mr / ct angiogram was performed on follow up showed preserved patency with no evidence of graft - related complications ( fig . follow up ct / mr angiograms of patients in the multicentric trials who underwent vascular reconstruction using test graft ( white arrows ) , showing good results following open repair of abdominal aortic aneurysm at 5 years ( a ) , aorto - femoral bypass for iliac occlusion at 4 years ( b ) , and open repair of thoraco - abdominal aortic aneurysm at 6 years ( c ) . one patient succumbed to stroke 18 months after aorto - femoral graft procedure ; and another to road traffic accident leading to irreversible brain damage three years after aaa repair . post - operative abi at 1 - 4 years were 0.9 or more in 15 , 0.8 in 4 , and 0.7 in 3 ( table iii ) . long - term follow up till date : follow up was complete in eight patients at > 10 yr after implantation of test vascular prosthesis in phase i trial and > 5 yr in 14 patients in phase ii trial . three patients who were around 70 yr of age in 1998 - 1999 died of old age and cardiac events beyond five years after the operation . present evaluation has confirmed excellent clinical status of the survivors and satisfactory graft function with no incidence of pseudoaneurysm , aneurysm , infection , thrombosis , dilatation or any other untoward sequelae with reference to implanted vascular prosthesis ( fig . 7)9 , except one 32 yr old patient with thoraco - abdominal aortic aneurysm due to takayasu 's disease was noted to have pseudoaneurysm at distal aortic anastomosis five years following surgery requiring endovascular aortic stent grafting . ct angiogram , volume - rendered image at 14 years following jump graft bypass ( white arrow ) from left subclavian artery to descending thoracic aorta for coarctation of aorta ( green arrow ) using 14 mm test graft . this study reports indigenously fabricated polyester graft developed and extensively tested in vitro , than tested in vivo using large animal , prior to clinical study by implantation in patients . each batch of grafts was put through physical ( yarn count , twist and tensile strength ) , chemical ( infra - red spectroscopy and thermal analysis ) and toxicity ( intracutaneous injection , haemolysin and intramuscular implantation in mice ) tests with satisfactory results in accordance with standards laid down by association for the advancement of medical instrumentation ( aami ) and american national standards institute ( ansi)5 . classic animal experimental data reported by adam wesolowski delineated pigs as the most suitable model for in vivo experiments10 . his original work on growing pigs weighing 20 - 39 kg formed the benchmark for graft research as implantation data of three months in growing pigs in terms of healing patterns and characteristics were noted to be equivalent to three years in man . pore size of prosthesis is essential for ingrowth of fibroblasts and capillary buds from peri - graft tissue to form and stabilize neointima in the graft body hence the optimal pore size is a trade off that minimizes blood loss through interstices while permitting ingrowth of fibroblasts and capillaries to augment neointimal healing11 . the woven test graft porosity was 200 50 ml / cm / min of normal saline at 120 mmhg . no bleeding problem was encountered through the prosthesis after blood pre - clotting when partial heparinisation ( 1 mg / kg ) was employed . for the experiment performed under cardio - pulmonary bypass , plasma pre - clotted and autoclaved test graft appeared impervious to blood during implantation and thereafter . subsequent to general concerns of survival , toxicity , allergy and rejection , in vivo animal experiments primarily focused on study of explanted vascular grafts at varying intervals after implantation . study of explanted graft , in turn , was further characterized by : ( i)structural integrity of the prosthesis and freedom from dilatation / pseudoaneurysm formation.(ii)patency in general , indicating the functional ability of graft in conducting blood for distal perfusion.(iii)perigraft capsule formation signifying the incorporation of foreign device into the host tissue.(iv)neointima formation , which constitutes the most critical determinant of optimal functioning of prosthetic graft in living body , the quality of which is inversely proportional to thrombogenicity . major sources of endothelialisation are as follows ; across suture lines by direct growth from either ends , by deposition of fibroblasts and wandering endothelial precursor cells ( epcs ) in the bloodstream onto the platelet - fibrin layer on the inner side , or by transmural ingrowth of fibroblasts and capillary beds from perigraft tissue entering through the pores of woven / knitted fabric12 patency in general , indicating the functional ability of graft in conducting blood for distal perfusion . neointima formation , which constitutes the most critical determinant of optimal functioning of prosthetic graft in living body , the quality of which is inversely proportional to thrombogenicity . major sources of endothelialisation are as follows ; across suture lines by direct growth from either ends , by deposition of fibroblasts and wandering endothelial precursor cells ( epcs ) in the bloodstream onto the platelet - fibrin layer on the inner side , or by transmural ingrowth of fibroblasts and capillary beds from perigraft tissue entering through the pores of woven / knitted fabric12 . structural integrity was well preserved and the anastomotic suture lines appeared covered with a transparent fibrin layer separating the flowing blood in the prosthesis . all grafts were incorporated into the perigraft capsule and developed a compact layer of neointima , with a thickness from 0.1 - 1.0 mm depending upon the diameter of graft , size of parent vessel , distal run - off and , to a certain extent , the surgical technique . in all short segment grafts , neointima appeared very thin , pink , shiny and intimately adherent as expected as endothelialisation is easily possible from both ends of native aorta . however , in long grafts , neointima was thicker ( 1 - 1.2 mm ) , whitish , slightly irregular but still clean and free from thrombus . in human studies , permanent implants like vascular prostheses must provide optimal safety for the recipient patient along with efficacy and performance for a long duration with uninterrupted functional integrity ( table iv)2 . reporting standards laid down by ad - hoc committee of the joint council of society of vascular surgery and international society of cardiovascular surgery , north american chapter , mandates a preliminary assessment at two years and final report at five years before multicentric trials can be resorted to2 . safety , efficacy and performance standards of the graft were found to be excellent in this study , even beyond six years and in some patients continuing beyond 14 years . similarly , patients in multicentric trial were followed up for duration of at least five years . reporting standards generated by an ad - hoc committee of joint councils of society for vascular surgery and international society for cardiovascular surgery , north american chapter2 all patients had optimal graft performance status on follow up with continued patency and absence of thrombus formation , pseudoaneurysm formation , loss of tensile strength , progressive dilatation or infection . there was only one incidence of anastomotic pseudoaneurysm in the entire series which involved a 32 yr old patient who developed distal anastomotic pseudoaneurysm four years after an intact repair of thoraco - abdominal aortic aneurysm , who also had a previous history of carotid pseudoaneurysm following carotid bypass . the occurance of such anastomotic pseudoaneurysms is not uncommon in takayasu 's disease13 especially in the setting of long - term steroid usage . this follow up study over a protracted period of 6 - 14 years has proven safety to patients . the results compared favourably with published data on long - term biostability of polyester vascular prosthesis which reported 95 per cent 10 yr patency rates and structural failure rate of 0.2 - 3.0 per cent141516 . in conclusion , indigenously designed , developed and thereafter comprehensively performed clinical trials have provided robust data regarding safety and efficacy of indigenously developed vascular prosthesis during an extended study period of over a decade . in a total of 32 patients , with diverse clinical conditions and ages thus studied in the clinical trials encompassing the technique of coating the prosthesis to render it impervious to blood at implantation17 , thus obviating the need for preclotting prior to implantation , was introduced to the chitra vascular graft as well . laboratory testing and in vivo experiments in 30 pig models were completed and the coated graft will undergo clinical trial to prove its safety and efficacy before making it available on shelf .

background & objectives : vascular illnesses are on the rise in india , due to increase in lifestyle diseases and demographic transition , requiring intervention to save life , organ or limbs using vascular prosthesis . the aim of this study was to develop indigenous large diameter vascular graft for treatment of patients with vascular pathologies.methods:the south india textile research association , at coimbatore , tamil nadu , india , developed seamless woven polyester ( polyethylene terephthalate ) graft at its research wing . further characterization and testing followed by clinical trials were conducted at sree chitra tirunal institute for medical sciences and technology , thiruvananthapuram , kerala , india . fifteen in vivo experiments were carried out in 1992 - 1994 in pigs as animal model . controlled ( phase i ) clinical trial in ten patients was performed along with control graft . thereafter , phase ii trial involved 22 patients who underwent multi - centre clinical trial in four centres across india.results:laboratory testing showed that polyester graft was non - toxic , non - leeching and non - haemolytic with preserved long - term quality , further confirming in pigs by implanting in thoracic aorta , comparable to control dacron grafts . perigraft incorporation and smooth neointima formation which are prime features of excellent healing characteristics , were noted at explantation at planned intervals . subsequently in the phase i and ii clinical trials , all patients had excellent recovery without mortality or device - related adverse events . patients receiving the test graft were followed up for 10 and 5 years , respectively . serial clinical , duplex scans and ct angiograms performed periodically confirmed excellent graft performance.interpretation & conclusions : indigenously developed chitra vascular graft was comparable to commercially available dacron graft , ready for clinical use at affordable cost to patients as against costly imported grafts .

Material & Methods Results None Pre-clinical animal testing Discussion

development & in vitro testing : designed and fabricated at south india textile research association ( sitra ) , coimbatore , the main testing and clinical work of woven grafts was carried out at sree chitra tirunal institute for medical sciences and technology ( sctimst ) , thiruvananthapuram , kerala , india . 1 ) to provide large diameter vascular graft with a porosity of 20050 ml / min / cm of normal saline at 120 mmhg . toxicity / biocompatibility studies such as acute systemic toxicity , intracutaneous irritation , in vitro haemolysis and implantation in muscle were studied as per international standards3 for the vascular graft material . grading of erythema and oedema of material in experimental and control rabbits were recorded at 24 , 48 and 72 h. in vitro haemolysis was carried out with material and extract of the material using centrifugation ( remi r-8c dx , india ) with rabbit blood . the implanted animals were sacrificed at the end of one , four and 12 wk , the tissue with the implanted materials were collected , sectioned using rm2255 microtome ( leica , biosystems , india ) and subjected to histopathological analysis ( axio imager z1 microscope , carl zeiss ) . the test vascular polyester polyethylene terephthalate ( pet ) graft with internal diameter ranging from 8 - 30 mm . pre - clinical animal testing : during 1982 - 1983 , as part of the pilot study4 , 30 pigs had received the test graft . further , in vivo experiments in 1992 - 1994 , reported herein , involved 15 pigs as per ethics committee requirements in accordance with draft document of association for the advancement of medical instrumentation5 . four to six months old large white yorkshire pigs ( source : government pig farm , parassala , thiruvananthapuram ) weighing 40 - 45 kg , were used for each experiment after due conditioning as per animal testing guidelines6 . left postero - lateral thoracotomy was performed and pleural cavity entered by excising 4 or 5 rib . replacement of segment of descending thoracic aorta in pig with the test graft using standard protocol via left postero - lateral thoracotomy ( a ) . gross healing characterstics of test vascular graft at explant post-6 month in vivo in pig in 8 , 4 and 12 cm lengths respectively ( b , c , d ) . grafts were explanted to study the healing characteristics , blood / tissue interactions and patency rates . phase i clinical trial : ( i ) design - prospective randomized study was designed to prove non - inferiority of the test prosthesis with concurrent controls using commercially available prosthetic grafts . objective patency assessment was made by non - invasive haemodynamic indices , duplex ultrasound and catheter angiography / ct angiography . ( ii ) parameters evaluated were ( a ) ease of pre - clotting ; ( b ) pliability / conformability ; ( c ) ease of suturing / suture retention ; and ( d ) short - term patency at one year , long - term patency at five years and beyond . ( iii ) institutional ethics committee ( iec ) gave permission to use large diameter prosthesis of length less than 10 cm for a total of 10 patients during phase i trial as a matter of abundant caution . ( iv ) methods - between september 1998 and november 1999 , 10 patients were included in phase i clinical trial using the test graft at sctimst , six of whom underwent repair for aaa and four for coa ( table ii ) . standard inclusion graft technique was performed using appropriate ( 14 - 20 mm internal diameter ) test graft using 3/0 monofilament polypropylene suture for proximal anastomosis and 4/0 suture for distal . excision of coarct segment and interposition graft placement using 14 - 16 mm i d test graft was done in one patient while left subclavian artery ( lsa ) to descending thoracic aorta bypass ( jump graft ) using 12 - 14 mm i d graft was done in the other three . in the control group , eight patients with aaa and two with coa underwent elective implantation of the control dacron graft . clinical details and results of phase i ( controlled ) clinical trial phase ii clinical trial : twenty two patients who underwent indexed vascular reconstructions at four designated hospitals viz . medwin hospital , hyderabad ; g. kuppuswamy naidu memorial hospital , coimbatore ; medical college hospital , and sctimst , thiruvananthapuram ; formed the basis of this part of the study conducted from august 2005 to september 2008 . patients were followed up for a maximum period of five years using clinical and imaging documentation of graft patency and integrity . clinical details and results of phase ii ( multicentric ) clinical trial long - term follow up till date : patients recruited in the clinical trials that involved implantation of the test graft at the institute as well as the control group were followed up by clinical examination and duplex ultrasound on yearly basis . hence the toxicity study concluded that the material was non toxic , non irritant , non haemolytic and biocompatible . ( i ) immediate - all animals , except one ( 15 ) , were extubated at the end of procedure and walked back to their cage 6 - 8 h thereafter . the third animal developed delayed paraplegia 24 h after operation that involved 45 min of aortic cross - clamping , had to be sacrificed and at autopsy the prosthetic graft was found to be patent and void of any thrombus . gross examination showed that the grafts were well incorporated in a 1 - 4 mm thick perigraft capsule . phase i clinical trial : first patient following repair of aaa was discharged from hospital on 10 day . all patients survived major surgical procedure and made satisfactory early recovery except one patient ( 4 ) who underwent surgical repair for complex post - subclavian coarctation of aorta with large heavily calcified post - coarct aneurysm , who developed paraplegia . all patients underwent duplex ultrasound and ct scan before discharge from hospital which showed satisfactory repair , patent graft and smooth regular luminal surface of prosthesis . contrast - enhanced abdominal ct scan axial section of a symptomatic patient in phase i clinical trial showing an infra - renal abdominal aortic aneurysm ( a ) . abdominal contrast enhanced ct scan of the same patient taken 8 years after repair showing intact and patent test vascular graft ( yellow arrow ) ( b ) . follow - up imaging of an adult patient with coarctation of aorta treated by interposition grafting with test vascular graft as part of phase i trial . digital subtraction angiogram ( dsa ) at 1 year ( c ) and ct angiogram at 6 years ( d ) showing preserved and patent graft ( red arrows ) . all patients who received the test implant survived the procedures and recovered well except the patient who developed paraplegia ; however , digital substraction angiography ( dsa ) at one year and ct aortogram at five years showed intact repair with patent graft . the luminal side of the grafts were clean and regular with no evidence of thrombus formation inside the test or control grafts . phase ii clinical trial : prosthesis was found to be surgeon friendly , with excellent suturing and suture retention qualities . all centres reported good graft handling qualities with pliability , lack of fraying , ease of pre - clotting and suturing and absence of excessive weeping following implantation similar to control dacron grafts . apart from regular clinical assessment , duplex scan evaluation was performed in patients with graft implantation in abdomen . check mr / ct angiogram was performed on follow up showed preserved patency with no evidence of graft - related complications ( fig . follow up ct / mr angiograms of patients in the multicentric trials who underwent vascular reconstruction using test graft ( white arrows ) , showing good results following open repair of abdominal aortic aneurysm at 5 years ( a ) , aorto - femoral bypass for iliac occlusion at 4 years ( b ) , and open repair of thoraco - abdominal aortic aneurysm at 6 years ( c ) . long - term follow up till date : follow up was complete in eight patients at > 10 yr after implantation of test vascular prosthesis in phase i trial and > 5 yr in 14 patients in phase ii trial . present evaluation has confirmed excellent clinical status of the survivors and satisfactory graft function with no incidence of pseudoaneurysm , aneurysm , infection , thrombosis , dilatation or any other untoward sequelae with reference to implanted vascular prosthesis ( fig . ct angiogram , volume - rendered image at 14 years following jump graft bypass ( white arrow ) from left subclavian artery to descending thoracic aorta for coarctation of aorta ( green arrow ) using 14 mm test graft . ( i ) immediate - all animals , except one ( 15 ) , were extubated at the end of procedure and walked back to their cage 6 - 8 h thereafter . phase i clinical trial : first patient following repair of aaa was discharged from hospital on 10 day . ct scan prior to discharge showed patent graft , intact suture lines , smooth luminal outline and patent inferior mesenteric artery . all patients survived major surgical procedure and made satisfactory early recovery except one patient ( 4 ) who underwent surgical repair for complex post - subclavian coarctation of aorta with large heavily calcified post - coarct aneurysm , who developed paraplegia . all patients underwent duplex ultrasound and ct scan before discharge from hospital which showed satisfactory repair , patent graft and smooth regular luminal surface of prosthesis . contrast - enhanced abdominal ct scan axial section of a symptomatic patient in phase i clinical trial showing an infra - renal abdominal aortic aneurysm ( a ) . follow - up imaging of an adult patient with coarctation of aorta treated by interposition grafting with test vascular graft as part of phase i trial . all patients who received the test implant survived the procedures and recovered well except the patient who developed paraplegia ; however , digital substraction angiography ( dsa ) at one year and ct aortogram at five years showed intact repair with patent graft . the luminal side of the grafts were clean and regular with no evidence of thrombus formation inside the test or control grafts . phase ii clinical trial : prosthesis was found to be surgeon friendly , with excellent suturing and suture retention qualities . all centres reported good graft handling qualities with pliability , lack of fraying , ease of pre - clotting and suturing and absence of excessive weeping following implantation similar to control dacron grafts . apart from regular clinical assessment , duplex scan evaluation was performed in patients with graft implantation in abdomen . check mr / ct angiogram was performed on follow up showed preserved patency with no evidence of graft - related complications ( fig . follow up ct / mr angiograms of patients in the multicentric trials who underwent vascular reconstruction using test graft ( white arrows ) , showing good results following open repair of abdominal aortic aneurysm at 5 years ( a ) , aorto - femoral bypass for iliac occlusion at 4 years ( b ) , and open repair of thoraco - abdominal aortic aneurysm at 6 years ( c ) . long - term follow up till date : follow up was complete in eight patients at > 10 yr after implantation of test vascular prosthesis in phase i trial and > 5 yr in 14 patients in phase ii trial . present evaluation has confirmed excellent clinical status of the survivors and satisfactory graft function with no incidence of pseudoaneurysm , aneurysm , infection , thrombosis , dilatation or any other untoward sequelae with reference to implanted vascular prosthesis ( fig . ct angiogram , volume - rendered image at 14 years following jump graft bypass ( white arrow ) from left subclavian artery to descending thoracic aorta for coarctation of aorta ( green arrow ) using 14 mm test graft . ( i ) immediate - all animals , except one ( 15 ) , were extubated at the end of procedure and walked back to their cage 6 - 8 h thereafter . another animal died on 2 post - operative day following hyperpyrexia of 104f and at autopsy graft was noted to be patent . phase i clinical trial : first patient following repair of aaa was discharged from hospital on 10 day . all patients survived major surgical procedure and made satisfactory early recovery except one patient ( 4 ) who underwent surgical repair for complex post - subclavian coarctation of aorta with large heavily calcified post - coarct aneurysm , who developed paraplegia . all patients underwent duplex ultrasound and ct scan before discharge from hospital which showed satisfactory repair , patent graft and smooth regular luminal surface of prosthesis . contrast - enhanced abdominal ct scan axial section of a symptomatic patient in phase i clinical trial showing an infra - renal abdominal aortic aneurysm ( a ) . follow - up imaging of an adult patient with coarctation of aorta treated by interposition grafting with test vascular graft as part of phase i trial . all patients who received the test implant survived the procedures and recovered well except the patient who developed paraplegia ; however , digital substraction angiography ( dsa ) at one year and ct aortogram at five years showed intact repair with patent graft . no untoward sequelae of excessive intimal hyperplasia or pseudoaneurysm formation were noted in and around the proximal and distal suture lines in these patients . phase ii clinical trial : prosthesis was found to be surgeon friendly , with excellent suturing and suture retention qualities . all centres reported good graft handling qualities with pliability , lack of fraying , ease of pre - clotting and suturing and absence of excessive weeping following implantation similar to control dacron grafts . two patients ( both taaa ) developed transient renal dysfunction ( increase in serum creatinine > 1 mg / dl higher than pre - operative level ) eventually normalising at discharge . apart from regular clinical assessment , duplex scan evaluation was performed in patients with graft implantation in abdomen . check mr / ct angiogram was performed on follow up showed preserved patency with no evidence of graft - related complications ( fig . follow up ct / mr angiograms of patients in the multicentric trials who underwent vascular reconstruction using test graft ( white arrows ) , showing good results following open repair of abdominal aortic aneurysm at 5 years ( a ) , aorto - femoral bypass for iliac occlusion at 4 years ( b ) , and open repair of thoraco - abdominal aortic aneurysm at 6 years ( c ) . long - term follow up till date : follow up was complete in eight patients at > 10 yr after implantation of test vascular prosthesis in phase i trial and > 5 yr in 14 patients in phase ii trial . present evaluation has confirmed excellent clinical status of the survivors and satisfactory graft function with no incidence of pseudoaneurysm , aneurysm , infection , thrombosis , dilatation or any other untoward sequelae with reference to implanted vascular prosthesis ( fig . 7)9 , except one 32 yr old patient with thoraco - abdominal aortic aneurysm due to takayasu 's disease was noted to have pseudoaneurysm at distal aortic anastomosis five years following surgery requiring endovascular aortic stent grafting . ct angiogram , volume - rendered image at 14 years following jump graft bypass ( white arrow ) from left subclavian artery to descending thoracic aorta for coarctation of aorta ( green arrow ) using 14 mm test graft . this study reports indigenously fabricated polyester graft developed and extensively tested in vitro , than tested in vivo using large animal , prior to clinical study by implantation in patients . classic animal experimental data reported by adam wesolowski delineated pigs as the most suitable model for in vivo experiments10 . the woven test graft porosity was 200 50 ml / cm / min of normal saline at 120 mmhg . subsequent to general concerns of survival , toxicity , allergy and rejection , in vivo animal experiments primarily focused on study of explanted vascular grafts at varying intervals after implantation . major sources of endothelialisation are as follows ; across suture lines by direct growth from either ends , by deposition of fibroblasts and wandering endothelial precursor cells ( epcs ) in the bloodstream onto the platelet - fibrin layer on the inner side , or by transmural ingrowth of fibroblasts and capillary beds from perigraft tissue entering through the pores of woven / knitted fabric12 patency in general , indicating the functional ability of graft in conducting blood for distal perfusion . major sources of endothelialisation are as follows ; across suture lines by direct growth from either ends , by deposition of fibroblasts and wandering endothelial precursor cells ( epcs ) in the bloodstream onto the platelet - fibrin layer on the inner side , or by transmural ingrowth of fibroblasts and capillary beds from perigraft tissue entering through the pores of woven / knitted fabric12 . structural integrity was well preserved and the anastomotic suture lines appeared covered with a transparent fibrin layer separating the flowing blood in the prosthesis . safety , efficacy and performance standards of the graft were found to be excellent in this study , even beyond six years and in some patients continuing beyond 14 years . similarly , patients in multicentric trial were followed up for duration of at least five years . reporting standards generated by an ad - hoc committee of joint councils of society for vascular surgery and international society for cardiovascular surgery , north american chapter2 all patients had optimal graft performance status on follow up with continued patency and absence of thrombus formation , pseudoaneurysm formation , loss of tensile strength , progressive dilatation or infection . the occurance of such anastomotic pseudoaneurysms is not uncommon in takayasu 's disease13 especially in the setting of long - term steroid usage . the results compared favourably with published data on long - term biostability of polyester vascular prosthesis which reported 95 per cent 10 yr patency rates and structural failure rate of 0.2 - 3.0 per cent141516 . in conclusion , indigenously designed , developed and thereafter comprehensively performed clinical trials have provided robust data regarding safety and efficacy of indigenously developed vascular prosthesis during an extended study period of over a decade . in a total of 32 patients , with diverse clinical conditions and ages thus studied in the clinical trials encompassing the technique of coating the prosthesis to render it impervious to blood at implantation17 , thus obviating the need for preclotting prior to implantation , was introduced to the chitra vascular graft as well . laboratory testing and in vivo experiments in 30 pig models were completed and the coated graft will undergo clinical trial to prove its safety and efficacy before making it available on shelf .

accidental falls in old age are an increasing problem in our graying society , and they have received a lot of attention in recent research . about 30% of persons aged 65 + years and about 50% of those aged 85 + years fall at least once a year and the probability of falling again increases after each fall [ 13 ] . early research addressed environmental hazards , sensorimotor deficits , and impaired balance as risk factors for accidental falls [ 4 , 5 ] , but more recent work focuses on the role of cognition [ 6 , 7 ] . according to this recent approach , age - related deficits of locomotion can be partly compensated by cognitive workaround strategies , thus replacing automated sensorimotor processing with effortful higher - order functions . this is a good example of neural plasticity , as it shows that deficits arising in one part of the nervous system can be overcome by engaging another part of that system . persons with a reduced cognitive capacity have only limited access to this compensation : according to empirical research , they are more likely to walk unsteadily and their risk of falling is higher . brain plasticity may help overcome the gait problems in old age , but there is a price to pay : cognitive resources allocated to seniors ' locomotion are no longer available for other activities while walking , such as obstacle avoidance , navigation along a planned route , watching for pedestrian and vehicular traffic , as well as engaging in gait - unrelated tasks . as a consequence , elderly persons often have larger problems than younger ones to walk and concurrently engage in another activity [ 6 , 7 ] . early studies in the 1990s were already able to show age - related changes in the human brain [ 911 ] . older people are affected by a general loss of brain mass and a distinctive atrophy of the frontal gray matter , as well as a white matter hyperintensity [ 12 , 13 ] . recent fmri studies showed a degradation of the cerebral cortex as a function of age . notably , these studies demonstrated a reduction of gray and white matter in the prefrontal cortex [ 14 , 15 ] and an age - related mass reduction of the frontal lobe [ 16 , 17 ] . additionally , a loss of central neurons and associated synaptic connections accrues , which leads to reduced processing speed and a deficit in the ability to handle several processes simultaneously . some authors state that these structural changes , especially the changes in white matter in the human brain , are caused by an age - related deterioration of the vascular system and the associated reduction in blood flow . compared with young people , the frontal cortex of people aged 65 + years is reduced by 1017% , while the temporal , parietal , and occipital cortices only show a reduction of approximately 1% . this selective shrinkage of the brain seems to affect higher - level cognitive functions [ 14 , 20 ] . probably most vulnerable to age - related decay are the so - called executive functions , that is , cognitive operations that include ( a ) the planning of strategies for different actions , ( b ) the monitoring of these actions , ( c ) adjusting future actions using feedback , alertness , and ( d ) the inhibition of task - irrelevant information . another age - dependent cognitive function is working memory , a set of mechanisms involved in the control , regulation , and active maintenance of task - relevant information in both novel and familiar tasks [ 2224 ] . executive functions and working memory are both thought to reside in the frontal lobes . recent experiments evaluated the neural activity in prefrontal brain structures while subjects handled tasks involving executive functions or working memory ( e.g. , the wisconsin card sorting test , a self - ordered pointing task , or a delayed - response task ) and found that these tasks are sensitive to prefrontal dysfunction [ 19 , 2628 ] . summing up , the available literature suggests that the anatomical decrease of brain mass , especially in the frontal lobe , contributes to a reduction of cognitive processing capacity with advancing age and thus limits to what extent neural plasticity can compensate for age - related decrements of locomotion . human walking , a task that people perform on a daily basis , involves complex processes that require the ongoing integration of visual , proprioceptive , and vestibular sensory information . for instance , joint positions have to be controlled , feedback from the terrain the person is walking on has to be integrated , and the environment the person is moving in needs to be observed . in addition , our everyday life affords numerous situations in which walking must be integrated with another activity , such as watching for traffic or using a mobile phone . this concurrence of locomotion and another activity , termed dual - task walking , has received a lot of attention in recent research [ 2933 ] . for example , walking speed and stride length decrease , while lateral sway and stride time variability increase with age [ 3436 ] . some of these changes are compensatory and are used to stabilize posture , while others are dysfunctional and correlate with the risk of accidental falls . the observed deterioration has been attributed to a variety of causal factors , notably to cognitive decline ; indeed , the critical role of cognition is supported by the fact that age - related gait changes are more pronounced in people with cognitive impairment [ 37 , 38 ] and that they are accentuated under dual - task conditions [ 6 , 39 ] . the additional tasks utilized in the literature are manifold and range from verbal response and memory tasks to more complex ones such as mathematical tasks or tasks involving visual or motor control . when two or more tasks need to be carried out concurrently , task performance declines at least in one of them . some studies on dual - task walking found an age - related decrease in dual - task performance when subjects were asked to walk at their preferred walking speed and simultaneously complete another task [ 3942 ] , whereas other studies were not able to create this kind of age - related deficit [ 4345 ] . this discrepancy might be due to the utilization of different secondary tasks used in these studies . some tasks interfere with walking while other tasks do not [ 46 , 47 ] . a meta - analysis conducted by al - yahya showed significant increases in age - related deficits while walking when the secondary task was associated with executive or memory functions , for example , verbal fluency tasks or mental imaging but not when the task added was rather simple , for example , reaction or discrimination tasks . this outcome indicates that the central ability to process walking requirements and cognitive demands simultaneously decreases with age , which might arise from insufficient central processing capabilities in older people or from disorders in the coordination of multiple sensory or motor information . a major problem of many studies on age - related decreases in dual - task performance is the inconsistency of methods . several studies addressing the influence of cognitive functions on locomotion in the elderly have considerable methodological flaws . for instance , some authors observed a decrease in motor performance under dual - task conditions in seniors but did not relate it to the performance of younger subjects and thus could not ascertain the presence of an age - related decrease [ 26 , 51 , 52 ] . other studies analyzed walking and disregarded the secondary task , or they focused on secondary task and ignored walking ; in consequence , they can not disambiguate changes of dual - task performance from those of task priority [ 38 , 40 , 45 , 53 ] . in principle , concurrent tasks can be given equal or different priorities such as to maximize gains or minimize risks ; while young people typically prioritize gait [ 55 , 56 ] , older people tend to assign higher priority to the secondary task . as a consequence studies that only evaluate walking will overestimate age - related dual - task deficits , and those that only evaluate the secondary task will underestimate those deficits . in experiments that considered both tasks and both age groups for their analysis , secondary tasks varied in their sensory demands , in their response requirements , or in their cognitive difficulty . table 1 shows the mean dual - task costs of young and older subjects taken as examples from recent studies , including the task characteristics of the additional task used . dual - task costs were determined according to the following formulas : ( 1)dtc = dss , where ( d : dual - task performance ; s : single task performance ) , using the mean values of each age group from the studies indicated . to express subjects ' dual - task ability irrespective of their individual task priorities , we calculated the dual - task costs across both tasks . the formula used is [ 57 , 58 ] ( 2)mdtc= dtc(task )+dtc(task )2 , where ( task : walking task ; task : additional task ) . thus calculated mean dual - task costs show a subject 's decrease in performance when completing two tasks simultaneously instead of one task alone . a high dtc value indicates a poorer performance under dual - task conditions compared with single - task conditions . as shown in figure 1 , one main difference between all these studies using dual - task paradigms is that different kinds of additional task demands lead to different levels of age - related dual - task costs ranging from 0.99 to 26.0% in young persons and from 2.6 to 44.0% in older aged . some studies found no deficits of dual - task walking in old age [ 29 , 59 , 60 ] , some observed small deficits [ 41 , 44 ] , and others substantial deficits [ 39 , 42 , 6164 ] . the wide divergence of age - related deficits in the above studies suggests that the magnitude of deficits is related to demands of the secondary task . evaluated the attentional requirements for maintaining posture and walking in eight young and eight older subjects . they combined a sitting , a standing , and a walking task with a verbal response task where reaction times were measured . subjects were asked to give a verbal response ( top ) to an auditory stimulus consisting of a 100 hz tone presented for 50 milliseconds . observation of the auditory task was conducted either in isolation or in combination with subjects walking along a path at their preferred walking speed . the outcome of this study showed a decline in reaction times and a decrease in walking speed in both age groups . ( 2006 ) , who investigated the effects of age on treadmill walking while performing a reaction time task . subjects had to walk at their preferred walking speed on a treadmill and were instructed to press a hand - held response button as soon as a letter was presented on a monitor in front of them . in single and dual - task condition , the mean walking speed of the young group was significantly faster than the walking speed of the older group , and the reaction times in both groups decreased while treadmill walking . these results suggest that completing psychomotor tasks while walking , irrespective of whether the response is verbal or manual , does not seem to be sensitive or challenging enough to give rise to age - related deficits in dual - task performance . springer and colleagues evaluated the possible age - related effects of tasks requiring executive functions on human walking . they combined walking with three different tasks : ( a ) listening to and remembering a simple text , ( b ) listening to and remembering a complex text , and ( c ) serial subtracting of seven , starting from 500 . moreover , this study subdivided the older group into fallers ( one or more fall in the previous six months ) and nonfallers ( no fall in the previous six months ) . the results identified no age - related effect of dual - task walking when the young group and the older nonfallers were compared . in contrast , when the fallers were compared with the young group , especially with regard to the arithmetic tasks , age - related deficits in dual - task behavior were clearly observable ( dual - task costs : old : 14% ; young : 9% ) . the simultaneous processing of arithmetic tasks ( subtracting serial 7s ) and its cognitive load while walking seems to destabilize the gait of elderly fallers but appears to have no effect in older non - fallers and young people . a further approach to evaluate the effects of cognitive load and memory function on dual - task walking was conducted by loevdn and colleagues . they manipulated working memory load using an n - back task while the subject was treadmill walking . their paradigm showed that cognitive processes used to solve n - back tasks are not related to the control of human locomotion . the extent of age - related dual - task deficit did not differ between the older and the young people . just like psychomotor speed , working memory , measured by n - back tasks , does not appear to be a factor in age - related deficits . in addition to that krampe et al . evaluated the impact of a semantic word fluency task on walking at a fast pace . subjects were asked to name as many words as possible related to a given category ( e.g. , the results indicated a decrease in walking speed for all age groups and this decrease was approximately 3.5% larger in the older group . recent studies that found a substantial increase of dual - task costs with advancing age were conducted by the groups of lindenberger et al . [ 39 , 42 ] and bock and beurskens [ 6164 ] . in the studies of the former group , subjects were given a visual memory task where words had to be memorized , and later reproduced , using visuo - spatial imagery . both studies found higher dual - task costs in the older group compared with younger people , accounted for by the memory task but not by the walking task . the age - related increase of dual - task costs amounted to about 15% , which is higher than that in most other studies . thus , the additional task used by the lindenberger group seems to be particularly sensitive to age - related deficits in cognitive performance . bock and colleagues tested this assumption explicitly by systematically investigating the influence of task characteristics on dual - task walking to find out which aspect of a task is responsible for the development of age - related deficits in dual - task conditions . the authors compared the single- and dual - task gait of young and elderly subjects with eight different combinations of walking and nonwalking tasks , and they found age - related deficits of dual - task walking for some but not for other task combinations . subjects either had to spell words of 18 to 21 letters , remember sequences of different symbols ( triangle , cross , ellipse , etc . ) , close buttons of nine different shapes and sizes on a jacket , accomplish a reaction time task , walk at maximum speed or on wide / narrow paths , walk on a treadmill , or avoid obstacles that were presented on a treadmill at irregular intervals . bock and colleagues found that dual - task costs were small in most experiments and did not differ between young and older participants . the only task combination in which dual - task performance was distinctly lower in older than in young subjects were experiments requiring the time - critical processing of visual information ( e.g. , obstacle avoidance in cooperation with a visual reaction time task ) . tasks showing the highest deficits in the elderly combined three main features : ( a ) the task was conducted on a treadmill , ( b ) subjects had to avoid obstacles , and ( c ) the walking and non - walking tasks required continuous visual control . in a second study , bock explored the effects of the different walking tasks by comparing walking on a treadmill with walking in a hallway . the results from both experiments were similar , overground and treadmill walking had similar effects on age - related dual - task decrements . this outcome leads to the suggestion that walking on a treadmill does not produce deficits in walking as a function of age . one secondary task required visual control ( i.e. , checking boxes on a sheet of paper ) and the other tasks required memory and attention resources ( i.e. , memorizing pictures ) . in these task combinations , obstacles did not give rise to any age - related deficits , and only the features of the secondary task led to age - related differences in dual - task performance . an increase in dual - task costs occurred mainly in a task requiring visual processing of information and managing two streams of visual information , one related to the checking task and the other one related to the walking task . these results are indirectly supported by a correlation of postural control and the degree of visual impairment that was found by jamet and colleagues . beurskens and bock extended the outcomes of bock and colleagues and showed that the visual component of a secondary task has a crucial influence on age - related deficits in dual - task walking [ 61 , 64 ] . the age - related increase of dual - task costs in their studies amounted to about 9% , which is higher than in most other studies but lower than in lindenberger 's experiments [ 39 , 42 ] . in contrast to the methods and tasks used in most of the presented studies , secondary tasks in our everyday life are hardly continuous , and they do not occupy a person for an extended period of time . usually , the tasks are rather brief , occur rarely and at unpredictable times , for example , stepping over wet spots on the street , watching street signs to work out the right direction or stopping to walk when a car approaches . to consider these kinds of situations , beurskens and bock recently compared young and older people 's walking behavior after short and unexpected distractions . eight monitors were arranged at irregular intervals on a straight floor , four to the subject 's left , and four to the right . participants walked the path ten times back and forth at their preferred speed , thus covering a total distance of 400 m and passing 160 times in front of a monitor . on twelve of those passes at the same time , a capital letter from the latin alphabet ( such as g or k ) was displayed for 2 seconds on that monitor . letters were presented mirror - reversed or nonreversed , at a rotation angle of 60% or 120 with respect to the vertical . if it was mirror - reversed , and no if it was not reversed . results showed that brief distractions did not influence younger people 's walking but significantly changed older people 's walking behavior by increasing step duration and decreasing the amplitude of a step . the age - related decrease in dual - task performance reached up to 14% , which is comparable to lindenberger 's findings with visual imagery . brief visual distracters , therefore , can be as disruptive for elderly persons as an ongoing demand on visual working memory . in addition to age - related cognitive disorders and a deterioration of cognitive functions with advancing age , there are other potential causes of changes in walking behavior and human locomotion . one further aspect often addressed in contemporary literature is the possible correlation of older people 's fear of falling with gait changes . some authors find correlations and state that the development of fear after a fall leads to changes in walking speed , step time , or the appropriate variability , which leads to recurrent falls during the following years [ 32 , 67 , 68 ] . however , other authors find no evidence that preceding falls increase the occurrence of subsequent falls [ 69 , 70 ] and can not show that the fear of falling is associated with a reduction of physical activity for safety reasons . in fact , the correlation of psychological functions and human walking is a multidimensional construct where several aspects , ranging from fear of falling , education , physical activity and stress , have to be taken into consideration . another possible confounding factor in this area of research is the role of eye movements . subjects typically focus both their gaze [ 72 , 73 ] and their attention on the goal of their activities [ 74 , 75 ] , which indicates that attention , eye and body movements are all closely interlinked . however , oculomotor behavior changes with advancing age : the latency and duration of saccades increase while their accuracy decreases , thus necessitating more corrective and re - fixation saccades [ 7678 ] . such deficits could complicate the navigation through visually defined space and its integration with another visual task , and thus contribute to impaired dual - task walking . yet another factor to be considered is the age - related shrinkage of the attention window , which can be observed not only with cognitive tasks [ 79 , 80 ] but also with manual ones : bimanual tracking deteriorates with display distance in older , but not in young subjects . however , the magnitude of this deterioration in a given elderly person is not related to that person 's dual - task costs when walking with a concurrent visuospatial task ( r = 0.04 ; p < 0.05 ) , which suggests that peripheral visual attention may not be an important factor in dual - task walking . when reviewing contemporary research on dual - task walking , motor control , and the role of cognitive functions , the topics of executive functions , and the role of the human frontal lobe have often been addressed . many studies associate the occurrence of dual - task deficits in the elderly while walking with the well - known decay of prefrontal cortical circuitry , the loss of prefrontal brain mass , and the associated deterioration of executive functions in old age [ 16 , 20 , 81 ] . the current review shows that such deficits are observable mainly with nonwalking tasks requiring substantial visual processing , and thus with the coordination of two independent visual streams of information . it is quite conceivable that this coordination depends critically on higher - level cognitive functions , such as executive functions and working memory , while peripheral visual attention does not seem to play a predominant role . this would fit well with norman and shallice 's concept of executive control , according to which shifts of attention from one task to another take place in a high - level supervisory attentional system . that being said , we must add that not all age - related deficits of dual - task walking can be explained by interference between two visual streams . deficits have also been reported for tasks without a visual component , although they were much smaller than those for tasks having a visual demand [ 29 , 41 , 59 ] or were limited to very old subjects ( 75 + years ) . it , therefore , appears that interference is not necessarily confined to the visual modality but rather can be intermodal , and increasingly so in very old age . summing up , we suggest that neural plasticity can partly compensate for age - related deficits of walking : it supplements deteriorated sensorimotor processes by cognitive processes . however , this compensation reduces the cognitive capacity available for concurrent tasks , and is limited by the age - related decay of the prefrontal cortex .

this review summarizes our present knowledge about elderly people 's problems with walking . we highlight the plastic changes in the brain that allow a partial compensation of these age - related deficits and discuss the associated costs and limitations . experimental evidence for the crucial role of executive functions and working memory is presented , leading us to the hypothesis that it is difficult for seniors to coordinate two streams of visual information , one related to navigation through visually defined space , and the other to a visually demanding second task . this hypothesis predicts that interventions aimed at the efficiency of visuovisual coordination in the elderly will ameliorate their deficits in dual - task walking .

1. Introduction 2. Anatomical Changes in the Human Brain as a Function of Age 3. The Role of Cognition in Human Locomotion 4. Additional Factors Influencing Dual-Task Walking Performance 5. Conclusions and Outlook

accidental falls in old age are an increasing problem in our graying society , and they have received a lot of attention in recent research . about 30% of persons aged 65 + years and about 50% of those aged 85 + years fall at least once a year and the probability of falling again increases after each fall [ 13 ] . early research addressed environmental hazards , sensorimotor deficits , and impaired balance as risk factors for accidental falls [ 4 , 5 ] , but more recent work focuses on the role of cognition [ 6 , 7 ] . according to this recent approach , age - related deficits of locomotion can be partly compensated by cognitive workaround strategies , thus replacing automated sensorimotor processing with effortful higher - order functions . this is a good example of neural plasticity , as it shows that deficits arising in one part of the nervous system can be overcome by engaging another part of that system . persons with a reduced cognitive capacity have only limited access to this compensation : according to empirical research , they are more likely to walk unsteadily and their risk of falling is higher . brain plasticity may help overcome the gait problems in old age , but there is a price to pay : cognitive resources allocated to seniors ' locomotion are no longer available for other activities while walking , such as obstacle avoidance , navigation along a planned route , watching for pedestrian and vehicular traffic , as well as engaging in gait - unrelated tasks . early studies in the 1990s were already able to show age - related changes in the human brain [ 911 ] . older people are affected by a general loss of brain mass and a distinctive atrophy of the frontal gray matter , as well as a white matter hyperintensity [ 12 , 13 ] . recent fmri studies showed a degradation of the cerebral cortex as a function of age . notably , these studies demonstrated a reduction of gray and white matter in the prefrontal cortex [ 14 , 15 ] and an age - related mass reduction of the frontal lobe [ 16 , 17 ] . additionally , a loss of central neurons and associated synaptic connections accrues , which leads to reduced processing speed and a deficit in the ability to handle several processes simultaneously . some authors state that these structural changes , especially the changes in white matter in the human brain , are caused by an age - related deterioration of the vascular system and the associated reduction in blood flow . compared with young people , the frontal cortex of people aged 65 + years is reduced by 1017% , while the temporal , parietal , and occipital cortices only show a reduction of approximately 1% . this selective shrinkage of the brain seems to affect higher - level cognitive functions [ 14 , 20 ] . probably most vulnerable to age - related decay are the so - called executive functions , that is , cognitive operations that include ( a ) the planning of strategies for different actions , ( b ) the monitoring of these actions , ( c ) adjusting future actions using feedback , alertness , and ( d ) the inhibition of task - irrelevant information . another age - dependent cognitive function is working memory , a set of mechanisms involved in the control , regulation , and active maintenance of task - relevant information in both novel and familiar tasks [ 2224 ] . executive functions and working memory are both thought to reside in the frontal lobes . recent experiments evaluated the neural activity in prefrontal brain structures while subjects handled tasks involving executive functions or working memory ( e.g. summing up , the available literature suggests that the anatomical decrease of brain mass , especially in the frontal lobe , contributes to a reduction of cognitive processing capacity with advancing age and thus limits to what extent neural plasticity can compensate for age - related decrements of locomotion . human walking , a task that people perform on a daily basis , involves complex processes that require the ongoing integration of visual , proprioceptive , and vestibular sensory information . for instance , joint positions have to be controlled , feedback from the terrain the person is walking on has to be integrated , and the environment the person is moving in needs to be observed . in addition , our everyday life affords numerous situations in which walking must be integrated with another activity , such as watching for traffic or using a mobile phone . this concurrence of locomotion and another activity , termed dual - task walking , has received a lot of attention in recent research [ 2933 ] . some of these changes are compensatory and are used to stabilize posture , while others are dysfunctional and correlate with the risk of accidental falls . the observed deterioration has been attributed to a variety of causal factors , notably to cognitive decline ; indeed , the critical role of cognition is supported by the fact that age - related gait changes are more pronounced in people with cognitive impairment [ 37 , 38 ] and that they are accentuated under dual - task conditions [ 6 , 39 ] . the additional tasks utilized in the literature are manifold and range from verbal response and memory tasks to more complex ones such as mathematical tasks or tasks involving visual or motor control . some studies on dual - task walking found an age - related decrease in dual - task performance when subjects were asked to walk at their preferred walking speed and simultaneously complete another task [ 3942 ] , whereas other studies were not able to create this kind of age - related deficit [ 4345 ] . this discrepancy might be due to the utilization of different secondary tasks used in these studies . some tasks interfere with walking while other tasks do not [ 46 , 47 ] . a meta - analysis conducted by al - yahya showed significant increases in age - related deficits while walking when the secondary task was associated with executive or memory functions , for example , verbal fluency tasks or mental imaging but not when the task added was rather simple , for example , reaction or discrimination tasks . this outcome indicates that the central ability to process walking requirements and cognitive demands simultaneously decreases with age , which might arise from insufficient central processing capabilities in older people or from disorders in the coordination of multiple sensory or motor information . a major problem of many studies on age - related decreases in dual - task performance is the inconsistency of methods . several studies addressing the influence of cognitive functions on locomotion in the elderly have considerable methodological flaws . for instance , some authors observed a decrease in motor performance under dual - task conditions in seniors but did not relate it to the performance of younger subjects and thus could not ascertain the presence of an age - related decrease [ 26 , 51 , 52 ] . other studies analyzed walking and disregarded the secondary task , or they focused on secondary task and ignored walking ; in consequence , they can not disambiguate changes of dual - task performance from those of task priority [ 38 , 40 , 45 , 53 ] . in principle , concurrent tasks can be given equal or different priorities such as to maximize gains or minimize risks ; while young people typically prioritize gait [ 55 , 56 ] , older people tend to assign higher priority to the secondary task . as a consequence studies that only evaluate walking will overestimate age - related dual - task deficits , and those that only evaluate the secondary task will underestimate those deficits . in experiments that considered both tasks and both age groups for their analysis , secondary tasks varied in their sensory demands , in their response requirements , or in their cognitive difficulty . table 1 shows the mean dual - task costs of young and older subjects taken as examples from recent studies , including the task characteristics of the additional task used . dual - task costs were determined according to the following formulas : ( 1)dtc = dss , where ( d : dual - task performance ; s : single task performance ) , using the mean values of each age group from the studies indicated . to express subjects ' dual - task ability irrespective of their individual task priorities , we calculated the dual - task costs across both tasks . thus calculated mean dual - task costs show a subject 's decrease in performance when completing two tasks simultaneously instead of one task alone . a high dtc value indicates a poorer performance under dual - task conditions compared with single - task conditions . as shown in figure 1 , one main difference between all these studies using dual - task paradigms is that different kinds of additional task demands lead to different levels of age - related dual - task costs ranging from 0.99 to 26.0% in young persons and from 2.6 to 44.0% in older aged . some studies found no deficits of dual - task walking in old age [ 29 , 59 , 60 ] , some observed small deficits [ 41 , 44 ] , and others substantial deficits [ 39 , 42 , 6164 ] . the wide divergence of age - related deficits in the above studies suggests that the magnitude of deficits is related to demands of the secondary task . evaluated the attentional requirements for maintaining posture and walking in eight young and eight older subjects . they combined a sitting , a standing , and a walking task with a verbal response task where reaction times were measured . subjects were asked to give a verbal response ( top ) to an auditory stimulus consisting of a 100 hz tone presented for 50 milliseconds . the outcome of this study showed a decline in reaction times and a decrease in walking speed in both age groups . subjects had to walk at their preferred walking speed on a treadmill and were instructed to press a hand - held response button as soon as a letter was presented on a monitor in front of them . in single and dual - task condition , the mean walking speed of the young group was significantly faster than the walking speed of the older group , and the reaction times in both groups decreased while treadmill walking . these results suggest that completing psychomotor tasks while walking , irrespective of whether the response is verbal or manual , does not seem to be sensitive or challenging enough to give rise to age - related deficits in dual - task performance . springer and colleagues evaluated the possible age - related effects of tasks requiring executive functions on human walking . they combined walking with three different tasks : ( a ) listening to and remembering a simple text , ( b ) listening to and remembering a complex text , and ( c ) serial subtracting of seven , starting from 500 . moreover , this study subdivided the older group into fallers ( one or more fall in the previous six months ) and nonfallers ( no fall in the previous six months ) . the results identified no age - related effect of dual - task walking when the young group and the older nonfallers were compared . in contrast , when the fallers were compared with the young group , especially with regard to the arithmetic tasks , age - related deficits in dual - task behavior were clearly observable ( dual - task costs : old : 14% ; young : 9% ) . a further approach to evaluate the effects of cognitive load and memory function on dual - task walking was conducted by loevdn and colleagues . they manipulated working memory load using an n - back task while the subject was treadmill walking . their paradigm showed that cognitive processes used to solve n - back tasks are not related to the control of human locomotion . the extent of age - related dual - task deficit did not differ between the older and the young people . just like psychomotor speed , working memory , measured by n - back tasks , does not appear to be a factor in age - related deficits . in addition to that krampe et al . evaluated the impact of a semantic word fluency task on walking at a fast pace . subjects were asked to name as many words as possible related to a given category ( e.g. , the results indicated a decrease in walking speed for all age groups and this decrease was approximately 3.5% larger in the older group . recent studies that found a substantial increase of dual - task costs with advancing age were conducted by the groups of lindenberger et al . in the studies of the former group , subjects were given a visual memory task where words had to be memorized , and later reproduced , using visuo - spatial imagery . both studies found higher dual - task costs in the older group compared with younger people , accounted for by the memory task but not by the walking task . the age - related increase of dual - task costs amounted to about 15% , which is higher than that in most other studies . thus , the additional task used by the lindenberger group seems to be particularly sensitive to age - related deficits in cognitive performance . bock and colleagues tested this assumption explicitly by systematically investigating the influence of task characteristics on dual - task walking to find out which aspect of a task is responsible for the development of age - related deficits in dual - task conditions . the authors compared the single- and dual - task gait of young and elderly subjects with eight different combinations of walking and nonwalking tasks , and they found age - related deficits of dual - task walking for some but not for other task combinations . , close buttons of nine different shapes and sizes on a jacket , accomplish a reaction time task , walk at maximum speed or on wide / narrow paths , walk on a treadmill , or avoid obstacles that were presented on a treadmill at irregular intervals . bock and colleagues found that dual - task costs were small in most experiments and did not differ between young and older participants . the only task combination in which dual - task performance was distinctly lower in older than in young subjects were experiments requiring the time - critical processing of visual information ( e.g. , obstacle avoidance in cooperation with a visual reaction time task ) . tasks showing the highest deficits in the elderly combined three main features : ( a ) the task was conducted on a treadmill , ( b ) subjects had to avoid obstacles , and ( c ) the walking and non - walking tasks required continuous visual control . in a second study , bock explored the effects of the different walking tasks by comparing walking on a treadmill with walking in a hallway . the results from both experiments were similar , overground and treadmill walking had similar effects on age - related dual - task decrements . this outcome leads to the suggestion that walking on a treadmill does not produce deficits in walking as a function of age . , checking boxes on a sheet of paper ) and the other tasks required memory and attention resources ( i.e. in these task combinations , obstacles did not give rise to any age - related deficits , and only the features of the secondary task led to age - related differences in dual - task performance . an increase in dual - task costs occurred mainly in a task requiring visual processing of information and managing two streams of visual information , one related to the checking task and the other one related to the walking task . these results are indirectly supported by a correlation of postural control and the degree of visual impairment that was found by jamet and colleagues . beurskens and bock extended the outcomes of bock and colleagues and showed that the visual component of a secondary task has a crucial influence on age - related deficits in dual - task walking [ 61 , 64 ] . the age - related increase of dual - task costs in their studies amounted to about 9% , which is higher than in most other studies but lower than in lindenberger 's experiments [ 39 , 42 ] . in contrast to the methods and tasks used in most of the presented studies , secondary tasks in our everyday life are hardly continuous , and they do not occupy a person for an extended period of time . to consider these kinds of situations , beurskens and bock recently compared young and older people 's walking behavior after short and unexpected distractions . eight monitors were arranged at irregular intervals on a straight floor , four to the subject 's left , and four to the right . on twelve of those passes at the same time , a capital letter from the latin alphabet ( such as g or k ) was displayed for 2 seconds on that monitor . letters were presented mirror - reversed or nonreversed , at a rotation angle of 60% or 120 with respect to the vertical . if it was mirror - reversed , and no if it was not reversed . results showed that brief distractions did not influence younger people 's walking but significantly changed older people 's walking behavior by increasing step duration and decreasing the amplitude of a step . the age - related decrease in dual - task performance reached up to 14% , which is comparable to lindenberger 's findings with visual imagery . brief visual distracters , therefore , can be as disruptive for elderly persons as an ongoing demand on visual working memory . in addition to age - related cognitive disorders and a deterioration of cognitive functions with advancing age , there are other potential causes of changes in walking behavior and human locomotion . one further aspect often addressed in contemporary literature is the possible correlation of older people 's fear of falling with gait changes . some authors find correlations and state that the development of fear after a fall leads to changes in walking speed , step time , or the appropriate variability , which leads to recurrent falls during the following years [ 32 , 67 , 68 ] . in fact , the correlation of psychological functions and human walking is a multidimensional construct where several aspects , ranging from fear of falling , education , physical activity and stress , have to be taken into consideration . another possible confounding factor in this area of research is the role of eye movements . subjects typically focus both their gaze [ 72 , 73 ] and their attention on the goal of their activities [ 74 , 75 ] , which indicates that attention , eye and body movements are all closely interlinked . however , oculomotor behavior changes with advancing age : the latency and duration of saccades increase while their accuracy decreases , thus necessitating more corrective and re - fixation saccades [ 7678 ] . such deficits could complicate the navigation through visually defined space and its integration with another visual task , and thus contribute to impaired dual - task walking . yet another factor to be considered is the age - related shrinkage of the attention window , which can be observed not only with cognitive tasks [ 79 , 80 ] but also with manual ones : bimanual tracking deteriorates with display distance in older , but not in young subjects . however , the magnitude of this deterioration in a given elderly person is not related to that person 's dual - task costs when walking with a concurrent visuospatial task ( r = 0.04 ; p < 0.05 ) , which suggests that peripheral visual attention may not be an important factor in dual - task walking . when reviewing contemporary research on dual - task walking , motor control , and the role of cognitive functions , the topics of executive functions , and the role of the human frontal lobe have often been addressed . many studies associate the occurrence of dual - task deficits in the elderly while walking with the well - known decay of prefrontal cortical circuitry , the loss of prefrontal brain mass , and the associated deterioration of executive functions in old age [ 16 , 20 , 81 ] . the current review shows that such deficits are observable mainly with nonwalking tasks requiring substantial visual processing , and thus with the coordination of two independent visual streams of information . it is quite conceivable that this coordination depends critically on higher - level cognitive functions , such as executive functions and working memory , while peripheral visual attention does not seem to play a predominant role . this would fit well with norman and shallice 's concept of executive control , according to which shifts of attention from one task to another take place in a high - level supervisory attentional system . that being said , we must add that not all age - related deficits of dual - task walking can be explained by interference between two visual streams . deficits have also been reported for tasks without a visual component , although they were much smaller than those for tasks having a visual demand [ 29 , 41 , 59 ] or were limited to very old subjects ( 75 + years ) . it , therefore , appears that interference is not necessarily confined to the visual modality but rather can be intermodal , and increasingly so in very old age . summing up , we suggest that neural plasticity can partly compensate for age - related deficits of walking : it supplements deteriorated sensorimotor processes by cognitive processes . however , this compensation reduces the cognitive capacity available for concurrent tasks , and is limited by the age - related decay of the prefrontal cortex .

human exposure assessment of environmental contaminants has been used as an important tool to establish environmental standards and public health policies . it requires a set of exposure factors reflecting the human behavioral characteristics that can influence human exposure such as air , water , food , and soil intakes , and the period and frequency of exposure in addition to the concentration of contaminants in the air , water , food , and soil . in 1989 , the united states environmental protection agency ( us epa ) devised a set of exposure factors and issued its first exposure factors handbook that offered recommended values . even though the exposure factors handbook of the epa contains a great deal of valuable information , its recommended values have a limited scope of application in other countries since they address the needs of the american people . not many nations , however , have so far succeeded in developing their own exposure factors handbooks since producing one involves a difficult development process . canada issued one and offered recommended values in 1994 ; the european commission joint research centre started to develop a database called expofacts , collecting data similar to those of epa 's exposure factors handbook in 2002 ; the national institute of advanced industrial science and technology of japan published its japanese version in 2007 ; and in recent years , china has been among the other countries implementing a national - level research project to set exposure factors and develop a handbook . korea took on a three - year research project called " development of the korean exposure factors handbook " in 2005 and issued the korean exposure factors handbook based on the findings in 2007 . korea comes second to none other than the us in terms of the scope of content and issuance time of such a handbook . once it was developed , the handbook has been cited and used in many domestic studies on exposure assessment [ 8 - 17 ] . following several recent developments including the increasing awareness of a need to develop an exposure factors handbook fit for each nation and the actual progress of its development in some nations , a need has surfaced to exchange information about the development process and to compare recommended values of exposure factors among nations . the korean exposure factors handbook , however , is facing some limitations regarding international citation and utilization since it is written in korean and has not been reported as a regular scientific paper . the journal of preventive medicine and public health thus decided to present a special review in the current issue , introducing the development process of the handbook and the major recommended values . the present paper serves as an introduction to the development process and significance of the handbook as well as an overview of its development . its three sister papers describe the development process of the exposure factors in the areas of general exposure , food ingestion , and human activity along with recommended values in each of the areas . they are " general factors of the korean exposure factors handbook , " " food ingestion factors of the korean exposure factors handbook , " and " activity factors of the korean exposure factors handbook . " since space is limited in the journal , the special review offers only the most representative recommended values of the exposure factors in the korean exposure factors handbook instead of them all . more recent data might have become available home and abroad after the development of the handbook , but the special review will consider the comparison of data at the time of its development when comparing the handbook data with those of its overseas counterparts since its main focus is to introduce the handbook . in korea , interest in and research on risk and exposure assessment became brisk in 2000 . researchers would adopt individual exposure factors needed for risk or exposure assessment at their own discretion or , in many cases , borrow the american exposure factors . as a result , exposure assessment results would vary among the researchers and lack certainty and reliability by using the exposure factors of a foreign country that did not reflect the exposure characteristics of korea based on the population 's ethnicity , environment , culture , and life habits . the need was thus identified to develop a reliable set of exposure factors to represent the entire nation of korea in order to ensure reliable risk assessment and establish all kinds of national criteria related to the environment . the korean exposure factors handbook , a kind of pilot study conducted as part of the " human exposure assessment for total risk assessment of environmental contaminants " in 2001 , was the nation 's first study that noted the need for recommended values of exposure factors and explored the potential for their development . the study reviewed the existing data available for the nation , concluded that there was a serious shortage of highly reliable nationally representative data , and proposed a full - blown study to generate recommended values of exposure factors at the national level . in addition , it offered provisional recommended values in use by the combination of some domestic data and the contents of the us epa exposure factors handbook . since then , a good number of national statistics were established with high reliability and representativeness under the leadership of various koreans ministries , and the core environmental technology development project for the next generation was launched to support basic environmental research . " development and application of korean exposure factors " was one of the studies that benefited from its support . the korean exposure factors handbook was finally developed based on the results of the three - year study conducted between december 2004 and november 2007 . the researchers reviewed the existing data and literature along with previous studies , confirmed a knowledge gap , and evaluated whether there were domestic data available for the development of exposure factors and identification of the reliability level of the data . they also set a host of criteria similar to the approach to examining the reliability of data in the us epa exposure factors handbook , evaluated them as high , medium , and low , and compiled the findings . in evaluating the reliability of the data , what was considered was verification of the data , reproducibility , association with exposure factors , connections with the population characteristics , primary data , recency , appropriateness of data collection period , size of the study , representativeness of the subjects , variability of the data , the research design , and reliability of measurement . of the exposure factors that were the top priority in the risk assessment , 24 exposure factors from the areas of general exposure , food ingestion , and activity factors were selected as the targets of research and development by taking into account whether there were reliable domestic data available or whether such data could be generated within a short period of time . compared with their counterparts in the us epa exposure factors handbook , the items of general exposure factors were the same ; to the food ingestion factors , however , were added the recommended values for the intakes of fats and oils , seaweeds , oilseeds and their products , seasonings , and sugars and sweeteners . activity factors also had difference between korea and the us in the items of factors . the next stage involved the review of the extent of domestic data accumulated on each of the 24 exposure factors and the categorization of development methods needed to calculate recommended values based on the results . type 1 ( direct use type ) exposure factors , such as life expectancy , are backed up by highly reliable national - level domestic data and can be used as exposure factors right away . type 2 ( reanalysis type ) exposure factors , including body weight and population and occupational mobility , are backed up by highly reliable national - level domestic data but needed statistical re - analysis before being used as exposure factors . type 3 ( conversion type ) exposure factors , including body surface area , food ingestion exposure factors , and time spent in activities , are backed up by highly reliable national - level domestic data but require full - scale reorganization , recategorization , and reanalysis of raw data before being used as exposure factors . finally , type 4 ( actual survey type ) exposure factors had no pre - existing domestic data that were highly reliable for the development of exposure factors , and new investigations were needed . these factors included inhalation rate , intake of drinking water and soil , time spent in various places , and time spent on transportation , washing hands , washing the face , showering , bathing , and swimming . table 1 presents the types of development methods and examples of exposure factors using each method according to the type of exposure factor . in the us , where many previous studies have been conducted on each exposure factor , the core task in the development of the us epa exposure factors handbook was a process of obtaining recommended values such as selecting studies of high representativeness and reliability . in korea , on the other hand , where very few studies on exposure factors have been published and in some cases , none , it was impossible to use the same development approach as the us . when there were previous studies available containing data useful for calculating recommended values by examining , analyzing , and evaluating the findings according to the exposure factors , the researchers were able to take advantage of them . when there were no such previous studies , however , they had to design and conduct first - hand investigations and use the results for setting exposure factor levels . when designing an investigation into exposure factors , they considered the selection criteria for the studies that were used to calculate recommended values with the highest representativeness and reliability in the development process of the us epa exposure factors handbook and tried to meet selection criteria at least as rigorous . the rigor of criteria was determined based on that of the number of subjects , subjects ' representativeness of the population , and methodologies . table 2 presents a sequential roadmap devised and implemented for the research tasks needed to develop the korean exposure factors handbook . in the first phase , the researchers calculated recommended values for which research data was available or could be calculated through relatively simple statistical reanalysis . these factors included life expectancy , body weight , population and occupational mobility , and body surface area . they also carried out a preliminary study for inhalation rate and intake of drinking water planned for the second phase , in which they investigated these two factors according to the methodologies developed and confirmed in the first phase preliminary study and developed a set of food ingestion exposure factors . they also conducted a preliminary study for time activity exposure factors to be developed in the third phase . in addition , they compiled the exposure factors developed in the first , second , and third phase , produced the korean exposure factors handbook , and built a web - based information system . after developing all the recommended values of each exposure factor , the researchers published the korean exposure factors handbook containing major information about the evaluation techniques related to the calculation of recommended values , principal statistical results , and available data . the handbook was comprised of an introduction and general , food ingestion , and activity exposure factors . the introduction addressed the need for an exposure factors handbook , plans for its utilization , and an overview of the research . the handbook presented statistical distribution values including means along with statistics by gender , age , and residential area according to the exposure factor . the body of the book consisted of an overview , calculation of recommended values , comparisons with cases from other countries , reliability , and limitations for each category of exposure factors . the overview covered the importance and significance of the subject exposure factors , overall content about the methodologies for the subject variables , and equations considering the usability of the exposure factors . the calculation of recommended values included explanations about the data used , methodologies , and principal statistics as well as the recommended values themselves . more detailed information on recommended values was provided through comparison with the values developed by other countries . finally , a web - based information system was established to increase public accessibility and utilization . the web - based information system consisted of a research introduction , user 's manual , general , food ingestion , and activity factors , and exposure assessment . it also had a download menu to help users easily download parts of or the entire content of the handbook . in korea , interest in and research on risk and exposure assessment became brisk in 2000 . researchers would adopt individual exposure factors needed for risk or exposure assessment at their own discretion or , in many cases , borrow the american exposure factors . as a result , exposure assessment results would vary among the researchers and lack certainty and reliability by using the exposure factors of a foreign country that did not reflect the exposure characteristics of korea based on the population 's ethnicity , environment , culture , and life habits . the need was thus identified to develop a reliable set of exposure factors to represent the entire nation of korea in order to ensure reliable risk assessment and establish all kinds of national criteria related to the environment . the korean exposure factors handbook , a kind of pilot study conducted as part of the " human exposure assessment for total risk assessment of environmental contaminants " in 2001 , was the nation 's first study that noted the need for recommended values of exposure factors and explored the potential for their development . the study reviewed the existing data available for the nation , concluded that there was a serious shortage of highly reliable nationally representative data , and proposed a full - blown study to generate recommended values of exposure factors at the national level . in addition , it offered provisional recommended values in use by the combination of some domestic data and the contents of the us epa exposure factors handbook . since then , a good number of national statistics were established with high reliability and representativeness under the leadership of various koreans ministries , and the core environmental technology development project for the next generation was launched to support basic environmental research . " development and application of korean exposure factors " was one of the studies that benefited from its support . the korean exposure factors handbook was finally developed based on the results of the three - year study conducted between december 2004 and november 2007 . the researchers reviewed the existing data and literature along with previous studies , confirmed a knowledge gap , and evaluated whether there were domestic data available for the development of exposure factors and identification of the reliability level of the data . they also set a host of criteria similar to the approach to examining the reliability of data in the us epa exposure factors handbook , evaluated them as high , medium , and low , and compiled the findings . in evaluating the reliability of the data , what was considered was verification of the data , reproducibility , association with exposure factors , connections with the population characteristics , primary data , recency , appropriateness of data collection period , size of the study , representativeness of the subjects , variability of the data , the research design , and reliability of measurement . of the exposure factors that were the top priority in the risk assessment , 24 exposure factors from the areas of general exposure , food ingestion , and activity factors were selected as the targets of research and development by taking into account whether there were reliable domestic data available or whether such data could be generated within a short period of time . compared with their counterparts in the us epa exposure factors handbook , the items of general exposure factors were the same ; to the food ingestion factors , however , were added the recommended values for the intakes of fats and oils , seaweeds , oilseeds and their products , seasonings , and sugars and sweeteners . activity factors also had difference between korea and the us in the items of factors . the next stage involved the review of the extent of domestic data accumulated on each of the 24 exposure factors and the categorization of development methods needed to calculate recommended values based on the results . type 1 ( direct use type ) exposure factors , such as life expectancy , are backed up by highly reliable national - level domestic data and can be used as exposure factors right away . type 2 ( reanalysis type ) exposure factors , including body weight and population and occupational mobility , are backed up by highly reliable national - level domestic data but needed statistical re - analysis before being used as exposure factors . type 3 ( conversion type ) exposure factors , including body surface area , food ingestion exposure factors , and time spent in activities , are backed up by highly reliable national - level domestic data but require full - scale reorganization , recategorization , and reanalysis of raw data before being used as exposure factors . finally , type 4 ( actual survey type ) exposure factors had no pre - existing domestic data that were highly reliable for the development of exposure factors , and new investigations were needed . these factors included inhalation rate , intake of drinking water and soil , time spent in various places , and time spent on transportation , washing hands , washing the face , showering , bathing , and swimming . table 1 presents the types of development methods and examples of exposure factors using each method according to the type of exposure factor . in the us , where many previous studies have been conducted on each exposure factor , the core task in the development of the us epa exposure factors handbook was a process of obtaining recommended values such as selecting studies of high representativeness and reliability . in korea , on the other hand , where very few studies on exposure factors have been published and in some cases , none , it was impossible to use the same development approach as the us . when there were previous studies available containing data useful for calculating recommended values by examining , analyzing , and evaluating the findings according to the exposure factors , the researchers were able to take advantage of them . when there were no such previous studies , however , they had to design and conduct first - hand investigations and use the results for setting exposure factor levels . when designing an investigation into exposure factors , they considered the selection criteria for the studies that were used to calculate recommended values with the highest representativeness and reliability in the development process of the us epa exposure factors handbook and tried to meet selection criteria at least as rigorous . the rigor of criteria was determined based on that of the number of subjects , subjects ' representativeness of the population , and methodologies . table 2 presents a sequential roadmap devised and implemented for the research tasks needed to develop the korean exposure factors handbook . in the first phase , the researchers calculated recommended values for which research data was available or could be calculated through relatively simple statistical reanalysis . these factors included life expectancy , body weight , population and occupational mobility , and body surface area . they also carried out a preliminary study for inhalation rate and intake of drinking water planned for the second phase , in which they investigated these two factors according to the methodologies developed and confirmed in the first phase preliminary study and developed a set of food ingestion exposure factors . they also conducted a preliminary study for time activity exposure factors to be developed in the third phase . in addition , they compiled the exposure factors developed in the first , second , and third phase , produced the korean exposure factors handbook , and built a web - based information system . after developing all the recommended values of each exposure factor , the researchers published the korean exposure factors handbook containing major information about the evaluation techniques related to the calculation of recommended values , principal statistical results , and available data . the handbook was comprised of an introduction and general , food ingestion , and activity exposure factors . the introduction addressed the need for an exposure factors handbook , plans for its utilization , and an overview of the research . the handbook presented statistical distribution values including means along with statistics by gender , age , and residential area according to the exposure factor . the body of the book consisted of an overview , calculation of recommended values , comparisons with cases from other countries , reliability , and limitations for each category of exposure factors . the overview covered the importance and significance of the subject exposure factors , overall content about the methodologies for the subject variables , and equations considering the usability of the exposure factors . the calculation of recommended values included explanations about the data used , methodologies , and principal statistics as well as the recommended values themselves . more detailed information on recommended values was provided through comparison with the values developed by other countries . finally , a web - based information system was established to increase public accessibility and utilization . the web - based information system consisted of a research introduction , user 's manual , general , food ingestion , and activity factors , and exposure assessment . it also had a download menu to help users easily download parts of or the entire content of the handbook . since the handbook is a national index for demonstrating the physiological , food ingestion , and behavioral characteristics of the korean people , it can serve various purposes in addition to the environment including risk assessment . other asian countries have to borrow exposure factors from other countries for risk assessment until they can develop their own . it will benefit them more to consider the exposure factors of the korean exposure factors handbook and adjust them according to their situations rather than using the american counterparts as they are in spite of the huge differences in physiology , dietary habits , and life . the significance of exposure factors is multiplied when it comes to children since they react very sensitively to environmental contamination and chemicals due to incomplete development of nervous , respiratory and reproductive organs and have different food and water intakes , and breathing and metabolic rates per unit of body weight than adults . in 2002 , the us epa published its findings on an exposure factors handbook for children . in 2005 , the european union announced some recommended values for children 's exposure factors . the present study calculated children 's intakes for all of the 12 exposure factors in the food ingestion category and children 's recommended values for life expectancy , body weight , and intake of soil in the general exposure category . this study , however , failed to obtain first - hand data on body surface area , inhalation rate , intake of drinking water , and all the exposure factors of activity for children category due to limitations of data and other circumstances . research should be conducted on the exposure factors left out of the study to develop an exposure factors handbook for children like that of the us . this study used the body measurements of korean people by the korean agency for technology and standards under the ministry of commerce , industry , and energy , the census , the data of the national statistical office , the basic statistical survey on the wage structure of workers , the national health and nutrition examination survey and other . the present study used the latest data at the time of completion , but one can also consider a need for revision reflecting the updates to the data . however , frequent updates are not necessarily recommended since exposure factors have a different utility and nature from common statistics in that the standard values should be used to reduce the uncertainty of risk assessment . korea , however , has the potential for rapid economic and social changes and thus rapid changes to the exposure factors , which means that changes in the statistical values based on national data should be reviewed to judge whether a revision is needed or not . performing additional investigations is necessary to compensate for the exposure factors that have been omitted , such as the characteristics of the residential environment , due to various limitations including restricted research time and budget and to evaluate the exposure factors that have characteristics unique to korea .

a set of exposure factors that reflects the characteristics of individual behavior capable of influencing exposure is essential for risk and exposure assessment . in 2007 , the korean exposure factors handbook was , therefore , issued , driven by the need to develop reliable exposure factors representing the korean population . the purpose of this study was to overview the development process of the korean exposure factors handbook and major recommended exposure values for the korean population to allow information exchanges and comparison of recommended values among nations . the researchers reviewed the domestic data that could be used in the development of exposure factors , confirmed a knowledge gap , and set a priority of development by phases . a methodology to measure exposure factors was established to develop measuring techniques and test their validity . data were processed or a survey was conducted according to the availability of data . the study thus produced recommended values for 24 exposure factors grouped by general exposure factors , food ingestion factors , and activity factors by setting up a database of exposure factors and carrying out statistical analysis . the study has significantly contributed to reducing the potential uncertainty of the risk and exposure assessment derived by the application of foreign data or research findings lacking representativeness or grounds by developing a set of exposure factors reflecting the characteristics of the korean people . it will be necessary to conduct revisions in light of the changing statistical values of national data and the exposure factors based on korean characteristics .

INTRODUCTION DEVELOPMENT PROCESS Brief History Development Process Publication of the Korean Exposure Factors Handbook and Establishment of a Web-based Information System CONCLUSION

it requires a set of exposure factors reflecting the human behavioral characteristics that can influence human exposure such as air , water , food , and soil intakes , and the period and frequency of exposure in addition to the concentration of contaminants in the air , water , food , and soil . in 1989 , the united states environmental protection agency ( us epa ) devised a set of exposure factors and issued its first exposure factors handbook that offered recommended values . even though the exposure factors handbook of the epa contains a great deal of valuable information , its recommended values have a limited scope of application in other countries since they address the needs of the american people . canada issued one and offered recommended values in 1994 ; the european commission joint research centre started to develop a database called expofacts , collecting data similar to those of epa 's exposure factors handbook in 2002 ; the national institute of advanced industrial science and technology of japan published its japanese version in 2007 ; and in recent years , china has been among the other countries implementing a national - level research project to set exposure factors and develop a handbook . korea took on a three - year research project called " development of the korean exposure factors handbook " in 2005 and issued the korean exposure factors handbook based on the findings in 2007 . once it was developed , the handbook has been cited and used in many domestic studies on exposure assessment [ 8 - 17 ] . following several recent developments including the increasing awareness of a need to develop an exposure factors handbook fit for each nation and the actual progress of its development in some nations , a need has surfaced to exchange information about the development process and to compare recommended values of exposure factors among nations . the korean exposure factors handbook , however , is facing some limitations regarding international citation and utilization since it is written in korean and has not been reported as a regular scientific paper . the journal of preventive medicine and public health thus decided to present a special review in the current issue , introducing the development process of the handbook and the major recommended values . the present paper serves as an introduction to the development process and significance of the handbook as well as an overview of its development . its three sister papers describe the development process of the exposure factors in the areas of general exposure , food ingestion , and human activity along with recommended values in each of the areas . they are " general factors of the korean exposure factors handbook , " " food ingestion factors of the korean exposure factors handbook , " and " activity factors of the korean exposure factors handbook . " since space is limited in the journal , the special review offers only the most representative recommended values of the exposure factors in the korean exposure factors handbook instead of them all . more recent data might have become available home and abroad after the development of the handbook , but the special review will consider the comparison of data at the time of its development when comparing the handbook data with those of its overseas counterparts since its main focus is to introduce the handbook . in korea , interest in and research on risk and exposure assessment became brisk in 2000 . researchers would adopt individual exposure factors needed for risk or exposure assessment at their own discretion or , in many cases , borrow the american exposure factors . as a result , exposure assessment results would vary among the researchers and lack certainty and reliability by using the exposure factors of a foreign country that did not reflect the exposure characteristics of korea based on the population 's ethnicity , environment , culture , and life habits . the need was thus identified to develop a reliable set of exposure factors to represent the entire nation of korea in order to ensure reliable risk assessment and establish all kinds of national criteria related to the environment . the korean exposure factors handbook , a kind of pilot study conducted as part of the " human exposure assessment for total risk assessment of environmental contaminants " in 2001 , was the nation 's first study that noted the need for recommended values of exposure factors and explored the potential for their development . the study reviewed the existing data available for the nation , concluded that there was a serious shortage of highly reliable nationally representative data , and proposed a full - blown study to generate recommended values of exposure factors at the national level . in addition , it offered provisional recommended values in use by the combination of some domestic data and the contents of the us epa exposure factors handbook . since then , a good number of national statistics were established with high reliability and representativeness under the leadership of various koreans ministries , and the core environmental technology development project for the next generation was launched to support basic environmental research . " development and application of korean exposure factors " was one of the studies that benefited from its support . the korean exposure factors handbook was finally developed based on the results of the three - year study conducted between december 2004 and november 2007 . the researchers reviewed the existing data and literature along with previous studies , confirmed a knowledge gap , and evaluated whether there were domestic data available for the development of exposure factors and identification of the reliability level of the data . they also set a host of criteria similar to the approach to examining the reliability of data in the us epa exposure factors handbook , evaluated them as high , medium , and low , and compiled the findings . in evaluating the reliability of the data , what was considered was verification of the data , reproducibility , association with exposure factors , connections with the population characteristics , primary data , recency , appropriateness of data collection period , size of the study , representativeness of the subjects , variability of the data , the research design , and reliability of measurement . of the exposure factors that were the top priority in the risk assessment , 24 exposure factors from the areas of general exposure , food ingestion , and activity factors were selected as the targets of research and development by taking into account whether there were reliable domestic data available or whether such data could be generated within a short period of time . compared with their counterparts in the us epa exposure factors handbook , the items of general exposure factors were the same ; to the food ingestion factors , however , were added the recommended values for the intakes of fats and oils , seaweeds , oilseeds and their products , seasonings , and sugars and sweeteners . activity factors also had difference between korea and the us in the items of factors . the next stage involved the review of the extent of domestic data accumulated on each of the 24 exposure factors and the categorization of development methods needed to calculate recommended values based on the results . type 1 ( direct use type ) exposure factors , such as life expectancy , are backed up by highly reliable national - level domestic data and can be used as exposure factors right away . type 2 ( reanalysis type ) exposure factors , including body weight and population and occupational mobility , are backed up by highly reliable national - level domestic data but needed statistical re - analysis before being used as exposure factors . type 3 ( conversion type ) exposure factors , including body surface area , food ingestion exposure factors , and time spent in activities , are backed up by highly reliable national - level domestic data but require full - scale reorganization , recategorization , and reanalysis of raw data before being used as exposure factors . finally , type 4 ( actual survey type ) exposure factors had no pre - existing domestic data that were highly reliable for the development of exposure factors , and new investigations were needed . table 1 presents the types of development methods and examples of exposure factors using each method according to the type of exposure factor . in the us , where many previous studies have been conducted on each exposure factor , the core task in the development of the us epa exposure factors handbook was a process of obtaining recommended values such as selecting studies of high representativeness and reliability . when there were previous studies available containing data useful for calculating recommended values by examining , analyzing , and evaluating the findings according to the exposure factors , the researchers were able to take advantage of them . when designing an investigation into exposure factors , they considered the selection criteria for the studies that were used to calculate recommended values with the highest representativeness and reliability in the development process of the us epa exposure factors handbook and tried to meet selection criteria at least as rigorous . the rigor of criteria was determined based on that of the number of subjects , subjects ' representativeness of the population , and methodologies . table 2 presents a sequential roadmap devised and implemented for the research tasks needed to develop the korean exposure factors handbook . in the first phase , the researchers calculated recommended values for which research data was available or could be calculated through relatively simple statistical reanalysis . they also carried out a preliminary study for inhalation rate and intake of drinking water planned for the second phase , in which they investigated these two factors according to the methodologies developed and confirmed in the first phase preliminary study and developed a set of food ingestion exposure factors . in addition , they compiled the exposure factors developed in the first , second , and third phase , produced the korean exposure factors handbook , and built a web - based information system . after developing all the recommended values of each exposure factor , the researchers published the korean exposure factors handbook containing major information about the evaluation techniques related to the calculation of recommended values , principal statistical results , and available data . the handbook was comprised of an introduction and general , food ingestion , and activity exposure factors . the introduction addressed the need for an exposure factors handbook , plans for its utilization , and an overview of the research . the handbook presented statistical distribution values including means along with statistics by gender , age , and residential area according to the exposure factor . the body of the book consisted of an overview , calculation of recommended values , comparisons with cases from other countries , reliability , and limitations for each category of exposure factors . the overview covered the importance and significance of the subject exposure factors , overall content about the methodologies for the subject variables , and equations considering the usability of the exposure factors . the calculation of recommended values included explanations about the data used , methodologies , and principal statistics as well as the recommended values themselves . finally , a web - based information system was established to increase public accessibility and utilization . the web - based information system consisted of a research introduction , user 's manual , general , food ingestion , and activity factors , and exposure assessment . in korea , interest in and research on risk and exposure assessment became brisk in 2000 . researchers would adopt individual exposure factors needed for risk or exposure assessment at their own discretion or , in many cases , borrow the american exposure factors . as a result , exposure assessment results would vary among the researchers and lack certainty and reliability by using the exposure factors of a foreign country that did not reflect the exposure characteristics of korea based on the population 's ethnicity , environment , culture , and life habits . the need was thus identified to develop a reliable set of exposure factors to represent the entire nation of korea in order to ensure reliable risk assessment and establish all kinds of national criteria related to the environment . the korean exposure factors handbook , a kind of pilot study conducted as part of the " human exposure assessment for total risk assessment of environmental contaminants " in 2001 , was the nation 's first study that noted the need for recommended values of exposure factors and explored the potential for their development . the study reviewed the existing data available for the nation , concluded that there was a serious shortage of highly reliable nationally representative data , and proposed a full - blown study to generate recommended values of exposure factors at the national level . in addition , it offered provisional recommended values in use by the combination of some domestic data and the contents of the us epa exposure factors handbook . since then , a good number of national statistics were established with high reliability and representativeness under the leadership of various koreans ministries , and the core environmental technology development project for the next generation was launched to support basic environmental research . " development and application of korean exposure factors " was one of the studies that benefited from its support . the korean exposure factors handbook was finally developed based on the results of the three - year study conducted between december 2004 and november 2007 . the researchers reviewed the existing data and literature along with previous studies , confirmed a knowledge gap , and evaluated whether there were domestic data available for the development of exposure factors and identification of the reliability level of the data . they also set a host of criteria similar to the approach to examining the reliability of data in the us epa exposure factors handbook , evaluated them as high , medium , and low , and compiled the findings . in evaluating the reliability of the data , what was considered was verification of the data , reproducibility , association with exposure factors , connections with the population characteristics , primary data , recency , appropriateness of data collection period , size of the study , representativeness of the subjects , variability of the data , the research design , and reliability of measurement . of the exposure factors that were the top priority in the risk assessment , 24 exposure factors from the areas of general exposure , food ingestion , and activity factors were selected as the targets of research and development by taking into account whether there were reliable domestic data available or whether such data could be generated within a short period of time . compared with their counterparts in the us epa exposure factors handbook , the items of general exposure factors were the same ; to the food ingestion factors , however , were added the recommended values for the intakes of fats and oils , seaweeds , oilseeds and their products , seasonings , and sugars and sweeteners . activity factors also had difference between korea and the us in the items of factors . the next stage involved the review of the extent of domestic data accumulated on each of the 24 exposure factors and the categorization of development methods needed to calculate recommended values based on the results . type 1 ( direct use type ) exposure factors , such as life expectancy , are backed up by highly reliable national - level domestic data and can be used as exposure factors right away . type 2 ( reanalysis type ) exposure factors , including body weight and population and occupational mobility , are backed up by highly reliable national - level domestic data but needed statistical re - analysis before being used as exposure factors . type 3 ( conversion type ) exposure factors , including body surface area , food ingestion exposure factors , and time spent in activities , are backed up by highly reliable national - level domestic data but require full - scale reorganization , recategorization , and reanalysis of raw data before being used as exposure factors . finally , type 4 ( actual survey type ) exposure factors had no pre - existing domestic data that were highly reliable for the development of exposure factors , and new investigations were needed . table 1 presents the types of development methods and examples of exposure factors using each method according to the type of exposure factor . in the us , where many previous studies have been conducted on each exposure factor , the core task in the development of the us epa exposure factors handbook was a process of obtaining recommended values such as selecting studies of high representativeness and reliability . when there were previous studies available containing data useful for calculating recommended values by examining , analyzing , and evaluating the findings according to the exposure factors , the researchers were able to take advantage of them . when designing an investigation into exposure factors , they considered the selection criteria for the studies that were used to calculate recommended values with the highest representativeness and reliability in the development process of the us epa exposure factors handbook and tried to meet selection criteria at least as rigorous . the rigor of criteria was determined based on that of the number of subjects , subjects ' representativeness of the population , and methodologies . table 2 presents a sequential roadmap devised and implemented for the research tasks needed to develop the korean exposure factors handbook . in the first phase , the researchers calculated recommended values for which research data was available or could be calculated through relatively simple statistical reanalysis . they also carried out a preliminary study for inhalation rate and intake of drinking water planned for the second phase , in which they investigated these two factors according to the methodologies developed and confirmed in the first phase preliminary study and developed a set of food ingestion exposure factors . in addition , they compiled the exposure factors developed in the first , second , and third phase , produced the korean exposure factors handbook , and built a web - based information system . after developing all the recommended values of each exposure factor , the researchers published the korean exposure factors handbook containing major information about the evaluation techniques related to the calculation of recommended values , principal statistical results , and available data . the handbook was comprised of an introduction and general , food ingestion , and activity exposure factors . the introduction addressed the need for an exposure factors handbook , plans for its utilization , and an overview of the research . the handbook presented statistical distribution values including means along with statistics by gender , age , and residential area according to the exposure factor . the body of the book consisted of an overview , calculation of recommended values , comparisons with cases from other countries , reliability , and limitations for each category of exposure factors . the overview covered the importance and significance of the subject exposure factors , overall content about the methodologies for the subject variables , and equations considering the usability of the exposure factors . the calculation of recommended values included explanations about the data used , methodologies , and principal statistics as well as the recommended values themselves . finally , a web - based information system was established to increase public accessibility and utilization . the web - based information system consisted of a research introduction , user 's manual , general , food ingestion , and activity factors , and exposure assessment . since the handbook is a national index for demonstrating the physiological , food ingestion , and behavioral characteristics of the korean people , it can serve various purposes in addition to the environment including risk assessment . other asian countries have to borrow exposure factors from other countries for risk assessment until they can develop their own . it will benefit them more to consider the exposure factors of the korean exposure factors handbook and adjust them according to their situations rather than using the american counterparts as they are in spite of the huge differences in physiology , dietary habits , and life . the significance of exposure factors is multiplied when it comes to children since they react very sensitively to environmental contamination and chemicals due to incomplete development of nervous , respiratory and reproductive organs and have different food and water intakes , and breathing and metabolic rates per unit of body weight than adults . in 2002 , the us epa published its findings on an exposure factors handbook for children . in 2005 , the european union announced some recommended values for children 's exposure factors . the present study calculated children 's intakes for all of the 12 exposure factors in the food ingestion category and children 's recommended values for life expectancy , body weight , and intake of soil in the general exposure category . this study , however , failed to obtain first - hand data on body surface area , inhalation rate , intake of drinking water , and all the exposure factors of activity for children category due to limitations of data and other circumstances . research should be conducted on the exposure factors left out of the study to develop an exposure factors handbook for children like that of the us . this study used the body measurements of korean people by the korean agency for technology and standards under the ministry of commerce , industry , and energy , the census , the data of the national statistical office , the basic statistical survey on the wage structure of workers , the national health and nutrition examination survey and other . however , frequent updates are not necessarily recommended since exposure factors have a different utility and nature from common statistics in that the standard values should be used to reduce the uncertainty of risk assessment . korea , however , has the potential for rapid economic and social changes and thus rapid changes to the exposure factors , which means that changes in the statistical values based on national data should be reviewed to judge whether a revision is needed or not . performing additional investigations is necessary to compensate for the exposure factors that have been omitted , such as the characteristics of the residential environment , due to various limitations including restricted research time and budget and to evaluate the exposure factors that have characteristics unique to korea .

the tropical parasite trypanosoma brucei is the causative agent of both human african trypanosomiasis ( hat , or sleeping sickness ) and the livestock disease , nagana . over 50,000 people throughout 36 countries in sub - saharan africa are currently estimated to be infected . the initial stage of disease , established when an individual is bitten by an infected tsetse fly , generates a local chancre at the bite site with this being followed by the proliferation of the parasite in the bloodstream resulting in fever and joint pain . at this stage , the disease is often undiagnosed and , as such , untreated , despite the availability of ( albeit rather old ) drugs such as suramin ( for trypanosoma brucei rhodesiense or acute sleeping sickness ) and pentamidine ( for trypanosoma brucei gambiense or chronic sleeping sickness ) . brain barrier to infect the central nervous system , causing disturbance to the patient 's sleep patterns , as well as causing confusion and difficulty with coordination . the current drug treatments for the later stage of the disease are difficult to administer , requiring a rigorous hospital regime , and all have associated toxicity . for example , melarsoprol is an arsenic - based treatment developed in 1949 that kills 310% of patients and has increasing incidence of drug resistance in the parasite population . a more recently developed treatment for t. brucei gambiense only , eflornithine , is less toxic , but requires high doses and intravenous administration . nonetheless , without treatment , trypanosome infection is always fatal . the ability of trypanosomes to infect both domestic and wild animals means that non - human hosts in affected regions can act as a reservoir for the parasite , confounding control of the disease . moreover , vaccination approaches have not been considered feasible because these parasites evade mammalian immune responses by frequently changing the proteins on their surface in an extreme form of antigenic variation . due to the severity and prevalence of the disease , as well as the lack of efficient disease management , african trypanosomiasis remains an important public health issue and is considered by the world health organisation to be a neglected tropical disease ( http://www.who.int/en ) . trypanosomes differentiate between distinct life stages in order to prepare for , and adapt to , the different environments they encounter during their life cycle [ 2 , 3 ] . during the bloodstream stage of the life cycle , trypanosomes exist as either proliferative slender forms or non - proliferative , transmissible , stumpy forms [ 2 , 3 ] ( fig . 1 ) , with transitional forms between these two types being described as intermediate forms . the proportion of these types changes during the course of infection , this apparently being governed by the density of parasites in the blood . the bloodstream trypanosome population is adapted to promote parasite maintenance in the mammalian blood and transmission to tsetse flies . thus , the proliferation of slender forms contributes to the establishment of parasite numbers in the blood and , through antigen variation , to immune evasion . in contrast , the irreversible division - arrest of stumpy forms acts to control the expansion of parasite numbers in the mammalian bloodstream and so prolongs host survival . this , combined with the preferential survival and the adaptations for differentiation of stumpy forms upon tsetse uptake , increases the probability of disease transmission . fig . 1a simplified depiction of the t. brucei life cycle . in the bloodstream of the mammalian host , the parasites produce stumpy induction factor ( sif ) which induces differentiation of a proportion of slender forms to stumpy forms . stumpy forms are pre - adapted to life in the tsetse vector : they are cell cycle - arrested and have elaborated mitochondrial activity . upon uptake in a tsetse blood - meal , stumpy forms differentiate into the proliferative procyclic forms in response to cold shock and cis - aconitate or citrate ( cca ) in the tsetse midgut . other signals such as proteases or ph stress may also contribute , as does inhibition of the tyrosine phosphatase , tbptp1 . further differentiation events occur in the tsetse to produce mature metacyclic forms in the salivary glands . these are pre - adapted to life in the mammalian host a simplified depiction of the t. brucei life cycle . in the bloodstream of the mammalian host , slender forms proliferate . as density increases , the parasites produce stumpy induction factor ( sif ) which induces differentiation of a proportion of slender forms to stumpy forms . stumpy forms are pre - adapted to life in the tsetse vector : they are cell cycle - arrested and have elaborated mitochondrial activity . upon uptake in a tsetse blood - meal , stumpy forms differentiate into the proliferative procyclic forms in response to cold shock and cis - aconitate or citrate ( cca ) in the tsetse midgut . other signals such as proteases or ph stress may also contribute , as does inhibition of the tyrosine phosphatase , tbptp1 . further differentiation events occur in the tsetse to produce mature metacyclic forms in the salivary glands . these are pre - adapted to life in the mammalian host as the dynamic balance between slender and stumpy forms in mammalian hosts plays a pivotal role in trypanosome infection and transmission , understanding the molecular events implicit in the slender to stumpy form transition and their subsequent differentiation to tsetse midgut forms ( procyclic forms ) is of interest and importance , though poorly understood . when a tsetse fly ingests a trypanosome - infected blood - meal , both slender and stumpy form parasites enter the tsetse midgut . although subject to some controversy , it is generally thought that stumpy forms are pre - adapted to life in the tsetse midgut , and only they differentiate efficiently to the tsetse midgut procyclic form . cytological evidence based on in vitro differentiation experiments suggests that bloodstream forms can only transform to procyclic forms from a given point in the cell cycle , namely within the g1 , or the quiescent g0 , phase . as stumpy forms are uniformly arrested as a population in g0/g1 and can accumulate to near homogeneity in experimental infections , this allows for their efficient and synchronous differentiation to procyclic forms . in contrast , proliferative slender cell populations are asynchronous in their cell cycle and must reach g0/g1 before differentiation . additionally , as cells enter the tsetse midgut , they are exposed to harmful proteases and changes in their environmental ph . the stumpy forms are more resistant than slender forms to the effects of such stresses and , as such , are more likely to endure the transition from the bloodstream to the tsetse midgut . moreover , in the glucose - rich mammalian bloodstream , slender cells obtain their energy entirely via glycolysis and , as a result , the mitochondrion of slender cells is relatively inactive . in intermediate and stumpy forms , however , the mitochondrion is elaborated and mitochondrial enzymes are partially expressed [ 3 , 9 ] . this enables them to quickly begin oxidative phosphorylation as they enter the glucose - poor tsetse midgut [ 3 , 9 ] . stumpy cells also persist longer than slender cells in the face of the developing immune response in the mammalian bloodstream [ 10 , 11 ] , as well as in the tsetse midgut . this is probably not caused by antigenic variation : being irreversibly arrested in the bloodstream , stumpy forms are unlikely to be able to undergo the dna recombination events important for antigen switching . rather , they withstand antibody clearance more effectively than slender cells because they preferentially clear bound antibody by hydrodynamic flow , whereby parasite - bound immunoglobulin is swept backward by the swimming action of the trypanosome in the blood and is then internalised and degraded . the molecular mechanism by which bloodstream trypanosomes perceive their change in environment and initiate differentiation to procyclic forms once in the tsetse fly has been elusive , until recently . it has long been known that the use of high concentrations ( > 3 mm ) of citrate or cis - aconitate ( cca ) could induce differentiation of bloodstream form trypanosomes to procyclic forms in vitro [ 1315 ] . the biological relevance of this signal was in doubt , however , as these levels exceed the concentrations of cca that a trypanosome would be exposed to in the tsetse midgut . however , in 2004 , engstler and boshart discovered that a drop in temperature from 37c to 20c could significantly increase trypanosome sensitivity to cca , potentially matching levels present within the tsetse blood - meal . since this temperature drop was compatible with that encountered by trypanosomes as they entered tsetse flies feeding at the cooler ambient temperatures of dusk or dawn , cca could be considered physiologically relevant . since temperature drop also allows some molecules regulated access to the parasite surface , engstler and boshart proposed a model whereby an unknown surface protein responsible for the reception of the differentiation signal would be retained within the cell in the bloodstream ( i.e. when at 37c ) and then trafficked to the cell surface in response to the cooler temperatures associated with tsetse uptake [ 16 , 17 ] . the molecular basis of this model , however , was unknown until a gene family encoding surface carboxylate transporters known as pad proteins ( proteins associated with differentiation ) was recently identified . indeed , two of the encoded pad proteins ( pad1 and pad2 ) were shown to be expressed in stumpy forms but not in slender forms and were found to be required for perception of cca at physiological concentrations . matching the predictions of engstler and boshart , pad2 was also demonstrated to be confined to the parasite 's flagellar pocket region at 37c but released to the cell surface at 20c , thereby demonstrating the redistribution of the transporter in response to cold shock [ 16 , 18 ] . such thermoregulatory events are increasingly recognised as being important in insect - borne parasites , including in leishmania and malaria [ 19 , 20 ] . the differential expression and localisation of the pad proteins provide two levels of control during the differentiation to procyclic forms ( fig . 2 ) . first , differential expression of pad proteins between slender and stumpy forms ensures only the pre - adapted transmissible stumpy forms are able to receive the differentiation signal . second , the differential localisation of pad2 between stumpy cells at 37c and 20c ensures that only cells in the appropriate environment ( i.e. the tsetse midgut ) are able to receive the signal . as citrate is present in blood at 130 m , this secondary control point could be crucial to prevent premature differentiation to procyclic forms whilst within the host , this signal only being detected upon temperature reduction in the tsetse blood - meal . a summary of the expression of pad proteins and the consequences for trypanosome differentiation events , based on the predictive model of engstler and boshart ( 2004 ) . slender cells proliferate in the bloodstream of the mammalian host . as parasite density increases , stumpy forms express pad proteins ( denoted by x ) , whereas slender forms do not . this ensures that only the transmissible stumpy forms are able to detect the differentiation signal . upon ingestion in a tsetse blood - meal and exposure to a drop in temperature , there is up - regulation and a relocation of at least one pad protein ( pad2 ) to the cell surface . retention of pad2 within the cell prior to cold shock ensures stumpy forms do not differentiate prematurely . slender forms do not perceive the signal and are sensitive to proteolytic and potential ph stress in the tsetse midgut and therefore do not survive pad protein expression and localisation act as checkpoints for differentiation . a summary of the expression of pad proteins and the consequences for trypanosome differentiation events , based on the predictive model of engstler and boshart ( 2004 ) . slender cells proliferate in the bloodstream of the mammalian host . as parasite density increases , stumpy forms express pad proteins ( denoted by x ) , whereas slender forms do not . this ensures that only the transmissible stumpy forms are able to detect the differentiation signal . upon ingestion in a tsetse blood - meal and exposure to a drop in temperature , there is up - regulation and a relocation of at least one pad protein ( pad2 ) to the cell surface . retention of pad2 within the cell prior to cold shock ensures stumpy forms do not differentiate prematurely . slender forms do not perceive the signal and are sensitive to proteolytic and potential ph stress in the tsetse midgut and therefore do not survive before the discovery of pad proteins , it was already established that the activity of a protein tyrosine phosphatase , tbptp1 , inhibits the differentiation of stumpy forms whilst in the bloodstream , this block being removed upon uptake by the tsetse fly . hence , it may be that tbptp1 acts downstream of the pad proteins as an effector of the cca signal ( fig . 1 ) or it may act via a parallel pathway , providing additional stringent control of differentiation . similarly , exposure to proteases [ 15 , 23 ] or ph stress in the tsetse midgut might also trigger differentiation , either in a complementary manner or as independent cues . although the role of these other environmental cues is yet to be fully understood , the discovery of the pad proteins provides the first molecular insight into the environmental sensing mechanisms used by the stumpy form to perceive transmission and so initiate differentiation . trypanosomes differentiate between distinct life stages in order to prepare for , and adapt to , the different environments they encounter during their life cycle [ 2 , 3 ] . during the bloodstream stage of the life cycle , trypanosomes exist as either proliferative slender forms or non - proliferative , transmissible , stumpy forms [ 2 , 3 ] ( fig . 1 ) , with transitional forms between these two types being described as intermediate forms . the proportion of these types changes during the course of infection , this apparently being governed by the density of parasites in the blood . the bloodstream trypanosome population is adapted to promote parasite maintenance in the mammalian blood and transmission to tsetse flies . thus , the proliferation of slender forms contributes to the establishment of parasite numbers in the blood and , through antigen variation , to immune evasion . in contrast , the irreversible division - arrest of stumpy forms acts to control the expansion of parasite numbers in the mammalian bloodstream and so prolongs host survival . this , combined with the preferential survival and the adaptations for differentiation of stumpy forms upon tsetse uptake , increases the probability of disease transmission . 1a simplified depiction of the t. brucei life cycle . in the bloodstream of the mammalian host , the parasites produce stumpy induction factor ( sif ) which induces differentiation of a proportion of slender forms to stumpy forms . stumpy forms are pre - adapted to life in the tsetse vector : they are cell cycle - arrested and have elaborated mitochondrial activity . upon uptake in a tsetse blood - meal , stumpy forms differentiate into the proliferative procyclic forms in response to cold shock and cis - aconitate or citrate ( cca ) in the tsetse midgut . other signals such as proteases or ph stress may also contribute , as does inhibition of the tyrosine phosphatase , tbptp1 . further differentiation events occur in the tsetse to produce mature metacyclic forms in the salivary glands . these are pre - adapted to life in the mammalian host a simplified depiction of the t. brucei life cycle . in the bloodstream of the mammalian host , slender forms proliferate . as density increases , the parasites produce stumpy induction factor ( sif ) which induces differentiation of a proportion of slender forms to stumpy forms . stumpy forms are pre - adapted to life in the tsetse vector : they are cell cycle - arrested and have elaborated mitochondrial activity . upon uptake in a tsetse blood - meal , stumpy forms differentiate into the proliferative procyclic forms in response to cold shock and cis - aconitate or citrate ( cca ) in the tsetse midgut . other signals such as proteases or ph stress may also contribute , as does inhibition of the tyrosine phosphatase , tbptp1 . further differentiation events occur in the tsetse to produce mature metacyclic forms in the salivary glands . these are pre - adapted to life in the mammalian host as the dynamic balance between slender and stumpy forms in mammalian hosts plays a pivotal role in trypanosome infection and transmission , understanding the molecular events implicit in the slender to stumpy form transition and their subsequent differentiation to tsetse midgut forms ( procyclic forms ) is of interest and importance , though poorly understood . when a tsetse fly ingests a trypanosome - infected blood - meal , both slender and stumpy form parasites enter the tsetse midgut . although subject to some controversy , it is generally thought that stumpy forms are pre - adapted to life in the tsetse midgut , and only they differentiate efficiently to the tsetse midgut procyclic form . cytological evidence based on in vitro differentiation experiments suggests that bloodstream forms can only transform to procyclic forms from a given point in the cell cycle , namely within the g1 , or the quiescent g0 , phase . as stumpy forms are uniformly arrested as a population in g0/g1 and can accumulate to near homogeneity in experimental infections , this allows for their efficient and synchronous differentiation to procyclic forms . in contrast , proliferative slender cell populations are asynchronous in their cell cycle and must reach g0/g1 before differentiation . additionally , as cells enter the tsetse midgut , they are exposed to harmful proteases and changes in their environmental ph . the stumpy forms are more resistant than slender forms to the effects of such stresses and , as such , are more likely to endure the transition from the bloodstream to the tsetse midgut . moreover , in the glucose - rich mammalian bloodstream , slender cells obtain their energy entirely via glycolysis and , as a result , the mitochondrion of slender cells is relatively inactive . in intermediate and stumpy forms , however , the mitochondrion is elaborated and mitochondrial enzymes are partially expressed [ 3 , 9 ] . this enables them to quickly begin oxidative phosphorylation as they enter the glucose - poor tsetse midgut [ 3 , 9 ] . stumpy cells also persist longer than slender cells in the face of the developing immune response in the mammalian bloodstream [ 10 , 11 ] , as well as in the tsetse midgut . this is probably not caused by antigenic variation : being irreversibly arrested in the bloodstream , stumpy forms are unlikely to be able to undergo the dna recombination events important for antigen switching . rather , they withstand antibody clearance more effectively than slender cells because they preferentially clear bound antibody by hydrodynamic flow , whereby parasite - bound immunoglobulin is swept backward by the swimming action of the trypanosome in the blood and is then internalised and degraded . the molecular mechanism by which bloodstream trypanosomes perceive their change in environment and initiate differentiation to procyclic forms once in the tsetse fly has been elusive , until recently . it has long been known that the use of high concentrations ( > 3 mm ) of citrate or cis - aconitate ( cca ) could induce differentiation of bloodstream form trypanosomes to procyclic forms in vitro [ 1315 ] . the biological relevance of this signal was in doubt , however , as these levels exceed the concentrations of cca that a trypanosome would be exposed to in the tsetse midgut . however , in 2004 , engstler and boshart discovered that a drop in temperature from 37c to 20c could significantly increase trypanosome sensitivity to cca , potentially matching levels present within the tsetse blood - meal . since this temperature drop was compatible with that encountered by trypanosomes as they entered tsetse flies feeding at the cooler ambient temperatures of dusk or dawn , cca could be considered physiologically relevant . since temperature drop also allows some molecules regulated access to the parasite surface , engstler and boshart proposed a model whereby an unknown surface protein responsible for the reception of the differentiation signal would be retained within the cell in the bloodstream ( i.e. when at 37c ) and then trafficked to the cell surface in response to the cooler temperatures associated with tsetse uptake [ 16 , 17 ] . the molecular basis of this model , however , was unknown until a gene family encoding surface carboxylate transporters known as pad proteins ( proteins associated with differentiation ) was recently identified . indeed , two of the encoded pad proteins ( pad1 and pad2 ) were shown to be expressed in stumpy forms but not in slender forms and were found to be required for perception of cca at physiological concentrations . matching the predictions of engstler and boshart , pad2 was also demonstrated to be confined to the parasite 's flagellar pocket region at 37c but released to the cell surface at 20c , thereby demonstrating the redistribution of the transporter in response to cold shock [ 16 , 18 ] . such thermoregulatory events are increasingly recognised as being important in insect - borne parasites , including in leishmania and malaria [ 19 , 20 ] . the differential expression and localisation of the pad proteins provide two levels of control during the differentiation to procyclic forms ( fig . first , differential expression of pad proteins between slender and stumpy forms ensures only the pre - adapted transmissible stumpy forms are able to receive the differentiation signal . second , the differential localisation of pad2 between stumpy cells at 37c and 20c ensures that only cells in the appropriate environment ( i.e. the tsetse midgut ) are able to receive the signal . as citrate is present in blood at 130 m , this secondary control point could be crucial to prevent premature differentiation to procyclic forms whilst within the host , this signal only being detected upon temperature reduction in the tsetse blood - meal . a summary of the expression of pad proteins and the consequences for trypanosome differentiation events , based on the predictive model of engstler and boshart ( 2004 ) . slender cells proliferate in the bloodstream of the mammalian host . as parasite density increases , stumpy forms express pad proteins ( denoted by x ) , whereas slender forms do not . this ensures that only the transmissible stumpy forms are able to detect the differentiation signal . upon ingestion in a tsetse blood - meal and exposure to a drop in temperature , there is up - regulation and a relocation of at least one pad protein ( pad2 ) to the cell surface . retention of pad2 within the cell prior to cold shock ensures stumpy forms do not differentiate prematurely . slender forms do not perceive the signal and are sensitive to proteolytic and potential ph stress in the tsetse midgut and therefore do not survive pad protein expression and localisation act as checkpoints for differentiation . a summary of the expression of pad proteins and the consequences for trypanosome differentiation events , based on the predictive model of engstler and boshart ( 2004 ) . slender cells proliferate in the bloodstream of the mammalian host . as parasite density increases , stumpy forms express pad proteins ( denoted by x ) , whereas slender forms do not . this ensures that only the transmissible stumpy forms are able to detect the differentiation signal . upon ingestion in a tsetse blood - meal and exposure to a drop in temperature , there is up - regulation and a relocation of at least one pad protein ( pad2 ) to the cell surface . retention of pad2 within the cell prior to cold shock ensures stumpy forms do not differentiate prematurely . slender forms do not perceive the signal and are sensitive to proteolytic and potential ph stress in the tsetse midgut and therefore do not survive before the discovery of pad proteins , it was already established that the activity of a protein tyrosine phosphatase , tbptp1 , inhibits the differentiation of stumpy forms whilst in the bloodstream , this block being removed upon uptake by the tsetse fly . hence , it may be that tbptp1 acts downstream of the pad proteins as an effector of the cca signal ( fig . 1 ) or it may act via a parallel pathway , providing additional stringent control of differentiation . similarly , exposure to proteases [ 15 , 23 ] or ph stress in the tsetse midgut might also trigger differentiation , either in a complementary manner or as independent cues . although the role of these other environmental cues is yet to be fully understood , the discovery of the pad proteins provides the first molecular insight into the environmental sensing mechanisms used by the stumpy form to perceive transmission and so initiate differentiation . one of the key reasons that the differentiation from bloodstream forms to procyclic forms has been so well - studied is that there are clear molecular markers to distinguish each developmental stage . furthermore , the development between the bloodstream and procyclic forms can be easily mimicked in vitro . contrasting with this , the absence of markers distinguishing slender from stumpy forms has hindered our understanding of the transmission biology of trypanosomes , as has the difficulty in successfully generating stumpy forms in culture . in earlier studies , various markers have been used to identify stumpy forms , including morphology , mitochondrial activation , cell cycle arrest and the capacity of cells to differentiate synchronously to the procyclic form [ 2529 ] . for example , analysis by morphology is subjective and time - consuming and is complicated by the presence of morphologically intermediate forms in the population . similarly , differentiation analyses are time - dependent and require population assays , rather than the analysis of individual cells . finally , mitochondrial activation , often visualised through the activity of dihydrolipoamide dehydrogenase in the diaphorase assay , may also be activated under non - physiological stress conditions . with the identification of pad1 as a surface molecule that identifies cells that are functionally competent for transmission , as well as developments in parasite culture and transfection , the route has been opened up to dissect trypanosome transmissibility at the level of individual cells as well as in the infecting parasite population . differentiation between life cycle stages and the progression of the cell cycle are intimately linked in trypanosomes [ 7 , 22 ] . as mentioned above , stumpy forms are cell cycle - arrested in g0/g1 but derived from proliferative slender forms . this transition , therefore , requires exit from the cell cycle , morphological change and stumpy - specific gene expression and has been studied both experimentally and by mathematical modelling [ 7 , 29 , 31 ] . from these studies , it appears that the commitment of a slender cell to differentiate to a stumpy form precedes a final cell division and morphological change [ 7 , 29 ] . however , the discovery of pad1 as a molecular marker for stumpy forms enables the events of cell cycle exit and stumpy formation to be tracked at the level of an individual cell , providing a temporal map of the steps leading to transmission competence . similarly , the signalling pathways that generate stumpy forms from slender forms can be investigated . this transition can occur independently of the host [ 32 , 33 ] and correlates with increased cell density [ 28 , 33 , 34 ] , with parasite parasite signalling operating through a form of quorum sensing . this is proposed to occur through the production by slender forms of a signal , termed stumpy induction factor ( sif ) , which accumulates and stimulates stumpy formation as parasite numbers increase . the identity of sif and the molecular mechanisms of its reception are currently unknown . it was thought that the sif signal was transmitted via the camp pathway , since cell - permeable camp can mimic the action of sif ( fig . 1 ) . however , more recent evidence suggests that hydrolysis products of camp also function , arguing against a conventional camp signalling pathway . additional compounds such as troglitazone and z - phe - ala - chn2 have also been proposed to mimic sif activity in vitro , inhibiting cell growth and generating morphologically stumpy - like forms . the biological significance of these different treatments , however , has been difficult to assess in the absence of a functional molecular marker for stumpy forms . hence , the identification of pad1 and its detection either by antibody or in transgenic reporter assays now enables sif , or sif mimics , to be tracked in high - throughput assays and identified . pad1 also provides a route to analyse the proportions of slender and stumpy forms in infections and to understand how the dynamics of these change throughout the course of a classical chronic infection . as trypanosome infections last from months to years , each infected individual can act as a reservoir for the disease for a considerable period of time . however , the degree of periodicity in their transmission capacity is poorly understood but potentially vital in a context where tsetse bites are rare or show seasonality . furthermore , through mathematic modelling , it has been predicted that the production of stumpy forms is a key contributor to the ordered expression of distinct antigenic variants that sustain chronic trypanosome infections . this , combined with the likelihood of co - infection with different trypanosome strains during the course of a chronic infection , generates the potential for complex infection dynamics with important possible implications for disease progression and transmission in endemic areas . with the availability of a marker for the transmission stage , quantitative models for the dynamics of slender and stumpy parasites as a proportion of the total parasite numbers over time can be generated and the consequences of potential therapeutic strategies predicted . the identification of a molecular marker that distinguishes the non - proliferative stumpy form provides the ability to develop high - throughput screens for compounds that accelerate stumpy formation at abnormally low parasite density . genetically modified reporter lines could provide simple assays for enhanced stumpy formation accessible to screening with small molecule or natural product libraries . if administrable compounds were identified that are able to force the irreversible differentiation of slender forms to stumpy forms at low levels of parasitaemia , then this could have potential therapeutic consequences . although accelerated parasite development has the potential to reduce host parasitaemia , it also has the potential to increase the transmission potential of the population through increasing the proportion of stumpy forms . the extent of this risk depends on several factors : the density of parasites in the bloodstream , the number of parasites required to infect a tsetse fly and the efficiency of accelerated development . assuming a typical parasitaemia in cattle of 10 parasites / ml and an infective dose to tsetse under optimal conditions of one trypanosome , then a reduction in parasite numbers of at least three orders of magnitude would be required to minimise the probability of parasite transmission in a tsetse blood - meal . however , in the field , chronic infections of cattle can exhibit refractory periods when there are insufficient parasites for tsetse transmission , suggesting that the parasites may be on the threshold of transmissibility , particularly when compounded by seasonal effects . moreover , if stumpy formation could be driven to 100% efficiency , then the irreversible division - arrest of these forms would ensure complete parasite elimination through cell senescence within only a few days , limiting the risks of enhanced transmission and providing a curative regime . clearly , without better understanding of the transmission biology of trypanosomes in the field , the prospects for a transmission blocking approach are difficult to assess . it is satisfying , therefore , that the identification of stumpy marker proteins such as pad offers the ability to both screen for new drugs to accelerate stumpy formation and to refine the transmission models required to optimise their potential deployment in the field . differentiation between life cycle stages and the progression of the cell cycle are intimately linked in trypanosomes [ 7 , 22 ] . as mentioned above , stumpy forms are cell cycle - arrested in g0/g1 but derived from proliferative slender forms . this transition , therefore , requires exit from the cell cycle , morphological change and stumpy - specific gene expression and has been studied both experimentally and by mathematical modelling [ 7 , 29 , 31 ] . from these studies , it appears that the commitment of a slender cell to differentiate to a stumpy form precedes a final cell division and morphological change [ 7 , 29 ] . however , the discovery of pad1 as a molecular marker for stumpy forms enables the events of cell cycle exit and stumpy formation to be tracked at the level of an individual cell , providing a temporal map of the steps leading to transmission competence . similarly , the signalling pathways that generate stumpy forms from slender forms can be investigated . this transition can occur independently of the host [ 32 , 33 ] and correlates with increased cell density [ 28 , 33 , 34 ] , with parasite parasite signalling operating through a form of quorum sensing . this is proposed to occur through the production by slender forms of a signal , termed stumpy induction factor ( sif ) , which accumulates and stimulates stumpy formation as parasite numbers increase . it was thought that the sif signal was transmitted via the camp pathway , since cell - permeable camp can mimic the action of sif ( fig . 1 ) . however , more recent evidence suggests that hydrolysis products of camp also function , arguing against a conventional camp signalling pathway . additional compounds such as troglitazone and z - phe - ala - chn2 have also been proposed to mimic sif activity in vitro , inhibiting cell growth and generating morphologically stumpy - like forms . the biological significance of these different treatments , however , has been difficult to assess in the absence of a functional molecular marker for stumpy forms . hence , the identification of pad1 and its detection either by antibody or in transgenic reporter assays now enables sif , or sif mimics , to be tracked in high - throughput assays and identified . pad1 also provides a route to analyse the proportions of slender and stumpy forms in infections and to understand how the dynamics of these change throughout the course of a classical chronic infection . as trypanosome infections last from months to years , each infected individual can act as a reservoir for the disease for a considerable period of time . however , the degree of periodicity in their transmission capacity is poorly understood but potentially vital in a context where tsetse bites are rare or show seasonality . furthermore , through mathematic modelling , it has been predicted that the production of stumpy forms is a key contributor to the ordered expression of distinct antigenic variants that sustain chronic trypanosome infections . this , combined with the likelihood of co - infection with different trypanosome strains during the course of a chronic infection , generates the potential for complex infection dynamics with important possible implications for disease progression and transmission in endemic areas . with the availability of a marker for the transmission stage , quantitative models for the dynamics of slender and stumpy parasites as a proportion of the total parasite numbers over time can be generated and the consequences of potential therapeutic strategies predicted . the identification of a molecular marker that distinguishes the non - proliferative stumpy form provides the ability to develop high - throughput screens for compounds that accelerate stumpy formation at abnormally low parasite density . genetically modified reporter lines could provide simple assays for enhanced stumpy formation accessible to screening with small molecule or natural product libraries . if administrable compounds were identified that are able to force the irreversible differentiation of slender forms to stumpy forms at low levels of parasitaemia , then this could have potential therapeutic consequences . although accelerated parasite development has the potential to reduce host parasitaemia , it also has the potential to increase the transmission potential of the population through increasing the proportion of stumpy forms . the extent of this risk depends on several factors : the density of parasites in the bloodstream , the number of parasites required to infect a tsetse fly and the efficiency of accelerated development . assuming a typical parasitaemia in cattle of 10 parasites / ml and an infective dose to tsetse under optimal conditions of one trypanosome , then a reduction in parasite numbers of at least three orders of magnitude would be required to minimise the probability of parasite transmission in a tsetse blood - meal . however , in the field , chronic infections of cattle can exhibit refractory periods when there are insufficient parasites for tsetse transmission , suggesting that the parasites may be on the threshold of transmissibility , particularly when compounded by seasonal effects . this might make transmission blocking feasible . moreover , if stumpy formation could be driven to 100% efficiency , then the irreversible division - arrest of these forms would ensure complete parasite elimination through cell senescence within only a few days , limiting the risks of enhanced transmission and providing a curative regime . clearly , without better understanding of the transmission biology of trypanosomes in the field , the prospects for a transmission blocking approach are difficult to assess . it is satisfying , therefore , that the identification of stumpy marker proteins such as pad offers the ability to both screen for new drugs to accelerate stumpy formation and to refine the transmission models required to optimise their potential deployment in the field . considerable progress has been made in recent years in understanding trypanosome transmission at the level of basic biology . however , these studies also have direct application in the search for new therapies for trypanosomes in the field . in particular , the ability of pad proteins to discriminate stumpy forms enables high - throughput screens for compounds that accelerate stumpy formation at low parasite density . by inducing stumpy formation at low parasitaemia , the potential for parasite transmission may be reduced ( fig . 3 ) . furthermore , with a reduced parasite load , the host immune system may be able to control the infection . importantly , genetically modified reporter lines could provide simple assays for enhanced stumpy formation accessible to screening with small molecule or natural product libraries . this offers great potential for disrupting the normal life cycle progression of the parasite and thereby restricting both pathogenicity and transmissibility of these important pathogens . as so often , an investment in basic knowledge has provided an unexpected opportunity for the rational screening for , and development of , novel therapeutic approaches . in the normal course of infection , slender cells generate stumpy forms once parasite numbers are sufficient to ensure transmission if taken up during a tsetse blood - meal . by accelerating stumpy formation at a lower parasitaemia furthermore , the consequent limitation in parasite numbers would reduce the potential for parasite transmission during a tsetse blood - meal accelerated parasite development as a route to limiting parasite transmission and parasitaemia . in the normal course of infection , slender cells generate stumpy forms once parasite numbers are sufficient to ensure transmission if taken up during a tsetse blood - meal . by accelerating stumpy formation at a lower parasitaemia furthermore , the consequent limitation in parasite numbers would reduce the potential for parasite transmission during a tsetse blood - meal

the sleeping sickness parasite , trypanosoma brucei , must differentiate in response to the changing environments that it encounters during its complex life cycle . one developmental form , the bloodstream stumpy stage , plays an important role in infection dynamics and transmission of the parasite . recent advances have shed light on the molecular mechanisms by which these stumpy forms differentiate as they are transmitted from the mammalian host to the insect vector of sleeping sickness , tsetse flies . these molecular advances now provide improved experimental tools for the study of stumpy formation and function within the mammalian bloodstream . they also offer new routes to therapy via high - throughput screens for agents that accelerate parasite development . here , we shall discuss the recent advances that have been made and the prospects for future research now available .

Sleeping sickness: an ancient and current disease The biology of disease spread Adaptation of stumpy forms for parasite transmission Molecules, models and medicines Differentiation of the individual parasite Differentiation of the population Accelerated parasite development as a therapeutic tool Perspectives

the tropical parasite trypanosoma brucei is the causative agent of both human african trypanosomiasis ( hat , or sleeping sickness ) and the livestock disease , nagana . the initial stage of disease , established when an individual is bitten by an infected tsetse fly , generates a local chancre at the bite site with this being followed by the proliferation of the parasite in the bloodstream resulting in fever and joint pain . at this stage , the disease is often undiagnosed and , as such , untreated , despite the availability of ( albeit rather old ) drugs such as suramin ( for trypanosoma brucei rhodesiense or acute sleeping sickness ) and pentamidine ( for trypanosoma brucei gambiense or chronic sleeping sickness ) . the current drug treatments for the later stage of the disease are difficult to administer , requiring a rigorous hospital regime , and all have associated toxicity . the ability of trypanosomes to infect both domestic and wild animals means that non - human hosts in affected regions can act as a reservoir for the parasite , confounding control of the disease . due to the severity and prevalence of the disease , as well as the lack of efficient disease management , african trypanosomiasis remains an important public health issue and is considered by the world health organisation to be a neglected tropical disease ( http://www.who.int/en ) . trypanosomes differentiate between distinct life stages in order to prepare for , and adapt to , the different environments they encounter during their life cycle [ 2 , 3 ] . during the bloodstream stage of the life cycle , trypanosomes exist as either proliferative slender forms or non - proliferative , transmissible , stumpy forms [ 2 , 3 ] ( fig . the bloodstream trypanosome population is adapted to promote parasite maintenance in the mammalian blood and transmission to tsetse flies . in contrast , the irreversible division - arrest of stumpy forms acts to control the expansion of parasite numbers in the mammalian bloodstream and so prolongs host survival . this , combined with the preferential survival and the adaptations for differentiation of stumpy forms upon tsetse uptake , increases the probability of disease transmission . 1a simplified depiction of the t. brucei life cycle . in the bloodstream of the mammalian host , the parasites produce stumpy induction factor ( sif ) which induces differentiation of a proportion of slender forms to stumpy forms . stumpy forms are pre - adapted to life in the tsetse vector : they are cell cycle - arrested and have elaborated mitochondrial activity . upon uptake in a tsetse blood - meal , stumpy forms differentiate into the proliferative procyclic forms in response to cold shock and cis - aconitate or citrate ( cca ) in the tsetse midgut . these are pre - adapted to life in the mammalian host a simplified depiction of the t. brucei life cycle . in the bloodstream of the mammalian host , slender forms proliferate . as density increases , the parasites produce stumpy induction factor ( sif ) which induces differentiation of a proportion of slender forms to stumpy forms . stumpy forms are pre - adapted to life in the tsetse vector : they are cell cycle - arrested and have elaborated mitochondrial activity . upon uptake in a tsetse blood - meal , stumpy forms differentiate into the proliferative procyclic forms in response to cold shock and cis - aconitate or citrate ( cca ) in the tsetse midgut . these are pre - adapted to life in the mammalian host as the dynamic balance between slender and stumpy forms in mammalian hosts plays a pivotal role in trypanosome infection and transmission , understanding the molecular events implicit in the slender to stumpy form transition and their subsequent differentiation to tsetse midgut forms ( procyclic forms ) is of interest and importance , though poorly understood . although subject to some controversy , it is generally thought that stumpy forms are pre - adapted to life in the tsetse midgut , and only they differentiate efficiently to the tsetse midgut procyclic form . the stumpy forms are more resistant than slender forms to the effects of such stresses and , as such , are more likely to endure the transition from the bloodstream to the tsetse midgut . moreover , in the glucose - rich mammalian bloodstream , slender cells obtain their energy entirely via glycolysis and , as a result , the mitochondrion of slender cells is relatively inactive . in intermediate and stumpy forms , however , the mitochondrion is elaborated and mitochondrial enzymes are partially expressed [ 3 , 9 ] . stumpy cells also persist longer than slender cells in the face of the developing immune response in the mammalian bloodstream [ 10 , 11 ] , as well as in the tsetse midgut . this is probably not caused by antigenic variation : being irreversibly arrested in the bloodstream , stumpy forms are unlikely to be able to undergo the dna recombination events important for antigen switching . the molecular mechanism by which bloodstream trypanosomes perceive their change in environment and initiate differentiation to procyclic forms once in the tsetse fly has been elusive , until recently . since this temperature drop was compatible with that encountered by trypanosomes as they entered tsetse flies feeding at the cooler ambient temperatures of dusk or dawn , cca could be considered physiologically relevant . since temperature drop also allows some molecules regulated access to the parasite surface , engstler and boshart proposed a model whereby an unknown surface protein responsible for the reception of the differentiation signal would be retained within the cell in the bloodstream ( i.e. when at 37c ) and then trafficked to the cell surface in response to the cooler temperatures associated with tsetse uptake [ 16 , 17 ] . indeed , two of the encoded pad proteins ( pad1 and pad2 ) were shown to be expressed in stumpy forms but not in slender forms and were found to be required for perception of cca at physiological concentrations . matching the predictions of engstler and boshart , pad2 was also demonstrated to be confined to the parasite 's flagellar pocket region at 37c but released to the cell surface at 20c , thereby demonstrating the redistribution of the transporter in response to cold shock [ 16 , 18 ] . a summary of the expression of pad proteins and the consequences for trypanosome differentiation events , based on the predictive model of engstler and boshart ( 2004 ) . slender cells proliferate in the bloodstream of the mammalian host . retention of pad2 within the cell prior to cold shock ensures stumpy forms do not differentiate prematurely . a summary of the expression of pad proteins and the consequences for trypanosome differentiation events , based on the predictive model of engstler and boshart ( 2004 ) . slender cells proliferate in the bloodstream of the mammalian host . retention of pad2 within the cell prior to cold shock ensures stumpy forms do not differentiate prematurely . slender forms do not perceive the signal and are sensitive to proteolytic and potential ph stress in the tsetse midgut and therefore do not survive before the discovery of pad proteins , it was already established that the activity of a protein tyrosine phosphatase , tbptp1 , inhibits the differentiation of stumpy forms whilst in the bloodstream , this block being removed upon uptake by the tsetse fly . although the role of these other environmental cues is yet to be fully understood , the discovery of the pad proteins provides the first molecular insight into the environmental sensing mechanisms used by the stumpy form to perceive transmission and so initiate differentiation . trypanosomes differentiate between distinct life stages in order to prepare for , and adapt to , the different environments they encounter during their life cycle [ 2 , 3 ] . during the bloodstream stage of the life cycle , trypanosomes exist as either proliferative slender forms or non - proliferative , transmissible , stumpy forms [ 2 , 3 ] ( fig . the bloodstream trypanosome population is adapted to promote parasite maintenance in the mammalian blood and transmission to tsetse flies . thus , the proliferation of slender forms contributes to the establishment of parasite numbers in the blood and , through antigen variation , to immune evasion . in contrast , the irreversible division - arrest of stumpy forms acts to control the expansion of parasite numbers in the mammalian bloodstream and so prolongs host survival . this , combined with the preferential survival and the adaptations for differentiation of stumpy forms upon tsetse uptake , increases the probability of disease transmission . 1a simplified depiction of the t. brucei life cycle . in the bloodstream of the mammalian host , the parasites produce stumpy induction factor ( sif ) which induces differentiation of a proportion of slender forms to stumpy forms . stumpy forms are pre - adapted to life in the tsetse vector : they are cell cycle - arrested and have elaborated mitochondrial activity . upon uptake in a tsetse blood - meal , stumpy forms differentiate into the proliferative procyclic forms in response to cold shock and cis - aconitate or citrate ( cca ) in the tsetse midgut . these are pre - adapted to life in the mammalian host a simplified depiction of the t. brucei life cycle . in the bloodstream of the mammalian host , slender forms proliferate . as density increases , the parasites produce stumpy induction factor ( sif ) which induces differentiation of a proportion of slender forms to stumpy forms . stumpy forms are pre - adapted to life in the tsetse vector : they are cell cycle - arrested and have elaborated mitochondrial activity . upon uptake in a tsetse blood - meal , stumpy forms differentiate into the proliferative procyclic forms in response to cold shock and cis - aconitate or citrate ( cca ) in the tsetse midgut . these are pre - adapted to life in the mammalian host as the dynamic balance between slender and stumpy forms in mammalian hosts plays a pivotal role in trypanosome infection and transmission , understanding the molecular events implicit in the slender to stumpy form transition and their subsequent differentiation to tsetse midgut forms ( procyclic forms ) is of interest and importance , though poorly understood . although subject to some controversy , it is generally thought that stumpy forms are pre - adapted to life in the tsetse midgut , and only they differentiate efficiently to the tsetse midgut procyclic form . the stumpy forms are more resistant than slender forms to the effects of such stresses and , as such , are more likely to endure the transition from the bloodstream to the tsetse midgut . moreover , in the glucose - rich mammalian bloodstream , slender cells obtain their energy entirely via glycolysis and , as a result , the mitochondrion of slender cells is relatively inactive . in intermediate and stumpy forms , however , the mitochondrion is elaborated and mitochondrial enzymes are partially expressed [ 3 , 9 ] . stumpy cells also persist longer than slender cells in the face of the developing immune response in the mammalian bloodstream [ 10 , 11 ] , as well as in the tsetse midgut . this is probably not caused by antigenic variation : being irreversibly arrested in the bloodstream , stumpy forms are unlikely to be able to undergo the dna recombination events important for antigen switching . the molecular mechanism by which bloodstream trypanosomes perceive their change in environment and initiate differentiation to procyclic forms once in the tsetse fly has been elusive , until recently . since this temperature drop was compatible with that encountered by trypanosomes as they entered tsetse flies feeding at the cooler ambient temperatures of dusk or dawn , cca could be considered physiologically relevant . since temperature drop also allows some molecules regulated access to the parasite surface , engstler and boshart proposed a model whereby an unknown surface protein responsible for the reception of the differentiation signal would be retained within the cell in the bloodstream ( i.e. when at 37c ) and then trafficked to the cell surface in response to the cooler temperatures associated with tsetse uptake [ 16 , 17 ] . indeed , two of the encoded pad proteins ( pad1 and pad2 ) were shown to be expressed in stumpy forms but not in slender forms and were found to be required for perception of cca at physiological concentrations . matching the predictions of engstler and boshart , pad2 was also demonstrated to be confined to the parasite 's flagellar pocket region at 37c but released to the cell surface at 20c , thereby demonstrating the redistribution of the transporter in response to cold shock [ 16 , 18 ] . a summary of the expression of pad proteins and the consequences for trypanosome differentiation events , based on the predictive model of engstler and boshart ( 2004 ) . slender cells proliferate in the bloodstream of the mammalian host . retention of pad2 within the cell prior to cold shock ensures stumpy forms do not differentiate prematurely . a summary of the expression of pad proteins and the consequences for trypanosome differentiation events , based on the predictive model of engstler and boshart ( 2004 ) . slender cells proliferate in the bloodstream of the mammalian host . retention of pad2 within the cell prior to cold shock ensures stumpy forms do not differentiate prematurely . slender forms do not perceive the signal and are sensitive to proteolytic and potential ph stress in the tsetse midgut and therefore do not survive before the discovery of pad proteins , it was already established that the activity of a protein tyrosine phosphatase , tbptp1 , inhibits the differentiation of stumpy forms whilst in the bloodstream , this block being removed upon uptake by the tsetse fly . although the role of these other environmental cues is yet to be fully understood , the discovery of the pad proteins provides the first molecular insight into the environmental sensing mechanisms used by the stumpy form to perceive transmission and so initiate differentiation . furthermore , the development between the bloodstream and procyclic forms can be easily mimicked in vitro . contrasting with this , the absence of markers distinguishing slender from stumpy forms has hindered our understanding of the transmission biology of trypanosomes , as has the difficulty in successfully generating stumpy forms in culture . in earlier studies , various markers have been used to identify stumpy forms , including morphology , mitochondrial activation , cell cycle arrest and the capacity of cells to differentiate synchronously to the procyclic form [ 2529 ] . differentiation between life cycle stages and the progression of the cell cycle are intimately linked in trypanosomes [ 7 , 22 ] . however , the discovery of pad1 as a molecular marker for stumpy forms enables the events of cell cycle exit and stumpy formation to be tracked at the level of an individual cell , providing a temporal map of the steps leading to transmission competence . similarly , the signalling pathways that generate stumpy forms from slender forms can be investigated . the identity of sif and the molecular mechanisms of its reception are currently unknown . hence , the identification of pad1 and its detection either by antibody or in transgenic reporter assays now enables sif , or sif mimics , to be tracked in high - throughput assays and identified . furthermore , through mathematic modelling , it has been predicted that the production of stumpy forms is a key contributor to the ordered expression of distinct antigenic variants that sustain chronic trypanosome infections . this , combined with the likelihood of co - infection with different trypanosome strains during the course of a chronic infection , generates the potential for complex infection dynamics with important possible implications for disease progression and transmission in endemic areas . with the availability of a marker for the transmission stage , quantitative models for the dynamics of slender and stumpy parasites as a proportion of the total parasite numbers over time can be generated and the consequences of potential therapeutic strategies predicted . the identification of a molecular marker that distinguishes the non - proliferative stumpy form provides the ability to develop high - throughput screens for compounds that accelerate stumpy formation at abnormally low parasite density . although accelerated parasite development has the potential to reduce host parasitaemia , it also has the potential to increase the transmission potential of the population through increasing the proportion of stumpy forms . the extent of this risk depends on several factors : the density of parasites in the bloodstream , the number of parasites required to infect a tsetse fly and the efficiency of accelerated development . clearly , without better understanding of the transmission biology of trypanosomes in the field , the prospects for a transmission blocking approach are difficult to assess . it is satisfying , therefore , that the identification of stumpy marker proteins such as pad offers the ability to both screen for new drugs to accelerate stumpy formation and to refine the transmission models required to optimise their potential deployment in the field . differentiation between life cycle stages and the progression of the cell cycle are intimately linked in trypanosomes [ 7 , 22 ] . however , the discovery of pad1 as a molecular marker for stumpy forms enables the events of cell cycle exit and stumpy formation to be tracked at the level of an individual cell , providing a temporal map of the steps leading to transmission competence . similarly , the signalling pathways that generate stumpy forms from slender forms can be investigated . hence , the identification of pad1 and its detection either by antibody or in transgenic reporter assays now enables sif , or sif mimics , to be tracked in high - throughput assays and identified . furthermore , through mathematic modelling , it has been predicted that the production of stumpy forms is a key contributor to the ordered expression of distinct antigenic variants that sustain chronic trypanosome infections . this , combined with the likelihood of co - infection with different trypanosome strains during the course of a chronic infection , generates the potential for complex infection dynamics with important possible implications for disease progression and transmission in endemic areas . with the availability of a marker for the transmission stage , quantitative models for the dynamics of slender and stumpy parasites as a proportion of the total parasite numbers over time can be generated and the consequences of potential therapeutic strategies predicted . the identification of a molecular marker that distinguishes the non - proliferative stumpy form provides the ability to develop high - throughput screens for compounds that accelerate stumpy formation at abnormally low parasite density . although accelerated parasite development has the potential to reduce host parasitaemia , it also has the potential to increase the transmission potential of the population through increasing the proportion of stumpy forms . the extent of this risk depends on several factors : the density of parasites in the bloodstream , the number of parasites required to infect a tsetse fly and the efficiency of accelerated development . clearly , without better understanding of the transmission biology of trypanosomes in the field , the prospects for a transmission blocking approach are difficult to assess . it is satisfying , therefore , that the identification of stumpy marker proteins such as pad offers the ability to both screen for new drugs to accelerate stumpy formation and to refine the transmission models required to optimise their potential deployment in the field . in particular , the ability of pad proteins to discriminate stumpy forms enables high - throughput screens for compounds that accelerate stumpy formation at low parasite density . this offers great potential for disrupting the normal life cycle progression of the parasite and thereby restricting both pathogenicity and transmissibility of these important pathogens . by accelerating stumpy formation at a lower parasitaemia furthermore , the consequent limitation in parasite numbers would reduce the potential for parasite transmission during a tsetse blood - meal accelerated parasite development as a route to limiting parasite transmission and parasitaemia . by accelerating stumpy formation at a lower parasitaemia furthermore , the consequent limitation in parasite numbers would reduce the potential for parasite transmission during a tsetse blood - meal

spinal stenosis is defined as a narrowing of spinal canal with encroachment on the neural structures by surrounding bone and soft issue.1 in the framingham study , prevalence of symptomatic lumbar spinal stenosis was estimated to be 27.2%.2 the three most common causes of low back and leg pain are spinal stenosis , disc herniation , and degenerative spondylolisthesis , for which surgery is usually performed.1,35 moreover , in patients older than 65 years , lumbar spinal stenosis has been regarded as the most frequent indication for spinal surgery.46 numerous modalities of treatment , including surgery with or without fusion , interventional techniques , and conservative approaches have been applied in managing lumbar central spinal stenosis.4,79 surgical interventions or epidural injections are commonly used for treatment of symptomatic spinal stenosis when conservative treatment fails.1,35,10,11 in the spine patient outcomes research trials ( sport ) , tosteson et al3 found that patients who received surgical intervention for spinal stenosis without degenerative spondylolisthesis achieved significant improvement in all primary outcomes compared to those undergoing nonsurgical treatment . moreover , in a systematic review that compared conservative care with surgery , surgery was shown to provide beneficial effects in managing pain and quality of life , but not ambulation.12 however , for patients with mild to moderate spinal stenosis , who are not candidates for surgery , or for those who do not respond well to surgery , epidural injections may be the only interventional treatment . consequently , apart from surgical intervention , epidural injections are the most commonly performed intervention in the management of chronic low back pain secondary to central spinal stenosis.13,14 manchikanti et al conducted a trial that compared the effect of epidural injections with or without steroids in the treatment of spinal stenosis.15 the authors found that 46% of the patients who were treated with local anesthetic and steroids achieved significant pain relief and an improvement in functional status of more than 50% , whereas the proportion of patients who improved after receiving only local anesthetic was 48%.15 in another trial , pain relief and improved functional status were observed in 73% of the patients who received both steroids and local anesthetic , and in 72% of those who received local anesthetic alone.16 no meta - analysis has yet been conducted to compare the effectiveness of epidural injections of local anesthetic with or without steroids in the treatment of patients with spinal stenosis , leaving the relative effectiveness of these treatments open to question . to address this need , we performed a meta - analysis based on randomized controlled trials ( rcts ) . we followed the preferred reported items for systematic and meta - analysis guideline ( prisma ) in this study.17 a comprehensive literature search was conducted to identify all the published rcts that compared epidural injection with or without steroids in the management of chronic low back pain in spinal stenosis . pubmed , embase , web of science , and cochrane library databases were searched from their inception through february 5 , 2015 . the following search terms were used : ( spinal stenosis [ mesh terms ] ) or ( spinal [ all fields ] and stenosis [ all fields ] ) or ( spinal stenosis [ all fields ] ) and ( injections , epidural [ mesh terms ] ) or ( injections [ all fields ] and epidural [ all fields ] ) or ( epidural injections [ all fields ] ) or ( epidural [ all fields ] and injection [ all fields ] ) or ( epidural injection [ all fields ] ) and ( anesthetics , local [ pharmacological action ] or anesthetics , local [ mesh terms ] ) or ( anesthetics [ all fields ] and local[all fields ] ) or ( local anesthetics [ all fields ] ) or ( local [ all fields ] and anesthetic [ all fields ] ) or ( local anesthetic [ all fields ] ) or ( anesthesia , local [ mesh terms ] ) or ( anesthesia [ all fields ] and local [ all fields ] ) or ( local anesthesia [ all fields ] ) or ( local [ all fields ] and anesthetic [ all fields ] ) and ( steroids [ mesh terms ] or steroids [ all fields ] or steroid [ all fields ] ) . we did not impose any language limitation in the search strategy . reference lists of the included studies were also manually screened until no more potential studies could be found . studies that met the following selection criteria were included in this meta - analysis : 1 ) study design : rct ; 2 ) study subjects : patients diagnosed with spinal stenosis with radicular pain who had a history of chronic function - limiting low back pain and lower extremity pain ; 3 ) study intervention : patients in the study arm received epidural injections of local anesthetic with steroids , whereas patients in the control arm received local anesthetic alone ; 4 ) outcomes : pain relief , numeric rating scale ( nrs ) of the pain rating scores , functional improvement , oswestry disability index ( odi ) of functional ability , opioid intake , average number of injections per year , total relief per year , and weight changes . two independent investigators ( nan su and hai meng ) extracted the following information from each included study : first author , year of publication , number of patients ( intervention / control arms ) , baseline characteristics , number of patients with significant ( 50% ) pain relief or improvement in functional status in each group , average number of injections per year in each group , total relief time per year in each group , opioid intake , and weight changes . the methodological quality of all the included studies was assessed using the jadad scale.18 the scale consists of three items in the report of an rct . the quality scale ranges from 0 to 5 points , with a higher score indicating a better quality study . articles with a score of more than 2 are deemed to be of high quality.19 this meta - analysis was conducted based on the data from 13 randomized trials . pain relief and functional status improvement were treated as dichotomous variables and were expressed as a risk ratio ( rr ) with a 95% confidence interval ( ci ) . the nrs , odi , total relief per year , opioid intake , average number of injections per year , and weight change were treated as continuous variables and were expressed as a weight mean difference ( wmd ) with 95% ci . before the data were pooled , a cochrane q chi - square test and i statistic were used to test the heterogeneity between studies , in which a p - value of less than 0.1 or i value of more than 50% were considered indicators of significant heterogeneity.20 a fixed effects model was used to pool the estimates when substantial heterogeneity existed;21 otherwise a random effects model was preferred to summarize the data.22 the presence of publication bias was assessed by using the begg22 and egger test.23 a p - value < 0.05 was judged as statistically significant , except where otherwise specified . all analyses were performed using stata version 12.0 ( stata corporation , college station , tx , usa ) . we followed the preferred reported items for systematic and meta - analysis guideline ( prisma ) in this study.17 a comprehensive literature search was conducted to identify all the published rcts that compared epidural injection with or without steroids in the management of chronic low back pain in spinal stenosis . pubmed , embase , web of science , and cochrane library databases were searched from their inception through february 5 , 2015 . the following search terms were used : ( spinal stenosis [ mesh terms ] ) or ( spinal [ all fields ] and stenosis [ all fields ] ) or ( spinal stenosis [ all fields ] ) and ( injections , epidural [ mesh terms ] ) or ( injections [ all fields ] and epidural [ all fields ] ) or ( epidural injections [ all fields ] ) or ( epidural [ all fields ] and injection [ all fields ] ) or ( epidural injection [ all fields ] ) and ( anesthetics , local [ pharmacological action ] or anesthetics , local [ mesh terms ] ) or ( anesthetics [ all fields ] and local[all fields ] ) or ( local anesthetics [ all fields ] ) or ( local [ all fields ] and anesthetic [ all fields ] ) or ( local anesthetic [ all fields ] ) or ( anesthesia , local [ mesh terms ] ) or ( anesthesia [ all fields ] and local [ all fields ] ) or ( local anesthesia [ all fields ] ) or ( local [ all fields ] and anesthetic [ all fields ] ) and ( steroids [ mesh terms ] or steroids [ all fields ] or steroid [ all fields ] ) . we did not impose any language limitation in the search strategy . reference lists of the included studies were also manually screened until no more potential studies could be found . studies that met the following selection criteria were included in this meta - analysis : 1 ) study design : rct ; 2 ) study subjects : patients diagnosed with spinal stenosis with radicular pain who had a history of chronic function - limiting low back pain and lower extremity pain ; 3 ) study intervention : patients in the study arm received epidural injections of local anesthetic with steroids , whereas patients in the control arm received local anesthetic alone ; 4 ) outcomes : pain relief , numeric rating scale ( nrs ) of the pain rating scores , functional improvement , oswestry disability index ( odi ) of functional ability , opioid intake , average number of injections per year , total relief per year , and weight changes . two independent investigators ( nan su and hai meng ) extracted the following information from each included study : first author , year of publication , number of patients ( intervention / control arms ) , baseline characteristics , number of patients with significant ( 50% ) pain relief or improvement in functional status in each group , average number of injections per year in each group , total relief time per year in each group , opioid intake , and weight changes . the methodological quality of all the included studies was assessed using the jadad scale.18 the scale consists of three items in the report of an rct . the quality scale ranges from 0 to 5 points , with a higher score indicating a better quality study . pain relief and functional status improvement were treated as dichotomous variables and were expressed as a risk ratio ( rr ) with a 95% confidence interval ( ci ) . the nrs , odi , total relief per year , opioid intake , average number of injections per year , and weight change were treated as continuous variables and were expressed as a weight mean difference ( wmd ) with 95% ci . before the data were pooled , a cochrane q chi - square test and i statistic were used to test the heterogeneity between studies , in which a p - value of less than 0.1 or i value of more than 50% were considered indicators of significant heterogeneity.20 a fixed effects model was used to pool the estimates when substantial heterogeneity existed;21 otherwise a random effects model was preferred to summarize the data.22 the presence of publication bias was assessed by using the begg22 and egger test.23 a p - value < 0.05 was judged as statistically significant , except where otherwise specified . all analyses were performed using stata version 12.0 ( stata corporation , college station , tx , usa ) . the initial search yielded 586 studies from the three databases ( table 1 ) , of which 376 were excluded because of duplicate records . after the title and abstract review , 195 were excluded , leaving 15 for the full - text review . of those remaining , two studies were excluded because one was a study protocol,24 and the other25 contained data redundant with that in a later , completed version of the study . ultimately , 13 rcts with a total of 1,465 patients that met the inclusion criteria were included in this meta - analysis.8,9,2636 a flowchart of the literature search strategy is shown in figure 1 . the main characteristics of the 13 rcts included in our analysis are summarized in table 2 . sample sizes in the included studies ranged from 19 to 400 . of the 13 studies , five8,2730 used the caudal route to administer anesthetic or steroids , three9,35,36 used an interlaminar route , two used a transforaminal route , and the remaining three31,33,34 did not report the administration route . in majority of the studies , the nrs was used to evaluate pain rating scores , and the odi was used to measure functional ability , with lower scores indicating less severe symptoms . a 50% or greater reduction in nrs or odi scores the median jadad score of the included studies was 4 ( range from 4 to 5 ) . pain relief data were reported in nine studies.8,9,2630,33,36 pooling these rcts , significant pain relief was demonstrated in 51.5% of patients in both groups at 3 months , 56.6% in both groups at 6 months , and 53.7% in group 1 and 56.4% in group 2 at 12 months . meta - analysis of nine rcts using a fixed effects model showed that there were no significant differences between the two groups in terms of pain relief at 3 months ( rr = 1.0 , 95% ci : 0.9 , 1.2 ; p=0.975 ) , 6 months ( rr = 1.0 , 95% ci : 0.9 , 1.1 ; p=1.000 ) , or 12 months ( rr = 1.0 , 95% ci : 0.9 , 1.2 ; p=1.000 ) ( figure 2 ) . data of nrs pain scores were available from ten studies.8,9,2631,33,35 pooled estimates indicated that in group 1 , patients at 3 , 6 , and 12 months had a 5.3 score reduction ( wmd = 5.3 , 95% ci : 6.7 , 3.8 ; p=0.000 ) , 4.0 score reduction ( wmd = 4.0 , 95% ci : 5.0 , 3.0 ; p=0.000 ) , and 3.7 score reduction ( wmd = 3.7 , 95% ci : 4.0 , 3.4 ; p=0.000 ) , respectively ; whereas , patients in group 2 had a 4.9 score reduction ( wmd = 4.9 , 95% ci : 6.4 , 3.3 ; p=0.000 ) , 4.5 score reduction ( wmd = 4.5 , 95% ci : 5.8 , 3.1 ; p=0.000 ) , and 3.6 score reduction ( wmd = 3.6 , 95% ci : 4.4 , 2.8 ; p=0.000 ) , respectively . however , there were no significant differences in terms of nrs pain scores between the two groups at 3 months ( wmd = 0.2 , 95% ci : 0.8 , 0.5 ; p=0.586 ) , 6 months ( wmd = 0.2 , 95% ci : 1.0 , 0.6 ; p=0.607 ) , or 12 months ( wmd = 0.3 , 95% ci : 1.3 , 0.6 ; p=0.490 ) ( figure 3 ) . seven studies reported data on functional assessment.8,9,2630 the pooled estimates showed that in group 1 and group 2 , 42.7% and 40.3% of patients at 3 months , 58.5% and 60.0% of patients at 6 months , and 65.2% and 63.1% of patients at 12 months , respectively , achieved a significant functional improvement . however , no significant differences of functional improvement were found between the two groups at 3 months ( rr = 1.0 , 95% ci : 0.9 , 1.2 ; p=0.669 ) , 6 months ( rr = 1.0 , 95% ci : 0.9 , 1.2 ; p=1.000 ) , or 12 months ( rr = 1.0 , 95% ci : 0.8 , 1.1 ; p=0.761 ) ( figure 4 ) . data on odi were available from ten studies.8,9,2632,34 pooled estimates suggest that in group 1 , patients at 3 , 6 , and 12 months had a 13.2 point reduction ( wmd = 13.2 , 95% ci : 18.6 , 7.7 ; p=0.000 ) , 12.1 point reduction ( wmd = 12.1 , 95% ci : 19.5 , 4.8 ; p=0.001 ) , and 13.8 point reduction ( wmd = 13.8 , 95% ci : 16.2 , 11.5 ; p=0.000 ) , respectively ; whereas , patients in group 2 had a 12.9 point reduction ( wmd = 12.9 , 95% ci : 18.6 , 7.3 ; p=0.000 ) , 12.8 point reduction ( wmd = 12.8 , 95% ci : 20.2 , 5.3 ; p=0.001 ) , and 14.5 point reduction ( wmd = 14.5 , 95% ci : 16.1 , 12.8 ; p=0.000 ) , respectively . however , there were no significant differences in terms of odi between the two groups at 3 months ( wmd = 0.1 , 95% ci : 0.2 , 0.0 ; p=0.096 ) , 6 months ( wmd = 0.1 , 95% ci : 0.0 , 0.2 ; p=0.123 ) , or 12 months ( wmd = 0.3 , 95% ci : 1.0 , 0.4 ; p=0.439 ) ( figure 5 ) . six studies provided data on opioid intake.8,9,2730 significant reductions from baseline were observed in group 1 , by 11.8 mg ( wmd = 11.8 , 95% ci : 18.0 , 5.6 ; p= 0.000 ) , 12.3 mg ( wmd = 12.3 , 95% ci : 18.6 , 6.0 ; p=0.001 ) , and 12.4 mg ( wmd = 12.4 , 95% ci : 18.7 , 6.0 ; p=0.000 ) at 3 , 6 , and 12 months , respectively ; whereas in group 2 , reductions were 8.4 mg ( wmd = 8.4 , 95% ci : 14.0 , 2.9 ; p=0.000 ) , 8.2 mg ( wmd = 8.2 , 95% ci : 13.8 , 2.6 ; p=0.001 ) , and 7.8 mg ( wmd = 7.8 , 95% ci : 13.4 , 2.2 ; p=0.000 ) , respectively . no significant differences between the two groups were found at 3 months ( wmd = 0.0 , 95% ci : 5.0 , 4.9 ; p=0.096 ) , 6 months ( wmd = 0.7 , 95% ci : 3.7 , 5.0 ; p=0.123 ) , or 12 months ( wmd = 0.6 , 95% ci : 3.6 , 4.9 ; p=0.439 ) ( figure 6 ) . six studies reported data on the frequency of injections.8,9,2730 pooled estimates using a fixed effects model showed that there was no significant difference in the average number of injections per year between the two groups ( wmd = 0.2 , 95% ci : 0.4 , 0.0 ; p=0.099 ) . six studies reported data on total relief time.8,9,2730 pooled analysis using a fixed effects model indicated that patients in group 1 had 4.5 weeks of average total relief per year less than those in group 2 ( wmd = 4.5 , 95% ci : 8.2 , 0.7 ; p=0.019 ) . five studies reported data on changes in weight.8,9,27,29,30 pooled estimates using a fixed effects model showed that there was no significant difference in weight change between the two groups ( wmd = 1.7 , 95% ci : 0.2 , 3.5 ; p=0.077 ) . egger s and begg s tests were used to assess publication bias among the included studies . we found no potential publication bias according to either egger s ( t=0.6 , p=0.566 ) or begg s tests ( z=0.4 , p=0.700 ) . the initial search yielded 586 studies from the three databases ( table 1 ) , of which 376 were excluded because of duplicate records . after the title and abstract review , 195 were excluded , leaving 15 for the full - text review . of those remaining , two studies were excluded because one was a study protocol,24 and the other25 contained data redundant with that in a later , completed version of the study . ultimately , 13 rcts with a total of 1,465 patients that met the inclusion criteria were included in this meta - analysis.8,9,2636 a flowchart of the literature search strategy is shown in figure 1 . the main characteristics of the 13 rcts included in our analysis are summarized in table 2 . sample sizes in the included studies ranged from 19 to 400 . of the 13 studies , five8,2730 used the caudal route to administer anesthetic or steroids , three9,35,36 used an interlaminar route , two used a transforaminal route , and the remaining three31,33,34 did not report the administration route . in majority of the studies , the nrs was used to evaluate pain rating scores , and the odi was used to measure functional ability , with lower scores indicating less severe symptoms . a 50% or greater reduction in nrs or odi scores the median jadad score of the included studies was 4 ( range from 4 to 5 ) . pain relief data were reported in nine studies.8,9,2630,33,36 pooling these rcts , significant pain relief was demonstrated in 51.5% of patients in both groups at 3 months , 56.6% in both groups at 6 months , and 53.7% in group 1 and 56.4% in group 2 at 12 months . meta - analysis of nine rcts using a fixed effects model showed that there were no significant differences between the two groups in terms of pain relief at 3 months ( rr = 1.0 , 95% ci : 0.9 , 1.2 ; p=0.975 ) , 6 months ( rr = 1.0 , 95% ci : 0.9 , 1.1 ; p=1.000 ) , or 12 months ( rr = 1.0 , 95% ci : 0.9 , 1.2 ; p=1.000 ) ( figure 2 ) . data of nrs pain scores were available from ten studies.8,9,2631,33,35 pooled estimates indicated that in group 1 , patients at 3 , 6 , and 12 months had a 5.3 score reduction ( wmd = 5.3 , 95% ci : 6.7 , 3.8 ; p=0.000 ) , 4.0 score reduction ( wmd = 4.0 , 95% ci : 5.0 , 3.0 ; p=0.000 ) , and 3.7 score reduction ( wmd = 3.7 , 95% ci : 4.0 , 3.4 ; p=0.000 ) , respectively ; whereas , patients in group 2 had a 4.9 score reduction ( wmd = 4.9 , 95% ci : 6.4 , 3.3 ; p=0.000 ) , 4.5 score reduction ( wmd = 4.5 , 95% ci : 5.8 , 3.1 ; p=0.000 ) , and 3.6 score reduction ( wmd = 3.6 , 95% ci : 4.4 , 2.8 ; p=0.000 ) , respectively . however , there were no significant differences in terms of nrs pain scores between the two groups at 3 months ( wmd = 0.2 , 95% ci : 0.8 , 0.5 ; p=0.586 ) , 6 months ( wmd = 0.2 , 95% ci : 1.0 , 0.6 ; p=0.607 ) , or 12 months ( wmd = 0.3 , 95% ci : 1.3 , 0.6 ; p=0.490 ) ( figure 3 ) . seven studies reported data on functional assessment.8,9,2630 the pooled estimates showed that in group 1 and group 2 , 42.7% and 40.3% of patients at 3 months , 58.5% and 60.0% of patients at 6 months , and 65.2% and 63.1% of patients at 12 months , respectively , achieved a significant functional improvement . however , no significant differences of functional improvement were found between the two groups at 3 months ( rr = 1.0 , 95% ci : 0.9 , 1.2 ; p=0.669 ) , 6 months ( rr = 1.0 , 95% ci : 0.9 , 1.2 ; p=1.000 ) , or 12 months ( rr = 1.0 , 95% ci : 0.8 , 1.1 ; p=0.761 ) ( figure 4 ) . data on odi were available from ten studies.8,9,2632,34 pooled estimates suggest that in group 1 , patients at 3 , 6 , and 12 months had a 13.2 point reduction ( wmd = 13.2 , 95% ci : 18.6 , 7.7 ; p=0.000 ) , 12.1 point reduction ( wmd = 12.1 , 95% ci : 19.5 , 4.8 ; p=0.001 ) , and 13.8 point reduction ( wmd = 13.8 , 95% ci : 16.2 , 11.5 ; p=0.000 ) , respectively ; whereas , patients in group 2 had a 12.9 point reduction ( wmd = 12.9 , 95% ci : 18.6 , 7.3 ; p=0.000 ) , 12.8 point reduction ( wmd = 12.8 , 95% ci : 20.2 , 5.3 ; p=0.001 ) , and 14.5 point reduction ( wmd = 14.5 , 95% ci : 16.1 , 12.8 ; p=0.000 ) , respectively . however , there were no significant differences in terms of odi between the two groups at 3 months ( wmd = 0.1 , 95% ci : 0.2 , 0.0 ; p=0.096 ) , 6 months ( wmd = 0.1 , 95% ci : 0.0 , 0.2 ; p=0.123 ) , or 12 months ( wmd = 0.3 , 95% ci : 1.0 , 0.4 ; p=0.439 ) ( figure 5 ) . six studies provided data on opioid intake.8,9,2730 significant reductions from baseline were observed in group 1 , by 11.8 mg ( wmd = 11.8 , 95% ci : 18.0 , 5.6 ; p= 0.000 ) , 12.3 mg ( wmd = 12.3 , 95% ci : 18.6 , 6.0 ; p=0.001 ) , and 12.4 mg ( wmd = 12.4 , 95% ci : 18.7 , 6.0 ; p=0.000 ) at 3 , 6 , and 12 months , respectively ; whereas in group 2 , reductions were 8.4 mg ( wmd = 8.4 , 95% ci : 14.0 , 2.9 ; p=0.000 ) , 8.2 mg ( wmd = 8.2 , 95% ci : 13.8 , 2.6 ; p=0.001 ) , and 7.8 mg ( wmd = 7.8 , 95% ci : 13.4 , 2.2 ; p=0.000 ) , respectively . no significant differences between the two groups were found at 3 months ( wmd = 0.0 , 95% ci : 5.0 , 4.9 ; p=0.096 ) , 6 months ( wmd = 0.7 , 95% ci : 3.7 , 5.0 ; p=0.123 ) , or 12 months ( wmd = 0.6 , 95% ci : 3.6 , 4.9 ; p=0.439 ) ( figure 6 ) . six studies reported data on the frequency of injections.8,9,2730 pooled estimates using a fixed effects model showed that there was no significant difference in the average number of injections per year between the two groups ( wmd = 0.2 , 95% ci : 0.4 , 0.0 ; p=0.099 ) . six studies reported data on total relief time.8,9,2730 pooled analysis using a fixed effects model indicated that patients in group 1 had 4.5 weeks of average total relief per year less than those in group 2 ( wmd = 4.5 , 95% ci : 8.2 , 0.7 ; p=0.019 ) . five studies reported data on changes in weight.8,9,27,29,30 pooled estimates using a fixed effects model showed that there was no significant difference in weight change between the two groups ( wmd = 1.7 , 95% ci : 0.2 , 3.5 ; p=0.077 ) . egger s and begg s tests were used to assess publication bias among the included studies . we found no potential publication bias according to either egger s ( t=0.6 , p=0.566 ) or begg s tests ( z=0.4 , p=0.700 ) . to the best of our knowledge , this is the first meta - analysis to assess the effectiveness of epidural injections with or without steroids in the treatment of chronic pain related to spinal stenosis . our analysis of 13 rcts showed that significant pain relief ( 50% ) was demonstrated in 53.7% of patients in group 1 and 56.4% of patients in group 2 . patients showed a reduction in nrs pain score of 3.7 and 3.6 in the two groups , respectively . significant functional improvement was achieved in 65.2% and 63.1% of patients in groups 1 and 2 , with an odi reduction of 13.8 points and 14.5 points , respectively . the overall number of injections per year was 3.21.3 and 3.41.2 with average total relief per year of 29.319.7 and 33.819.3 weeks in groups 1 and 2 , respectively , and the opioid intakes decreased from baseline by 12.4 and 7.8 mg , respectively . however , there were no significant differences between the two groups in terms of the outcomes mentioned earlier except in the total relief time . it is challenging to treat the disabling pain associated with spinal stenosis , with or without surgery . however , no studies with the criteria of contemporary interventional pain management have been conducted to assess the conservative management approach used in the past . consequently , the most effective management strategy for spinal stenosis has not been still determined , even though the condition was first reported in 1954 , when it was characterized by the narrowing of spinal canal , neurogenic spinal claudication , and radicular pain.37 reports indicate that only a subgroup of patients has a good response to surgical intervention for spinal stenosis , whereas other subgroups of patients respond well to epidural injections.7,8 however , until now no studies could explicitly delineate the characteristics of these subgroups . the mechanisms by which epidural injections with steroids produce beneficial effects in pain relief and functional status improvement are not entirely known . it is hypothesized that the anti - inflammatory properties of steroids are responsible for the neural blockade.3841 emerging evidence demonstrates that local anesthetics may be as effective as steroids in the management of low back pain , without disc herniation originating at the facet joint , and in some other types of nerve block.4245 this was also verified in a recently published systematic review and meta - analysis.46 in that study , the authors summarized ten rcts to compare the effectiveness of epidural steroid injection to that of local anesthetic in patients with lumbar spinal stenosis . the pooled results showed minimal or no significant difference between the epidural steroid injection group and the local anesthetic injection group for short - term benefit , specifically , changes in leg pain vas score ( wmd = 7.00 , 95% ci : 12.73 , 1.27 ; p=0.02 ) , changes in back pain vas score ( wmd = 0.60 , 95% ci : 0.07 , 1.13 ; p=0.03 ) , and swiss spinal stenosis questionnaire ( sssq ) subscales for symptoms ( wmd = 0.2 , 95% ci : 0.34 , 0.06 ; p=0.05).46 therefore , the authors concluded that compared with local anesthetic , epidural steroid injection therapy provided no statistically significant improvement in pain symptoms or walking ability in lumbar spinal stenosis patients.46 on the basis of their report , patients with spinal stenosis would be expected to improve symptomatically with epidural injection of local anesthetic or with steroids . additionally , emphasis should be placed on the targeted delivery of the injectate to the site of pathology . it is reported that the use of blind approach to epidural procedures is the main reason for failed responses . according to the previous publications , inappropriate placement of drug because of inaccurate injection had occurred in 20%38% of the patients.4749 the use of fluoroscopy to guide epidural injections is therefore essential to ensure accurate and consistent drug placement at the target site . there are some limitations in this meta - analysis , which should be taken into account when interpreting our results . first , we acknowledge that the studies we included lack homogeneity in their approaches to epidural injection , as well as in dosing , injection frequency , and length of follow - up . second , our meta - analysis included several studies with relatively small numbers of patients ( n<50 in three studies ) , and in some the relevant data was incomplete . third , injection routes are important factors that influence the treatment effect of epidural injections with steroids , and there are debates about the effects of three epidural injection routes ( transforaminal epidural injection , caudal epidural injection , and interlaminar approach ) . because of limited reporting across the trials , we did not compare the effects or long - term effectiveness of epidural injection with steroids between the different injection routes . this study shows that epidural injections , with or without steroids , are effective in managing chronic low back pain secondary to spinal stenosis . however , the inclusion of steroids does not provide any additional benefit compared with injection of local anesthetic alone . therefore , on the basis of the current findings , there is a lack of evidence to support that the addition of steroids to epidural injections provides benefit in patients with spinal stenosis . further , large - scale , well - conducted rcts are required to clearly determine the effectiveness of epidural injections with steroids in the treatment of spinal stenosis .

backgroundepidural injections of anesthetic with or without steroids are widely used for treating lumbar spinal stenosis , a common cause of chronic low back pain , but there is a lack of rigorous data comparing the effectiveness of epidural injections of anesthetic with and without steroids . this meta - analysis presents a current , comprehensive picture of how epidural injections of anesthetic with steroids compare with those using local anesthetic alone.methodspubmed , embase , web of science , and cochrane library databases were searched from their inception through february 5 , 2015 . weight mean difference , risk ratio , and 95% confidence intervals were calculated . a random effects model or fixed effects model was used to pool the estimates , according to the heterogeneity between the included studies.resultswe included 13 randomized controlled trials , involving 1,465 patients . significant pain relief ( 50% ) was demonstrated in 53.7% of patients administered with epidural injections of anesthetic with steroids ( group 1 ) and in 56.4% of those administered with local anesthetic alone ( group 2 ) . patients showed a reduction in numeric rating scale pain score of 3.7 and 3.6 in the two groups , respectively . significant functional improvement was achieved in 65.2% of patients in group 1 and 63.1% of patients in group 2 , with oswestry disability index reductions of 13.8 and 14.5 points , respectively . the overall number of injections per year was 3.21.3 and 3.41.2 with average total relief per year of 29.319.7 and 33.819.3 weeks , respectively . the opioid intakes decreased from baseline by 12.4 and 7.8 mg , respectively . among the outcomes listed , only total relief time differed significantly between the two groups.conclusionboth epidural injections with steroids or with local anesthetic alone provide significant pain relief and functional improvement in managing chronic low back pain secondary to lumbar spinal stenosis , and the inclusion of steroids confers no advantage compared to local anesthetic alone .

Introduction Methods and materials Search strategy and study inclusion Data extraction and quality assessment Statistical analysis Results Identification of eligible studies Study characteristics and quality assessment Pain relief Functional assessment Opioid intake Therapeutic procedural characteristics Publication bias Discussion Conclusion

spinal stenosis is defined as a narrowing of spinal canal with encroachment on the neural structures by surrounding bone and soft issue.1 in the framingham study , prevalence of symptomatic lumbar spinal stenosis was estimated to be 27.2%.2 the three most common causes of low back and leg pain are spinal stenosis , disc herniation , and degenerative spondylolisthesis , for which surgery is usually performed.1,35 moreover , in patients older than 65 years , lumbar spinal stenosis has been regarded as the most frequent indication for spinal surgery.46 numerous modalities of treatment , including surgery with or without fusion , interventional techniques , and conservative approaches have been applied in managing lumbar central spinal stenosis.4,79 surgical interventions or epidural injections are commonly used for treatment of symptomatic spinal stenosis when conservative treatment fails.1,35,10,11 in the spine patient outcomes research trials ( sport ) , tosteson et al3 found that patients who received surgical intervention for spinal stenosis without degenerative spondylolisthesis achieved significant improvement in all primary outcomes compared to those undergoing nonsurgical treatment . consequently , apart from surgical intervention , epidural injections are the most commonly performed intervention in the management of chronic low back pain secondary to central spinal stenosis.13,14 manchikanti et al conducted a trial that compared the effect of epidural injections with or without steroids in the treatment of spinal stenosis.15 the authors found that 46% of the patients who were treated with local anesthetic and steroids achieved significant pain relief and an improvement in functional status of more than 50% , whereas the proportion of patients who improved after receiving only local anesthetic was 48%.15 in another trial , pain relief and improved functional status were observed in 73% of the patients who received both steroids and local anesthetic , and in 72% of those who received local anesthetic alone.16 no meta - analysis has yet been conducted to compare the effectiveness of epidural injections of local anesthetic with or without steroids in the treatment of patients with spinal stenosis , leaving the relative effectiveness of these treatments open to question . we followed the preferred reported items for systematic and meta - analysis guideline ( prisma ) in this study.17 a comprehensive literature search was conducted to identify all the published rcts that compared epidural injection with or without steroids in the management of chronic low back pain in spinal stenosis . pubmed , embase , web of science , and cochrane library databases were searched from their inception through february 5 , 2015 . studies that met the following selection criteria were included in this meta - analysis : 1 ) study design : rct ; 2 ) study subjects : patients diagnosed with spinal stenosis with radicular pain who had a history of chronic function - limiting low back pain and lower extremity pain ; 3 ) study intervention : patients in the study arm received epidural injections of local anesthetic with steroids , whereas patients in the control arm received local anesthetic alone ; 4 ) outcomes : pain relief , numeric rating scale ( nrs ) of the pain rating scores , functional improvement , oswestry disability index ( odi ) of functional ability , opioid intake , average number of injections per year , total relief per year , and weight changes . two independent investigators ( nan su and hai meng ) extracted the following information from each included study : first author , year of publication , number of patients ( intervention / control arms ) , baseline characteristics , number of patients with significant ( 50% ) pain relief or improvement in functional status in each group , average number of injections per year in each group , total relief time per year in each group , opioid intake , and weight changes . the nrs , odi , total relief per year , opioid intake , average number of injections per year , and weight change were treated as continuous variables and were expressed as a weight mean difference ( wmd ) with 95% ci . before the data were pooled , a cochrane q chi - square test and i statistic were used to test the heterogeneity between studies , in which a p - value of less than 0.1 or i value of more than 50% were considered indicators of significant heterogeneity.20 a fixed effects model was used to pool the estimates when substantial heterogeneity existed;21 otherwise a random effects model was preferred to summarize the data.22 the presence of publication bias was assessed by using the begg22 and egger test.23 a p - value < 0.05 was judged as statistically significant , except where otherwise specified . we followed the preferred reported items for systematic and meta - analysis guideline ( prisma ) in this study.17 a comprehensive literature search was conducted to identify all the published rcts that compared epidural injection with or without steroids in the management of chronic low back pain in spinal stenosis . pubmed , embase , web of science , and cochrane library databases were searched from their inception through february 5 , 2015 . studies that met the following selection criteria were included in this meta - analysis : 1 ) study design : rct ; 2 ) study subjects : patients diagnosed with spinal stenosis with radicular pain who had a history of chronic function - limiting low back pain and lower extremity pain ; 3 ) study intervention : patients in the study arm received epidural injections of local anesthetic with steroids , whereas patients in the control arm received local anesthetic alone ; 4 ) outcomes : pain relief , numeric rating scale ( nrs ) of the pain rating scores , functional improvement , oswestry disability index ( odi ) of functional ability , opioid intake , average number of injections per year , total relief per year , and weight changes . two independent investigators ( nan su and hai meng ) extracted the following information from each included study : first author , year of publication , number of patients ( intervention / control arms ) , baseline characteristics , number of patients with significant ( 50% ) pain relief or improvement in functional status in each group , average number of injections per year in each group , total relief time per year in each group , opioid intake , and weight changes . the nrs , odi , total relief per year , opioid intake , average number of injections per year , and weight change were treated as continuous variables and were expressed as a weight mean difference ( wmd ) with 95% ci . before the data were pooled , a cochrane q chi - square test and i statistic were used to test the heterogeneity between studies , in which a p - value of less than 0.1 or i value of more than 50% were considered indicators of significant heterogeneity.20 a fixed effects model was used to pool the estimates when substantial heterogeneity existed;21 otherwise a random effects model was preferred to summarize the data.22 the presence of publication bias was assessed by using the begg22 and egger test.23 a p - value < 0.05 was judged as statistically significant , except where otherwise specified . pain relief data were reported in nine studies.8,9,2630,33,36 pooling these rcts , significant pain relief was demonstrated in 51.5% of patients in both groups at 3 months , 56.6% in both groups at 6 months , and 53.7% in group 1 and 56.4% in group 2 at 12 months . data of nrs pain scores were available from ten studies.8,9,2631,33,35 pooled estimates indicated that in group 1 , patients at 3 , 6 , and 12 months had a 5.3 score reduction ( wmd = 5.3 , 95% ci : 6.7 , 3.8 ; p=0.000 ) , 4.0 score reduction ( wmd = 4.0 , 95% ci : 5.0 , 3.0 ; p=0.000 ) , and 3.7 score reduction ( wmd = 3.7 , 95% ci : 4.0 , 3.4 ; p=0.000 ) , respectively ; whereas , patients in group 2 had a 4.9 score reduction ( wmd = 4.9 , 95% ci : 6.4 , 3.3 ; p=0.000 ) , 4.5 score reduction ( wmd = 4.5 , 95% ci : 5.8 , 3.1 ; p=0.000 ) , and 3.6 score reduction ( wmd = 3.6 , 95% ci : 4.4 , 2.8 ; p=0.000 ) , respectively . seven studies reported data on functional assessment.8,9,2630 the pooled estimates showed that in group 1 and group 2 , 42.7% and 40.3% of patients at 3 months , 58.5% and 60.0% of patients at 6 months , and 65.2% and 63.1% of patients at 12 months , respectively , achieved a significant functional improvement . data on odi were available from ten studies.8,9,2632,34 pooled estimates suggest that in group 1 , patients at 3 , 6 , and 12 months had a 13.2 point reduction ( wmd = 13.2 , 95% ci : 18.6 , 7.7 ; p=0.000 ) , 12.1 point reduction ( wmd = 12.1 , 95% ci : 19.5 , 4.8 ; p=0.001 ) , and 13.8 point reduction ( wmd = 13.8 , 95% ci : 16.2 , 11.5 ; p=0.000 ) , respectively ; whereas , patients in group 2 had a 12.9 point reduction ( wmd = 12.9 , 95% ci : 18.6 , 7.3 ; p=0.000 ) , 12.8 point reduction ( wmd = 12.8 , 95% ci : 20.2 , 5.3 ; p=0.001 ) , and 14.5 point reduction ( wmd = 14.5 , 95% ci : 16.1 , 12.8 ; p=0.000 ) , respectively . six studies provided data on opioid intake.8,9,2730 significant reductions from baseline were observed in group 1 , by 11.8 mg ( wmd = 11.8 , 95% ci : 18.0 , 5.6 ; p= 0.000 ) , 12.3 mg ( wmd = 12.3 , 95% ci : 18.6 , 6.0 ; p=0.001 ) , and 12.4 mg ( wmd = 12.4 , 95% ci : 18.7 , 6.0 ; p=0.000 ) at 3 , 6 , and 12 months , respectively ; whereas in group 2 , reductions were 8.4 mg ( wmd = 8.4 , 95% ci : 14.0 , 2.9 ; p=0.000 ) , 8.2 mg ( wmd = 8.2 , 95% ci : 13.8 , 2.6 ; p=0.001 ) , and 7.8 mg ( wmd = 7.8 , 95% ci : 13.4 , 2.2 ; p=0.000 ) , respectively . six studies reported data on the frequency of injections.8,9,2730 pooled estimates using a fixed effects model showed that there was no significant difference in the average number of injections per year between the two groups ( wmd = 0.2 , 95% ci : 0.4 , 0.0 ; p=0.099 ) . six studies reported data on total relief time.8,9,2730 pooled analysis using a fixed effects model indicated that patients in group 1 had 4.5 weeks of average total relief per year less than those in group 2 ( wmd = 4.5 , 95% ci : 8.2 , 0.7 ; p=0.019 ) . pain relief data were reported in nine studies.8,9,2630,33,36 pooling these rcts , significant pain relief was demonstrated in 51.5% of patients in both groups at 3 months , 56.6% in both groups at 6 months , and 53.7% in group 1 and 56.4% in group 2 at 12 months . meta - analysis of nine rcts using a fixed effects model showed that there were no significant differences between the two groups in terms of pain relief at 3 months ( rr = 1.0 , 95% ci : 0.9 , 1.2 ; p=0.975 ) , 6 months ( rr = 1.0 , 95% ci : 0.9 , 1.1 ; p=1.000 ) , or 12 months ( rr = 1.0 , 95% ci : 0.9 , 1.2 ; p=1.000 ) ( figure 2 ) . data of nrs pain scores were available from ten studies.8,9,2631,33,35 pooled estimates indicated that in group 1 , patients at 3 , 6 , and 12 months had a 5.3 score reduction ( wmd = 5.3 , 95% ci : 6.7 , 3.8 ; p=0.000 ) , 4.0 score reduction ( wmd = 4.0 , 95% ci : 5.0 , 3.0 ; p=0.000 ) , and 3.7 score reduction ( wmd = 3.7 , 95% ci : 4.0 , 3.4 ; p=0.000 ) , respectively ; whereas , patients in group 2 had a 4.9 score reduction ( wmd = 4.9 , 95% ci : 6.4 , 3.3 ; p=0.000 ) , 4.5 score reduction ( wmd = 4.5 , 95% ci : 5.8 , 3.1 ; p=0.000 ) , and 3.6 score reduction ( wmd = 3.6 , 95% ci : 4.4 , 2.8 ; p=0.000 ) , respectively . seven studies reported data on functional assessment.8,9,2630 the pooled estimates showed that in group 1 and group 2 , 42.7% and 40.3% of patients at 3 months , 58.5% and 60.0% of patients at 6 months , and 65.2% and 63.1% of patients at 12 months , respectively , achieved a significant functional improvement . data on odi were available from ten studies.8,9,2632,34 pooled estimates suggest that in group 1 , patients at 3 , 6 , and 12 months had a 13.2 point reduction ( wmd = 13.2 , 95% ci : 18.6 , 7.7 ; p=0.000 ) , 12.1 point reduction ( wmd = 12.1 , 95% ci : 19.5 , 4.8 ; p=0.001 ) , and 13.8 point reduction ( wmd = 13.8 , 95% ci : 16.2 , 11.5 ; p=0.000 ) , respectively ; whereas , patients in group 2 had a 12.9 point reduction ( wmd = 12.9 , 95% ci : 18.6 , 7.3 ; p=0.000 ) , 12.8 point reduction ( wmd = 12.8 , 95% ci : 20.2 , 5.3 ; p=0.001 ) , and 14.5 point reduction ( wmd = 14.5 , 95% ci : 16.1 , 12.8 ; p=0.000 ) , respectively . six studies provided data on opioid intake.8,9,2730 significant reductions from baseline were observed in group 1 , by 11.8 mg ( wmd = 11.8 , 95% ci : 18.0 , 5.6 ; p= 0.000 ) , 12.3 mg ( wmd = 12.3 , 95% ci : 18.6 , 6.0 ; p=0.001 ) , and 12.4 mg ( wmd = 12.4 , 95% ci : 18.7 , 6.0 ; p=0.000 ) at 3 , 6 , and 12 months , respectively ; whereas in group 2 , reductions were 8.4 mg ( wmd = 8.4 , 95% ci : 14.0 , 2.9 ; p=0.000 ) , 8.2 mg ( wmd = 8.2 , 95% ci : 13.8 , 2.6 ; p=0.001 ) , and 7.8 mg ( wmd = 7.8 , 95% ci : 13.4 , 2.2 ; p=0.000 ) , respectively . six studies reported data on the frequency of injections.8,9,2730 pooled estimates using a fixed effects model showed that there was no significant difference in the average number of injections per year between the two groups ( wmd = 0.2 , 95% ci : 0.4 , 0.0 ; p=0.099 ) . six studies reported data on total relief time.8,9,2730 pooled analysis using a fixed effects model indicated that patients in group 1 had 4.5 weeks of average total relief per year less than those in group 2 ( wmd = 4.5 , 95% ci : 8.2 , 0.7 ; p=0.019 ) . to the best of our knowledge , this is the first meta - analysis to assess the effectiveness of epidural injections with or without steroids in the treatment of chronic pain related to spinal stenosis . our analysis of 13 rcts showed that significant pain relief ( 50% ) was demonstrated in 53.7% of patients in group 1 and 56.4% of patients in group 2 . patients showed a reduction in nrs pain score of 3.7 and 3.6 in the two groups , respectively . significant functional improvement was achieved in 65.2% and 63.1% of patients in groups 1 and 2 , with an odi reduction of 13.8 points and 14.5 points , respectively . the overall number of injections per year was 3.21.3 and 3.41.2 with average total relief per year of 29.319.7 and 33.819.3 weeks in groups 1 and 2 , respectively , and the opioid intakes decreased from baseline by 12.4 and 7.8 mg , respectively . it is hypothesized that the anti - inflammatory properties of steroids are responsible for the neural blockade.3841 emerging evidence demonstrates that local anesthetics may be as effective as steroids in the management of low back pain , without disc herniation originating at the facet joint , and in some other types of nerve block.4245 this was also verified in a recently published systematic review and meta - analysis.46 in that study , the authors summarized ten rcts to compare the effectiveness of epidural steroid injection to that of local anesthetic in patients with lumbar spinal stenosis . the pooled results showed minimal or no significant difference between the epidural steroid injection group and the local anesthetic injection group for short - term benefit , specifically , changes in leg pain vas score ( wmd = 7.00 , 95% ci : 12.73 , 1.27 ; p=0.02 ) , changes in back pain vas score ( wmd = 0.60 , 95% ci : 0.07 , 1.13 ; p=0.03 ) , and swiss spinal stenosis questionnaire ( sssq ) subscales for symptoms ( wmd = 0.2 , 95% ci : 0.34 , 0.06 ; p=0.05).46 therefore , the authors concluded that compared with local anesthetic , epidural steroid injection therapy provided no statistically significant improvement in pain symptoms or walking ability in lumbar spinal stenosis patients.46 on the basis of their report , patients with spinal stenosis would be expected to improve symptomatically with epidural injection of local anesthetic or with steroids . this study shows that epidural injections , with or without steroids , are effective in managing chronic low back pain secondary to spinal stenosis .

the application of new technologies to identify protective antigens , to optimally present antigens to the immune system , and to manufacture vaccines using highly characterized , synthetic methods of production has greatly facilitated the discovery and development of new and improved vaccines.1 genetic vaccines involve direct immunization with rna or dna encoding the antigen(s ) of interest and have as one of their main advantages the simplicity and purity with which they can be produced.2 the use of nucleic acid - based vaccines to combine the benefits of in situ expression of antigens with the safety of inactivated and subunit vaccines has been a key advancement . upon their discovery more than 20 years ago , nucleic acid vaccines promised to be a safe and effective means to mimic immunization with a live organism vaccine , particularly for induction of t - cell immunity.3 gene - based vaccines are under development for a broad variety of applications , ranging from vaccines to immunotherapies for infectious diseases , cancer , autoimmune diseases , and allergy . nucleic acid vaccines have significant advantages over conventional vaccines,4 including : safety , since living organisms and potent adjuvants are not required ; effectiveness , since after immunization they express antigens in situ , mimicking a true infection ; induction of both b and t - cell responses ( including cytotoxic t lymphocytes ) ; specificity , since they induce the immune response only to the antigen of interest ; and relatively low production cost , high stability , ease of manipulation , and the possibility of expressing complex antigens such as transmembrane proteins.5 dna vaccines are bacterial plasmids constructed to express an encoded protein following in vivo administration and subsequent transfection of cells.6 initially , these plasmids were thought to function simply as a shuttle system for the gene , resulting in the in situ production of antigen ( for vaccines ) or therapeutic protein ( for gene therapy applications ) . since the initial demonstration that such a plasmid encoding a viral protein could result in both cellular and antibody immunity , as well as protection from viral challenge ( including cross - strain challenge by a strain of virus quite different from the strain from which the gene was cloned ) , the technology has been used for wide - ranging applications , from a laboratory tool to licensed veterinary vaccines , and is under development for a variety of human biomedical applications . besides dna , the naturally transient and cytosolically active messenger rna ( mrna ) molecules are seen as a possibly safer and more potent alternative to dna for gene vaccination . optimized mrna was demonstrated to be a potent gene vaccination vehicle when delivered naked , in liposomes , or coated on particles . mrna can also be utilized for transfecting dendritic cells ex vivo , which are then transferred back to the patient or directly administered in vivo . for instance , in a phase i clinical trial , it was shown that tumor - associated antigen - mrna - electroporated mature dendritic cells are capable of inducing tumor - associated antigen - t - cell responses in multiple myeloma patients after stem cell transplantation . moreover , vaccination was well tolerated with limited toxicity.7 the proof of concept of the utility of rna in vaccination was demonstrated when intramuscular injection of mrna in mice resulted in local production of an encoded protein8 and induction of immune responses against an encoded antigen.9 a large body of nonclinical data and a considerable number of clinical studies have demonstrated the excellent safety profile of mrna - based immunotherapeutic strategies , mainly in tumor vaccination.10 moreover , induction of target - specific immune responses has been established in vaccinated patients , albeit at weak levels in most cases . the use of rna in vaccination has been studied mainly with mrna or nonamplifying rna , and to a lesser extent , with self - amplifying rna generated with modified rna from virus . moreover , rna interference ( rnai ) has also been studied as a possible strategy in vaccination . rnai is a natural process based on complementarity between rna and its target mrna to cause destruction of the target.11 in a previous study , rnai - mediated chemokine receptor 5 ( ccr5 ) silencing prevented human immunodeficiency virus ( hiv ) infection in blt mice and , therefore , it may be useful as anti - hiv prophylaxis.12 the blockage of negative regulatory molecules on dendritic cells with rnai has also been studied to prevent and treat chronic virus infections.13 one of the highly potential applications for the clinical use of rnai is cancer vaccination ; the suppression of gene expression ( for instance , certain cytokines ) in dendritic cells has been shown to enhance their immunostimulatory capacity and to result in an enhanced antitumor response.14 gene targets for vaccination with rna include reporter genes,15 tumor antigens,16 viral antigens,17 and allergens.18 table 1 presents several diseases that can potentially be treated with rna vaccines . regardless of the encoded antigen , viruses with an rna genome are able to induce the innate immune response of the host.19 this means that certain rna molecules may cause a strong innate response that leads to induction of a broad and efficient adaptative immune response.20 for instance , double - stranded rna , normally not present in cells but synthesized during viral replication , is recognized by host cells as a signal to mount a strong immune response.21 it has been shown that induction of antigen - specific immunity can be achieved by administering rna vaccines through several routes , ie , intravenous , intradermal , subcutaneous , intranodal , and intrasplenic.1 additionally , mrna has also been used via intradermal application to induce its uptake by langerhans cells and dermal dendritic cells for further transport to the lymph node . although attention has been mainly focused on plasmid - based dna vaccines , use of rna also has advantages.5 first , the theoretical risk of vector integration into the host genome and subsequent malignant cell transformation is omitted . second , due to the relatively short half - life of the rna molecule , its expression is transient and , therefore , exposure to the antigen is more controlled . consequently , the risk when using tumor - associated antigen genes such as proto - oncogenes for immunization decreases . moreover , this transient expression minimizes the risk of induction of tolerance.17 third , rna must be released into the cytoplasm to be transcribed into the protein , rendering its application much safer.22 however , dna must be delivered into the nucleus for transcription into mrna , which is transported back to the cytoplasm to be translated ; therefore , dna must cross the nuclear membrane , a limiting step that is avoided with rna . several studies have shown that microinjections of plasmid dna into the cytoplasm of nondividing cells resulted in very low levels of gene expression , but direct intranuclear injection of the same number of plasmid dna copies led to efficient transfection.23 therefore , an additional advantage of rna - based vaccines is the possibility to transfect slow or nondividing cells , the nuclear membrane of which is much more difficult to cross than rapidly dividing cells.24 rna can be produced by a cell - free enzymatic transcription reaction , thus avoiding the use of micro - organisms or cultured cells in manufacturing , with associated quality and safety issues . this method enables simple downstream purification and very rapid and cost - effective manufacturing.25 lyophilization studies have shown that rna vaccines are not less stable than conventional vaccines that require a cold chain to be effective.26 therefore , mrna can be produced in large amounts and with good manufacturing practice quality , thus allowing further development of mrna - based therapies . in europe , all mrna - based therapies fall under the scope of the regulation for advanced therapy medicinal products.27,28 according to the european regulation,28 a gene therapy medicinal product is an active substance which contains or consists of a recombinant nucleic acid used in or administered to human beings ; therefore , mrna is categorized as gene therapy . in contrast , in the usa , mrna - based vaccination therapies are not considered as gene therapy since they do not imply the same risks as they are not capable of integrating into the genome.10 this means that , at least in the usa , clinical translation of mrna - based medicinal products is simpler and faster than that of dna - based therapies . in spite of the advantages of rna vaccines in comparison with those that are dna - based , few studies have explored approaches to increase the delivery and effectiveness of mrna vaccines in vivo . as a result , knowledge concerning the potency of nonviral delivery of mrna vaccines is scarce and data describing their immunological properties are largely lacking.17 clinical experience with rna vaccines is limited . there are some experiences in phase i / ii clinical vaccination trials with mrna that codes for tumor antigens;29,30 these studies have shown that the treatment is feasible and safe , although the clinical effectiveness for antitumor immunotherapy must be evaluated in further trials . self - replicating rna vectors ( termed rna replicons ) have been developed based on advancements in the molecular virology of single - stranded rna viruses . they are a form of a nucleic acid - based vaccine derived from either positive - strand or negative - strand rna viruses . the gene sequences encoding structural proteins in these rna viruses are replaced by mrna encoding antigens of interest as well as by the rna polymerase for rna replicon replication and transcription.31 the foreign genes inserted in place of the structural protein gene region , when introduced into cells , will replicate and express the heterologous genes , inducing synthesis of large amounts of the foreign gene product within the cell , reaching levels of 15%20% of total cell protein.32 self - amplifying rna can be regarded as disabled virus vectors that are capable of amplifying within the cytoplasm of host cells for a prolonged period but are unable to produce infectious progeny.33 they may be potentially more potent than mrna vaccines . figure 1 shows a schematic representation of a self - amplifying rna . as rna replicons encode gene sequences of structural proteins and an rna transcript form , their size is larger than that of conventional nonamplifying mrna.4 for vaccine application , the genes encoding the structural proteins are replaced by the antigen or antigens of interest . their production under special conditions ( eg , packaging cell lines ) leads to formation of single - round infectious particles carrying rnas encoding the antigens.34 in this way , transient high levels of antigen production can be achieved without the use of a live spreading viral infection . the most studied rna molecules of this class are derived from alphavirus,4 such as sindbis , semliki forest , and venezuelan equine encephalitis viruses , and from flaviviruses35 ( for instance , kunjin virus and yellow fever virus ) . a major distinction between the alphavirus and flavivirus replicons is their cytopathic nature ; alphavirus replicons are generally cytopathic and flavivirus replicons are generally noncytopathic , although whether cytopathicity is an advantage or disadvantage still remains unclear . in a previous study,35 semliki forest virus ( alphavirus ) and kunjin virus ( flavivirus ) , when delivered as naked rnas , elicited comparable cd8 + t - cell responses , but the semliki forest virus vectors elicited greater humoral responses to an encoded cytoplasmic antigen , - galactosidase . studies in major histocompatibility complex class ii - deficient mice revealed that neither vector could overcome the dependence of cd4 + t - cell help in development of humoral and cellular responses following immunization . these studies indicate that the distinct biology of the two replicon systems may differentially impact the adaptive immune response , and this may need to be considered when designing vaccination strategies . therefore , replicons derived from different rna viruses differ with regard to levels and duration of heterologous gene expression , allowing generation of a versatile toolbox for vaccine or gene therapy applications.36 as mrna , rna replicons are effective at eliciting antigen - specific humoral and cellular immune responses in animal models of infectious and noninfectious diseases.4 like dna vaccines and viral vectors , the rna vaccine candidate induces both cd4 + and cd8 + t - cell responses , as transfection of host cells can facilitate antigen presentation by major histocompatibility complex molecules and priming of naive t lymphocytes . self - amplifying rna vaccines have been successfully assayed with many different antigens as vaccine candidates , and have been shown to be potent in several animal species , including mice,5 nonhuman primates,37 and humans.38 in a phase i trial of an alphavirus replicon vaccine for cytomegalovirus in cytomegalovirus - seronegative adult volunteers , the vaccine was safe and induced neutralizing antibody and multifunctional t - cell responses against three cytomegalovirus antigens that are important targets for protective immunity.38 to the best of our knowledge , this has been the only clinical trial of self - amplifying mrna for vaccination . although the possibility of an infection is eliminated because of removal of the genes encoding structural proteins , more research on the potential for induction of a harmful infection is needed . on the other hand , this kind of vaccine is more complex than nonamplifying mrna vaccines , and their greater size and complexity make it difficult to design an efficient delivery system . a key challenge in realizing the broad potential of rna - based therapeutics is finding safe and effective delivery methods . unmodified rna is unstable in the bloodstream , can be immunogenic , and does not readily cross membranes to enter cells . therefore , chemical modifications and/or delivery material are required to bring rna to its site of action without adverse effects.39 in some applications , effective delivery of naked rnas , without a carrier , may be possible.40 however , systemic delivery to many tissues , including the liver , requires a vehicle to provide protection and to transport the rna to the cells of interest . therefore , the most important , and most difficult challenge in therapy using nucleic acids , including self - amplifying rna vaccines , is the issue of delivery.41 lately , there has been an important development of nonviral delivery systems for rnai,39 but the experience with mrna or self - amplifying rna is very limited . however , most of the systems studied for delivery of the former can be applied to the latter . ideally , an rna nanocarrier should provide protection from blood nucleases and extended blood circulation , which ultimately would increase the possibility of reaching the target tissue.42 the delivery system containing the rna must then be internalized by the target cell and , upon receptor - mediated endocytosis , must be able to escape from the endosomal compartment into the cell cytoplasm where the rna machinery is located , while avoiding degradation by lysosomal enzymes.43 further , delivery systems for the systemic administration of rna ought to be well tolerated upon administration . they should be safe , enabling the multiadministration treatment modalities required for improved clinical outcomes.44 from a manufacturing point of view , production of large batches with reproducible specifications is also desirable . a broad diversity of materials is under exploration to address the challenges of in vivo delivery , including viral and nonviral vectors . although viral vectors have been extensively studied for delivery of nucleic acids , safety ( immunogenicity and oncogenicity ) concerns limit their clinical application . nonviral vectors include inorganic particles , polymeric - based , cationic lipid - based , and peptide - based vectors , and physical methods such as electroporation , sonoporation , and magnetofection.41 despite the delivery success achieved by some of these carriers , advances are necessary to allow the fullest application of rna at the clinical level . self - amplifying rna vaccines have been delivered mainly as naked rna or with viral vectors . the practical utility of viral vectors , however , is limited by manufacturing considerations , cost - effectiveness , and potential adverse health effects.45 very limited data have been published on nonviral delivery of rna replicons , although it is a highly attractive approach . moreover , and as mentioned in the section self - amplifying rna vaccines , rna replicons usually have very large sizes ( 9 kb ) compared with mrna ( 2 kb ) , which adds an additional challenge to the development of drug delivery vehicles . lipid - based formulations have been increasingly recognized as one of the most promising delivery systems for rna due to their biocompatibility and their ease of large - scale production.45,46 cationic lipids have been widely studied as synthetic materials for delivery of rna . after mixing together , nucleic acids are condensed by cationic lipids to form lipid / nucleic acid complexes known as lipoplexes . these lipid complexes are able to protect genetic material from the action of nucleases and deliver it into cells by interacting with the negatively charged cell membrane.47 many research groups focus their work on the design of new cationic lipids as candidates for gene delivery,48 and some of them have been assayed in clinical trials.49 lipoplexes can be prepared by directly mixing positively charged lipids at physiological ph with negatively charged nucleic acids , so their preparation is simple . more complex lipid - based formulations including liposomes , nanoemulsions , and solid lipid nanoparticles , have also been studied in depth for nucleic acid delivery.47 to deliver rna efficiently , several limiting steps must be overcome , including dissociation from the nanocarrier , cellular uptake , endosomal escape , and appropriate intracellular disassembly.50 the relationship between the physicochemical properties of the nanocarrier ( particle size , rna entrapment , acid dissociation constant ) and biological barriers ( for instance , cellular uptake ) , and that between biological barriers and rna activity remain unclear.51 this lack of clarity is one of the reasons for poor in vitro in vivo translation . this type of analysis aids in the identification of meaningful structure function activity relationships , improves the in vitro screening process before in vivo use , and facilitates the future design of potent nanocarriers . in spite of that , significant progress has been made toward developing lipid - based systems for nucleic acids , such as immunostimulatory oligonucleotides , plasmid dna containing therapeutic genes , and antisense oligonucleotides and rna.4143 cationic lipids used in vitro and in vivo to prepare lipid - based formulations to deliver nucleic acids may have toxic effects , although there is little to no effect on organ function or tissue architecture if they are used at relatively low doses . however , acute inflammation and tissue damage may occur at higher doses.52 the toxic effect is due mainly to the cationic nature of the vector , although the biodegradability of the compound is also important.47 upon intravenous administration , positively charged nanoparticles interact with negatively charged serum proteins , forming aggregates that accumulate mainly in the lungs , liver , and spleen . moreover , lipoplexes often induce pulmonary toxicity associated with complement activation and inflammation.42 therefore , formulations for rna delivery must have adequate properties to minimize these effects . despite tremendous efforts to develop lipid - based formulations for drug delivery , only a few formulations are approved for clinical use , and none of them has included nucleic acids . efforts must be focused primarily on two areas:53 1 ) technical aspects , such as manufacturing strategies , development of techniques for reproducible formulations , large - scale production , and conjugation of targeting molecules ; and 2 ) novel concepts and approaches to accomplish on - demand release of active molecules from the lipid nanocarrier ( based on the unique properties of the assembly components of lipid - based nanocarriers ) . another factor that limits clinical application is the lack of well defined mandatory preclinical investigations for rna - based approaches . liposomes are colloidal lipid - based and surfactant - based delivery systems composed of a phospholipid bilayer surrounding an aqueous compartment . they may present as spherical vesicles and can range in size from 20 nm to a few microns . cationic lipid - based liposomes are able to complex with negatively charged nucleic acids via electrostatic interactions , resulting in complexes that offer biocompatibility , low toxicity , and the possibility of the large - scale production required for in vivo clinical applications.54 the lipid to rna ratio , and overall lipid concentration used in forming these complexes , are very important for efficient gene delivery and vary depending on applications . liposomes can fuse with the plasma membrane for uptake ; once inside the cell , the liposomes are processed via the endocytic pathway and the genetic material is then released from the endosome / carrier into the cytoplasm . compared with polymeric nanoparticles , liposomes have long been perceived as better drug delivery vehicles because of their superior biocompatibility , given that liposomes are basically analogs of biological membranes , and can be prepared from both natural and synthetic phospholipids.54 several studies have shown the usefulness of liposomes in delivery of mrna for vaccination . for instance , liposomes containing mrna encoding the influenza virus nucleoprotein induced anti - influenza cytotoxic t lymphocytes , which were identical to those obtained in vivo with infectious virus in terms of specificity , lysing both peptide - sensitized and virus - infected targets.9 intravenous delivery of mrna encapsulated into liposomes has been shown to be the most efficient route of vaccination , although intradermal and eventually subcutaneous injections could also induce immunity.22 in a recent study , a liposomal formulation loaded with mrna encoding tumor - associated antigens induced a dendritic cell - mediated antitumor response in vivo and a 35-fold decrease in the number of lung metastases when compared with a control group . these pulsed dendritic cells caused a slight proinflammatory response in mice which was balanced by a positive effect of the dendritic cell - induced antitumor cytokine response.55 liposomes can be combined with polymers to form lipopolyplexes , which are also useful for vaccination with nucleic acids . in a recent study , mice receiving systemic injections of a new tumor antigen mrna ( mart1 ) formulated in a polyethylene glycol ( peg)ylated derivative of histidylated polylysine and l - histidine-(n , n - di - n - hexadecylamine ) ethylamide liposomes were specifically and significantly protected against melanoma tumor progression.56 although many studies using liposomes for gene therapy have been published , it was not until recently that geall et al57 confirmed the delivery of self - amplifying rna via liposomes . in a model of respiratory syncytial virus infection , the authors showed that a self - amplifying rna vaccine formulated in liposomes potently induced neutralizing antibodies in cotton rats , as well as antigen - specific interferon--producing cd4 + and cd8 + t - cells in mice . these responses were comparable with or exceeded those elicited by rna delivered via viral particles or electroporation of plasmid dna and provided protection against subsequent respiratory syncytial virus infection . even though cationic lipid - based liposomes have shown good gene transfection ability and biocompatibility in vitro , and although a few therapeutic clinical trials are underway , the clinical applications of liposomes have been limited because of in vivo instability.58 it has been reported that intravenously injected liposome / dna complexes form large aggregates with blood components and that these aggregates become entrapped in the lung capillary bed.59 therefore , cationic lipid - based liposomes usually require addition of components to enhance their in vivo stability after exposure to blood components . for example , hydrophobic cholesterol , nonionic surfactants , or peg have been used to increase the physical stability of cationic lipid - based liposomes under such conditions.58 it has been reported that such stability - enhanced liposomes have much better transfection efficiency , especially under in vivo conditions.60 despite these drawbacks , liposomes are very promising systems for nonviral gene delivery . for example , allovectin-7 ( vical , san diego , ca , usa ) , a plasmid dna carrying major histocompatibility complex , class 1 , b ( hla - b ) and 2-microglobulin genes complexed with 1,2-dimyristyloxy - propyl-3-dimethyl - hydroxy ethyl ammonium bromide / dioleoylphosphatidylethanolamine ( dmrie / dope ) liposomes , has been assessed for safety and efficacy in phase i and ii clinical trials.61,62 however , further research is necessary to improve their shape , size , cytotoxicity , efficiency , and biocompatibility , which may yield an effective method of gene delivery in the future . nanoemulsions are thermodynamically stable isotropic systems in which two immiscible liquids ( water and oil ) are mixed to form a single phase by means of appropriate surfactants , with a droplet diameter in the approximate range of 0.5100 m.63 depending on the nature of the core , ie , water or oil , the emulsions can be water - in - oil or oil - in - water systems , respectively . since a nanoemulsion is a stable isotropic system , careful balance of the three phases is essential to achieve a thermodynamically stable state . nanoemulsions often have a negatively surfaced charge , so are not suitable for gene therapy . however , by using cationic surfactants or cationic lipids , it is possible to prepare cationic nanoemulsions . the extent of the electrostatic interaction between the nucleic acid and the nanoemulsion depends on the cationic lipid ( nature of the cationic polar head group and the acyl chains ) and the nucleic acid structures ( lengths and base composition).64 stearylamine , 1,2-dioleoyl - sn - glycero-3-trimethylammonium - propane ( dotap ) , and 3-[n-(n,n-dimethylaminoethane)-carbamoyl]cholesterol ( dc - cholesterol ) are examples of cationic surfactants used to prepare cationic nanoemulsions able to interact electrostatically with negatively charged nucleic acids.63 nevertheless , these cationic surfactants were also noted to exhibit toxicity . new cationic lipids , which have an aspartate or glutamate backbone , a lysine head - group , and two alkyl tails , yield efficient gene expression with lower cytotoxicity . in addition , amino acid - modified fatty acids , which mimic natural lipoamino acids , have been developed . for example , lauroyl - arginine methyl ester was reported to be a cationic surfactant with rich self - aggregation properties , biodegradability , and low toxicity , and can be used in food , pharmaceutical , and cosmetic applications.65 the colipid dope is extensively used because of its fusogenic properties to improve the ability of emulsions and cationic liposomes to transfect cells.66 this is partially explained by the fact that the amine group of dope interacts with dna phosphate groups , thus weakening the binding affinity between cationic lipids and dna . cationic nanoemulsions have been described as a promising strategy to improve transfection of nucleic acids to mammalian cells , protecting them against nuclease attack . for instance , bruxel et al67 prepared a cationic nanoemulsion with dotap as a delivery system for antisense oligonucleotides targeting malarial topoisomerase ii . the physical characteristics and serum - resistant properties of the nanoemulsion complexes indicate that cationic nanoemulsions could be a more efficient carrier system for nucleic acids and/or immunogene delivery than liposomes.66 the fact that transfection is efficient in the presence of serum is an important advantage . one of the reasons for the serum - resistant properties of the cationic lipid nanoemulsion may be the stability of the nanoemulsion / nucleic acid complex . in several in vivo studies , cationic nanoemulsions were found to be more suitable for gene delivery than liposomes because of the higher transfection and lower toxicity . compared with liposomes , emulsions also have the advantages of easy processing and low production costs.68 additionally , large - scale production of emulsions can be performed in a cost - effective and simple way using high - pressure homogenization.69 the introduction of nonionic surfactants with a branched peg head group , such as tween 80 , increments nanoemulsion stability , probably because of their steric hindrance and generation of a hydrophilic surface , which prevents physical aggregation of the nucleic acid / nanoemulsion complexes . moreover , due to their hydrophilicity and lack of aggregates , emulsions decorated with peg avoid protein adsorption . therefore , incorporation of peg derivatives into the cationic lipid nanoemulsions may prevent them from enzymatic degradation , resulting in prolonged circulation in the blood . further , nanoemulsions with a hydrophilic surfactant are taken up slowly by phagocytic cells.66 in spite of the advantages of nanoemulsions for the delivery of nucleic acids , only a few attempts have been made to use this new delivery system for rna , and all of them have been made with rnai . for instance , kaneda et al70 showed the potential application of cationic nanoemulsion prepared with dotap , dope , and cholesterol for delivery of small interfering rna . transfection complexes with a mean particle diameter of approximately 300 nm were able to suppress expression of upregulated vascular adhesion molecules by endothelial cells . to the best of our knowledge , no studies with mrna or self - amplifying rna have been published . solid lipid nanoparticles ( sln ) comprise a variety of systems with a particle diameter of 501,000 nm . they are colloidal particles made up of a relatively rigid biocompatible and biodegradable matrix of hydrophobic lipids that are solid at room and body temperatures . sln were developed in the early 1990s in an attempt to combine the advantages of solid particles , emulsions , and liposomes . on the one hand , solid particles protect their incorporated active compounds and are more flexible in modulating the release of these compounds . on the other hand , as with liposomes and emulsions , they are composed of nontoxic excipients , and their large - scale production may be easily accomplished.71 sln can be prepared by using high - pressure homogenization , so use of organic solvents is avoided ; this is an important advantage from the point of view of industrial production.72 moreover , sln possess very good stability,73 and they can be lyophilized.74 in a previous study reported by our group,75 we showed long permanence of the lipid nanocarriers in blood and tissues after intravenous administration to rats . the particle size , surface charge , and surfactant content of the nanoparticles affected their tissue biodistribution profile . although sln have undergone constant development over recent years as drug delivery systems , there is not much literature concerning their application in gene therapy . however , their capacity to transfect different cell lines has been demonstrated in vitro76,77 and in vivo.78,79 their ability to condense and protect genetic material , and their efficiency when entering cells , and once inside , to release dna or rna , make this nanoparticulate system an interesting vector for gene therapy.80,81 the suitability of sln for gene therapy depends on the ratio between the nanoparticles and the nucleic acid ; an equilibrium between the binding forces to achieve protection without hampering posterior release at the site of action is necessary.82 figure 2 shows an sln prepared by our group using precirol ato5 as the solid lipid and dotap and tween as surfactants . after intravenous administration , positively charged nanoparticles interact with negatively charged serum proteins , forming aggregates that accumulate mainly in the lungs , liver , and spleen.83 in order to avoid this problem , as with liposomes and emulsions , significant advances have been seen in the development of different types of peg - grafted sln.43 the hydrophilic nature of peg provides an aqueous shield around the nanoparticle surface , thus decreasing the extent of opsonization and subsequent recognition by macrophages of the mononuclear phagocytic system , that ultimately leads to an increase in nanoparticle blood residence time.43 peg has been used extensively by the pharmaceutical industry to improve the pharmacokinetic properties of different therapeutic agents and drug nanocarriers . in order to prolong circulation of the nanoparticles in blood , other strategies can be considered . for instance , in a previous study , sln were prepared with dextran and protamine , and their in vivo transfection capacity improved.78 the improved transfection profile was explained by the presence of negatively charged dextran on the nanoparticle surface . this polyanion - biocompatible polysaccharide hampers strong interactions with other components such as serum proteins,84 so may induce a longer circulation time of the vector in blood . moreover , the high condensation of the nucleic acid due to protamine , which contributes to nuclease resistance , may also result in an extended stay of the plasmid in the organism . for instance , sln can incorporate cell - penetrating peptides , such as the dimeric hiv-1 transactivator of transcription ( tat ) peptide85 or the synthetic sweet arrow peptide.86 cationic sln produced by microemulsion with compritol ato 888 as matrix lipid , pluronic f68 as surfactant , and dimethyldioctadecylammonium bromide as cationic lipid were able to protect mrna against enzymatic degradation . moreover , toxicity studies in a living organism ( eggs of the sea urchin paracentrotus lividus , an organism very sensitive to a number of chemical and environmental agents ) revealed that sln allows normal embryonic development , high viability , a regular cell cycle , and correct morphogenesis . the authors observed correct biodistribution of the rna , suggesting that the complexes allow an efficient transfer of mrna into the cell , thus maintaining its functionality.87 liposomes are colloidal lipid - based and surfactant - based delivery systems composed of a phospholipid bilayer surrounding an aqueous compartment . they may present as spherical vesicles and can range in size from 20 nm to a few microns . cationic lipid - based liposomes are able to complex with negatively charged nucleic acids via electrostatic interactions , resulting in complexes that offer biocompatibility , low toxicity , and the possibility of the large - scale production required for in vivo clinical applications.54 the lipid to rna ratio , and overall lipid concentration used in forming these complexes , are very important for efficient gene delivery and vary depending on applications . liposomes can fuse with the plasma membrane for uptake ; once inside the cell , the liposomes are processed via the endocytic pathway and the genetic material is then released from the endosome / carrier into the cytoplasm . compared with polymeric nanoparticles , liposomes have long been perceived as better drug delivery vehicles because of their superior biocompatibility , given that liposomes are basically analogs of biological membranes , and can be prepared from both natural and synthetic phospholipids.54 several studies have shown the usefulness of liposomes in delivery of mrna for vaccination . for instance , liposomes containing mrna encoding the influenza virus nucleoprotein induced anti - influenza cytotoxic t lymphocytes , which were identical to those obtained in vivo with infectious virus in terms of specificity , lysing both peptide - sensitized and virus - infected targets.9 intravenous delivery of mrna encapsulated into liposomes has been shown to be the most efficient route of vaccination , although intradermal and eventually subcutaneous injections could also induce immunity.22 in a recent study , a liposomal formulation loaded with mrna encoding tumor - associated antigens induced a dendritic cell - mediated antitumor response in vivo and a 35-fold decrease in the number of lung metastases when compared with a control group . these pulsed dendritic cells caused a slight proinflammatory response in mice which was balanced by a positive effect of the dendritic cell - induced antitumor cytokine response.55 liposomes can be combined with polymers to form lipopolyplexes , which are also useful for vaccination with nucleic acids . in a recent study , mice receiving systemic injections of a new tumor antigen mrna ( mart1 ) formulated in a polyethylene glycol ( peg)ylated derivative of histidylated polylysine and l - histidine-(n , n - di - n - hexadecylamine ) ethylamide liposomes were specifically and significantly protected against melanoma tumor progression.56 although many studies using liposomes for gene therapy have been published , it was not until recently that geall et al57 confirmed the delivery of self - amplifying rna via liposomes . in a model of respiratory syncytial virus infection , the authors showed that a self - amplifying rna vaccine formulated in liposomes potently induced neutralizing antibodies in cotton rats , as well as antigen - specific interferon--producing cd4 + and cd8 + t - cells in mice . these responses were comparable with or exceeded those elicited by rna delivered via viral particles or electroporation of plasmid dna and provided protection against subsequent respiratory syncytial virus infection . even though cationic lipid - based liposomes have shown good gene transfection ability and biocompatibility in vitro , and although a few therapeutic clinical trials are underway , the clinical applications of liposomes have been limited because of in vivo instability.58 it has been reported that intravenously injected liposome / dna complexes form large aggregates with blood components and that these aggregates become entrapped in the lung capillary bed.59 therefore , cationic lipid - based liposomes usually require addition of components to enhance their in vivo stability after exposure to blood components . for example , hydrophobic cholesterol , nonionic surfactants , or peg have been used to increase the physical stability of cationic lipid - based liposomes under such conditions.58 it has been reported that such stability - enhanced liposomes have much better transfection efficiency , especially under in vivo conditions.60 despite these drawbacks , liposomes are very promising systems for nonviral gene delivery . for example , allovectin-7 ( vical , san diego , ca , usa ) , a plasmid dna carrying major histocompatibility complex , class 1 , b ( hla - b ) and 2-microglobulin genes complexed with 1,2-dimyristyloxy - propyl-3-dimethyl - hydroxy ethyl ammonium bromide / dioleoylphosphatidylethanolamine ( dmrie / dope ) liposomes , has been assessed for safety and efficacy in phase i and ii clinical trials.61,62 however , further research is necessary to improve their shape , size , cytotoxicity , efficiency , and biocompatibility , which may yield an effective method of gene delivery in the future . nanoemulsions are thermodynamically stable isotropic systems in which two immiscible liquids ( water and oil ) are mixed to form a single phase by means of appropriate surfactants , with a droplet diameter in the approximate range of 0.5100 m.63 depending on the nature of the core , ie , water or oil , the emulsions can be water - in - oil or oil - in - water systems , respectively . since a nanoemulsion is a stable isotropic system , careful balance of the three phases is essential to achieve a thermodynamically stable state . nanoemulsions often have a negatively surfaced charge , so are not suitable for gene therapy . however , by using cationic surfactants or cationic lipids , it is possible to prepare cationic nanoemulsions . the extent of the electrostatic interaction between the nucleic acid and the nanoemulsion depends on the cationic lipid ( nature of the cationic polar head group and the acyl chains ) and the nucleic acid structures ( lengths and base composition).64 stearylamine , 1,2-dioleoyl - sn - glycero-3-trimethylammonium - propane ( dotap ) , and 3-[n-(n,n-dimethylaminoethane)-carbamoyl]cholesterol ( dc - cholesterol ) are examples of cationic surfactants used to prepare cationic nanoemulsions able to interact electrostatically with negatively charged nucleic acids.63 nevertheless , these cationic surfactants were also noted to exhibit toxicity . new cationic lipids , which have an aspartate or glutamate backbone , a lysine head - group , and two alkyl tails , yield efficient gene expression with lower cytotoxicity . in addition , amino acid - modified fatty acids , which mimic natural lipoamino acids , have been developed . for example , lauroyl - arginine methyl ester was reported to be a cationic surfactant with rich self - aggregation properties , biodegradability , and low toxicity , and can be used in food , pharmaceutical , and cosmetic applications.65 the colipid dope is extensively used because of its fusogenic properties to improve the ability of emulsions and cationic liposomes to transfect cells.66 this is partially explained by the fact that the amine group of dope interacts with dna phosphate groups , thus weakening the binding affinity between cationic lipids and dna . cationic nanoemulsions have been described as a promising strategy to improve transfection of nucleic acids to mammalian cells , protecting them against nuclease attack . for instance , bruxel et al67 prepared a cationic nanoemulsion with dotap as a delivery system for antisense oligonucleotides targeting malarial topoisomerase ii . the physical characteristics and serum - resistant properties of the nanoemulsion complexes indicate that cationic nanoemulsions could be a more efficient carrier system for nucleic acids and/or immunogene delivery than liposomes.66 the fact that transfection is efficient in the presence of serum is an important advantage . one of the reasons for the serum - resistant properties of the cationic lipid nanoemulsion may be the stability of the nanoemulsion / nucleic acid complex . in several in vivo studies , cationic nanoemulsions were found to be more suitable for gene delivery than liposomes because of the higher transfection and lower toxicity . compared with liposomes , emulsions also have the advantages of easy processing and low production costs.68 additionally , large - scale production of emulsions can be performed in a cost - effective and simple way using high - pressure homogenization.69 the introduction of nonionic surfactants with a branched peg head group , such as tween 80 , increments nanoemulsion stability , probably because of their steric hindrance and generation of a hydrophilic surface , which prevents physical aggregation of the nucleic acid / nanoemulsion complexes . moreover , due to their hydrophilicity and lack of aggregates , emulsions decorated with peg avoid protein adsorption . therefore , incorporation of peg derivatives into the cationic lipid nanoemulsions may prevent them from enzymatic degradation , resulting in prolonged circulation in the blood . further , nanoemulsions with a hydrophilic surfactant are taken up slowly by phagocytic cells.66 in spite of the advantages of nanoemulsions for the delivery of nucleic acids , only a few attempts have been made to use this new delivery system for rna , and all of them have been made with rnai . for instance , kaneda et al70 showed the potential application of cationic nanoemulsion prepared with dotap , dope , and cholesterol for delivery of small interfering rna . transfection complexes with a mean particle diameter of approximately 300 nm were able to suppress expression of upregulated vascular adhesion molecules by endothelial cells . to the best of our knowledge , no studies with mrna or self - amplifying rna have been published . solid lipid nanoparticles ( sln ) comprise a variety of systems with a particle diameter of 501,000 nm . they are colloidal particles made up of a relatively rigid biocompatible and biodegradable matrix of hydrophobic lipids that are solid at room and body temperatures . sln were developed in the early 1990s in an attempt to combine the advantages of solid particles , emulsions , and liposomes . on the one hand , solid particles protect their incorporated active compounds and are more flexible in modulating the release of these compounds . on the other hand , as with liposomes and emulsions , they are composed of nontoxic excipients , and their large - scale production may be easily accomplished.71 sln can be prepared by using high - pressure homogenization , so use of organic solvents is avoided ; this is an important advantage from the point of view of industrial production.72 moreover , sln possess very good stability,73 and they can be lyophilized.74 in a previous study reported by our group,75 we showed long permanence of the lipid nanocarriers in blood and tissues after intravenous administration to rats . the particle size , surface charge , and surfactant content of the nanoparticles affected their tissue biodistribution profile . although sln have undergone constant development over recent years as drug delivery systems , there is not much literature concerning their application in gene therapy . however , their capacity to transfect different cell lines has been demonstrated in vitro76,77 and in vivo.78,79 their ability to condense and protect genetic material , and their efficiency when entering cells , and once inside , to release dna or rna , make this nanoparticulate system an interesting vector for gene therapy.80,81 the suitability of sln for gene therapy depends on the ratio between the nanoparticles and the nucleic acid ; an equilibrium between the binding forces to achieve protection without hampering posterior release at the site of action is necessary.82 figure 2 shows an sln prepared by our group using precirol ato5 as the solid lipid and dotap and tween as surfactants . after intravenous administration , positively charged nanoparticles interact with negatively charged serum proteins , forming aggregates that accumulate mainly in the lungs , liver , and spleen.83 in order to avoid this problem , as with liposomes and emulsions , significant advances have been seen in the development of different types of peg - grafted sln.43 the hydrophilic nature of peg provides an aqueous shield around the nanoparticle surface , thus decreasing the extent of opsonization and subsequent recognition by macrophages of the mononuclear phagocytic system , that ultimately leads to an increase in nanoparticle blood residence time.43 peg has been used extensively by the pharmaceutical industry to improve the pharmacokinetic properties of different therapeutic agents and drug nanocarriers . in order to prolong circulation of the nanoparticles in blood , for instance , in a previous study , sln were prepared with dextran and protamine , and their in vivo transfection capacity improved.78 the improved transfection profile was explained by the presence of negatively charged dextran on the nanoparticle surface . this polyanion - biocompatible polysaccharide hampers strong interactions with other components such as serum proteins,84 so may induce a longer circulation time of the vector in blood . moreover , the high condensation of the nucleic acid due to protamine , which contributes to nuclease resistance , may also result in an extended stay of the plasmid in the organism . for instance , sln can incorporate cell - penetrating peptides , such as the dimeric hiv-1 transactivator of transcription ( tat ) peptide85 or the synthetic sweet arrow peptide.86 cationic sln produced by microemulsion with compritol ato 888 as matrix lipid , pluronic f68 as surfactant , and dimethyldioctadecylammonium bromide as cationic lipid were able to protect mrna against enzymatic degradation . moreover , toxicity studies in a living organism ( eggs of the sea urchin paracentrotus lividus , an organism very sensitive to a number of chemical and environmental agents ) revealed that sln allows normal embryonic development , high viability , a regular cell cycle , and correct morphogenesis . the authors observed correct biodistribution of the rna , suggesting that the complexes allow an efficient transfer of mrna into the cell , thus maintaining its functionality.87 vaccines based on nucleic acids ( both dna and rna ) have been broadly investigated for several years , but have not yet resulted in a commercial product for human use . self - amplifying rna vaccines have been shown to induce both humoral and cellular immune responses . ideally , the delivery vectors should be specific , effective , long - lasting , and safe . lipid nanoparticles , including liposomes and sln , are two of the most promising delivery systems for self - amplifying rna vaccines due to their biocompatibility and the ease of large - scale production . therefore , this approach could provide a potential generic platform for rapid development of potent and versatile vaccines .

self - amplifying rna or rna replicon is a form of nucleic acid - based vaccine derived from either positive - strand or negative - strand rna viruses . the gene sequences encoding structural proteins in these rna viruses are replaced by mrna encoding antigens of interest as well as by rna polymerase for replication and transcription . this kind of vaccine has been successfully assayed with many different antigens as vaccines candidates , and has been shown to be potent in several animal species , including mice , nonhuman primates , and humans . a key challenge to realizing the broad potential of self - amplifying vaccines is the need for safe and effective delivery methods . ideally , an rna nanocarrier should provide protection from blood nucleases and extended blood circulation , which ultimately would increase the possibility of reaching the target tissue . the delivery system must then be internalized by the target cell and , upon receptor - mediated endocytosis , must be able to escape from the endosomal compartment into the cell cytoplasm , where the rna machinery is located , while avoiding degradation by lysosomal enzymes . further , delivery systems for systemic administration ought to be well tolerated upon administration . they should be safe , enabling the multiadministration treatment modalities required for improved clinical outcomes and , from a developmental point of view , production of large batches with reproducible specifications is also desirable . in this review , the concept of self - amplifying rna vaccines and the most promising lipid - based delivery systems are discussed .

Nucleic acid vaccines Self-amplifying RNA vaccines Delivery systems for self-amplifying RNA Lipid-based formulations as vehicles for self-amplifying RNA Liposomes Nanoemulsions Solid lipid nanoparticles Conclusion

the application of new technologies to identify protective antigens , to optimally present antigens to the immune system , and to manufacture vaccines using highly characterized , synthetic methods of production has greatly facilitated the discovery and development of new and improved vaccines.1 genetic vaccines involve direct immunization with rna or dna encoding the antigen(s ) of interest and have as one of their main advantages the simplicity and purity with which they can be produced.2 the use of nucleic acid - based vaccines to combine the benefits of in situ expression of antigens with the safety of inactivated and subunit vaccines has been a key advancement . upon their discovery more than 20 years ago , nucleic acid vaccines promised to be a safe and effective means to mimic immunization with a live organism vaccine , particularly for induction of t - cell immunity.3 gene - based vaccines are under development for a broad variety of applications , ranging from vaccines to immunotherapies for infectious diseases , cancer , autoimmune diseases , and allergy . nucleic acid vaccines have significant advantages over conventional vaccines,4 including : safety , since living organisms and potent adjuvants are not required ; effectiveness , since after immunization they express antigens in situ , mimicking a true infection ; induction of both b and t - cell responses ( including cytotoxic t lymphocytes ) ; specificity , since they induce the immune response only to the antigen of interest ; and relatively low production cost , high stability , ease of manipulation , and the possibility of expressing complex antigens such as transmembrane proteins.5 dna vaccines are bacterial plasmids constructed to express an encoded protein following in vivo administration and subsequent transfection of cells.6 initially , these plasmids were thought to function simply as a shuttle system for the gene , resulting in the in situ production of antigen ( for vaccines ) or therapeutic protein ( for gene therapy applications ) . since the initial demonstration that such a plasmid encoding a viral protein could result in both cellular and antibody immunity , as well as protection from viral challenge ( including cross - strain challenge by a strain of virus quite different from the strain from which the gene was cloned ) , the technology has been used for wide - ranging applications , from a laboratory tool to licensed veterinary vaccines , and is under development for a variety of human biomedical applications . moreover , vaccination was well tolerated with limited toxicity.7 the proof of concept of the utility of rna in vaccination was demonstrated when intramuscular injection of mrna in mice resulted in local production of an encoded protein8 and induction of immune responses against an encoded antigen.9 a large body of nonclinical data and a considerable number of clinical studies have demonstrated the excellent safety profile of mrna - based immunotherapeutic strategies , mainly in tumor vaccination.10 moreover , induction of target - specific immune responses has been established in vaccinated patients , albeit at weak levels in most cases . the use of rna in vaccination has been studied mainly with mrna or nonamplifying rna , and to a lesser extent , with self - amplifying rna generated with modified rna from virus . rnai is a natural process based on complementarity between rna and its target mrna to cause destruction of the target.11 in a previous study , rnai - mediated chemokine receptor 5 ( ccr5 ) silencing prevented human immunodeficiency virus ( hiv ) infection in blt mice and , therefore , it may be useful as anti - hiv prophylaxis.12 the blockage of negative regulatory molecules on dendritic cells with rnai has also been studied to prevent and treat chronic virus infections.13 one of the highly potential applications for the clinical use of rnai is cancer vaccination ; the suppression of gene expression ( for instance , certain cytokines ) in dendritic cells has been shown to enhance their immunostimulatory capacity and to result in an enhanced antitumor response.14 gene targets for vaccination with rna include reporter genes,15 tumor antigens,16 viral antigens,17 and allergens.18 table 1 presents several diseases that can potentially be treated with rna vaccines . regardless of the encoded antigen , viruses with an rna genome are able to induce the innate immune response of the host.19 this means that certain rna molecules may cause a strong innate response that leads to induction of a broad and efficient adaptative immune response.20 for instance , double - stranded rna , normally not present in cells but synthesized during viral replication , is recognized by host cells as a signal to mount a strong immune response.21 it has been shown that induction of antigen - specific immunity can be achieved by administering rna vaccines through several routes , ie , intravenous , intradermal , subcutaneous , intranodal , and intrasplenic.1 additionally , mrna has also been used via intradermal application to induce its uptake by langerhans cells and dermal dendritic cells for further transport to the lymph node . several studies have shown that microinjections of plasmid dna into the cytoplasm of nondividing cells resulted in very low levels of gene expression , but direct intranuclear injection of the same number of plasmid dna copies led to efficient transfection.23 therefore , an additional advantage of rna - based vaccines is the possibility to transfect slow or nondividing cells , the nuclear membrane of which is much more difficult to cross than rapidly dividing cells.24 rna can be produced by a cell - free enzymatic transcription reaction , thus avoiding the use of micro - organisms or cultured cells in manufacturing , with associated quality and safety issues . they are a form of a nucleic acid - based vaccine derived from either positive - strand or negative - strand rna viruses . the gene sequences encoding structural proteins in these rna viruses are replaced by mrna encoding antigens of interest as well as by the rna polymerase for rna replicon replication and transcription.31 the foreign genes inserted in place of the structural protein gene region , when introduced into cells , will replicate and express the heterologous genes , inducing synthesis of large amounts of the foreign gene product within the cell , reaching levels of 15%20% of total cell protein.32 self - amplifying rna can be regarded as disabled virus vectors that are capable of amplifying within the cytoplasm of host cells for a prolonged period but are unable to produce infectious progeny.33 they may be potentially more potent than mrna vaccines . as rna replicons encode gene sequences of structural proteins and an rna transcript form , their size is larger than that of conventional nonamplifying mrna.4 for vaccine application , the genes encoding the structural proteins are replaced by the antigen or antigens of interest . therefore , replicons derived from different rna viruses differ with regard to levels and duration of heterologous gene expression , allowing generation of a versatile toolbox for vaccine or gene therapy applications.36 as mrna , rna replicons are effective at eliciting antigen - specific humoral and cellular immune responses in animal models of infectious and noninfectious diseases.4 like dna vaccines and viral vectors , the rna vaccine candidate induces both cd4 + and cd8 + t - cell responses , as transfection of host cells can facilitate antigen presentation by major histocompatibility complex molecules and priming of naive t lymphocytes . self - amplifying rna vaccines have been successfully assayed with many different antigens as vaccine candidates , and have been shown to be potent in several animal species , including mice,5 nonhuman primates,37 and humans.38 in a phase i trial of an alphavirus replicon vaccine for cytomegalovirus in cytomegalovirus - seronegative adult volunteers , the vaccine was safe and induced neutralizing antibody and multifunctional t - cell responses against three cytomegalovirus antigens that are important targets for protective immunity.38 to the best of our knowledge , this has been the only clinical trial of self - amplifying mrna for vaccination . on the other hand , this kind of vaccine is more complex than nonamplifying mrna vaccines , and their greater size and complexity make it difficult to design an efficient delivery system . a key challenge in realizing the broad potential of rna - based therapeutics is finding safe and effective delivery methods . therefore , chemical modifications and/or delivery material are required to bring rna to its site of action without adverse effects.39 in some applications , effective delivery of naked rnas , without a carrier , may be possible.40 however , systemic delivery to many tissues , including the liver , requires a vehicle to provide protection and to transport the rna to the cells of interest . therefore , the most important , and most difficult challenge in therapy using nucleic acids , including self - amplifying rna vaccines , is the issue of delivery.41 lately , there has been an important development of nonviral delivery systems for rnai,39 but the experience with mrna or self - amplifying rna is very limited . ideally , an rna nanocarrier should provide protection from blood nucleases and extended blood circulation , which ultimately would increase the possibility of reaching the target tissue.42 the delivery system containing the rna must then be internalized by the target cell and , upon receptor - mediated endocytosis , must be able to escape from the endosomal compartment into the cell cytoplasm where the rna machinery is located , while avoiding degradation by lysosomal enzymes.43 further , delivery systems for the systemic administration of rna ought to be well tolerated upon administration . they should be safe , enabling the multiadministration treatment modalities required for improved clinical outcomes.44 from a manufacturing point of view , production of large batches with reproducible specifications is also desirable . self - amplifying rna vaccines have been delivered mainly as naked rna or with viral vectors . moreover , and as mentioned in the section self - amplifying rna vaccines , rna replicons usually have very large sizes ( 9 kb ) compared with mrna ( 2 kb ) , which adds an additional challenge to the development of drug delivery vehicles . lipid - based formulations have been increasingly recognized as one of the most promising delivery systems for rna due to their biocompatibility and their ease of large - scale production.45,46 cationic lipids have been widely studied as synthetic materials for delivery of rna . more complex lipid - based formulations including liposomes , nanoemulsions , and solid lipid nanoparticles , have also been studied in depth for nucleic acid delivery.47 to deliver rna efficiently , several limiting steps must be overcome , including dissociation from the nanocarrier , cellular uptake , endosomal escape , and appropriate intracellular disassembly.50 the relationship between the physicochemical properties of the nanocarrier ( particle size , rna entrapment , acid dissociation constant ) and biological barriers ( for instance , cellular uptake ) , and that between biological barriers and rna activity remain unclear.51 this lack of clarity is one of the reasons for poor in vitro in vivo translation . in spite of that , significant progress has been made toward developing lipid - based systems for nucleic acids , such as immunostimulatory oligonucleotides , plasmid dna containing therapeutic genes , and antisense oligonucleotides and rna.4143 cationic lipids used in vitro and in vivo to prepare lipid - based formulations to deliver nucleic acids may have toxic effects , although there is little to no effect on organ function or tissue architecture if they are used at relatively low doses . efforts must be focused primarily on two areas:53 1 ) technical aspects , such as manufacturing strategies , development of techniques for reproducible formulations , large - scale production , and conjugation of targeting molecules ; and 2 ) novel concepts and approaches to accomplish on - demand release of active molecules from the lipid nanocarrier ( based on the unique properties of the assembly components of lipid - based nanocarriers ) . cationic lipid - based liposomes are able to complex with negatively charged nucleic acids via electrostatic interactions , resulting in complexes that offer biocompatibility , low toxicity , and the possibility of the large - scale production required for in vivo clinical applications.54 the lipid to rna ratio , and overall lipid concentration used in forming these complexes , are very important for efficient gene delivery and vary depending on applications . liposomes can fuse with the plasma membrane for uptake ; once inside the cell , the liposomes are processed via the endocytic pathway and the genetic material is then released from the endosome / carrier into the cytoplasm . for instance , liposomes containing mrna encoding the influenza virus nucleoprotein induced anti - influenza cytotoxic t lymphocytes , which were identical to those obtained in vivo with infectious virus in terms of specificity , lysing both peptide - sensitized and virus - infected targets.9 intravenous delivery of mrna encapsulated into liposomes has been shown to be the most efficient route of vaccination , although intradermal and eventually subcutaneous injections could also induce immunity.22 in a recent study , a liposomal formulation loaded with mrna encoding tumor - associated antigens induced a dendritic cell - mediated antitumor response in vivo and a 35-fold decrease in the number of lung metastases when compared with a control group . in a recent study , mice receiving systemic injections of a new tumor antigen mrna ( mart1 ) formulated in a polyethylene glycol ( peg)ylated derivative of histidylated polylysine and l - histidine-(n , n - di - n - hexadecylamine ) ethylamide liposomes were specifically and significantly protected against melanoma tumor progression.56 although many studies using liposomes for gene therapy have been published , it was not until recently that geall et al57 confirmed the delivery of self - amplifying rna via liposomes . in a model of respiratory syncytial virus infection , the authors showed that a self - amplifying rna vaccine formulated in liposomes potently induced neutralizing antibodies in cotton rats , as well as antigen - specific interferon--producing cd4 + and cd8 + t - cells in mice . even though cationic lipid - based liposomes have shown good gene transfection ability and biocompatibility in vitro , and although a few therapeutic clinical trials are underway , the clinical applications of liposomes have been limited because of in vivo instability.58 it has been reported that intravenously injected liposome / dna complexes form large aggregates with blood components and that these aggregates become entrapped in the lung capillary bed.59 therefore , cationic lipid - based liposomes usually require addition of components to enhance their in vivo stability after exposure to blood components . further , nanoemulsions with a hydrophilic surfactant are taken up slowly by phagocytic cells.66 in spite of the advantages of nanoemulsions for the delivery of nucleic acids , only a few attempts have been made to use this new delivery system for rna , and all of them have been made with rnai . the authors observed correct biodistribution of the rna , suggesting that the complexes allow an efficient transfer of mrna into the cell , thus maintaining its functionality.87 liposomes are colloidal lipid - based and surfactant - based delivery systems composed of a phospholipid bilayer surrounding an aqueous compartment . cationic lipid - based liposomes are able to complex with negatively charged nucleic acids via electrostatic interactions , resulting in complexes that offer biocompatibility , low toxicity , and the possibility of the large - scale production required for in vivo clinical applications.54 the lipid to rna ratio , and overall lipid concentration used in forming these complexes , are very important for efficient gene delivery and vary depending on applications . liposomes can fuse with the plasma membrane for uptake ; once inside the cell , the liposomes are processed via the endocytic pathway and the genetic material is then released from the endosome / carrier into the cytoplasm . for instance , liposomes containing mrna encoding the influenza virus nucleoprotein induced anti - influenza cytotoxic t lymphocytes , which were identical to those obtained in vivo with infectious virus in terms of specificity , lysing both peptide - sensitized and virus - infected targets.9 intravenous delivery of mrna encapsulated into liposomes has been shown to be the most efficient route of vaccination , although intradermal and eventually subcutaneous injections could also induce immunity.22 in a recent study , a liposomal formulation loaded with mrna encoding tumor - associated antigens induced a dendritic cell - mediated antitumor response in vivo and a 35-fold decrease in the number of lung metastases when compared with a control group . in a recent study , mice receiving systemic injections of a new tumor antigen mrna ( mart1 ) formulated in a polyethylene glycol ( peg)ylated derivative of histidylated polylysine and l - histidine-(n , n - di - n - hexadecylamine ) ethylamide liposomes were specifically and significantly protected against melanoma tumor progression.56 although many studies using liposomes for gene therapy have been published , it was not until recently that geall et al57 confirmed the delivery of self - amplifying rna via liposomes . in a model of respiratory syncytial virus infection , the authors showed that a self - amplifying rna vaccine formulated in liposomes potently induced neutralizing antibodies in cotton rats , as well as antigen - specific interferon--producing cd4 + and cd8 + t - cells in mice . even though cationic lipid - based liposomes have shown good gene transfection ability and biocompatibility in vitro , and although a few therapeutic clinical trials are underway , the clinical applications of liposomes have been limited because of in vivo instability.58 it has been reported that intravenously injected liposome / dna complexes form large aggregates with blood components and that these aggregates become entrapped in the lung capillary bed.59 therefore , cationic lipid - based liposomes usually require addition of components to enhance their in vivo stability after exposure to blood components . further , nanoemulsions with a hydrophilic surfactant are taken up slowly by phagocytic cells.66 in spite of the advantages of nanoemulsions for the delivery of nucleic acids , only a few attempts have been made to use this new delivery system for rna , and all of them have been made with rnai . however , their capacity to transfect different cell lines has been demonstrated in vitro76,77 and in vivo.78,79 their ability to condense and protect genetic material , and their efficiency when entering cells , and once inside , to release dna or rna , make this nanoparticulate system an interesting vector for gene therapy.80,81 the suitability of sln for gene therapy depends on the ratio between the nanoparticles and the nucleic acid ; an equilibrium between the binding forces to achieve protection without hampering posterior release at the site of action is necessary.82 figure 2 shows an sln prepared by our group using precirol ato5 as the solid lipid and dotap and tween as surfactants . self - amplifying rna vaccines have been shown to induce both humoral and cellular immune responses . ideally , the delivery vectors should be specific , effective , long - lasting , and safe . lipid nanoparticles , including liposomes and sln , are two of the most promising delivery systems for self - amplifying rna vaccines due to their biocompatibility and the ease of large - scale production .

a food allergy is an adverse health effect arising from a specific immune response that occurs reproducibly on exposure to a given food1 . food allergy is an important health concern as it affects both children and adults with increasing prevalence worldwide . estimates of food allergy 's true prevalence have been difficult to establish as most studies are based on self - report and not on established diagnosis via skin or blood testing and food challenge . in a meta - analysis by rona et al2 , self - reported prevalence of food allergy varied from 1.2 to 17 per cent for milk , 0.2 to 7 per cent for egg , 0 to 2 per cent for peanuts and fish , 0 to 10 per cent for shellfish , and 3 to 35 per cent for any food . prevalence in the united states was recently estimated at approximately 2.5 per cent for peanut , milk , egg , and/or shrimp when taking into account food specific ige levels finding an increased association with childhood age , male sex , and non - hispanic black race / ethnicity3 . along similar lines , a recent australian study of challenge proven food allergy in 12 month old children found a prevalence of 3 per cent for peanut , 8.9 per cent for egg and 0.8 per cent for sesame allergy4 . in asia , prevalence figures remain similar to those in the western world , despite the perception that allergy is less prevalent5 . given this continued increase in prevalence , the potentially fatal outcome of food - induced anaphylaxis and the lack of standardized therapy , food allergy demands further attention and study . though many adverse reactions to food exist , this review focuses solely on immunoglobulin e ( ige ) mediated food allergy . hallmark manifestations of ige - mediated food allergy include urticaria , angioedema , pruritus , difficulty in breathing , laryngeal oedema , vomiting , diarrhoea and/or hypotension . while cutaneous symptoms are the most common sign of reactions , up to 20 per cent of cases may not present with skin findings , especially as the patient ages , and , therefore , a lack of rash does not rule out anaphylaxis67 . these symptoms typically develop within minutes to two hours of ingestion of the food allergen and are reproducible upon each ingestion . the constellation of symptoms involving two or more organ symptoms leading to anaphylaxis can be fatal if not treated promptly . the goal of this review is to highlight the diagnosis , current management and evolving therapeutics for ige - mediated food allergy . though food allergy has been noted in the urbanized western world for sometime , it is becoming increasingly prevalent throughout the world . however , a few studies are published estimating prevalence data and there appears to be a difference in which allergens are more prevalent in each country . in the western world , cow 's milk and egg are the most common allergens in infants and young children8 . similar to the united states ( us ) and the united kingdom ( uk ) , in most of asia egg and milk remain the most common allergens in young children5 . however , in older children and adults , shellfish is the most common food allergen11 . yet in korea , wheat and buckwheat are the most common causes of food - induced anaphylaxis12 . allergens that may seem atypical in some areas of the world are widespread in certain countries like singapore where the delicacy bird 's nest soup is the most common food allergen13 . these differences likely are reflective of differences in allergen exposure , handling and processing , though differences may also be affected by environmental levels of exposure or genetic differences14 . a detailed and careful patient history is of utmost importance in the diagnosis of food allergy . a focused physical examination should be performed though is often unrevealing outside of reactions , asides from manifestations of potential co - morbid atopic conditions such as rhinitis and atopic dermatitis . all reports of food allergy should be confirmed as recommended by the national institute of allergy and infectious diseases ( niaid ) expert panel report published in 20101 as multiple studies demonstrate that much of presumed food allergy is in error15 . should the history be suggestive of a typical food reaction , confirmation with in vivo / in vitro testing is recommended . both skin prick testing ( spt ) and in vitro serum ige testing are used routinely in practice to confirm a food allergy once reported . unproven tests for food allergy include serum igg4 testing which can be performed to many foods , however , its presence is not known to play a role in the immunology of food allergy16 . spt : spt involves the introduction of the food protein in the superficial layer of the skin and requires the patient to be free of antihistamines . the testing is inexpensive and results are immediately available while the patient is in the office . positive testing alone by skin or blood testing , however , does not confirm food allergy as neither mode of testing has a high predictive value . though skin testing is quite sensitive , it is not very specific17 . a negative test mostly confirms a lack of ige - mediated allergy if performed correctly with histamine and saline control tests18 . presence of food specific ige without a suspicious reaction indicates sensitization , but is not always representative of a true allergy . however , larger wheal size on skin testing has been correlated with an increased likelihood of clinical allergy upon open food challenge19 . serum specific - ige testing : in vitro serum ige testing quantifies the amount of ige to the food - specific protein via an enzymatic assay . serum testing is readily available and does not require that the patient be free of antihistamines ; however , there is a higher cost and the need to obtain a blood sample . several commercial assays are available however , significant differences in the measured ige levels have been found between assays using identical serum samples20 analogous to the trends noted with skin prick testing , largely positive food - specific ige levels have been found to correlate with a likelihood of clinical reaction1821 . specialists have data at hand with levels that are > 95 per cent predictive of reaction among children for common allergens such as egg , peanut and milk2122 . in an effort to distinguish true allergy from sensitization new testing , termed component - resolved diagnostics ( crd ) , has been established . thus far , component testing has been noted to be particularly helpful in differentiating between asymptomatic sensitization and peanut allergy . positivity to storage proteins rara h 1 , rara h 2 , and rara h 3 are more associated with immediate , clinical reactions to peanut , whereas positivity to rara h 8 ( a bet v 1 , or birch pollen , homologue ) is more associated with a milder birch pollen related oral allergy syndrome24 . however , the clinical relevance is not yet well established for other food proteins and some studies show increase specificity with testing , but decreased sensitivity when compared to traditional testing20 . oral food challenges : a food allergy diagnosis is supported when reaction symptoms and testing correlate . to confirm the diagnosis of food allergy in some instances or to determine if tolerance to an allergen has been achieved ( i.e. a food allergy outgrown ) , an oral food challenge ( ofc ) may be performed . in an ofc , the offending food allergen is given to the patient in escalating doses either in an open or blinded fashion . typically , the challenge occurs in the clinic setting under direct observation so that possible reactions may be treated promptly . for the gold standard diagnosis of food allergy , the challenge should be performed in a double - blind placebo controlled fashion though this is costly and often difficult in practice . therefore , many allergy specialists choose to employ open feeding challenges . in a survey of american academy of asthma , allergy & immunology members practicing in the us , 85.5 per cent of allergists reported that they perform challenges , however , a very small number of allergists ( 5.6% ) performed more than 10 ofcs per month , while 70 per cent performed only 1 to 5 ofcs per month25 . the decision to have a patient undergo an ofc may be dependent on several factors including severity of past reactions , patient age , disease comorbidities and patient anxiety26 . these ofcs have been determined to be safe when performed on selected patients in the hands of experienced personnel and biphasic , or delayed / late phase reactions , have been noted to be rare in these instances27 . avoidance : once the diagnosis of a food allergy is established the patient should be instructed to completely avoid the allergen . unfortunately , avoidance is easier said than done and as hidden allergens pose a possible fatal risk , the patient should be provided with an emergency plan for medications and actions to be given should accidental ingestion occur . in a study of peanut allergic children in quebec , an annual incidence rate of accidental exposure was found to be 14.3 per cent with the majority of ingestions occurring at the patient 's home or at the home of a friend or relative28 . this was followed by a nationwide survey in canada finding an annual peanut accidental ingestion incidence of 12.5 per cent when excluding recently diagnosed individuals29 . while cutaneous contact with some allergens such as peanut can cause reactions , the majority of severe or systemic reactions must come from direct ingestion of the allergen3031 . therefore , the us has strict labelling laws for the eight most common food allergens when present as ingredients . however , reporting use of allergens on shared equipment and in processing are at the discretion of the manufacturer . among items declaring these advisory statement labels in the us , ford et al32 found that 5.3 per cent contained detectable residues of allergenic food ( ranging in concentration from 3222 ppm ) versus 1.9 per cent among products that lacked a precautionary statement . treatment of acute reactions : prompt administration of intramuscular epinephrine is the first - line therapy for management of food - induced anaphylaxis . epinephrine is classified by the world allergy organization ( wao ) as an essential medication for the treatment of anaphylaxis33 . injection of a dose of 0.01 mg / kg of 1:1,000 ( 1 mg / ml ) solution to a maximum of 0.5 mg in the lateral thigh ( vastus lateralis muscle ) is preferred and has been shown to achieve a faster , higher serum medication peak when compared to the administration in the deltoid muscle intramuscularly and to subcutaneous administration34 . milder , isolated , non - progressive cutaneous reactions may be treated simply with h1- receptor antagonists . these medications may help to relieve pruritus , hives , angioedema and conjunctivitis , yet in an anaphylactic reaction , these do not aid with upper airway obstruction , hypotension or shock and are , therefore , not a substitute for epinephrine33 . inhaled beta-2 adrenergic agonists have been used for the relief of cough , wheezing and shortness of breath in conjunction with epinephrine , but once again are not a substitute for epinephrine33 . other adjuncts include supplemental oxygen and fluids which may be considered after the administration of epinephrine depending on the patient 's symptoms . oral or intravascular corticosteroids are often administered during an acute reaction with the intent of preventing protracted or biphasic episodes of anaphylaxis . biphasic reactions are defined as a recurrence of reaction symptoms after the initial anaphylactic reaction has appeared to resolve and occur in 5 - 7 per cent of all anaphylactic reactions3536 . however , retrospective studies are discrepant in whether there is a difference in the rate of biphasic reaction with or without steroid dosing3536 . a cochrane review was unable to identify any quality , relevant studies with evidence for the use of corticosteroids in anaphylaxis and felt unable to either recommend or refute the use of corticosteroids in this instance37 . it has been noted that the time between onset of symptoms and the initial dose of epinephrine was significantly longer for those patients who did have biphasic reactions3638 . long - term outpatient recommendations : it is the current recommendation that once diagnosed with food allergy the individual avoids all traces of the offending food as well as possible contamination . patient education should focus on preventing accidental exposures and reading labels to aid in identifying allergenic foods . given that foods are the most common trigger of anaphylaxis in children , adolescents , and young adults33 , patients must receive instruction on emergency medications and managing home reactions . an emergency healthcare plan in a useful tool to relay this information if presented in a simple , clear manner39 . each patient diagnosed with a food allergy should be prescribed self - injectable epinephrine for use in an anaphylactic reaction . in addition , proper device use must be reviewed as one study indicated that although 86 per cent of parents of food allergic children carried the device , only one third were able to correctly administer the medication40 . in a prospective study of 122 food allergic children prescribed epinephrine , 69 per cent of parents were unable to use the device , did not have it available , or did not know when it should be administered41 . families were six times more likely to have received a practical demonstration on epinephrine autoinjector use if the prescribing doctor was a specialist as compared to a general practitioner and 17 times more likely if the physician was an allergy specialist41 . as reaction severity can not be predicted from prior reactions or testing , the importance of having self - injectable epinephrine on hand at all times must be imparted to the patient . risk factors for fatal and near fatal anaphylactic reactions include failure to have epinephrine available , history of prior severe reactions , known food allergy , adolescent age and most importantly asthma38 . comorbid asthma , especially poorly controlled asthma , is most often linked to severe reactions42 . multiple studies have demonstrated food allergy 's effect on quality of life ( qol ) measures . in particular , females with food allergy , those with a larger number of food allergies , history of more previous reactions and those with co - existing atopic diseases report poorer qol43 . it is to be noted that the prescribing of epinephrine autoinjectors has been found to reduce anxiety in both parents and children though this improvement in anxiety does not improve adherence in carrying the autoinjector44 . prevention : the american academy of pediatrics ( aap ) published updated recommendations in 2008 reviewing nutritional choices in pregnancy , lactation and in the first year of life which may affect development of atopic disease in infants and children45 . no definitive conclusions have been found regarding maternal dietary exposures during pregnancy and lactation contributing significantly to the development of food allergy in the infant . therefore , no adjustments to the maternal diet are recommended at this time . while current evidence does support exclusive breastfeeding in high risk infants for at least four months to decrease atopic dermatitis and cow milk allergy in the first two years of life , overall there is little literature supporting the role of breastfeeding in either preventing or delaying the onset of specific food allergies4546 . the recommendations note that there is modest evidence that atopic dermatitis may be delayed or prevented by the use of extensively or partially hydrolyzed formulas , compared with cow milk formula , in children unable to be breastfed for the first 4 to 6 months of life45 . yet , the data have not been impressive for the use in the prevention of food allergy . timing of the introduction of complementary or solid foods has also been pursued as a factor in the prevention of food allergy in children . however , there is no evidence supporting that delaying introduction beyond 4 to 6 months of age will affect atopy or development of food allergy . studies looking at early weaning and the development of food allergy in children have actually found a potential protective effect in early weaning . in one study , children introduced to solids at or after 16 wk of age were more likely to have food hypersensitivity and sensitization at one year of age than those weaned prior to 16 wk47 . more recent data also suggest that delaying the introduction of foods considered highly allergenic may in fact increase the incidence of allergy to these foods4849 . the most convincing evidence for early introduction of highly allergenic food proteins and possible oral tolerance comes from a study of the prevalence of peanut allergy in jewish children in the uk and israel . jewish children in the uk were found to have a 10-fold higher prevalence of peanut allergy than those in israel ( 1.85 and 0.17% , respectively ) where 69 per cent of children ingest peanut by the age of 9 months48 . the safest way recommended to introduce foods thought to be highly allergenic is at home , in gradually increasing amounts with a rate of introduction of one new food every 3 to 5 days50 . several trials underway are addressing this gap in the literature as to whether food introduction plays a role in primary prevention of food allergy including the starting time for allergy reduction ( star ) , learning early about peanut ( leap ) , preventing peanut allergy in atopic dermatitis ( peaad ) , starting time for egg protein ( step ) and beating egg allergy trials ( beat)50 . early results from the star trial indicate that a high proportion of high risk infants with eczema already have sensitization to foods as well as clinical reactivity prior to the introduction of solid foods at 4 to 5 months of age indicating the possible need for interventions prior to the introduction to solid foods to prevent food allergy51 . emerging therapeutics : the possibility of severe fatal reactions with accidental ingestions and lack of standard treatment has led to a strong push to find a viable therapy option for patients . several small studies have previously been published and now larger , multi - center trials are underway . however , caution must be used when interpreting study results as there is a difference in the outcomes of desensitization and tolerance . desensitization is a change in the threshold or amount of the allergen needed to induce a reaction whereas tolerance is achieved when one can ingest the allergen on an ad lib basis without reaction . tolerance is a long lasting immunity while with desensitization one must continue to ingest the allergen daily or the immunologic changes may be lost . allergen specific therapies include oral immunotherapy ( oit ) , sublingual immunotherapy ( slit ) and epicutaneous immunotherapy ( epit ) . oit involves introducing the food allergen in initial quite low oral doses , and typically escalating the dose over a day or two of rapid dose increase , followed by a slower incremental dose increase over weeks and months . varying oit protocols have been used in several small trials showing promise for desensitization to food allergens525354 . a multi - center double - blind , randomized , placebo controlled study of oit in egg allergic children showed both safety and efficacy with a desensitization rate of 75 per cent in the oit receiving subjects after 22 months of therapy52 . peanut oit has been a particular focus given that peanut is a major cause of food - induced anaphylaxis . however , a recent cochrane review of peanut oit trials found only one randomized control trial that resulted in desensitization , yet with a risk of clinically - significant adverse reactions55 . though many adverse events have been noted in the studies , especially in the more rapid dose escalation phases , no life threatening event or death has been reported thus far56 . however , a proportion of patients ( ~10 - 20% ) in each study appear to be unable to tolerate desensitization due to the side effects of therapy57 . the cochrane review authors note that due to the risk of adverse events and current lack of evidence of long - term benefits , peanut oit can not currently be recommended without further study55 . subsets of children who are reactive to unheated or lightly cooked egg and milk have been noted to have tolerance of items containing these allergens that are extensively heated . the food protein is thought to be denaturated , with the heat labile protein undergoing a conformational change secondary to the high heat of cooking rendering it to be non allergenic to some patients . studies have shown that introduction of extensively heated milk and egg appears to hasten the development of tolerance to the unheated food protein585960 . introduction of extensively heated egg and milk in tolerant children may have immunomodulatory benefit and may potentially be safe in inducing tolerance in the traditional oit61 . similar to oit , slit uses small escalating doses of the food allergen ; however , doses are given under the tongue via an extract vehicle . studies have shown slit thus far to be quite safe62 , yet concerns for its efficacy exist as slit is unable to achieve the high doses that appear to be necessary in oit to induce desensitization . the first multi - center , randomized , placebo - controlled study of peanut slit showed a modest desensitization at ofc after 44 wk of therapy with 14 of 20 subjects receiving peanut slit being able to consume at least a 10-fold increase in the amount of peanut powder they were able to consume when compared to their baseline ofc63 . the majority of adverse reactions to doses involved the oral - pharyngeal mucosa with only one subject receiving epinephrine at home for oral - pharyngeal symptoms that progressed to cough after antihistamine dosing63 . in a direct comparison study of slit versus oit for cow milk allergy , systemic reactions were more common with oit , however , oit was more efficacious than slit alone64 . however , slit remains an appealing therapy to study given it is less likely to cause serious adverse reactions . fewer trials so far , have evaluated the administration of allergen via the skin in a patch form in epit . in a pilot study using epit for the treatment of cow milk allergy , a trend to improvement was noted , but there was no significant increase in the cumulative total dose of cow milk tolerated after three months of therapy with only local reactions being noted65 . a phase ib trial of peanut epit in humans showed mostly local , cutaneous adverse events with no significant difference in systemic reactions between the epit and placebo groups68 . phase ii trials are ongoing ( clintrials.gov identifier : nct01675882 ) . therapies for food allergy in general that are not directed to a specific allergen are also in the midst of study . the food allergy herbal formula-2 ( fahf-2 ) is a capsule containing a chinese herbal formula found to abolish anaphylaxis in mice with peanut allergy69 . subsequent extended phase i trials in humans showed dosing of the nine herb formula to be both safe and well tolerated over a 6 month period70 . ( clintrials.gov identifiers : nct00602160 , nct01197053 ) . available from : http://clinicaltrials.gov/show/nct00602160 nlm identifiernct00602160 . the monoclonal anti - ige antibody omalizumab has also been found to be a potential non - specific allergen therapy . a multi - center , randomized , double - blind , placebo - controlled phase ii trial using omalizumab in the treatment of peanut allergy completed 14 of the planned 150 subjects prior to early discontinuation due to severe anaphylactic reactions during the qualifying ofcs71 . of the 14 completing the study , subjects receiving omalizumab trended in tolerating an increased amount of peanut protein following the 24 wk of therapy when compared to placebo71 . while the data are limited on using omalizumab as a single agent for the treatment of food allergy , focus has shifted to study if omalizumab has a role in oit , as an adjunct to limit side effects seen in oit dose escalation . a pilot study using omalizumab as an adjunct to cow milk oit demonstrated that omalizumab permitted rapid milk dose escalation in a majority of subjects72 . food allergy remains an important health concern due to increasing prevalence worldwide , potentially fatal reactions and current lack of curative therapy . importance must be placed on proper diagnosis which may at times be difficult given the limitations of current available testing . avoidance of the offending allergen and prompt treatment of acute reactions are the current mainstays of food allergy management . present data indicate that there is no benefit in delaying the introduction of allergenic foods to the diet of children . however , much still must be elucidated to understand the optimal timing of allergenic food introduction . while little headway has been made in the prevention of food allergy , multiple therapeutic options including both allergen specific and non - specific therapies are in human efficacy trials . though the initial results are promising , no single therapy is considered ready for common use until further data on optimal dosing and long - term safety are available .

ige - mediated food allergy is an important health concern with increasing prevalence worldwide . manifestations of ige - mediated food allergy include urticaria , angioedema , pruritus , difficulty in breathing , laryngeal oedema , vomiting , diarrhoea and/or hypotension within minutes to two hours of the offending food 's ingestion . diagnosis requires both a careful history and supportive testing with laboratory studies and possibly oral food challenges . current treatment of food allergy focuses on avoidance of the allergen and prompt emergency management of reactions . epinephrine autoinjectors are provided to patients for the treatment of severe reactions . more research is needed to determine the optimal timing with which to introduce common allergens into a child 's diet to possibly prevent the development of food allergy . novel therapies are under investigation given the difficulty of allergen avoidance and the potentially fatal nature of reactions . both allergen specific therapies such as oral , sublingual and epicutaneous immunotherapy and allergen non - specific therapies such the chinese herbal formula fahf-2 and omalizumab show promise though more data on efficacy and long - term safety are needed before these therapies become mainstream .

Introduction World experience Diagnosis Management Prevention and treatment Conclusion

a food allergy is an adverse health effect arising from a specific immune response that occurs reproducibly on exposure to a given food1 . food allergy is an important health concern as it affects both children and adults with increasing prevalence worldwide . estimates of food allergy 's true prevalence have been difficult to establish as most studies are based on self - report and not on established diagnosis via skin or blood testing and food challenge . in a meta - analysis by rona et al2 , self - reported prevalence of food allergy varied from 1.2 to 17 per cent for milk , 0.2 to 7 per cent for egg , 0 to 2 per cent for peanuts and fish , 0 to 10 per cent for shellfish , and 3 to 35 per cent for any food . prevalence in the united states was recently estimated at approximately 2.5 per cent for peanut , milk , egg , and/or shrimp when taking into account food specific ige levels finding an increased association with childhood age , male sex , and non - hispanic black race / ethnicity3 . along similar lines , a recent australian study of challenge proven food allergy in 12 month old children found a prevalence of 3 per cent for peanut , 8.9 per cent for egg and 0.8 per cent for sesame allergy4 . in asia , prevalence figures remain similar to those in the western world , despite the perception that allergy is less prevalent5 . given this continued increase in prevalence , the potentially fatal outcome of food - induced anaphylaxis and the lack of standardized therapy , food allergy demands further attention and study . though many adverse reactions to food exist , this review focuses solely on immunoglobulin e ( ige ) mediated food allergy . hallmark manifestations of ige - mediated food allergy include urticaria , angioedema , pruritus , difficulty in breathing , laryngeal oedema , vomiting , diarrhoea and/or hypotension . while cutaneous symptoms are the most common sign of reactions , up to 20 per cent of cases may not present with skin findings , especially as the patient ages , and , therefore , a lack of rash does not rule out anaphylaxis67 . these symptoms typically develop within minutes to two hours of ingestion of the food allergen and are reproducible upon each ingestion . the constellation of symptoms involving two or more organ symptoms leading to anaphylaxis can be fatal if not treated promptly . the goal of this review is to highlight the diagnosis , current management and evolving therapeutics for ige - mediated food allergy . though food allergy has been noted in the urbanized western world for sometime , it is becoming increasingly prevalent throughout the world . in the western world , cow 's milk and egg are the most common allergens in infants and young children8 . similar to the united states ( us ) and the united kingdom ( uk ) , in most of asia egg and milk remain the most common allergens in young children5 . yet in korea , wheat and buckwheat are the most common causes of food - induced anaphylaxis12 . allergens that may seem atypical in some areas of the world are widespread in certain countries like singapore where the delicacy bird 's nest soup is the most common food allergen13 . a detailed and careful patient history is of utmost importance in the diagnosis of food allergy . a focused physical examination should be performed though is often unrevealing outside of reactions , asides from manifestations of potential co - morbid atopic conditions such as rhinitis and atopic dermatitis . all reports of food allergy should be confirmed as recommended by the national institute of allergy and infectious diseases ( niaid ) expert panel report published in 20101 as multiple studies demonstrate that much of presumed food allergy is in error15 . should the history be suggestive of a typical food reaction , confirmation with in vivo / in vitro testing is recommended . both skin prick testing ( spt ) and in vitro serum ige testing are used routinely in practice to confirm a food allergy once reported . unproven tests for food allergy include serum igg4 testing which can be performed to many foods , however , its presence is not known to play a role in the immunology of food allergy16 . spt : spt involves the introduction of the food protein in the superficial layer of the skin and requires the patient to be free of antihistamines . the testing is inexpensive and results are immediately available while the patient is in the office . positive testing alone by skin or blood testing , however , does not confirm food allergy as neither mode of testing has a high predictive value . a negative test mostly confirms a lack of ige - mediated allergy if performed correctly with histamine and saline control tests18 . presence of food specific ige without a suspicious reaction indicates sensitization , but is not always representative of a true allergy . serum specific - ige testing : in vitro serum ige testing quantifies the amount of ige to the food - specific protein via an enzymatic assay . serum testing is readily available and does not require that the patient be free of antihistamines ; however , there is a higher cost and the need to obtain a blood sample . several commercial assays are available however , significant differences in the measured ige levels have been found between assays using identical serum samples20 analogous to the trends noted with skin prick testing , largely positive food - specific ige levels have been found to correlate with a likelihood of clinical reaction1821 . specialists have data at hand with levels that are > 95 per cent predictive of reaction among children for common allergens such as egg , peanut and milk2122 . thus far , component testing has been noted to be particularly helpful in differentiating between asymptomatic sensitization and peanut allergy . however , the clinical relevance is not yet well established for other food proteins and some studies show increase specificity with testing , but decreased sensitivity when compared to traditional testing20 . oral food challenges : a food allergy diagnosis is supported when reaction symptoms and testing correlate . to confirm the diagnosis of food allergy in some instances or to determine if tolerance to an allergen has been achieved ( i.e. a food allergy outgrown ) , an oral food challenge ( ofc ) may be performed . in an ofc , the offending food allergen is given to the patient in escalating doses either in an open or blinded fashion . for the gold standard diagnosis of food allergy , the challenge should be performed in a double - blind placebo controlled fashion though this is costly and often difficult in practice . therefore , many allergy specialists choose to employ open feeding challenges . in a survey of american academy of asthma , allergy & immunology members practicing in the us , 85.5 per cent of allergists reported that they perform challenges , however , a very small number of allergists ( 5.6% ) performed more than 10 ofcs per month , while 70 per cent performed only 1 to 5 ofcs per month25 . avoidance : once the diagnosis of a food allergy is established the patient should be instructed to completely avoid the allergen . unfortunately , avoidance is easier said than done and as hidden allergens pose a possible fatal risk , the patient should be provided with an emergency plan for medications and actions to be given should accidental ingestion occur . in a study of peanut allergic children in quebec , an annual incidence rate of accidental exposure was found to be 14.3 per cent with the majority of ingestions occurring at the patient 's home or at the home of a friend or relative28 . this was followed by a nationwide survey in canada finding an annual peanut accidental ingestion incidence of 12.5 per cent when excluding recently diagnosed individuals29 . while cutaneous contact with some allergens such as peanut can cause reactions , the majority of severe or systemic reactions must come from direct ingestion of the allergen3031 . therefore , the us has strict labelling laws for the eight most common food allergens when present as ingredients . however , reporting use of allergens on shared equipment and in processing are at the discretion of the manufacturer . among items declaring these advisory statement labels in the us , ford et al32 found that 5.3 per cent contained detectable residues of allergenic food ( ranging in concentration from 3222 ppm ) versus 1.9 per cent among products that lacked a precautionary statement . treatment of acute reactions : prompt administration of intramuscular epinephrine is the first - line therapy for management of food - induced anaphylaxis . epinephrine is classified by the world allergy organization ( wao ) as an essential medication for the treatment of anaphylaxis33 . injection of a dose of 0.01 mg / kg of 1:1,000 ( 1 mg / ml ) solution to a maximum of 0.5 mg in the lateral thigh ( vastus lateralis muscle ) is preferred and has been shown to achieve a faster , higher serum medication peak when compared to the administration in the deltoid muscle intramuscularly and to subcutaneous administration34 . milder , isolated , non - progressive cutaneous reactions may be treated simply with h1- receptor antagonists . these medications may help to relieve pruritus , hives , angioedema and conjunctivitis , yet in an anaphylactic reaction , these do not aid with upper airway obstruction , hypotension or shock and are , therefore , not a substitute for epinephrine33 . inhaled beta-2 adrenergic agonists have been used for the relief of cough , wheezing and shortness of breath in conjunction with epinephrine , but once again are not a substitute for epinephrine33 . other adjuncts include supplemental oxygen and fluids which may be considered after the administration of epinephrine depending on the patient 's symptoms . biphasic reactions are defined as a recurrence of reaction symptoms after the initial anaphylactic reaction has appeared to resolve and occur in 5 - 7 per cent of all anaphylactic reactions3536 . however , retrospective studies are discrepant in whether there is a difference in the rate of biphasic reaction with or without steroid dosing3536 . a cochrane review was unable to identify any quality , relevant studies with evidence for the use of corticosteroids in anaphylaxis and felt unable to either recommend or refute the use of corticosteroids in this instance37 . it has been noted that the time between onset of symptoms and the initial dose of epinephrine was significantly longer for those patients who did have biphasic reactions3638 . long - term outpatient recommendations : it is the current recommendation that once diagnosed with food allergy the individual avoids all traces of the offending food as well as possible contamination . each patient diagnosed with a food allergy should be prescribed self - injectable epinephrine for use in an anaphylactic reaction . in addition , proper device use must be reviewed as one study indicated that although 86 per cent of parents of food allergic children carried the device , only one third were able to correctly administer the medication40 . in a prospective study of 122 food allergic children prescribed epinephrine , 69 per cent of parents were unable to use the device , did not have it available , or did not know when it should be administered41 . families were six times more likely to have received a practical demonstration on epinephrine autoinjector use if the prescribing doctor was a specialist as compared to a general practitioner and 17 times more likely if the physician was an allergy specialist41 . as reaction severity can not be predicted from prior reactions or testing , the importance of having self - injectable epinephrine on hand at all times must be imparted to the patient . risk factors for fatal and near fatal anaphylactic reactions include failure to have epinephrine available , history of prior severe reactions , known food allergy , adolescent age and most importantly asthma38 . multiple studies have demonstrated food allergy 's effect on quality of life ( qol ) measures . in particular , females with food allergy , those with a larger number of food allergies , history of more previous reactions and those with co - existing atopic diseases report poorer qol43 . it is to be noted that the prescribing of epinephrine autoinjectors has been found to reduce anxiety in both parents and children though this improvement in anxiety does not improve adherence in carrying the autoinjector44 . prevention : the american academy of pediatrics ( aap ) published updated recommendations in 2008 reviewing nutritional choices in pregnancy , lactation and in the first year of life which may affect development of atopic disease in infants and children45 . no definitive conclusions have been found regarding maternal dietary exposures during pregnancy and lactation contributing significantly to the development of food allergy in the infant . therefore , no adjustments to the maternal diet are recommended at this time . the recommendations note that there is modest evidence that atopic dermatitis may be delayed or prevented by the use of extensively or partially hydrolyzed formulas , compared with cow milk formula , in children unable to be breastfed for the first 4 to 6 months of life45 . yet , the data have not been impressive for the use in the prevention of food allergy . timing of the introduction of complementary or solid foods has also been pursued as a factor in the prevention of food allergy in children . however , there is no evidence supporting that delaying introduction beyond 4 to 6 months of age will affect atopy or development of food allergy . studies looking at early weaning and the development of food allergy in children have actually found a potential protective effect in early weaning . the most convincing evidence for early introduction of highly allergenic food proteins and possible oral tolerance comes from a study of the prevalence of peanut allergy in jewish children in the uk and israel . the safest way recommended to introduce foods thought to be highly allergenic is at home , in gradually increasing amounts with a rate of introduction of one new food every 3 to 5 days50 . several trials underway are addressing this gap in the literature as to whether food introduction plays a role in primary prevention of food allergy including the starting time for allergy reduction ( star ) , learning early about peanut ( leap ) , preventing peanut allergy in atopic dermatitis ( peaad ) , starting time for egg protein ( step ) and beating egg allergy trials ( beat)50 . emerging therapeutics : the possibility of severe fatal reactions with accidental ingestions and lack of standard treatment has led to a strong push to find a viable therapy option for patients . several small studies have previously been published and now larger , multi - center trials are underway . desensitization is a change in the threshold or amount of the allergen needed to induce a reaction whereas tolerance is achieved when one can ingest the allergen on an ad lib basis without reaction . tolerance is a long lasting immunity while with desensitization one must continue to ingest the allergen daily or the immunologic changes may be lost . allergen specific therapies include oral immunotherapy ( oit ) , sublingual immunotherapy ( slit ) and epicutaneous immunotherapy ( epit ) . oit involves introducing the food allergen in initial quite low oral doses , and typically escalating the dose over a day or two of rapid dose increase , followed by a slower incremental dose increase over weeks and months . varying oit protocols have been used in several small trials showing promise for desensitization to food allergens525354 . peanut oit has been a particular focus given that peanut is a major cause of food - induced anaphylaxis . however , a recent cochrane review of peanut oit trials found only one randomized control trial that resulted in desensitization , yet with a risk of clinically - significant adverse reactions55 . though many adverse events have been noted in the studies , especially in the more rapid dose escalation phases , no life threatening event or death has been reported thus far56 . however , a proportion of patients ( ~10 - 20% ) in each study appear to be unable to tolerate desensitization due to the side effects of therapy57 . the cochrane review authors note that due to the risk of adverse events and current lack of evidence of long - term benefits , peanut oit can not currently be recommended without further study55 . studies have shown that introduction of extensively heated milk and egg appears to hasten the development of tolerance to the unheated food protein585960 . introduction of extensively heated egg and milk in tolerant children may have immunomodulatory benefit and may potentially be safe in inducing tolerance in the traditional oit61 . similar to oit , slit uses small escalating doses of the food allergen ; however , doses are given under the tongue via an extract vehicle . the first multi - center , randomized , placebo - controlled study of peanut slit showed a modest desensitization at ofc after 44 wk of therapy with 14 of 20 subjects receiving peanut slit being able to consume at least a 10-fold increase in the amount of peanut powder they were able to consume when compared to their baseline ofc63 . in a direct comparison study of slit versus oit for cow milk allergy , systemic reactions were more common with oit , however , oit was more efficacious than slit alone64 . however , slit remains an appealing therapy to study given it is less likely to cause serious adverse reactions . fewer trials so far , have evaluated the administration of allergen via the skin in a patch form in epit . in a pilot study using epit for the treatment of cow milk allergy , a trend to improvement was noted , but there was no significant increase in the cumulative total dose of cow milk tolerated after three months of therapy with only local reactions being noted65 . therapies for food allergy in general that are not directed to a specific allergen are also in the midst of study . the food allergy herbal formula-2 ( fahf-2 ) is a capsule containing a chinese herbal formula found to abolish anaphylaxis in mice with peanut allergy69 . subsequent extended phase i trials in humans showed dosing of the nine herb formula to be both safe and well tolerated over a 6 month period70 . available from : http://clinicaltrials.gov/show/nct00602160 nlm identifiernct00602160 . the monoclonal anti - ige antibody omalizumab has also been found to be a potential non - specific allergen therapy . a multi - center , randomized , double - blind , placebo - controlled phase ii trial using omalizumab in the treatment of peanut allergy completed 14 of the planned 150 subjects prior to early discontinuation due to severe anaphylactic reactions during the qualifying ofcs71 . of the 14 completing the study , subjects receiving omalizumab trended in tolerating an increased amount of peanut protein following the 24 wk of therapy when compared to placebo71 . while the data are limited on using omalizumab as a single agent for the treatment of food allergy , focus has shifted to study if omalizumab has a role in oit , as an adjunct to limit side effects seen in oit dose escalation . a pilot study using omalizumab as an adjunct to cow milk oit demonstrated that omalizumab permitted rapid milk dose escalation in a majority of subjects72 . food allergy remains an important health concern due to increasing prevalence worldwide , potentially fatal reactions and current lack of curative therapy . importance must be placed on proper diagnosis which may at times be difficult given the limitations of current available testing . avoidance of the offending allergen and prompt treatment of acute reactions are the current mainstays of food allergy management . however , much still must be elucidated to understand the optimal timing of allergenic food introduction . while little headway has been made in the prevention of food allergy , multiple therapeutic options including both allergen specific and non - specific therapies are in human efficacy trials . though the initial results are promising , no single therapy is considered ready for common use until further data on optimal dosing and long - term safety are available .

measles is a highly contagious acute viral disease characterized by high fever , malaise , coryza , conjunctivitis , cough , and a maculopapular rash ( griffin , 2007 ) . patients with measles develop a severe and temporary immunosuppression , which is often accompanied by secondary bacterial infections ( griffin , 2007 ) . despite the availability of highly effective vaccines , measles - related deaths were estimated to be 164,000 worldwide in 2008 ( who , 2009 ) . the causative agent is measles virus ( mv ) , which belongs to the genus morbillivirus in the family paramyxoviridae . the virus particle is enveloped and contains a non - segmented negative - strand rna genome encoding six tandem linked genes , n , p / v / c , m , f , h , and l. the genome is encapsidated by the nucleocapsid ( n ) protein and associated with viral rna - dependent rna polymerases , forming a helical ribonucleoprotein complex ( rnp ) . on the envelope , the viral particle possesses two types of viral glycoprotein spikes , the hemagglutinin ( h ) and fusion ( f ) proteins ( griffin , 2007 ) . the h protein is responsible for binding to cellular receptors on the target host cells , and plays a key role in the determination of host cell specificity ( tropism ) of mv ( yanagi et al . , 2009 ) . binding of the h protein to a receptor triggers f protein - mediated membrane fusion between the virus envelope and the host cell plasma membrane , releasing the rnp into the cytoplasm . in cells infected with mv , the h and f proteins are expressed on the cell surface and cause cell - to - cell fusion , producing syncytia . therefore , the distribution pattern of its receptors is a key determinant of which cells become infected with mv ( yanagi et al . , 2009 ) . the initial discovery of an mv receptor came in 1993 ( dorig et al . , 1993 ; naniche et al . , two independent studies indicated that the receptor molecule for mv is the human membrane cofactor protein ( mcp / cd46 ) , a central component of the complement system , which is expressed ubiquitously on all organs and tissues throughout the human body ( dorig et al . these findings were highly welcomed from the viewpoint that mv causes a systemic infection . meanwhile , kobune et al . ( 1990 ) reported the isolation of lymphotropic mv strains , and subsequent studies indicated that these lymphotropic mv strains do not use mcp / cd46 as a receptor ( yanagi et al . , 2009 ) . importantly , kobune s isolates exhibited a high virulence in experimentally infected monkeys , whereas mcp / cd46-using classical mv isolates caused no or mild disease in monkeys ( kobune et al . , 1990 , 1996 ; takeda et al . , 1998 ) . hence , new two questions have arisen for mv researchers . what is the receptor for these lymphotropic strains ? which strains are the real wild - type ( wt ) mv strains ? in 2000 , using kobune s isolates , another receptor was identified ( tatsuo et al . , 2000 ) . this receptor is signaling lymphocyte activation molecule , also known as cd150 ( slam / cd150 ) , which is expressed on cells of the immune system ( tatsuo et al . , 2000 ) . subsequent studies clarified that slam / cd150 is a receptor for wt mv strains circulating in patients , and that mcp / cd46 does not act as a receptor for wt mv strains ( yanagi et al . , 2009 ) . mcp / cd46 acts as a receptor only for vaccine and some laboratory mv strains ( yanagi et al . , 2009 ) . currently , it is clear that these mv strains have acquired the ability to use mcp / cd46 as an alternative receptor to grow in laboratory cell lines lacking slam / cd150 expression ( yanagi et al . , 2009 ) . hence , it has become generally accepted that wt mv is a lymphotropic virus that specifically targets immune cells , similar to the case of human immunodeficiency virus ( hiv ) and human t cell lymphotropic virus type 1 ( htlv1 ) . in 2000 , a recombinant mv , ic323 , was generated based on kobune s first isolate ( takeda et al . , 2000 ) , and has greatly contributed to our understanding of the molecular bases for the pathogenesis of wt mv strains ( takeuchi et al . , 2005 ; de swart et al . , 2007 ; devaux et al . , 2008 , 2011 ; leonard et al . , 2008 ; nakatsu et al . , 2008 ; de vries et al . , 2010 ; ludlow et al . , 2010 ; mhlebach et al . , 2011 ; 2011 ) . at that time , only slam / cd150-positive cells were found to be susceptible to wt mv infections . however , it remained difficult to make a final conclusion that slam / cd150 is the sole receptor for wt mv , because histopathological examinations of measles patients and monkeys infected with mv have revealed considerable levels of mv protein expression in the epithelia of various organs , and histopathological changes are also evident in these epithelia ( nii et al . , 1964 ; lightwood and nolan , 1970 ; olding - stenkvist and bjorvatn , 1976 ; moench et al . , 1988 ; craighead , 2000 ; figure 1 ) . in 2003 , primary cultures of human small airway epithelial cells ( saecs ) were shown to be susceptible to wt mv infection ( takeuchi et al . , 2003 ) . upon mv infection , large syncytia developed in saecs via a slam - independent mechanism ( takeuchi et al . , 2003 ) . after searching many cell lines , several epithelial cell lines with high susceptibility to mv infection ( 2008 ) clearly demonstrated that wt mv infects epithelial cell lines that form tight junctions ( tjs ) using an unidentified receptor ( leonard et al . , 2008 ; tahara et al . , 2008 ) . using these cells and recombinant ic323 expressing green fluorescent protein ( ic323-egfp ; takeda et al . , 2000 ; hashimoto et al . , 2002 ) , a final answer was obtained for the receptor on epithelial cells . two groups independently demonstrated that nectin4 , which is expressed at adherens junctions ( ajs ) , acts as a receptor for mv ( mhlebach et al . interestingly , mv loses the ability to use either slam / cd150 or nectin4 when it possesses specific mutations in the h protein ( leonard et al . , 2008 ; tahara et al . , 2008 ; nectin4-blind mv still infects slam / cd150-positive immune cells efficiently ( slam / cd150-tropic ) , and conversely , slam / cd150-blind mv infects nectin4-positive epithelial cells efficiently ( nectin4-tropic ; leonard et al . , 2008 ; tahara et al . , 2008 ; figure 2 ) . in this regard , mv is intrinsically dual - tropic to immune cells and epithelial cells ( figure 2 ) . measles virus antigen and histopathological changes in bronchiolar epithelial cells . at 11 days post - infection , a cynomolgus monkey experimentally infected with wt mv ic323 strain was subjected to the histopathological examination . multinucleated cell were seen in the epithelial layer of the bronchiole with inflammatory infiltration of eosinophils and lymphocytes . ( b ) immunohistochemical double staining for the n protein of mv ( left , green ) and keratin ( center , red ) . multinucleated epithelial giant cells showing positive staining for the n protein are present in the bronchiole ( right , yellow in merged images ) . wt mv ( ic323 ) infects and produces syncytia in both slam - positive lymphoblastoid b95a and nectin4-positive epithelial ii-18 cells ( dual - tropic ) . ic323 with an r533a mutation in the h protein fails to spread in b95a cells ( slam - blind ) , but replicates well in ii-18 cells ( nectin4-tropic ) . on the contrary , ic323 with a y543s mutation in the h protein spreads in b95a cells ( slam - tropic ) , but fails to spread in ii-18 cells ( nectin4-blind ) . there is now no doubt that slam / cd150 and nectin4 are the major receptors for mv . however , other molecules may further support mv infection in vivo , being involved in the development of measles and its neurological sequela . for example , the mechanism that the c - type lectin dc - specific intercellular adhesion molecule 3-grabbing non - integrin ( dc - sign ) acts as an attachment receptor for mv , thereby promoting mv infection of dcs , may be ideal to understand the extraordinarily high transmissibility of measles ( de witte et al . it is well known that mv causes subacute sclerosing panencephalitis ( sspe ) , a persistent infection of the central nervous system ( cns ) with mv . this occurs with a mean latency period of 710 years after suffering from acute measles at a frequency of 1/5,0001/100,000 reported cases of acute measles ( takasu et al . , 2003 ; bellini et al . , although nectin4 is a possible candidate for an mv receptor in the cns , no ( or undetectable ) nectin4 expression was observed in the cns in humans ( reymond et al . , 2001 ; brancati et al . , 2010 ) , and some mv strains derived from sspe patients are likely to use nectin4 inefficiently ( seki et al . , 2011 ) . ( 2007 ) suggest that neurokinin-1 , a substance p receptor , supports trans - synaptic transmission of mv by acting as a receptor for the f protein . measles virus infection causes immunosuppression in patients and is often accompanied by secondary bacterial infections . typically , mv - induced immunosuppression is characterized by a marked lymphopenia , and an early th1 response followed by predominant and prolonged th2 response in patients , with suppression of mitogen - induced lymphocyte proliferation ex vivo ( griffin and ward , 1993 ; schneider - schaulies and schneider - schaulies , 2009 ) . some , if not all , of these immunological observations must be attributed either directly or indirectly to the fact that mv uses slam / cd150 as a receptor . slam / cd150 is a member of the slam - family receptors , which belong to the immunoglobulin ( ig ) superfamily ( veillette , 2010 ; ma and deenick , 2011 ) . the slam - family consists of nine members ( cannons et al . , 2011 ; ma and deenick , 2011 ) . the slam - family receptors are type i transmembrane proteins that typically possess an extracellular region with two ig - like domains ( an amino - terminal variable ( v)-like domain and a carboxy - terminal constant-2 ( c2)-like domain ) , a transmembrane region , and a cytoplasmic region that harbors multiple tyrosine - based motifs ( detre et al . , 2010 ; veillette , 2010 ; cannons et al . , 2011 ; ma and deenick , 2011 ) . these motifs are referred to as immunoreceptor tyrosine - based switch motifs ( itsms ; cannons et al . , 2011 ) . the slam - family receptors are expressed in a broad range of immune cells and play critical roles in immunity . in general , the receptors act as self - ligands and their homophilic trans - interactions occur between either heterotypic or homotypic immune cells ( veillette , 2010 ; ma and deenick , 2011 ) . slam / cd150 is expressed on thymocytes , subsets of b and t lymphocytes , mature dendritic cells ( dcs ) , macrophages , and platelets , and their expression is upregulated or induced in lymphocytes and monocytes upon activation ( detre et al . signaling lymphocyte activation molecule - associated protein ( sap)-family adaptors [ sap , ewing s sarcoma - associated transcript ( eat)-2 , and eat-2-related transducer ( ert ) ] play important roles for the signal transductions mediated by the slam - family receptors ( veillette , 2010 ; ma and deenick , 2011 ) . they are small proteins that consist of a single src homology 2 ( sh2 ) domain and a short carboxy - terminal region . sap associates intracellularly with the itsms in the cytoplasmic region of the slam - family receptors via the sh2 domain ( dong and veillette , 2010 ; veillette , 2010 ; ma and deenick , 2011 ) . sap has the ability to bind concomitantly to the src - family protein tyrosine kinase , fyn , thereby coupling the slam - family receptors with fyn ( dong and veillette , 2010 ; veillette , 2010 ; cannons et al . , 2011 ) . thereafter , fyn phosphorylates tyrosine residues at the cytoplasmic region of slam - family receptors and other intracellular effector molecules , activating the downstream signals ( detre et al . , 2010 ; dong and veillette , 2010 ; cannons et al . sap binding to the sh2 domain of slam - family receptors competes with the binding of other sh2 domain - containing molecules , thus modulating the slam - mediated signaling ( dong and veillette , 2010 ; veillette , 2010 ; cannons et al . via slam / cd150-sap fyn interactions play important roles in regulating t cell receptor - mediated induction of th2 cytokines , such as interleukin ( il)-4 and il-13 ( detre et al . , 2010 ; cannons et al . , 2011 ; ma and deenick , 2011 ) . eat-2 also mediates the signal transduction cascades of the slam - family receptors via a similar but distinct mechanism to that of sap ( cannons et al . , 2011 ) . similar to sap , eat-2 also associates with the itsms of slam - family receptors through its sh2 domain , but mediates the subsequent signal cascades via its own phosphorylated tyrosine in the short carboxy - terminal region ( veillette , 2010 ) . in general , the signals mediated by the sap - family adaptors induce the activation and differentiation of immune cells ( veillette , 2010 ) . however , if the sap - family adaptors are absent , the slam - family receptors mediate inhibitory signals to immune cells ( a switch - of - function effect ; dong and veillette , 2010 ; veillette , 2010 ) . roles for slam / cd150 in macrophage functions , cell adhesion , and nkt cell development have also been demonstrated , although many data were obtained in mice ( dong and veillette , 2010 ; veillette , 2010 ; cannons et al . , 2011 ; ma and deenick , 2011 ) . x - linked lymphoproliferative syndrome is a rare immunodeficiency disease typically caused by mutations in the sap - encoding gene , sh2d1a ( veillette , 2010 ; ma and deenick , 2011 ) . patients with this syndrome have various functional defects and impaired differentiation of immune cells , indicating crucial roles for sap in normal immunity ( dong and veillette , 2010 ) . barr virus infection are characteristics of the disease ( veillette , 2010 ; ma and deenick , 2011 ) . although slam - family receptors have some functional redundancy , each receptor plays specific roles in a variety of immune responses ( dong and veillette , 2010 ; veillette , 2010 ) . in general , lymphotropic viruses , such as hiv and htlv1 , can never be airborne , and are transmitted inefficiently even through direct contact with patients . in sharp contrast , mv transmits via aerosols , and has a highly contagious nature . therefore , this transmission style of mv can not be easily explained by the fact that the virus uses a lymphocytic molecule , slam / cd150 , as a receptor . the recent findings showing that mv uses nectin4 , a cell adhesion molecule ( cam ) expressed at the ajs of epithelia , may partly but nicely explain how and why mv transmits efficiently from a patient to other individuals . epithelial cells are connected with one another through the formation of several specialized cell cell junctions , such as tjs , ajs , desmosomes , and gap junctions . tjs function as a physical barrier that prevents the passage of soluble molecules through the intercellular gaps , and also blocks the lateral movement of lipids and membrane proteins across the tj barrier , thereby acting as the border of the apical and basolateral membranes . nectin4 is a member of the nectin family , which consists of four members ( nectin1 , 2 , 3 , and 4 ; takai et al . , nectin1 and nectin2 were originally identified as poliovirus receptor - related protein ( prr)-1 and prr-2 , respectively , and subsequently shown to support the entry of some herpes viruses ( takai et al . are also type i transmembrane proteins that belong to the ig superfamily ( takai et al . , they possess an extracellular region with three ig - like domains ( an amino - terminal v - like domain and two c - like domains ) , a transmembrane region , and a cytoplasmic region with a short afadin - binding motif ( takai et al . the consensus motif was reported to be e / a - x - y - v for nectin1 , 2 , and 3 , while nectin4 does not have this motif but still binds to afadin ( reymond et al . , 2001 ; ( 2001 ) proposed a new consensus motif , k / r - x - x - y / l - v , for all four nectins . afadin is an actin filament ( f - actin)-binding protein , and supports nectins to interact and co - operate with cadherins , other cams , and intracellular signaling molecules ( takai et al . , nectins are expressed as dimers , and interact in trans with other nectin dimers expressed on neighboring cells ( takai et al . all nectins show homophilic interactions , while heterophilic interactions are also observed between specific nectins , such as those between nectin1/nectin3 and nectin 2/nectin3 ( takai et al . nectin4 shows homophilic interactions as well as heterophilic interactions with nectin1 ( reymond et al . , 2001 ; the ig v - like domain is used for the trans - interaction ( reymond et al . , 2001 ; fabre et al . , 2002 ) . nectins play key roles in the initiation of aj formation , and regulate various physiological functions of epithelial cells , such as contact inhibition of cell movement and proliferation , survival , differentiation , and cell polarization ( takai et al . , 2008a , b ) . although nectin1 and nectin2 are expressed in a broad range of tissues , the expression of nectin3 and nectin4 is more specific ( reymond et al . , 2001 ) . ( 2010 ) showed that human nectin4 is expressed mainly in the placenta and to lesser extents in the trachea , prostate , lung , and stomach . ( 2010 ) demonstrated nectin4 expression in human keratinocytes , suprabasal nucleated layers of the epidermis , and non - keratinized structures of hair . some levels of expression in epithelial cells of the tonsil , oral mucosa , esophagus , and nasopharynx have also been reported ( www.proteinatlas.org ) . although , in many cases , nectin4 is expressed in low or undetectable levels in normal human tissues , many cancer cells are highly positive for nectin4 . thus , it has been proposed that nectin4 is a new tumor - associated marker ( fabre - lafay et al . these observations may provide a rationale for the use of mv as an oncolytic agent ( mhlebach et al . , 2011 ) . in humans , mutations in the pvrl4 gene encoding nectin4 cause ectodermal - dysplasia - syndactyly syndrome ( edss ) , in which patients have affected skin and skin appendages , such as hair , teeth , and nails ( brancati et al . , 2010 ; jelani et al . , 2011 ) . the pathology of measles can now be drawn with these two receptors ( figure 3 ) . although nectin4-expressing epithelial cells can be the initial targets of mv , no or very limited infection of epithelia was observed in monkeys experimentally infected with mv at the early days after infection ( ludlow et al . , 2010 ; lemon et al . , 2011 instead , mv initiates its infection via slam - mediated entry into alveolar macrophages and dcs in the lung or respiratory tracts ( de witte et al . , 2008 ; de vries et al . , 2010 . these infections may allow mv to penetrate into the human body and reach the lymphoid organs or tissues , where slam / cd150-expressing cells are abundant ( corry et al . after initial replication in these lymphoid organs or tissues , mv or mv - infected lymphocytes can easily enter the bloodstream . subsequently , a dramatic mv infection is observed in all lymphoid organs , such as the spleen , thymus , appendix , tonsil , and lymph nodes , throughout the body ( moench et al . , 1988 ; kobune et al . , 1996 ; de swart et al . , 2007 ; de vries et al . , 2010 ) . at the time when the mv infection of lymphoid organs reaches its peak , mv infection of epithelia , such as squamous stratified epithelia of the tongue and buccal mucosa and ciliated epithelia of the trachea , becomes evident ( nii et al . , 1964 ; olding - stenkvist and bjorvatn , 1976 ; moench et al . , 1988 ; this epithelial infection is probably led by mv - infected immune cells and initiated through the basolateral side , since monkeys infected with mv often show infectious foci in the epithelia with mv - infected lymphoid or myeloid cells in the subepithelial cell layers of the trachea , bronchus , and tongue ( de vries et al . , 2010 ; ludlow et al . , the h protein expressed on mv - infected immune cells that migrate through the epithelial cell layer likely recognizes nectin4 expressed at ajs , triggering f protein - mediated membrane fusion between the mv - infected immune cells and the target epithelial cells . ( 2011 ) demonstrated a correlation between nectin4 expression and mv infection in epithelia in vivo . importantly , mv has a mechanism that further facilitates virus shedding in the airway . in epithelia , mv selectively releases progeny virus particles from the apical membrane to the luminal side of the respiratory tract ( leonard et al . , 2008 ; leonard et al . ( 2008 ) showed that mv genetically engineered to use slam / cd150 , but not nectin4 ( nectin4-blind or slam / cd150-tropic ) , does not shed progeny viruses into the respiratory tract , although it does show systemic infection of lymphoid organs , similar to the case for wt mv . mv initiates its infection via slam - mediated entry into alveolar macrophages and dcs in the lung . after initial replication in regional lymph nodes , mv enters the bloodstream and subsequently spreads to all lymphoid organs , such as the spleen , thymus , appendix , tonsil , and lymph nodes , throughout the body . when the mv infection of the lymphoid organs reaches its peak , mv - infected immune cells transfer mv to epithelial cells through the basolateral side . at this step , mv uses nectin4 expressed at ajs as a receptor . in the epithelia , mv selectively releases its progeny virus particles from the apical membrane to the luminal side of the respiratory tract . membrane cofactor protein / cd46 was first identified as a receptor for mv ( dorig et al . , however , our current knowledge of mv receptors has been totally transformed . in 2000 , it was shown that slam / cd150 expressed on cells of the immune system , but not mcp / cd46 , is a real receptor for wt mv . however , recent studies further showed that slam / cd150 is not the sole receptor for mv . mv has an intrinsic ability to enter not only immune cells but also epithelial cells . in 2011 , a clear answer was obtained through the identification of the epithelial mv receptor nectin4 , which is expressed at ajs , thereby partly explaining why mv exhibits its highly contagious nature ( mhlebach et al . , 2011 ; noyce et al . , however , many aspects and molecular mechanisms underlying immunosuppressive effects and a highly contagious nature of mv still remain to be elucidated . analyses of physiological roles of mv receptors , slam / cd150 , and nectin4 , would provide deep insights into mv pathogenesis . 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 .

measles is a highly contagious disease that causes temporary and severe immunosuppression in patients . signaling lymphocyte activation molecule ( slam ) expressed on cells of the immune system functions as a receptor for measles virus ( mv ) . in addition to slam , vaccine strains of mv also use a ubiquitously expressed complement regulatory protein , cd46 , as a receptor , whereas wild - type ( wt ) mv strains do not use this receptor . however , recent studies have indicated that slam is not the sole receptor for wt mv strains . these strains have an intrinsic ability to enter both immune and epithelial cells using distinct receptor binding sites in their hemagglutinin ( h ) protein . recently , a clear answer was obtained through the identification of an epithelial mv receptor , nectin4 , expressed at adherens junctions , thereby greatly improving our knowledge of mv receptors . it is now clear that mv specifically targets two cell types , immune cells and epithelial cells , using slam and nectin4 , respectively . mv loses the ability to use either slam or nectin4 when it possesses specific mutations in the h protein . however , nectin4-blind mv still infects slam - positive immune cells efficiently ( slam - tropic ) , and conversely , slam - blind mv infects nectin4-positive epithelial cells efficiently ( nectin4-tropic ) . in this regard , mv is intrinsically dual - tropic to immune cells and epithelial cells . although many aspects and molecular mechanisms underlying immunosuppressive effects and a highly contagious nature of mv still remain to be elucidated , analyses of physiological functions of these two receptors would provide deep insights into mv pathogenesis .

Measles Virus Discoveries of Cellular Receptors for MV SLAM/CD150 Nectin4 Relevance of SLAM/CD150 and Nectin4 to MV Pathogenesis Concluding Remarks Conflict of Interest Statement

measles is a highly contagious acute viral disease characterized by high fever , malaise , coryza , conjunctivitis , cough , and a maculopapular rash ( griffin , 2007 ) . the causative agent is measles virus ( mv ) , which belongs to the genus morbillivirus in the family paramyxoviridae . the virus particle is enveloped and contains a non - segmented negative - strand rna genome encoding six tandem linked genes , n , p / v / c , m , f , h , and l. the genome is encapsidated by the nucleocapsid ( n ) protein and associated with viral rna - dependent rna polymerases , forming a helical ribonucleoprotein complex ( rnp ) . on the envelope , the viral particle possesses two types of viral glycoprotein spikes , the hemagglutinin ( h ) and fusion ( f ) proteins ( griffin , 2007 ) . the h protein is responsible for binding to cellular receptors on the target host cells , and plays a key role in the determination of host cell specificity ( tropism ) of mv ( yanagi et al . binding of the h protein to a receptor triggers f protein - mediated membrane fusion between the virus envelope and the host cell plasma membrane , releasing the rnp into the cytoplasm . in cells infected with mv , the h and f proteins are expressed on the cell surface and cause cell - to - cell fusion , producing syncytia . the initial discovery of an mv receptor came in 1993 ( dorig et al . , two independent studies indicated that the receptor molecule for mv is the human membrane cofactor protein ( mcp / cd46 ) , a central component of the complement system , which is expressed ubiquitously on all organs and tissues throughout the human body ( dorig et al . ( 1990 ) reported the isolation of lymphotropic mv strains , and subsequent studies indicated that these lymphotropic mv strains do not use mcp / cd46 as a receptor ( yanagi et al . importantly , kobune s isolates exhibited a high virulence in experimentally infected monkeys , whereas mcp / cd46-using classical mv isolates caused no or mild disease in monkeys ( kobune et al . which strains are the real wild - type ( wt ) mv strains ? this receptor is signaling lymphocyte activation molecule , also known as cd150 ( slam / cd150 ) , which is expressed on cells of the immune system ( tatsuo et al . subsequent studies clarified that slam / cd150 is a receptor for wt mv strains circulating in patients , and that mcp / cd46 does not act as a receptor for wt mv strains ( yanagi et al . mcp / cd46 acts as a receptor only for vaccine and some laboratory mv strains ( yanagi et al . currently , it is clear that these mv strains have acquired the ability to use mcp / cd46 as an alternative receptor to grow in laboratory cell lines lacking slam / cd150 expression ( yanagi et al . hence , it has become generally accepted that wt mv is a lymphotropic virus that specifically targets immune cells , similar to the case of human immunodeficiency virus ( hiv ) and human t cell lymphotropic virus type 1 ( htlv1 ) . in 2000 , a recombinant mv , ic323 , was generated based on kobune s first isolate ( takeda et al . , 2000 ) , and has greatly contributed to our understanding of the molecular bases for the pathogenesis of wt mv strains ( takeuchi et al . at that time , only slam / cd150-positive cells were found to be susceptible to wt mv infections . however , it remained difficult to make a final conclusion that slam / cd150 is the sole receptor for wt mv , because histopathological examinations of measles patients and monkeys infected with mv have revealed considerable levels of mv protein expression in the epithelia of various organs , and histopathological changes are also evident in these epithelia ( nii et al . in 2003 , primary cultures of human small airway epithelial cells ( saecs ) were shown to be susceptible to wt mv infection ( takeuchi et al . after searching many cell lines , several epithelial cell lines with high susceptibility to mv infection ( 2008 ) clearly demonstrated that wt mv infects epithelial cell lines that form tight junctions ( tjs ) using an unidentified receptor ( leonard et al . , 2002 ) , a final answer was obtained for the receptor on epithelial cells . two groups independently demonstrated that nectin4 , which is expressed at adherens junctions ( ajs ) , acts as a receptor for mv ( mhlebach et al . interestingly , mv loses the ability to use either slam / cd150 or nectin4 when it possesses specific mutations in the h protein ( leonard et al . , 2008 ; nectin4-blind mv still infects slam / cd150-positive immune cells efficiently ( slam / cd150-tropic ) , and conversely , slam / cd150-blind mv infects nectin4-positive epithelial cells efficiently ( nectin4-tropic ; leonard et al . in this regard , mv is intrinsically dual - tropic to immune cells and epithelial cells ( figure 2 ) . measles virus antigen and histopathological changes in bronchiolar epithelial cells . at 11 days post - infection , a cynomolgus monkey experimentally infected with wt mv ic323 strain was subjected to the histopathological examination . multinucleated cell were seen in the epithelial layer of the bronchiole with inflammatory infiltration of eosinophils and lymphocytes . wt mv ( ic323 ) infects and produces syncytia in both slam - positive lymphoblastoid b95a and nectin4-positive epithelial ii-18 cells ( dual - tropic ) . ic323 with an r533a mutation in the h protein fails to spread in b95a cells ( slam - blind ) , but replicates well in ii-18 cells ( nectin4-tropic ) . on the contrary , ic323 with a y543s mutation in the h protein spreads in b95a cells ( slam - tropic ) , but fails to spread in ii-18 cells ( nectin4-blind ) . there is now no doubt that slam / cd150 and nectin4 are the major receptors for mv . however , other molecules may further support mv infection in vivo , being involved in the development of measles and its neurological sequela . for example , the mechanism that the c - type lectin dc - specific intercellular adhesion molecule 3-grabbing non - integrin ( dc - sign ) acts as an attachment receptor for mv , thereby promoting mv infection of dcs , may be ideal to understand the extraordinarily high transmissibility of measles ( de witte et al . it is well known that mv causes subacute sclerosing panencephalitis ( sspe ) , a persistent infection of the central nervous system ( cns ) with mv . , although nectin4 is a possible candidate for an mv receptor in the cns , no ( or undetectable ) nectin4 expression was observed in the cns in humans ( reymond et al . , 2010 ) , and some mv strains derived from sspe patients are likely to use nectin4 inefficiently ( seki et al . ( 2007 ) suggest that neurokinin-1 , a substance p receptor , supports trans - synaptic transmission of mv by acting as a receptor for the f protein . measles virus infection causes immunosuppression in patients and is often accompanied by secondary bacterial infections . typically , mv - induced immunosuppression is characterized by a marked lymphopenia , and an early th1 response followed by predominant and prolonged th2 response in patients , with suppression of mitogen - induced lymphocyte proliferation ex vivo ( griffin and ward , 1993 ; schneider - schaulies and schneider - schaulies , 2009 ) . some , if not all , of these immunological observations must be attributed either directly or indirectly to the fact that mv uses slam / cd150 as a receptor . slam / cd150 is a member of the slam - family receptors , which belong to the immunoglobulin ( ig ) superfamily ( veillette , 2010 ; ma and deenick , 2011 ) . the slam - family consists of nine members ( cannons et al . the slam - family receptors are type i transmembrane proteins that typically possess an extracellular region with two ig - like domains ( an amino - terminal variable ( v)-like domain and a carboxy - terminal constant-2 ( c2)-like domain ) , a transmembrane region , and a cytoplasmic region that harbors multiple tyrosine - based motifs ( detre et al . the slam - family receptors are expressed in a broad range of immune cells and play critical roles in immunity . slam / cd150 is expressed on thymocytes , subsets of b and t lymphocytes , mature dendritic cells ( dcs ) , macrophages , and platelets , and their expression is upregulated or induced in lymphocytes and monocytes upon activation ( detre et al . signaling lymphocyte activation molecule - associated protein ( sap)-family adaptors [ sap , ewing s sarcoma - associated transcript ( eat)-2 , and eat-2-related transducer ( ert ) ] play important roles for the signal transductions mediated by the slam - family receptors ( veillette , 2010 ; ma and deenick , 2011 ) . they are small proteins that consist of a single src homology 2 ( sh2 ) domain and a short carboxy - terminal region . sap associates intracellularly with the itsms in the cytoplasmic region of the slam - family receptors via the sh2 domain ( dong and veillette , 2010 ; veillette , 2010 ; ma and deenick , 2011 ) . sap has the ability to bind concomitantly to the src - family protein tyrosine kinase , fyn , thereby coupling the slam - family receptors with fyn ( dong and veillette , 2010 ; veillette , 2010 ; cannons et al . eat-2 also mediates the signal transduction cascades of the slam - family receptors via a similar but distinct mechanism to that of sap ( cannons et al . similar to sap , eat-2 also associates with the itsms of slam - family receptors through its sh2 domain , but mediates the subsequent signal cascades via its own phosphorylated tyrosine in the short carboxy - terminal region ( veillette , 2010 ) . however , if the sap - family adaptors are absent , the slam - family receptors mediate inhibitory signals to immune cells ( a switch - of - function effect ; dong and veillette , 2010 ; veillette , 2010 ) . roles for slam / cd150 in macrophage functions , cell adhesion , and nkt cell development have also been demonstrated , although many data were obtained in mice ( dong and veillette , 2010 ; veillette , 2010 ; cannons et al . x - linked lymphoproliferative syndrome is a rare immunodeficiency disease typically caused by mutations in the sap - encoding gene , sh2d1a ( veillette , 2010 ; ma and deenick , 2011 ) . patients with this syndrome have various functional defects and impaired differentiation of immune cells , indicating crucial roles for sap in normal immunity ( dong and veillette , 2010 ) . barr virus infection are characteristics of the disease ( veillette , 2010 ; ma and deenick , 2011 ) . in sharp contrast , mv transmits via aerosols , and has a highly contagious nature . therefore , this transmission style of mv can not be easily explained by the fact that the virus uses a lymphocytic molecule , slam / cd150 , as a receptor . the recent findings showing that mv uses nectin4 , a cell adhesion molecule ( cam ) expressed at the ajs of epithelia , may partly but nicely explain how and why mv transmits efficiently from a patient to other individuals . epithelial cells are connected with one another through the formation of several specialized cell cell junctions , such as tjs , ajs , desmosomes , and gap junctions . tjs function as a physical barrier that prevents the passage of soluble molecules through the intercellular gaps , and also blocks the lateral movement of lipids and membrane proteins across the tj barrier , thereby acting as the border of the apical and basolateral membranes . nectin4 is a member of the nectin family , which consists of four members ( nectin1 , 2 , 3 , and 4 ; takai et al . , nectin1 and nectin2 were originally identified as poliovirus receptor - related protein ( prr)-1 and prr-2 , respectively , and subsequently shown to support the entry of some herpes viruses ( takai et al . , they possess an extracellular region with three ig - like domains ( an amino - terminal v - like domain and two c - like domains ) , a transmembrane region , and a cytoplasmic region with a short afadin - binding motif ( takai et al . the consensus motif was reported to be e / a - x - y - v for nectin1 , 2 , and 3 , while nectin4 does not have this motif but still binds to afadin ( reymond et al . afadin is an actin filament ( f - actin)-binding protein , and supports nectins to interact and co - operate with cadherins , other cams , and intracellular signaling molecules ( takai et al . , nectins are expressed as dimers , and interact in trans with other nectin dimers expressed on neighboring cells ( takai et al . nectins play key roles in the initiation of aj formation , and regulate various physiological functions of epithelial cells , such as contact inhibition of cell movement and proliferation , survival , differentiation , and cell polarization ( takai et al . ( 2010 ) showed that human nectin4 is expressed mainly in the placenta and to lesser extents in the trachea , prostate , lung , and stomach . ( 2010 ) demonstrated nectin4 expression in human keratinocytes , suprabasal nucleated layers of the epidermis , and non - keratinized structures of hair . some levels of expression in epithelial cells of the tonsil , oral mucosa , esophagus , and nasopharynx have also been reported ( www.proteinatlas.org ) . in humans , mutations in the pvrl4 gene encoding nectin4 cause ectodermal - dysplasia - syndactyly syndrome ( edss ) , in which patients have affected skin and skin appendages , such as hair , teeth , and nails ( brancati et al . the pathology of measles can now be drawn with these two receptors ( figure 3 ) . although nectin4-expressing epithelial cells can be the initial targets of mv , no or very limited infection of epithelia was observed in monkeys experimentally infected with mv at the early days after infection ( ludlow et al . , 2011 instead , mv initiates its infection via slam - mediated entry into alveolar macrophages and dcs in the lung or respiratory tracts ( de witte et al . subsequently , a dramatic mv infection is observed in all lymphoid organs , such as the spleen , thymus , appendix , tonsil , and lymph nodes , throughout the body ( moench et al . at the time when the mv infection of lymphoid organs reaches its peak , mv infection of epithelia , such as squamous stratified epithelia of the tongue and buccal mucosa and ciliated epithelia of the trachea , becomes evident ( nii et al . , 1988 ; this epithelial infection is probably led by mv - infected immune cells and initiated through the basolateral side , since monkeys infected with mv often show infectious foci in the epithelia with mv - infected lymphoid or myeloid cells in the subepithelial cell layers of the trachea , bronchus , and tongue ( de vries et al . , the h protein expressed on mv - infected immune cells that migrate through the epithelial cell layer likely recognizes nectin4 expressed at ajs , triggering f protein - mediated membrane fusion between the mv - infected immune cells and the target epithelial cells . importantly , mv has a mechanism that further facilitates virus shedding in the airway . in epithelia , mv selectively releases progeny virus particles from the apical membrane to the luminal side of the respiratory tract ( leonard et al . ( 2008 ) showed that mv genetically engineered to use slam / cd150 , but not nectin4 ( nectin4-blind or slam / cd150-tropic ) , does not shed progeny viruses into the respiratory tract , although it does show systemic infection of lymphoid organs , similar to the case for wt mv . mv initiates its infection via slam - mediated entry into alveolar macrophages and dcs in the lung . after initial replication in regional lymph nodes , mv enters the bloodstream and subsequently spreads to all lymphoid organs , such as the spleen , thymus , appendix , tonsil , and lymph nodes , throughout the body . when the mv infection of the lymphoid organs reaches its peak , mv - infected immune cells transfer mv to epithelial cells through the basolateral side . at this step , mv uses nectin4 expressed at ajs as a receptor . in the epithelia , mv selectively releases its progeny virus particles from the apical membrane to the luminal side of the respiratory tract . membrane cofactor protein / cd46 was first identified as a receptor for mv ( dorig et al . , however , our current knowledge of mv receptors has been totally transformed . in 2000 , it was shown that slam / cd150 expressed on cells of the immune system , but not mcp / cd46 , is a real receptor for wt mv . however , recent studies further showed that slam / cd150 is not the sole receptor for mv . mv has an intrinsic ability to enter not only immune cells but also epithelial cells . in 2011 , a clear answer was obtained through the identification of the epithelial mv receptor nectin4 , which is expressed at ajs , thereby partly explaining why mv exhibits its highly contagious nature ( mhlebach et al . , however , many aspects and molecular mechanisms underlying immunosuppressive effects and a highly contagious nature of mv still remain to be elucidated . analyses of physiological roles of mv receptors , slam / cd150 , and nectin4 , would provide deep insights into mv pathogenesis . 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 .

alzheimer 's disease ( ad ) is a neurodegenerative disorder clinically characterized by progressive dementia and neuropsychiatric symptoms . a breakdown in connectivity , both in the functional and structural system domain , the human brain is organized as a complex network , allowing for segregation and integration of information processing . by modeling the brain as a complex network , data from recent studies suggest that ad - associated brain network changes may be related to cognitive decline and neuropsychological impairment . . demonstrated the topological alterations in ad patients using interregional correlation of cortical thickness as a metric of the structural basis underlying brain dynamics in structural networks . reported diminished functional connectivity between the hippocampus and posterior cingulate cortex for functional networks that are prominently involved in the processes of episodic memory in patients at prodromal stages of ad . two basic topological measurements of a complex network are clustering coefficient ( cc ) and characteristic path length ( cpl ) . the cc quantifies the local efficiency of information transfer while cpl quantifies the global efficiency of parallel information transmission . although previous studies have indicated that the cognitive deficits may be due to topological deteriorations of the brain network , a consensus regarding the nature of the alteration pattern has not been reached . a study by stam et al . found that the network cc was unchanged in patients with ad although the average cpl was elongated . contradictorily , supekar et al . indicated that ad patients had a lower cc , but no changes in cpl compared with healthy controls . . demonstrated a higher cc and longer cpl in patients with ad . to account for these discrepancies , one suggestion is to compare structural and functional networks simultaneously in the same group of people . , most studies just examined the large - scale brain network properties at a specific frequency band . it is not clear whether the selection of a specific frequency band could impact the topological properties in patients with ad . recent studies have reported the frequency specificity of functional connectivity in multiple functional networks derived from resting - state functional magnetic resonance imaging ( rs - fmri ) signals , as well as frequency - specific correlations between hemodynamic changes and electroencephalography oscillations in healthy people . frequency - specific alterations of neuronal oscillations were revealed in patients suffering from schizophrenia using rs - fmri , and parkinson 's disease by simultaneous magnetoencephalography ( meg ) and local field potential recordings . in our study , we expected to find frequency - specific changes in the topological properties of ad patients . focusing on rs - fmri data only , large - scale topological properties of moderate ad patients and healthy controls were investigated at three distinct frequency bands ( 0.010.06 hz , 0.060.11 hz , and 0.110.25 hz ) . the global efficiency , the cc , the cpl and the small - world property of the subjects were quantified to describe the alterations in each frequency band . all participants were screened and enrolled according to the procedure used in former studies of the same group . finally , according to the nincds - adrda criteria for probable ad the patients were all clinically categorized as those at moderate stages of ad according to the clinical dementia rating ( cdr ) scale of 2 . ten right - handed healthy controls with no history of neurological disorder or head trauma and normal neurologic examination findings ( cdr = 0 ) were recruited from the local community . global cognitive function of all subjects was evaluated using the mini - mental state examination . subject characteristics ad : alzheimer s disease ; mmse : mini - mental state examination . a 3 t mr scanner ( signa hdxt , ge healthcare , usa ) with 8-channel head array coil was used in this study . each subject was scanned in a supine , head - first position with symmetrically placed cushions on both sides of the head to ensure stability . during the rs - fmri scanning , the subjects were instructed to keep as still as possible , with eyes closed , and think of nothing in particular while maintaining wakefulness . a gradient - echo planar sequence was performed for the acquisition of rs - fmri data , with the acquisition parameters as follows : repetition time ( tr)/echo time ( te ) 2000/30 ms , field of view ( fov ) 240 mm 240 mm , phase fov 1 , matrix 64 64 , slices 33 , slice thickness 4.0 mm , slice gap 0.5 mm , scan time 8 min . a three - dimensional fast spoiled gradient recalled - echo sequence covering the whole brain was used for structural data acquisition with tr / te / inversion time 6.5/2.1/400 ms , fov 256 mm 256 mm , phase fov 1 , matrix 256 256 , slice thickness 1.0 mm , slice gap 0 mm , number of signal averages ( nsa ) 1 , flip angle 15 , scan time 4 min 8 s. spm8 ( http://www.fil.ion.ucl.ac.uk/spm/ ) was used for the preprocessing of fmri datasets . after the first 10 time points were discarded , the remaining images of all subjects were then normalized to montreal neurological institute 152 space . all images were then smoothed with a 4 mm 4 mm 4 mm gaussian kernel . datasets were then drifted and filtered in three distinct frequency bands : 0.010.06 hz , 0.060.11 hz , and 0.110.25 hz . nuisance covariates of whole brain , white matter , and cerebrospinal fluid signals were removed using regression . the registered fmri data were segmented into 90 regions using an automated anatomical labeling template . the pearson correlation coefficients were calculated between any two functionally connected regions at three different frequency bands ( 0.010.06 hz , 0.060.11 hz and 0.110.25 hz ) . each subject was assigned a 90 90 correlation matrix at different filter bands . in order to detect the topological differences of functional brain network between ad patients and healthy controls , a wide range of thresholds specifically , if the pearson correlation coefficient was greater than the threshold , the correlation coefficient was set to 1 ; otherwise , it was set to 0 . the selected threshold ranged from 0.35 to 0.60 and increased by 0.01 at each step . in this study , the global efficiency , the cc , the cpl , and small - world property were calculated in a wide range of thresholds and averaged within each group . the definition of these parameters can be found in bullmore and sporns . to compare the degree distributions of functional brain network between ad patients and healthy controls , the node degree was then calculated in each node and averaged within each group to generate the degree distribution curve . network is characterized as a network with a shorter cpl than a regular network ( high cc and long cpl ) and a greater cc than a random network ( low cc and short cpl ) . to detect the differences of small - world properties between ad patients and healthy controls , the ratio of cc and cpl was quantified in each subject and averaged within each group . the differences of topological parameters between patients and controls were compared by two - sample t - test using spss 17.0 ( ssps inc . , chicago , il , usa ) software . all participants were screened and enrolled according to the procedure used in former studies of the same group . finally , according to the nincds - adrda criteria for probable ad the patients were all clinically categorized as those at moderate stages of ad according to the clinical dementia rating ( cdr ) scale of 2 . ten right - handed healthy controls with no history of neurological disorder or head trauma and normal neurologic examination findings ( cdr = 0 ) were recruited from the local community . global cognitive function of all subjects was evaluated using the mini - mental state examination . subject characteristics ad : alzheimer s disease ; mmse : mini - mental state examination . a 3 t mr scanner ( signa hdxt , ge healthcare , usa ) with 8-channel head array coil was used in this study . each subject was scanned in a supine , head - first position with symmetrically placed cushions on both sides of the head to ensure stability . during the rs - fmri scanning , the subjects were instructed to keep as still as possible , with eyes closed , and think of nothing in particular while maintaining wakefulness . a gradient - echo planar sequence was performed for the acquisition of rs - fmri data , with the acquisition parameters as follows : repetition time ( tr)/echo time ( te ) 2000/30 ms , field of view ( fov ) 240 mm 240 mm , phase fov 1 , matrix 64 64 , slices 33 , slice thickness 4.0 mm , slice gap 0.5 mm , scan time 8 min . a three - dimensional fast spoiled gradient recalled - echo sequence covering the whole brain was used for structural data acquisition with tr / te / inversion time 6.5/2.1/400 ms , fov 256 mm 256 mm , phase fov 1 , matrix 256 256 , slice thickness 1.0 mm , slice gap 0 mm , number of signal averages ( nsa ) 1 , flip angle 15 , scan time 4 min 8 s. after the first 10 time points were discarded , slice timing and head motion correction were performed . the remaining images of all subjects were then normalized to montreal neurological institute 152 space . all images were then smoothed with a 4 mm 4 mm 4 mm gaussian kernel . datasets were then drifted and filtered in three distinct frequency bands : 0.010.06 hz , 0.060.11 hz , and 0.110.25 hz . nuisance covariates of whole brain , white matter , and cerebrospinal fluid signals were removed using regression . the registered fmri data were segmented into 90 regions using an automated anatomical labeling template . the pearson correlation coefficients were calculated between any two functionally connected regions at three different frequency bands ( 0.010.06 hz , 0.060.11 hz and 0.110.25 hz ) . each subject was assigned a 90 90 correlation matrix at different filter bands . in order to detect the topological differences of functional brain network between ad patients and healthy controls , a wide range of thresholds specifically , if the pearson correlation coefficient was greater than the threshold , the correlation coefficient was set to 1 ; otherwise , it was set to 0 . the selected threshold ranged from 0.35 to 0.60 and increased by 0.01 at each step . in this study , the global efficiency , the cc , the cpl , and small - world property were calculated in a wide range of thresholds and averaged within each group . the definition of these parameters can be found in bullmore and sporns . to compare the degree distributions of functional brain network between ad patients and healthy controls , the node degree was then calculated in each node and averaged within each group to generate the degree distribution curve . network is characterized as a network with a shorter cpl than a regular network ( high cc and long cpl ) and a greater cc than a random network ( low cc and short cpl ) . to detect the differences of small - world properties between ad patients and healthy controls , the ratio of cc and cpl the differences of topological parameters between patients and controls were compared by two - sample t - test using spss 17.0 ( ssps inc . , chicago , il , usa ) software . the global efficiencies of ad patients and healthy controls at three different frequency bands are shown in figure 1 . the global efficiencies of ad patients were always lower than healthy controls at the frequency bands of 0.010.06 hz and 0.060.11 hz under all thresholds . no significant difference of global efficiency between groups at the frequency band of 0.110.25 hz was found at smaller thresholds . however , within a higher threshold ( more than 0.5 ) , the global efficiency was still lower in ad patients than in healthy controls . the global efficiencies of the functional brain networks in alzheimer 's disease patients ( marked in black ) and in healthy controls ( marked in red ) at the three different frequency bands . figure 2 shows the cc of the functional brain network of ad patients and healthy controls at different frequency bands . the results indicate that the cc was always lower in ad patients than in controls at lower frequency bands of 0.010.06 hz and 0.060.11 hz . for higher frequency band ( 0.110.25 hz ) , no significant difference was found between groups . the clustering coefficients of the functional brain networks in alzheimer 's disease patients ( marked in black ) and in healthy controls ( marked in red ) at the three different frequency bands . the average cpl of the healthy controls was much shorter at the frequency band of 0.110.25 hz if the threshold was greater than 0.5 . there was no significant difference in the cpl at lower frequency bands ( 0.010.06 hz , 0.060.11 hz ) . the average shortest path length of the functional brain networks in alzheimer 's disease patients ( marked in black ) and in healthy controls ( marked in red ) at the three different frequency bands . small - world figure 4 shows the small - world property of the healthy controls was always higher than that of ad patients under all the thresholds at three distinct frequency bands . properties of the functional brain networks in alzheimer 's disease patients ( marked in black ) and in healthy controls ( marked in red ) at the three different frequency bands . the degree of a node is the number of connections that link it to the rest of the network . the degree of each node was calculated at the frequency band of 0.060.11 hz with a threshold value of 0.45 in each subject and then averaged within each group . table 2 shows the average node degrees in patient group and control group . among all 90 nodes , the proportion of nodes with a node degree higher than 17 was just 5.56% in ad patient group while in healthy controls the proportion was as high as 28.89% . the probability of node degree in ad patient group and healthy control group ad : alzheimer s disease . previous studies have shown that the degree distribution of the functional brain network is described by a gauss function curve . figure 5 represents the degree distribution curve of ad patients and controls fitted with a gaussian distribution . the probability distribution of node degree in the healthy group was higher than in the ad group when the node degree was > 15 . the degree distribution of alzheimer 's disease group ( black curve ) and control group ( red curve ) after gaussian function curve - fitting . the differences of node degree between ad patients and healthy controls are further investigated [ figure 6 ] . the results demonstrate that a reduction of node degree was distributed in the left postcentral gyrus ( pocg.l ) , the right hippocampus ( hip.r ) , the left middle temporal gyrus ( mtg.l ) , the right middle occipital gyrus ( mog.r ) , the right orbital part of middle frontal gyrus ( orbsupmed.r ) , the right amygdala ( amyg.r ) , the left lingual gyrus ( ling.l ) , the medial part of right superior frontal gyrus ( sfgmed.r ) , and the medial part of left superior frontal gyrus ( sfgmed.l ) . only a few regions show increased node degree in ad patients : the left inferior temporal gyrus ( itg.l ) , the left orbital part of superior frontal gyrus ( orbsup.l ) , and the right mtg ( mtg.r ) . the structure - specific alterations of node degrees in ad patients . the red dots indicate increased node degrees in the ad group compared with healthy controls ; the blue dots indicate reduced node degrees in the ad group . ad : alzheimer 's disease ; amyg.r : right amygdala ; hip.r : right hippocampus ; itg.l : inferior temporal gyrus ; ling.l : left lingual gyrus ; mog.r : right middle occipital gyrus ; mtg.l : left middle temporal gyrus ; mtg.r : right middle temporal gyrus ; orbsupmed.r : right orbital part of middle frontal gyrus ; pocg.l : left postcentral gyrus ; sfgmed.l : medial part of left superior frontal gyrus ; sfgmed.r : medial part of right superior frontal gyrus . the global efficiencies of ad patients and healthy controls at three different frequency bands are shown in figure 1 . the global efficiencies of ad patients were always lower than healthy controls at the frequency bands of 0.010.06 hz and 0.060.11 hz under all thresholds . no significant difference of global efficiency between groups at the frequency band of 0.110.25 hz was found at smaller thresholds . however , within a higher threshold ( more than 0.5 ) , the global efficiency was still lower in ad patients than in healthy controls . the global efficiencies of the functional brain networks in alzheimer 's disease patients ( marked in black ) and in healthy controls ( marked in red ) at the three different frequency bands . figure 2 shows the cc of the functional brain network of ad patients and healthy controls at different frequency bands . the results indicate that the cc was always lower in ad patients than in controls at lower frequency bands of 0.010.06 hz and 0.060.11 hz . for higher frequency band ( 0.110.25 hz ) , no significant difference was found between groups . the clustering coefficients of the functional brain networks in alzheimer 's disease patients ( marked in black ) and in healthy controls ( marked in red ) at the three different frequency bands . the average cpl of the healthy controls was much shorter at the frequency band of 0.110.25 hz if the threshold was greater than 0.5 . there was no significant difference in the cpl at lower frequency bands ( 0.010.06 hz , 0.060.11 hz ) . the average shortest path length of the functional brain networks in alzheimer 's disease patients ( marked in black ) and in healthy controls ( marked in red ) at the three different frequency bands . small - world figure 4 shows the small - world property of the healthy controls was always higher than that of ad patients under all the thresholds at three distinct frequency bands . properties of the functional brain networks in alzheimer 's disease patients ( marked in black ) and in healthy controls ( marked in red ) at the three different frequency bands . the degree of a node is the number of connections that link it to the rest of the network . the degree of each node was calculated at the frequency band of 0.060.11 hz with a threshold value of 0.45 in each subject and then averaged within each group . table 2 shows the average node degrees in patient group and control group . among all 90 nodes , the proportion of nodes with a node degree higher than 17 was just 5.56% in ad patient group while in healthy controls the proportion was as high as 28.89% . the probability of node degree in ad patient group and healthy control group ad : alzheimer s disease . previous studies have shown that the degree distribution of the functional brain network is described by a gauss function curve . figure 5 represents the degree distribution curve of ad patients and controls fitted with a gaussian distribution . the probability distribution of node degree in the healthy group was higher than in the ad group when the node degree was > 15 . the degree distribution of alzheimer 's disease group ( black curve ) and control group ( red curve ) after gaussian function curve - fitting . the differences of node degree between ad patients and healthy controls are further investigated [ figure 6 ] . the results demonstrate that a reduction of node degree was distributed in the left postcentral gyrus ( pocg.l ) , the right hippocampus ( hip.r ) , the left middle temporal gyrus ( mtg.l ) , the right middle occipital gyrus ( mog.r ) , the right orbital part of middle frontal gyrus ( orbsupmed.r ) , the right amygdala ( amyg.r ) , the left lingual gyrus ( ling.l ) , the medial part of right superior frontal gyrus ( sfgmed.r ) , and the medial part of left superior frontal gyrus ( sfgmed.l ) . only a few regions show increased node degree in ad patients : the left inferior temporal gyrus ( itg.l ) , the left orbital part of superior frontal gyrus ( orbsup.l ) , and the right mtg ( mtg.r ) . the structure - specific alterations of node degrees in ad patients . the red dots indicate increased node degrees in the ad group compared with healthy controls ; the blue dots indicate reduced node degrees in the ad group . ad : alzheimer 's disease ; amyg.r : right amygdala ; hip.r : right hippocampus ; itg.l : inferior temporal gyrus ; ling.l : left lingual gyrus ; mog.r : right middle occipital gyrus ; mtg.l : left middle temporal gyrus ; mtg.r : right middle temporal gyrus ; orbsupmed.r : right orbital part of middle frontal gyrus ; pocg.l : left postcentral gyrus ; sfgmed.l : medial part of left superior frontal gyrus ; sfgmed.r : medial part of right superior frontal gyrus . the present study shows that the global efficiency , the cc , and the small - world properties in the ad patient group changes with different frequency bands , suggesting the existence of disrupted global and local functional organization associated with ad . specifically , at lower - frequency bands ( 0.010.06 hz , 0.060.11 hz ) , the global efficiency , the cc and the small - world properties of ad patients decreased compared with healthy controls while the cpl elongated within a wide range of thresholds . however , at higher - frequency bands ( 0.110.25 hz ) , the global efficiency and the cc showed no significant differences compared with healthy controls , while the small - world properties were lower and the cpl were much longer in ad than in controls , particularly at a higher threshold . comparisons of node degree indicate that the probability distribution of node degree in healthy groups was higher than in ad group if the node degree exceeded 15 . for structure - specific alterations , there was a significant reduction of node degree in brain regions of pocg.l , hip.r , mtg.l , mog.r , orbsupmed.r , amyg.r , ling.l , sfgmed.r , and sfgmed.l ; while in areas of itg.l , orbsup.l , and mtg.r , the node degree increased . the results of this study demonstrate that the topological property of large - scale functional brain networks in ad patients is frequency dependent . previous studies have suggested that the hemodynamic responses measured by rs - fmri reflect spontaneous neural activity which largely fluctuates at a lower - frequency band . it has been demonstrated that spontaneous low - frequency fluctuations ( 0.010.1 hz ) are physiologically meaningful . higher - frequency fluctuations are usually affected by respiratory signal frequency ( 0.10.5 hz ) and cardiovascular signal frequency ( 0.10.5 hz ) . in this study , the topological properties of ad patients and healthy controls exhibited different properties at different frequency bands . one possibility is that topological properties are frequency - dependent , which contributed to the discrepancies observed in previous studies . stam et al . demonstrated that within an extensively wide range of thresholds , ad patients exhibited a longer cpl than healthy controls but no significant changes in the cc were found . however , supekar et al . showed a decline of cc in patients with ad . meanwhile , in 2009 , using meg data , stam et al . demonstrated that ad patients had a lower cc and a longer cpl . in 2010 , using rs - fmri data , sanz - arigita et al . reported no significant changes in cc in ad but with a shorter cpl the inconsistent results of these studies may be caused by the selection of different frequency bands . our report agrees with reports from de haan et al . , which suggest that various frequency bands impact the topological properties of a functional brain network . global efficiency is one of the most pertinent parameters for quantifying the parallel information propagation capability of a network . in this study , the global efficiency of ad patients was always lower than healthy controls at lower - frequency bands ( 0.010.06 hz , 0.060.11 hz ) [ figure 1 ] , data which agrees with zhao et al . the decline of global efficiency in ad patients indicates that pathological changes in the brain have reduced connectivity strength and blocked information communication in remote functional regions . a wealth of studies has demonstrated that the global efficiency of the brain network declines if the brain is pathologically impaired . this may be one major factor contributing to the cognitive decline of patients with ad . however , compared with healthy controls , there was no significant difference of the global efficiency at a higher frequency band ( 0.110.25 hz ) . we suggest that higher - frequency fluctuations may result from coexisting noise during the acquisition of mri signals in both ad patients and healthy controls . the cc of a network quantifies the number of local connections that exist as a proportion of the maximum number of possible connections . our study demonstrates that at lower - frequency bands within a higher range of thresholds , the cc of ad patients is diminished compared with healthy controls , in line with previous studies . this finding indicates a decline in coupling , robustness , as well as the local functional connectivity strength between nodes in patients with ad . there was no significant difference in cpl between ad and controls , demonstrating that short - range connections are maintained in ad patients . short paths promote effective interactions between neuronal elements within and across cortical regions , and are essential for functional integration . in 1998 , watts and strogatz first explored the characteristic of a small - world network . it has been suggested that hierarchical organizations of ad and controls differ with each other , resulting in a significant decline in cc among ad patients . it not only facilitates both modularized and distributed information processing but also maximizes the efficiency of information transfer at a relatively low wiring cost . in a small - world network , there are many clusters ( small groups of nodes connected to each other ) or node clusters of which the number of connections is less than the number of clusters . in addition , any two nodes are usually connected by at least one short path . numerous studies have demonstrated that the small - world properties of ad patients suffer significantly deterioration . properties indicates that the efficiency of information transfer within different brain regions has also declined , a factor which may play a major role in the cognitive decline observed in ad patients . at a threshold of 0.45 , the structure - specific alterations of node degree in ad patients were further investigated at the frequency band of 0.060.11 hz . the results indicate that areas with node degree reduction are located mostly in the temporal lobe and frontal lobe . previous studies have demonstrated that a deposition in ad occurs preferentially in locations of cortical hubs ( nodes ) , accounting for the observed activity - dependent mechanism associated with connectional hubs . the cortical components of the medial temporal lobe and the frontoparietal lobe of these hubs are especially vulnerable to ad . furthermore , three regions show an increased node degree in ad patients ( itg.l , orbsup.l and mtg.r ) , indicating a coexisting functional compensation in the temporal lobe and frontal lobe . in conclusion , based on data analysis of resting - state functional networks , large - scale functional brain networks of ad patients and healthy controls were constructed and compared for a wide range of thresholds at different frequency bands . compared with healthy controls , the patients with ad exhibited lower global efficiency , lower cc , and also lower small - world furthermore , a decline in the node degree was observed in the large majority of pathologically attacked regions , with other areas increasing as functional compensations . our study accurately depicts the frequency - specific topological alterations in ad - related large - scale functional brain networks , providing fundamental support for optimal frequency selection in future related research . previous studies have suggested that the hemodynamic responses measured by rs - fmri reflect spontaneous neural activity which largely fluctuates at a lower - frequency band . it has been demonstrated that spontaneous low - frequency fluctuations ( 0.010.1 hz ) are physiologically meaningful . higher - frequency fluctuations are usually affected by respiratory signal frequency ( 0.10.5 hz ) and cardiovascular signal frequency ( 0.10.5 hz ) . in this study , the topological properties of ad patients and healthy controls exhibited different properties at different frequency bands . one possibility is that topological properties are frequency - dependent , which contributed to the discrepancies observed in previous studies . stam et al . demonstrated that within an extensively wide range of thresholds , ad patients exhibited a longer cpl than healthy controls but no significant changes in the cc were found . however , supekar et al . showed a decline of cc in patients with ad . meanwhile , in 2009 , using meg data , stam et al . demonstrated that ad patients had a lower cc and a longer cpl . in 2010 , using rs - fmri data , sanz - arigita et al . reported no significant changes in cc in ad but with a shorter cpl the inconsistent results of these studies may be caused by the selection of different frequency bands . our report agrees with reports from de haan et al . , which suggest that various frequency bands impact the topological properties of a functional brain network . global efficiency is one of the most pertinent parameters for quantifying the parallel information propagation capability of a network . in this study , the global efficiency of ad patients was always lower than healthy controls at lower - frequency bands ( 0.010.06 hz , 0.060.11 hz ) [ figure 1 ] , data which agrees with zhao et al . the decline of global efficiency in ad patients indicates that pathological changes in the brain have reduced connectivity strength and blocked information communication in remote functional regions . a wealth of studies has demonstrated that the global efficiency of the brain network declines if the brain is pathologically impaired . this may be one major factor contributing to the cognitive decline of patients with ad . however , compared with healthy controls , there was no significant difference of the global efficiency at a higher frequency band ( 0.110.25 hz ) . we suggest that higher - frequency fluctuations may result from coexisting noise during the acquisition of mri signals in both ad patients and healthy controls . the cc of a network quantifies the number of local connections that exist as a proportion of the maximum number of possible connections . our study demonstrates that at lower - frequency bands within a higher range of thresholds , the cc of ad patients is diminished compared with healthy controls , in line with previous studies . this finding indicates a decline in coupling , robustness , as well as the local functional connectivity strength between nodes in patients with ad . there was no significant difference in cpl between ad and controls , demonstrating that short - range connections are maintained in ad patients . short paths promote effective interactions between neuronal elements within and across cortical regions , and are essential for functional integration . in 1998 , watts and strogatz first explored the characteristic of a small - world network . it has been suggested that hierarchical organizations of ad and controls differ with each other , resulting in a significant decline in cc among ad patients . it not only facilitates both modularized and distributed information processing but also maximizes the efficiency of information transfer at a relatively low wiring cost . in a small - world network , there are many clusters ( small groups of nodes connected to each other ) or node clusters of which the number of connections is less than the number of clusters . in addition , any two nodes are usually connected by at least one short path . numerous studies have demonstrated that the small - world properties of ad patients suffer significantly deterioration . properties indicates that the efficiency of information transfer within different brain regions has also declined , a factor which may play a major role in the cognitive decline observed in ad patients . at a threshold of 0.45 , the structure - specific alterations of node degree in ad patients were further investigated at the frequency band of 0.060.11 hz . the results indicate that areas with node degree reduction are located mostly in the temporal lobe and frontal lobe . previous studies have demonstrated that a deposition in ad occurs preferentially in locations of cortical hubs ( nodes ) , accounting for the observed activity - dependent mechanism associated with connectional hubs . the cortical components of the medial temporal lobe and the frontoparietal lobe of these hubs are especially vulnerable to ad . furthermore , three regions show an increased node degree in ad patients ( itg.l , orbsup.l and mtg.r ) , indicating a coexisting functional compensation in the temporal lobe and frontal lobe . in conclusion , based on data analysis of resting - state functional networks , large - scale functional brain networks of ad patients and healthy controls were constructed and compared for a wide range of thresholds at different frequency bands . compared with healthy controls , the patients with ad exhibited lower global efficiency , lower cc , and also lower small - world furthermore , a decline in the node degree was observed in the large majority of pathologically attacked regions , with other areas increasing as functional compensations . our study accurately depicts the frequency - specific topological alterations in ad - related large - scale functional brain networks , providing fundamental support for optimal frequency selection in future related research .

background : previous studies have indicated that the cognitive deficits in patients with alzheimer 's disease ( ad ) may be due to topological deteriorations of the brain network . however , whether the selection of a specific frequency band could impact the topological properties is still not clear . our hypothesis is that the topological properties of ad patients are also frequency-specific.methods:resting state functional magnetic resonance imaging data from 10 right - handed moderate ad patients ( mean age : 64.3 years ; mean mini mental state examination [ mmse ] : 18.0 ) and 10 age and gender - matched healthy controls ( mean age : 63.6 years ; mean mmse : 28.2 ) were enrolled in this study . the global efficiency , the clustering coefficient ( cc ) , the characteristic path length ( cpl ) , and small - world property were calculated in a wide range of thresholds and averaged within each group , at three different frequency bands ( 0.010.06 hz , 0.060.11 hz , and 0.110.25 hz).results : at lower - frequency bands ( 0.010.06 hz , 0.060.11 hz ) , the global efficiency , the cc and the small - world properties of ad patients decreased compared to controls . while at higher - frequency bands ( 0.110.25 hz ) , the cpl was much longer , and the small - world property was disrupted in ad , particularly at a higher threshold . the topological properties changed with different frequency bands , suggesting the existence of disrupted global and local functional organization associated with ad.conclusions:this study demonstrates that the topological alterations of large - scale functional brain networks in ad patients are frequency dependent , thus providing fundamental support for optimal frequency selection in future related research .

I M Subjects Magnetic resonance imaging examination Data preprocessing Construction of brain functional network Topological properties of the functional brain network Statistics R Topological parameter comparisons at different frequency bands Changes of the node degree D Different topological properties at different frequency bands Topological organizations of large-scale functional brain network Regional alterations of node degree

alzheimer 's disease ( ad ) is a neurodegenerative disorder clinically characterized by progressive dementia and neuropsychiatric symptoms . demonstrated the topological alterations in ad patients using interregional correlation of cortical thickness as a metric of the structural basis underlying brain dynamics in structural networks . two basic topological measurements of a complex network are clustering coefficient ( cc ) and characteristic path length ( cpl ) . although previous studies have indicated that the cognitive deficits may be due to topological deteriorations of the brain network , a consensus regarding the nature of the alteration pattern has not been reached . found that the network cc was unchanged in patients with ad although the average cpl was elongated . demonstrated a higher cc and longer cpl in patients with ad . , most studies just examined the large - scale brain network properties at a specific frequency band . it is not clear whether the selection of a specific frequency band could impact the topological properties in patients with ad . recent studies have reported the frequency specificity of functional connectivity in multiple functional networks derived from resting - state functional magnetic resonance imaging ( rs - fmri ) signals , as well as frequency - specific correlations between hemodynamic changes and electroencephalography oscillations in healthy people . frequency - specific alterations of neuronal oscillations were revealed in patients suffering from schizophrenia using rs - fmri , and parkinson 's disease by simultaneous magnetoencephalography ( meg ) and local field potential recordings . in our study , we expected to find frequency - specific changes in the topological properties of ad patients . focusing on rs - fmri data only , large - scale topological properties of moderate ad patients and healthy controls were investigated at three distinct frequency bands ( 0.010.06 hz , 0.060.11 hz , and 0.110.25 hz ) . the global efficiency , the cc , the cpl and the small - world property of the subjects were quantified to describe the alterations in each frequency band . ten right - handed healthy controls with no history of neurological disorder or head trauma and normal neurologic examination findings ( cdr = 0 ) were recruited from the local community . datasets were then drifted and filtered in three distinct frequency bands : 0.010.06 hz , 0.060.11 hz , and 0.110.25 hz . the pearson correlation coefficients were calculated between any two functionally connected regions at three different frequency bands ( 0.010.06 hz , 0.060.11 hz and 0.110.25 hz ) . in order to detect the topological differences of functional brain network between ad patients and healthy controls , a wide range of thresholds specifically , if the pearson correlation coefficient was greater than the threshold , the correlation coefficient was set to 1 ; otherwise , it was set to 0 . in this study , the global efficiency , the cc , the cpl , and small - world property were calculated in a wide range of thresholds and averaged within each group . to compare the degree distributions of functional brain network between ad patients and healthy controls , the node degree was then calculated in each node and averaged within each group to generate the degree distribution curve . to detect the differences of small - world properties between ad patients and healthy controls , the ratio of cc and cpl was quantified in each subject and averaged within each group . ten right - handed healthy controls with no history of neurological disorder or head trauma and normal neurologic examination findings ( cdr = 0 ) were recruited from the local community . datasets were then drifted and filtered in three distinct frequency bands : 0.010.06 hz , 0.060.11 hz , and 0.110.25 hz . the pearson correlation coefficients were calculated between any two functionally connected regions at three different frequency bands ( 0.010.06 hz , 0.060.11 hz and 0.110.25 hz ) . in order to detect the topological differences of functional brain network between ad patients and healthy controls , a wide range of thresholds specifically , if the pearson correlation coefficient was greater than the threshold , the correlation coefficient was set to 1 ; otherwise , it was set to 0 . in this study , the global efficiency , the cc , the cpl , and small - world property were calculated in a wide range of thresholds and averaged within each group . to compare the degree distributions of functional brain network between ad patients and healthy controls , the node degree was then calculated in each node and averaged within each group to generate the degree distribution curve . to detect the differences of small - world properties between ad patients and healthy controls , the ratio of cc and cpl the differences of topological parameters between patients and controls were compared by two - sample t - test using spss 17.0 ( ssps inc . the global efficiencies of ad patients and healthy controls at three different frequency bands are shown in figure 1 . the global efficiencies of ad patients were always lower than healthy controls at the frequency bands of 0.010.06 hz and 0.060.11 hz under all thresholds . no significant difference of global efficiency between groups at the frequency band of 0.110.25 hz was found at smaller thresholds . however , within a higher threshold ( more than 0.5 ) , the global efficiency was still lower in ad patients than in healthy controls . the global efficiencies of the functional brain networks in alzheimer 's disease patients ( marked in black ) and in healthy controls ( marked in red ) at the three different frequency bands . figure 2 shows the cc of the functional brain network of ad patients and healthy controls at different frequency bands . the results indicate that the cc was always lower in ad patients than in controls at lower frequency bands of 0.010.06 hz and 0.060.11 hz . for higher frequency band ( 0.110.25 hz ) , no significant difference was found between groups . the clustering coefficients of the functional brain networks in alzheimer 's disease patients ( marked in black ) and in healthy controls ( marked in red ) at the three different frequency bands . the average cpl of the healthy controls was much shorter at the frequency band of 0.110.25 hz if the threshold was greater than 0.5 . there was no significant difference in the cpl at lower frequency bands ( 0.010.06 hz , 0.060.11 hz ) . the average shortest path length of the functional brain networks in alzheimer 's disease patients ( marked in black ) and in healthy controls ( marked in red ) at the three different frequency bands . small - world figure 4 shows the small - world property of the healthy controls was always higher than that of ad patients under all the thresholds at three distinct frequency bands . properties of the functional brain networks in alzheimer 's disease patients ( marked in black ) and in healthy controls ( marked in red ) at the three different frequency bands . the degree of each node was calculated at the frequency band of 0.060.11 hz with a threshold value of 0.45 in each subject and then averaged within each group . previous studies have shown that the degree distribution of the functional brain network is described by a gauss function curve . the results demonstrate that a reduction of node degree was distributed in the left postcentral gyrus ( pocg.l ) , the right hippocampus ( hip.r ) , the left middle temporal gyrus ( mtg.l ) , the right middle occipital gyrus ( mog.r ) , the right orbital part of middle frontal gyrus ( orbsupmed.r ) , the right amygdala ( amyg.r ) , the left lingual gyrus ( ling.l ) , the medial part of right superior frontal gyrus ( sfgmed.r ) , and the medial part of left superior frontal gyrus ( sfgmed.l ) . only a few regions show increased node degree in ad patients : the left inferior temporal gyrus ( itg.l ) , the left orbital part of superior frontal gyrus ( orbsup.l ) , and the right mtg ( mtg.r ) . the global efficiencies of ad patients and healthy controls at three different frequency bands are shown in figure 1 . the global efficiencies of ad patients were always lower than healthy controls at the frequency bands of 0.010.06 hz and 0.060.11 hz under all thresholds . no significant difference of global efficiency between groups at the frequency band of 0.110.25 hz was found at smaller thresholds . however , within a higher threshold ( more than 0.5 ) , the global efficiency was still lower in ad patients than in healthy controls . the global efficiencies of the functional brain networks in alzheimer 's disease patients ( marked in black ) and in healthy controls ( marked in red ) at the three different frequency bands . figure 2 shows the cc of the functional brain network of ad patients and healthy controls at different frequency bands . the results indicate that the cc was always lower in ad patients than in controls at lower frequency bands of 0.010.06 hz and 0.060.11 hz . for higher frequency band ( 0.110.25 hz ) , no significant difference was found between groups . the clustering coefficients of the functional brain networks in alzheimer 's disease patients ( marked in black ) and in healthy controls ( marked in red ) at the three different frequency bands . the average cpl of the healthy controls was much shorter at the frequency band of 0.110.25 hz if the threshold was greater than 0.5 . there was no significant difference in the cpl at lower frequency bands ( 0.010.06 hz , 0.060.11 hz ) . the average shortest path length of the functional brain networks in alzheimer 's disease patients ( marked in black ) and in healthy controls ( marked in red ) at the three different frequency bands . small - world figure 4 shows the small - world property of the healthy controls was always higher than that of ad patients under all the thresholds at three distinct frequency bands . properties of the functional brain networks in alzheimer 's disease patients ( marked in black ) and in healthy controls ( marked in red ) at the three different frequency bands . the degree of each node was calculated at the frequency band of 0.060.11 hz with a threshold value of 0.45 in each subject and then averaged within each group . previous studies have shown that the degree distribution of the functional brain network is described by a gauss function curve . the results demonstrate that a reduction of node degree was distributed in the left postcentral gyrus ( pocg.l ) , the right hippocampus ( hip.r ) , the left middle temporal gyrus ( mtg.l ) , the right middle occipital gyrus ( mog.r ) , the right orbital part of middle frontal gyrus ( orbsupmed.r ) , the right amygdala ( amyg.r ) , the left lingual gyrus ( ling.l ) , the medial part of right superior frontal gyrus ( sfgmed.r ) , and the medial part of left superior frontal gyrus ( sfgmed.l ) . only a few regions show increased node degree in ad patients : the left inferior temporal gyrus ( itg.l ) , the left orbital part of superior frontal gyrus ( orbsup.l ) , and the right mtg ( mtg.r ) . the present study shows that the global efficiency , the cc , and the small - world properties in the ad patient group changes with different frequency bands , suggesting the existence of disrupted global and local functional organization associated with ad . specifically , at lower - frequency bands ( 0.010.06 hz , 0.060.11 hz ) , the global efficiency , the cc and the small - world properties of ad patients decreased compared with healthy controls while the cpl elongated within a wide range of thresholds . however , at higher - frequency bands ( 0.110.25 hz ) , the global efficiency and the cc showed no significant differences compared with healthy controls , while the small - world properties were lower and the cpl were much longer in ad than in controls , particularly at a higher threshold . the results of this study demonstrate that the topological property of large - scale functional brain networks in ad patients is frequency dependent . previous studies have suggested that the hemodynamic responses measured by rs - fmri reflect spontaneous neural activity which largely fluctuates at a lower - frequency band . it has been demonstrated that spontaneous low - frequency fluctuations ( 0.010.1 hz ) are physiologically meaningful . higher - frequency fluctuations are usually affected by respiratory signal frequency ( 0.10.5 hz ) and cardiovascular signal frequency ( 0.10.5 hz ) . in this study , the topological properties of ad patients and healthy controls exhibited different properties at different frequency bands . one possibility is that topological properties are frequency - dependent , which contributed to the discrepancies observed in previous studies . demonstrated that within an extensively wide range of thresholds , ad patients exhibited a longer cpl than healthy controls but no significant changes in the cc were found . reported no significant changes in cc in ad but with a shorter cpl the inconsistent results of these studies may be caused by the selection of different frequency bands . , which suggest that various frequency bands impact the topological properties of a functional brain network . in this study , the global efficiency of ad patients was always lower than healthy controls at lower - frequency bands ( 0.010.06 hz , 0.060.11 hz ) [ figure 1 ] , data which agrees with zhao et al . the decline of global efficiency in ad patients indicates that pathological changes in the brain have reduced connectivity strength and blocked information communication in remote functional regions . a wealth of studies has demonstrated that the global efficiency of the brain network declines if the brain is pathologically impaired . this may be one major factor contributing to the cognitive decline of patients with ad . however , compared with healthy controls , there was no significant difference of the global efficiency at a higher frequency band ( 0.110.25 hz ) . we suggest that higher - frequency fluctuations may result from coexisting noise during the acquisition of mri signals in both ad patients and healthy controls . our study demonstrates that at lower - frequency bands within a higher range of thresholds , the cc of ad patients is diminished compared with healthy controls , in line with previous studies . this finding indicates a decline in coupling , robustness , as well as the local functional connectivity strength between nodes in patients with ad . in 1998 , watts and strogatz first explored the characteristic of a small - world network . numerous studies have demonstrated that the small - world properties of ad patients suffer significantly deterioration . at a threshold of 0.45 , the structure - specific alterations of node degree in ad patients were further investigated at the frequency band of 0.060.11 hz . previous studies have demonstrated that a deposition in ad occurs preferentially in locations of cortical hubs ( nodes ) , accounting for the observed activity - dependent mechanism associated with connectional hubs . furthermore , three regions show an increased node degree in ad patients ( itg.l , orbsup.l and mtg.r ) , indicating a coexisting functional compensation in the temporal lobe and frontal lobe . in conclusion , based on data analysis of resting - state functional networks , large - scale functional brain networks of ad patients and healthy controls were constructed and compared for a wide range of thresholds at different frequency bands . compared with healthy controls , the patients with ad exhibited lower global efficiency , lower cc , and also lower small - world furthermore , a decline in the node degree was observed in the large majority of pathologically attacked regions , with other areas increasing as functional compensations . our study accurately depicts the frequency - specific topological alterations in ad - related large - scale functional brain networks , providing fundamental support for optimal frequency selection in future related research . previous studies have suggested that the hemodynamic responses measured by rs - fmri reflect spontaneous neural activity which largely fluctuates at a lower - frequency band . it has been demonstrated that spontaneous low - frequency fluctuations ( 0.010.1 hz ) are physiologically meaningful . higher - frequency fluctuations are usually affected by respiratory signal frequency ( 0.10.5 hz ) and cardiovascular signal frequency ( 0.10.5 hz ) . in this study , the topological properties of ad patients and healthy controls exhibited different properties at different frequency bands . one possibility is that topological properties are frequency - dependent , which contributed to the discrepancies observed in previous studies . demonstrated that within an extensively wide range of thresholds , ad patients exhibited a longer cpl than healthy controls but no significant changes in the cc were found . reported no significant changes in cc in ad but with a shorter cpl the inconsistent results of these studies may be caused by the selection of different frequency bands . , which suggest that various frequency bands impact the topological properties of a functional brain network . in this study , the global efficiency of ad patients was always lower than healthy controls at lower - frequency bands ( 0.010.06 hz , 0.060.11 hz ) [ figure 1 ] , data which agrees with zhao et al . the decline of global efficiency in ad patients indicates that pathological changes in the brain have reduced connectivity strength and blocked information communication in remote functional regions . a wealth of studies has demonstrated that the global efficiency of the brain network declines if the brain is pathologically impaired . this may be one major factor contributing to the cognitive decline of patients with ad . however , compared with healthy controls , there was no significant difference of the global efficiency at a higher frequency band ( 0.110.25 hz ) . we suggest that higher - frequency fluctuations may result from coexisting noise during the acquisition of mri signals in both ad patients and healthy controls . our study demonstrates that at lower - frequency bands within a higher range of thresholds , the cc of ad patients is diminished compared with healthy controls , in line with previous studies . this finding indicates a decline in coupling , robustness , as well as the local functional connectivity strength between nodes in patients with ad . in 1998 , watts and strogatz first explored the characteristic of a small - world network . numerous studies have demonstrated that the small - world properties of ad patients suffer significantly deterioration . properties indicates that the efficiency of information transfer within different brain regions has also declined , a factor which may play a major role in the cognitive decline observed in ad patients . at a threshold of 0.45 , the structure - specific alterations of node degree in ad patients were further investigated at the frequency band of 0.060.11 hz . previous studies have demonstrated that a deposition in ad occurs preferentially in locations of cortical hubs ( nodes ) , accounting for the observed activity - dependent mechanism associated with connectional hubs . furthermore , three regions show an increased node degree in ad patients ( itg.l , orbsup.l and mtg.r ) , indicating a coexisting functional compensation in the temporal lobe and frontal lobe . in conclusion , based on data analysis of resting - state functional networks , large - scale functional brain networks of ad patients and healthy controls were constructed and compared for a wide range of thresholds at different frequency bands . compared with healthy controls , the patients with ad exhibited lower global efficiency , lower cc , and also lower small - world furthermore , a decline in the node degree was observed in the large majority of pathologically attacked regions , with other areas increasing as functional compensations . our study accurately depicts the frequency - specific topological alterations in ad - related large - scale functional brain networks , providing fundamental support for optimal frequency selection in future related research .

hepatitis c is a global health problem affecting over 170 - 200 million people and the virus is distributed worldwide with various prevalences from 0.2% to 40% in different countries ( 1 , 2 ) . worldwide , 130 to 170 million people are infected with hepatitis c virus ( hcv ) . hcv is the most common cause of chronic liver disease and responsible for 8,000 - 12,000 deaths per year . according to the world health organization ( who ) , the prevalence of antibodies against hepatitis c in the united states is close to 1.6% ( about 1.4 million ) ( 3 ) . hcv is the most common cause of cirrhosis and hepatocellular carcinoma ( hcc ) in america and japan . hcv becomes chronic hepatitis in 80% of the cases ; of this rate , 10% - 12% develop cirrhosis . about 1% - 5% of the patients with liver cirrhosis may develop liver cancer during the following 20 - 30 years ( 5 ) . hepatitis c is now the most common cause of chronic hepatitis and liver cirrhosis in iranian patients with hemophilia , thalassemia and kidney failure ( 5 , 6 ) . hepatitis c virus is spread parentally , either through intravenous drug use or , in the developing countries , through transfusion of blood products and contamination during medical procedures . despite the declining incidence of new infections , the burden of disease , both in terms of mortality and in terms of cost , is expected to increase over the next decade and hcv infection will be a potential cause of substantial morbidity and mortality in future ( 1 , 2 ) . hepatitis c as the first cause of liver disease necessitates further liver transplantation ( 7 ) . hcv can be easily transmitted through blood products and infected syringes , and infection rates are typically high among intravenous drug users ( idus ) ( 8) . idus are at high risk of acquiring parenteral transmitted diseases especially hiv and hcv infections ( 9 ) . patients with hemophilia and thalassemia are prone to be infected with hcv , too . hcv infection control is an important public health concern since the majority of infections is not resolved and leads to chronic infection ( 7 ) . nowadays , intravenous drug abuse is the major risk factor for hcv infection ( 10 ) . shaving by barbers , tattooing and ear - piercing known to be associated with hcv infection are common in the developing countries ( 11 ) . it is possible that chronic hcv infection may progress to cirrhosis and liver failure or hepatocellular carcinoma . in some patients it quickly causes cirrhosis , sometimes within 15 months after the first attack of hepatitis ( 12 ) . iran is located on the thalassemia belt and there are more than 25,000 cases of known thalassemia with hepatitis c ( 13 ) . according to epidemiological studies , 20% - 40% of these patients are infected with hepatitis c ( 13 ) . hcv may account for 40% of all chronic liver diseases , 20% - 30% of all liver transplants and 8,000 - 10,000 deaths annually ( 9 ) . the burden of disease and the death rate associated with hcv are expected to double over the next two decades as this large cohort of hcv - positive patients progress to cirrhosis , cancer and liver failure ( 14 ) . the current study examined the cost burden associated with hcv and hcv - related comorbidities for patients . to the authors best knowledge , no study estimated the economic costs of hcv in iran . the current study aimed to assess the direct and indirect expenses of chronic hcv infection and its related diseases . the economic burden of illness for patients referred to medical centers was estimated according to community perspectives in iran in 2015 . considering the burden of infection in the world and iran and lack of a comprehensive economic analysis of the disease in the country , it was necessary to conduct the study . this descriptive cross - sectional analytical study was conducted in 2015 on the patients associated with hcv infection sequel ( all patients with chronic hepatitis c infection , liver cirrhosis and hepatocellular carcinoma ) referred to the gastroenterohepatology research center , shiraz university of medical sciences , shiraz , iran . this center is a referral center of hepatitis network in the southern iran ; there is also nemazee hospital affiliated to shiraz university of medical sciences , whose liver transplantation center is the main treatment hub of the country . the sample size was determined by statistical consultation based on pilot sampling . with respect to the risk of the disease in iran , 166 patients with chronic hepatitis c , 24 patients with cirrhosis and 10 patients with hcc were included . random sampling of the patients enrolled in the gastroenterohepatology research center was done using the table of random digits . the inclusion criteria were having chronic hepatitis c infection , liver cirrhosis or hepatocellular carcinoma due to chronic hepatitis c infection . the billing system and patient records were used as the data source and a standardized data collection form was prepared . the standard questionnaire included data on recalling information about the condition and use of inpatient and outpatient services as well as their characteristics ( such as age , gender , education , occupation and insurance ) , disease status ( disease stage , elapsed time of diagnosis , follow - up results and symptoms ) and expenses of the medical treatment ( number of physician visits , levels of facility use , types of checkups , prescribed drugs , hospital stay , surgery , other treatments such as liver transplantation and self - treatment ) . moreover , days of absence from work ( patient or his / her caregiver ) and other services used outside the health system were documented . a standard questionnaire was translated from english to persian and then its reliability and validity were checked by the help of expert statistician . the questionnaire was matched with patient s situation and information recall about the use of outpatient services . the economic burden of disease was estimated based on the prevalence with an emphasis on direct medical costs . patients with chronic hcv , cirrhosis and hepatocellular carcinoma were followed up for three months and the costs were estimated using a bottom - up approach . input costs are collected per service by the bottom - up approach . in this case , the input data at the patient level was used to estimate the number and the type of services provided at each center . since major differences might exist between iran and other countries with respect to the costs and the patients social and economic status , specific cost information was evaluated for iran . the process of analyzing the costs of hcv infection sequel was performed in three parts as follows : a ) the first part determined and estimated the direct costs imposed on patients by hcv infection at its different stages . c ) in the third part , using a combination of data obtained from the two previous parts an estimate was made ; it was multiplied by the number of patients with hcv infection at its various stages to calculate the overall financial burden of the disease in the country . total economic burden of chronic hepatitis c infection consisted of two parts , the direct economic burden ( medical costs and non - medical costs ) and the indirect costs during one year . it was tried to use the patients and specialists viewpoints and inpatient and outpatient medical records simultaneously from january 1 , 2015 to march 31 , 2015 . to estimate the costs at different stages of the disease , the patients were classified into three groups as follows : chronic hcv , cirrhosis and hcc . with the use of economic resources of healthcare , direct medical costs could be measured including the average outpatient and inpatient costs , the cost of drugs purchased from pharmacies outside the hospital and other medical procedures . annual outpatient or inpatient cost per patient was determined by the rate of visits and average cost of every visit . the formulas were : dmc = aoe + aie + aesm in which , aoe is annual outpatient expenditure per patient ; aie is annual inpatient expenditure per patient ; aesm is the annual expenditure of self - medication per patient . aoe and aie were also calculated as follow : aoe = average outpatient expense at a visit the average rate of outpatient visits in three months 4 aie = average inpatient expense at a time at the certain hospital level proportion of inpatients at the certain hospital level ) annual rate of hospital admissions per patient to estimate the related costs , the patient s self - report was used during the study period . since the vast majority of patients referred to the mentioned treatment centers were living outside shiraz , items such as travel time , costs of travel to these centers to receive health care , and accommodation and meals costs were considered as important components of direct non - medical costs . cost of transportation and extra health products due to illness were summed up to obtain the direct non - medical cost . the average yearly amount of consumption could be measured using the mean monthly consumption stated in the questionnaire . cost data were collected via face - to - face or telephone interviews with the patients receiving inpatient and outpatient services from the mentioned treatment centers during the study period . moreover , the informed consent was obtained from all patients . with respect to the indirect costs , the human capital approach was used ( 16 ) . this technique measures health by improving productivity and income ( especially through the reduction of work absenteeism and increased life expectancy ) . it should be noted that the income of housewives and people who were unemployed before the disease was considered zero in this study . the indirect cost of each family depended on the daily income and days of sick leave of the patient , and the average daily income per caregiver , as well as the duration of their absence from work for the sake of nursing and caring . after estimating direct and indirect costs for every stage of the disease separately , two types of financial burden were estimated for every stage of the disease . first , after collection of data about the prevalence rate of the disease , the population of the country , the average cost of each stage of disease and the potential financial burden of the disease in patients in the country was calculated using the following formula : financial burden = total cost ( direct medical + direct non - medical + indirect costs ) estimated number of infected subjects in iran second , in order to calculate more accurately , it was attempted to estimate the costs of treatments for patients with chc at different stages of the disease . accordingly , to calculate the real costs imposed on health system due to this disease per year , the estimated and multiplied by the cost of the disease for each stage of the disease . the total annual cost imposed on treated patients at each stage of the disease = the average number of treated patients the average cost imposed on a patient all costs were calculated based on purchasing power parity ( ppp ) usd . spss software version 16 was used for data analysis including mean , percentage and range calculation . this descriptive cross - sectional analytical study was conducted in 2015 on the patients associated with hcv infection sequel ( all patients with chronic hepatitis c infection , liver cirrhosis and hepatocellular carcinoma ) referred to the gastroenterohepatology research center , shiraz university of medical sciences , shiraz , iran . this center is a referral center of hepatitis network in the southern iran ; there is also nemazee hospital affiliated to shiraz university of medical sciences , whose liver transplantation center is the main treatment hub of the country . the sample size was determined by statistical consultation based on pilot sampling . with respect to the risk of the disease in iran , 166 patients with chronic hepatitis c , 24 patients with cirrhosis and 10 patients with hcc were included . random sampling of the patients enrolled in the gastroenterohepatology research center was done using the table of random digits . the inclusion criteria were having chronic hepatitis c infection , liver cirrhosis or hepatocellular carcinoma due to chronic hepatitis c infection . the billing system and patient records were used as the data source and a standardized data collection form was prepared . the standard questionnaire included data on recalling information about the condition and use of inpatient and outpatient services as well as their characteristics ( such as age , gender , education , occupation and insurance ) , disease status ( disease stage , elapsed time of diagnosis , follow - up results and symptoms ) and expenses of the medical treatment ( number of physician visits , levels of facility use , types of checkups , prescribed drugs , hospital stay , surgery , other treatments such as liver transplantation and self - treatment ) . moreover , days of absence from work ( patient or his / her caregiver ) and other services used outside the health system were documented . a standard questionnaire was translated from english to persian and then its reliability and validity were checked by the help of expert statistician . the questionnaire was matched with patient s situation and information recall about the use of outpatient services . the economic burden of disease was estimated based on the prevalence with an emphasis on direct medical costs . patients with chronic hcv , cirrhosis and hepatocellular carcinoma were followed up for three months and the costs were estimated using a bottom - up approach . input costs are collected per service by the bottom - up approach . in this case , the input data at the patient level was used to estimate the number and the type of services provided at each center . since major differences might exist between iran and other countries with respect to the costs and the patients social and economic status , specific cost information was evaluated for iran . the process of analyzing the costs of hcv infection sequel was performed in three parts as follows : a ) the first part determined and estimated the direct costs imposed on patients by hcv infection at its different stages . c ) in the third part , using a combination of data obtained from the two previous parts an estimate was made ; it was multiplied by the number of patients with hcv infection at its various stages to calculate the overall financial burden of the disease in the country . total economic burden of chronic hepatitis c infection consisted of two parts , the direct economic burden ( medical costs and non - medical costs ) and the indirect costs during one year . it was tried to use the patients and specialists viewpoints and inpatient and outpatient medical records simultaneously from january 1 , 2015 to march 31 , 2015 . to estimate the costs at different stages of the disease , the patients were classified into three groups as follows : chronic hcv , cirrhosis and hcc . with the use of economic resources of healthcare , direct medical costs could be measured including the average outpatient and inpatient costs , the cost of drugs purchased from pharmacies outside the hospital and other medical procedures . annual outpatient or inpatient cost per patient was determined by the rate of visits and average cost of every visit . the formulas were : dmc = aoe + aie + aesm in which , aoe is annual outpatient expenditure per patient ; aie is annual inpatient expenditure per patient ; aesm is the annual expenditure of self - medication per patient . aoe and aie were also calculated as follow : aoe = average outpatient expense at a visit the average rate of outpatient visits in three months 4 aie = average inpatient expense at a time at the certain hospital level proportion of inpatients at the certain hospital level ) annual rate of hospital admissions per patient to estimate the related costs , the patient s self - report was used during the study period . since the vast majority of patients referred to the mentioned treatment centers were living outside shiraz , items such as travel time , costs of travel to these centers to receive health care , and accommodation and meals costs were considered as important components of direct non - medical costs . cost of transportation and extra health products due to illness were summed up to obtain the direct non - medical cost . the average yearly amount of consumption could be measured using the mean monthly consumption stated in the questionnaire . cost data were collected via face - to - face or telephone interviews with the patients receiving inpatient and outpatient services from the mentioned treatment centers during the study period . moreover , the informed consent was obtained from all patients . with respect to the indirect costs , the human capital approach was used ( 16 ) . this technique measures health by improving productivity and income ( especially through the reduction of work absenteeism and increased life expectancy ) . it should be noted that the income of housewives and people who were unemployed before the disease was considered zero in this study . the indirect cost of each family depended on the daily income and days of sick leave of the patient , and the average daily income per caregiver , as well as the duration of their absence from work for the sake of nursing and caring . after estimating direct and indirect costs for every stage of the disease separately , two types of financial burden were estimated for every stage of the disease . first , after collection of data about the prevalence rate of the disease , the population of the country , the average cost of each stage of disease and the potential financial burden of the disease in patients in the country was calculated using the following formula : financial burden = total cost ( direct medical + direct non - medical + indirect costs ) estimated number of infected subjects in iran second , in order to calculate more accurately , it was attempted to estimate the costs of treatments for patients with chc at different stages of the disease . accordingly , to calculate the real costs imposed on health system due to this disease per year , the estimated and multiplied by the cost of the disease for each stage of the disease . the total annual cost imposed on treated patients at each stage of the disease = the average number of treated patients the average cost imposed on a patient spss software version 16 was used for data analysis including mean , percentage and range calculation . in the current study , 200 patients at different stages of the disease ( 166 patients with chronic hepatitis c infection , 24 with cirrhosis and 10 with hcc ) were enrolled respectively , with a mean sd age of 41 2.1 years ( range : 1 - 72 years ) . an increase was found in the total direct annual medical costs for patients with advanced disease . most of the direct costs of medical services were related to diagnostic costs for chronic hepatitis c infection . however , for later stages of the disease ( liver cirrhosis and hepatocellular carcinoma ) , hospitalization admission costs comprised a large portion of direct medical costs . the estimated annual cost of treatment per patient at different stages of the disease is shown in table 1 . in 2015 , the estimated annual direct medical cost per patient at different stages of chronic hepatitis c infection was averagely usd 4591.5 . given the severity of the disease , there will be more costs ; the high cost of annual direct medical costs due to liver cirrhosis and he - patocellular carcinoma in patients . abbreviations : chc , chronic hepatitis c ; dmc , direct medical costs ; dnmc , direct non - medical costs ; hcc , hepatocellular carcinoma . direct non - medical costs and indirect costs per person at different stages of the disease are shown in table 1 . the table also presents the annual costs of utilization of treatment and diagnostic services ( direct medical costs ) and the costs of other services ( direct non - medical costs ) for chronic hepatitis c infection per patient for each stage of the disease . as shown , the minimum cost ( about usd 613.9 ) was observed in patients with chronic hepatitis c infection and the maximum cost ( usd 8573.7 ) in patients with hcc . table 1 also shows that the annual direct medical costs of treatment increased with disease progression . in all cases , except for patients with chronic hepatitis c infection , the costs of medicine accounted for the greatest share of healthcare costs ; therefore , 83% and 68% of direct health care costs were spent on hospitalization required for cirrhosis and hcc stages , respectively . non - direct medical costs : since many patients come from neighboring provinces and their travel time is often a full working day or more , the costs of travelling , meals , especially accommodation , increases with progression of the disease to more severe stages ( table 2 ) . abbreviations : chc , chronic hepatitis c ; dmc , direct medical costs ; dnmc , direct non - medical costs ; hcc , hepatocellular carcinoma ; ic , indirect cost . as it is shown , indirect annual treatment costs increase with the progression of the disease , and indirect costs for hcc patients have the greatest share of health care costs , which is about usd 1513.00 ( table 2 ) . the estimated annual indirect costs for all stages of disease per patient with chronic hepatitis c infection were usd 1053.6 . figure 1 presents a summary of the direct medical costs , direct non - medical costs , and indirect costs per patient and for different stages of the disease in southern iran . the total cost of chronic hepatitis c infection for different stages of the disease among the active hepatitis c population and patients receiving treatment in the country was usd 6263.3 . in addition , the ratio of direct to indirect costs increases with the progress of the disease . the results showed that the direct medical costs are the main component of the total cost of treating patients with hcv infection and encompasses about 73% of the costs for active hepatitis c population and patients receiving treatment . ic , indirect cost ; dmc , direct medical costs ; dnmc , direct non - medical costs . direct non - medical costs and indirect costs per person at different stages of the disease are shown in table 1 . the table also presents the annual costs of utilization of treatment and diagnostic services ( direct medical costs ) and the costs of other services ( direct non - medical costs ) for chronic hepatitis c infection per patient for each stage of the disease . as shown , the minimum cost ( about usd 613.9 ) was observed in patients with chronic hepatitis c infection and the maximum cost ( usd 8573.7 ) in patients with hcc . table 1 also shows that the annual direct medical costs of treatment increased with disease progression . in all cases , except for patients with chronic hepatitis c infection , the costs of medicine accounted for the greatest share of healthcare costs ; therefore , 83% and 68% of direct health care costs were spent on hospitalization required for cirrhosis and hcc stages , respectively . non - direct medical costs : since many patients come from neighboring provinces and their travel time is often a full working day or more , the costs of travelling , meals , especially accommodation , increases with progression of the disease to more severe stages ( table 2 ) . abbreviations : chc , chronic hepatitis c ; dmc , direct medical costs ; dnmc , direct non - medical costs ; hcc , hepatocellular carcinoma ; ic , indirect cost . as it is shown , indirect annual treatment costs increase with the progression of the disease , and indirect costs for hcc patients have the greatest share of health care costs , which is about usd 1513.00 ( table 2 ) . the estimated annual indirect costs for all stages of disease per patient with chronic hepatitis c infection were usd 1053.6 . figure 1 presents a summary of the direct medical costs , direct non - medical costs , and indirect costs per patient and for different stages of the disease in southern iran . the total cost of chronic hepatitis c infection for different stages of the disease among the active hepatitis c population and patients receiving treatment in the country was usd 6263.3 . in addition , the ratio of direct to indirect costs increases with the progress of the disease . the results showed that the direct medical costs are the main component of the total cost of treating patients with hcv infection and encompasses about 73% of the costs for active hepatitis c population and patients receiving treatment . ic , indirect cost ; dmc , direct medical costs ; dnmc , direct non - medical costs . given that the reliable prevalence data of different stages of hepatitis was not readily available for many countries ( 17 ) ; authors used information from various sources to estimate the prevalence . according to the study by malekzadeh et al . hcv becomes chronic hepatitis in 80% of the cases and among them 10% - 12% of the patients develop cirrhosis . about 1% - 5% of the patients with liver cirrhosis may develop liver cancer during the following 20 - 30 years ( 5 ) . according to the latest figures released by the office for prevention of viral hepatitis in the ministry of health and medical education , the prevalence of hcv in iran therefore , considering an estimated population of 78 million people in iran in 2015 ( 19 ) , the total number of hcv carriers was estimated to be 390,000 people . despite the growing evidence that hepatitis c is an urgent public health issue , few countries have developed strategic national responses to address the hepatitis c epidemics within their populations ( 9 ) . the current study was the first attempt to estimate the direct costs in iran and indirect costs in the terms of lost productivity or absence from work due to chronic hepatitis c infection , cirrhosis and hepatocellular carcinoma . the study found that chronic infection with hepatitis c and related diseases imposes great financial burden on the health system and society in iran . the study by ashtari et al . estimated diagnosis and treatment costs of hepatitis c based on the type of treatment in iran . they proved that hepatitis c is a disease that imposes a heavy burden on the health system and used a large part of the resources to the health sector ( 20 ) . in the current study , due to the high financial burden of the disease ( average usd 6263.3 per person in a year ) , many of the patients were obliged to borrow from relatives , sell their assets , or get a loan , and consequently , their treatment had greatly influenced their household income . in the study by razavi et al . burden of chronic hepatitis c was estimated in the united states of america , the total cost of the disease was 5.6 billion dollars by 2011 , and also it came to the conclusion that america will reach 1.9 billion dollars by 2024 . cost of illness per person was calculated at usd 64.490 in 2011 ( 21 ) . early stage of the disease is less expensive for treatment but later stages and their complications are associated with higher treatment costs . in patients with chronic hepatitis c infection , nearly 68% of direct costs are allocated to medical diagnostic services ( such as laboratory tests , biopsy takings , ultrasonography , etc . ) , followed by the cost of drugs ( 16% ) . however , hospital admissions for patients in the later stages of the disease ( cirrhosis and hepatocellular carcinoma ) were among the highest direct costs . this can be attributed to the duration of admissions and stay in the intensive care units ( icus ) . many patients with chronic hepatitis c infection are referred from suburban areas or nearby provinces . also patients with liver cirrhosis and hepatocellular carcinoma are referred to the study centers for liver transplantation from all over the country ; therefore , direct non - medical costs and indirect costs are considered as part of the cost of illness . the study found that the average medical expenditures associated with having an hcv diagnosis peak early after the first - observed diagnosis , but that diagnosis may come relatively late in the disease , leading to high costs . more specifically , inpatient and outpatient costs were over usd 10,000 per year in the first two years following the diagnosis ( 22 ) . the present study showed that chronic hepatitis c and its associated complications can impose a considerable financial burden on the country health system and community in general . similar to other published studies ( 22 , 23 ) ; the current study demonstrated that severe stages of the disease , including cirrhosis and hcc , are very expensive for patients . when patients were asked whether they had to spend their own or their family members saving or loans , almost all participants in more severe stages of the disease , including cirrhosis and hcc , they also said that the disease had imposed heavy financial burden on patients and their families , and most of them had many difficulties to cover the cost of treatment . the problem was more significant for services not covered by insurance , such as certain medications or receiving services from nongovernmental centers ( 24 ) , which is considered as a catastrophic cost . considering the abovementioned problems , it should be tried to prevent the progress of the disease to more severe stages via proper and timely diagnosis and treatment . in general , accurate data about the economic impact of hepatitis c infection is essential for health policy - makers with respect to prevention and treatment of this disease . the results of the current study can be considered in the governmental policy , and treatment expenses need to be considered with priority . indeed , one of the priorities of ministry of health and medical education in recent years is supporting the cost of illness research . the current study had several limitations ; firstly the sample may not be a good representative of the society . many patients with chronic hepatitis c who lived in rural areas were farmers or had low - income jobs ; therefore , going to the clinic for outpatient visits on a regular basis was difficult for them . moreover , the data were only collected from limited centers in shiraz and the results were generalized at the national level . even with the limitations listed here , this is the only study in iran that estimated the cost burden of chronic hepatitis c infection and can provide valuable information for health system managers and policy makers to plan and allocate resources . this study had one limitation in the indirect cost calculation which was the lack of any estimation of the transportation and residency costs . also , since the direct cost is based on self - report , it would be seen as a conservative estimation . despite this limitation , to reduce the financial burden of diseases associated with chronic hepatitis c infection in iran , the following recommendations can be provided : the use of screening programs and patients access to timely detection of hepatitis c infection , improving the quality and quantity of health insurance for people with hepatitis c infections , providing subsidies by the government to reduce costs , management and resource allocation and proportional to the tariff , and improving comprehensive services and public health systems based on primary health care .

backgroundhepatitis c virus ( hcv ) infection is a major blood - borne infection which imposes high economic cost on the patients.objectivesthe current study aimed to evaluate the total annual cost due to chronic hcv related diseases imposed on each patient and their family in southern iran.patients and methodseconomic burden of chronic hepatitis c - related liver diseases ( chronic hepatitis c , cirrhosis and hepatocellular carcinoma ) were examined . the current retrospective study evaluated 200 iranian patients for their socioeconomic status , utilization ( direct and indirect costs ) and treatment costs and work days lost due to illness by a structured questionnaire in 2015 . costs of hospital admissions were extracted from databases of nemazee hospital , shiraz , iran . the outpatient expenditure per patient was measured through the rate of outpatient visits and average cost per visit reported by the patients ; while the inpatient costs were calculated through annual rate of hospital admissions and average expenditure . self - medication and direct non - medical costs were also reported . the human capital approach was used to measure the work loss cost.resultsthe total annual cost per patient for chronic hepatitis c , cirrhosis and hepatocellular carcinoma ( hcc ) based on purchasing power parity ( ppp ) were usd 1625.50 , usd 6117.2 , and usd 11047.2 in 2015 , respectively.conclusionschronic hepatitis c - related liver diseases impose a substantial economic burden on patients , families and the society . the current study provides useful information on cost of treatment and work loss for different disease states , which can be further used in cost - effectiveness evaluations .

1. Background 2. Objectives 3. Patients and Methods 3.1. Patients 3.2. Economic Burden Calculation 3.3. Measurement of Direct Medical Cost 3.4. Measurement of Direct Non-Medical Cost 3.5. Calculation of Direct Non-Medical Cost 3.6. Measurement of Indirect Cost 3.7. Financial Burden Imposed on all Patients With Chronic Hepatitis C at Different Stages 3.8. Statistical Analysis 4. Results 4.1. Direct Medical and Non-Medical Costs 5. Discussion

hepatitis c is a global health problem affecting over 170 - 200 million people and the virus is distributed worldwide with various prevalences from 0.2% to 40% in different countries ( 1 , 2 ) . worldwide , 130 to 170 million people are infected with hepatitis c virus ( hcv ) . hcv is the most common cause of cirrhosis and hepatocellular carcinoma ( hcc ) in america and japan . hepatitis c is now the most common cause of chronic hepatitis and liver cirrhosis in iranian patients with hemophilia , thalassemia and kidney failure ( 5 , 6 ) . it is possible that chronic hcv infection may progress to cirrhosis and liver failure or hepatocellular carcinoma . the current study examined the cost burden associated with hcv and hcv - related comorbidities for patients . the current study aimed to assess the direct and indirect expenses of chronic hcv infection and its related diseases . the economic burden of illness for patients referred to medical centers was estimated according to community perspectives in iran in 2015 . this descriptive cross - sectional analytical study was conducted in 2015 on the patients associated with hcv infection sequel ( all patients with chronic hepatitis c infection , liver cirrhosis and hepatocellular carcinoma ) referred to the gastroenterohepatology research center , shiraz university of medical sciences , shiraz , iran . this center is a referral center of hepatitis network in the southern iran ; there is also nemazee hospital affiliated to shiraz university of medical sciences , whose liver transplantation center is the main treatment hub of the country . with respect to the risk of the disease in iran , 166 patients with chronic hepatitis c , 24 patients with cirrhosis and 10 patients with hcc were included . the inclusion criteria were having chronic hepatitis c infection , liver cirrhosis or hepatocellular carcinoma due to chronic hepatitis c infection . the economic burden of disease was estimated based on the prevalence with an emphasis on direct medical costs . patients with chronic hcv , cirrhosis and hepatocellular carcinoma were followed up for three months and the costs were estimated using a bottom - up approach . in this case , the input data at the patient level was used to estimate the number and the type of services provided at each center . since major differences might exist between iran and other countries with respect to the costs and the patients social and economic status , specific cost information was evaluated for iran . the process of analyzing the costs of hcv infection sequel was performed in three parts as follows : a ) the first part determined and estimated the direct costs imposed on patients by hcv infection at its different stages . total economic burden of chronic hepatitis c infection consisted of two parts , the direct economic burden ( medical costs and non - medical costs ) and the indirect costs during one year . to estimate the costs at different stages of the disease , the patients were classified into three groups as follows : chronic hcv , cirrhosis and hcc . with the use of economic resources of healthcare , direct medical costs could be measured including the average outpatient and inpatient costs , the cost of drugs purchased from pharmacies outside the hospital and other medical procedures . annual outpatient or inpatient cost per patient was determined by the rate of visits and average cost of every visit . the formulas were : dmc = aoe + aie + aesm in which , aoe is annual outpatient expenditure per patient ; aie is annual inpatient expenditure per patient ; aesm is the annual expenditure of self - medication per patient . aoe and aie were also calculated as follow : aoe = average outpatient expense at a visit the average rate of outpatient visits in three months 4 aie = average inpatient expense at a time at the certain hospital level proportion of inpatients at the certain hospital level ) annual rate of hospital admissions per patient to estimate the related costs , the patient s self - report was used during the study period . since the vast majority of patients referred to the mentioned treatment centers were living outside shiraz , items such as travel time , costs of travel to these centers to receive health care , and accommodation and meals costs were considered as important components of direct non - medical costs . cost of transportation and extra health products due to illness were summed up to obtain the direct non - medical cost . with respect to the indirect costs , the human capital approach was used ( 16 ) . the indirect cost of each family depended on the daily income and days of sick leave of the patient , and the average daily income per caregiver , as well as the duration of their absence from work for the sake of nursing and caring . after estimating direct and indirect costs for every stage of the disease separately , two types of financial burden were estimated for every stage of the disease . first , after collection of data about the prevalence rate of the disease , the population of the country , the average cost of each stage of disease and the potential financial burden of the disease in patients in the country was calculated using the following formula : financial burden = total cost ( direct medical + direct non - medical + indirect costs ) estimated number of infected subjects in iran second , in order to calculate more accurately , it was attempted to estimate the costs of treatments for patients with chc at different stages of the disease . accordingly , to calculate the real costs imposed on health system due to this disease per year , the estimated and multiplied by the cost of the disease for each stage of the disease . the total annual cost imposed on treated patients at each stage of the disease = the average number of treated patients the average cost imposed on a patient all costs were calculated based on purchasing power parity ( ppp ) usd . this descriptive cross - sectional analytical study was conducted in 2015 on the patients associated with hcv infection sequel ( all patients with chronic hepatitis c infection , liver cirrhosis and hepatocellular carcinoma ) referred to the gastroenterohepatology research center , shiraz university of medical sciences , shiraz , iran . this center is a referral center of hepatitis network in the southern iran ; there is also nemazee hospital affiliated to shiraz university of medical sciences , whose liver transplantation center is the main treatment hub of the country . with respect to the risk of the disease in iran , 166 patients with chronic hepatitis c , 24 patients with cirrhosis and 10 patients with hcc were included . the inclusion criteria were having chronic hepatitis c infection , liver cirrhosis or hepatocellular carcinoma due to chronic hepatitis c infection . the economic burden of disease was estimated based on the prevalence with an emphasis on direct medical costs . patients with chronic hcv , cirrhosis and hepatocellular carcinoma were followed up for three months and the costs were estimated using a bottom - up approach . in this case , the input data at the patient level was used to estimate the number and the type of services provided at each center . since major differences might exist between iran and other countries with respect to the costs and the patients social and economic status , specific cost information was evaluated for iran . the process of analyzing the costs of hcv infection sequel was performed in three parts as follows : a ) the first part determined and estimated the direct costs imposed on patients by hcv infection at its different stages . total economic burden of chronic hepatitis c infection consisted of two parts , the direct economic burden ( medical costs and non - medical costs ) and the indirect costs during one year . to estimate the costs at different stages of the disease , the patients were classified into three groups as follows : chronic hcv , cirrhosis and hcc . with the use of economic resources of healthcare , direct medical costs could be measured including the average outpatient and inpatient costs , the cost of drugs purchased from pharmacies outside the hospital and other medical procedures . annual outpatient or inpatient cost per patient was determined by the rate of visits and average cost of every visit . the formulas were : dmc = aoe + aie + aesm in which , aoe is annual outpatient expenditure per patient ; aie is annual inpatient expenditure per patient ; aesm is the annual expenditure of self - medication per patient . aoe and aie were also calculated as follow : aoe = average outpatient expense at a visit the average rate of outpatient visits in three months 4 aie = average inpatient expense at a time at the certain hospital level proportion of inpatients at the certain hospital level ) annual rate of hospital admissions per patient to estimate the related costs , the patient s self - report was used during the study period . since the vast majority of patients referred to the mentioned treatment centers were living outside shiraz , items such as travel time , costs of travel to these centers to receive health care , and accommodation and meals costs were considered as important components of direct non - medical costs . cost of transportation and extra health products due to illness were summed up to obtain the direct non - medical cost . with respect to the indirect costs , the human capital approach was used ( 16 ) . the indirect cost of each family depended on the daily income and days of sick leave of the patient , and the average daily income per caregiver , as well as the duration of their absence from work for the sake of nursing and caring . after estimating direct and indirect costs for every stage of the disease separately , two types of financial burden were estimated for every stage of the disease . first , after collection of data about the prevalence rate of the disease , the population of the country , the average cost of each stage of disease and the potential financial burden of the disease in patients in the country was calculated using the following formula : financial burden = total cost ( direct medical + direct non - medical + indirect costs ) estimated number of infected subjects in iran second , in order to calculate more accurately , it was attempted to estimate the costs of treatments for patients with chc at different stages of the disease . accordingly , to calculate the real costs imposed on health system due to this disease per year , the estimated and multiplied by the cost of the disease for each stage of the disease . the total annual cost imposed on treated patients at each stage of the disease = the average number of treated patients the average cost imposed on a patient spss software version 16 was used for data analysis including mean , percentage and range calculation . in the current study , 200 patients at different stages of the disease ( 166 patients with chronic hepatitis c infection , 24 with cirrhosis and 10 with hcc ) were enrolled respectively , with a mean sd age of 41 2.1 years ( range : 1 - 72 years ) . most of the direct costs of medical services were related to diagnostic costs for chronic hepatitis c infection . however , for later stages of the disease ( liver cirrhosis and hepatocellular carcinoma ) , hospitalization admission costs comprised a large portion of direct medical costs . the estimated annual cost of treatment per patient at different stages of the disease is shown in table 1 . in 2015 , the estimated annual direct medical cost per patient at different stages of chronic hepatitis c infection was averagely usd 4591.5 . given the severity of the disease , there will be more costs ; the high cost of annual direct medical costs due to liver cirrhosis and he - patocellular carcinoma in patients . abbreviations : chc , chronic hepatitis c ; dmc , direct medical costs ; dnmc , direct non - medical costs ; hcc , hepatocellular carcinoma . direct non - medical costs and indirect costs per person at different stages of the disease are shown in table 1 . the table also presents the annual costs of utilization of treatment and diagnostic services ( direct medical costs ) and the costs of other services ( direct non - medical costs ) for chronic hepatitis c infection per patient for each stage of the disease . as shown , the minimum cost ( about usd 613.9 ) was observed in patients with chronic hepatitis c infection and the maximum cost ( usd 8573.7 ) in patients with hcc . table 1 also shows that the annual direct medical costs of treatment increased with disease progression . in all cases , except for patients with chronic hepatitis c infection , the costs of medicine accounted for the greatest share of healthcare costs ; therefore , 83% and 68% of direct health care costs were spent on hospitalization required for cirrhosis and hcc stages , respectively . non - direct medical costs : since many patients come from neighboring provinces and their travel time is often a full working day or more , the costs of travelling , meals , especially accommodation , increases with progression of the disease to more severe stages ( table 2 ) . abbreviations : chc , chronic hepatitis c ; dmc , direct medical costs ; dnmc , direct non - medical costs ; hcc , hepatocellular carcinoma ; ic , indirect cost . as it is shown , indirect annual treatment costs increase with the progression of the disease , and indirect costs for hcc patients have the greatest share of health care costs , which is about usd 1513.00 ( table 2 ) . the estimated annual indirect costs for all stages of disease per patient with chronic hepatitis c infection were usd 1053.6 . figure 1 presents a summary of the direct medical costs , direct non - medical costs , and indirect costs per patient and for different stages of the disease in southern iran . the total cost of chronic hepatitis c infection for different stages of the disease among the active hepatitis c population and patients receiving treatment in the country was usd 6263.3 . the results showed that the direct medical costs are the main component of the total cost of treating patients with hcv infection and encompasses about 73% of the costs for active hepatitis c population and patients receiving treatment . ic , indirect cost ; dmc , direct medical costs ; dnmc , direct non - medical costs . direct non - medical costs and indirect costs per person at different stages of the disease are shown in table 1 . the table also presents the annual costs of utilization of treatment and diagnostic services ( direct medical costs ) and the costs of other services ( direct non - medical costs ) for chronic hepatitis c infection per patient for each stage of the disease . as shown , the minimum cost ( about usd 613.9 ) was observed in patients with chronic hepatitis c infection and the maximum cost ( usd 8573.7 ) in patients with hcc . table 1 also shows that the annual direct medical costs of treatment increased with disease progression . in all cases , except for patients with chronic hepatitis c infection , the costs of medicine accounted for the greatest share of healthcare costs ; therefore , 83% and 68% of direct health care costs were spent on hospitalization required for cirrhosis and hcc stages , respectively . non - direct medical costs : since many patients come from neighboring provinces and their travel time is often a full working day or more , the costs of travelling , meals , especially accommodation , increases with progression of the disease to more severe stages ( table 2 ) . abbreviations : chc , chronic hepatitis c ; dmc , direct medical costs ; dnmc , direct non - medical costs ; hcc , hepatocellular carcinoma ; ic , indirect cost . as it is shown , indirect annual treatment costs increase with the progression of the disease , and indirect costs for hcc patients have the greatest share of health care costs , which is about usd 1513.00 ( table 2 ) . the estimated annual indirect costs for all stages of disease per patient with chronic hepatitis c infection were usd 1053.6 . figure 1 presents a summary of the direct medical costs , direct non - medical costs , and indirect costs per patient and for different stages of the disease in southern iran . the total cost of chronic hepatitis c infection for different stages of the disease among the active hepatitis c population and patients receiving treatment in the country was usd 6263.3 . the results showed that the direct medical costs are the main component of the total cost of treating patients with hcv infection and encompasses about 73% of the costs for active hepatitis c population and patients receiving treatment . ic , indirect cost ; dmc , direct medical costs ; dnmc , direct non - medical costs . according to the latest figures released by the office for prevention of viral hepatitis in the ministry of health and medical education , the prevalence of hcv in iran therefore , considering an estimated population of 78 million people in iran in 2015 ( 19 ) , the total number of hcv carriers was estimated to be 390,000 people . the current study was the first attempt to estimate the direct costs in iran and indirect costs in the terms of lost productivity or absence from work due to chronic hepatitis c infection , cirrhosis and hepatocellular carcinoma . the study found that chronic infection with hepatitis c and related diseases imposes great financial burden on the health system and society in iran . estimated diagnosis and treatment costs of hepatitis c based on the type of treatment in iran . they proved that hepatitis c is a disease that imposes a heavy burden on the health system and used a large part of the resources to the health sector ( 20 ) . in the current study , due to the high financial burden of the disease ( average usd 6263.3 per person in a year ) , many of the patients were obliged to borrow from relatives , sell their assets , or get a loan , and consequently , their treatment had greatly influenced their household income . burden of chronic hepatitis c was estimated in the united states of america , the total cost of the disease was 5.6 billion dollars by 2011 , and also it came to the conclusion that america will reach 1.9 billion dollars by 2024 . however , hospital admissions for patients in the later stages of the disease ( cirrhosis and hepatocellular carcinoma ) were among the highest direct costs . also patients with liver cirrhosis and hepatocellular carcinoma are referred to the study centers for liver transplantation from all over the country ; therefore , direct non - medical costs and indirect costs are considered as part of the cost of illness . the present study showed that chronic hepatitis c and its associated complications can impose a considerable financial burden on the country health system and community in general . similar to other published studies ( 22 , 23 ) ; the current study demonstrated that severe stages of the disease , including cirrhosis and hcc , are very expensive for patients . when patients were asked whether they had to spend their own or their family members saving or loans , almost all participants in more severe stages of the disease , including cirrhosis and hcc , they also said that the disease had imposed heavy financial burden on patients and their families , and most of them had many difficulties to cover the cost of treatment . in general , accurate data about the economic impact of hepatitis c infection is essential for health policy - makers with respect to prevention and treatment of this disease . the results of the current study can be considered in the governmental policy , and treatment expenses need to be considered with priority . the current study had several limitations ; firstly the sample may not be a good representative of the society . many patients with chronic hepatitis c who lived in rural areas were farmers or had low - income jobs ; therefore , going to the clinic for outpatient visits on a regular basis was difficult for them . even with the limitations listed here , this is the only study in iran that estimated the cost burden of chronic hepatitis c infection and can provide valuable information for health system managers and policy makers to plan and allocate resources . despite this limitation , to reduce the financial burden of diseases associated with chronic hepatitis c infection in iran , the following recommendations can be provided : the use of screening programs and patients access to timely detection of hepatitis c infection , improving the quality and quantity of health insurance for people with hepatitis c infections , providing subsidies by the government to reduce costs , management and resource allocation and proportional to the tariff , and improving comprehensive services and public health systems based on primary health care .

distributed water treatment systems offer a potential means of exploiting alternative water sources , including municipal wastewater effluent , roof water , stormwater , and water from shallow aquifers . unfortunately , alternative water sources often contain trace concentrations of organic contaminants ( e.g. , pesticides , solvents , pharmaceuticals , disinfection byproducts ) . as a result , previous attempts to develop point - of - use treatment systems capable of removing trace organic contaminants prior to nonpotable reuse have employed electrochemical processes , but these systems suffer from limitations including the production of toxic byproducts , an inability to remove recalcitrant compounds and high cost of treatment . trace organic contaminants can be removed from water by exposure to hydroxyl radicals ( ho ) in advanced oxidation processes ( aops ) . in full - scale potable water reuse systems , trace organic contaminants are frequently removed by addition of a modest concentration of h2o2 ( e.g. , 3 mg / l ) followed by exposure to ultraviolet ( uv ) light . this approach offers numerous benefits over other aops in terms of energy consumption , reliability , and production of toxic byproducts . although uv / h2o2 is a well established technology in centralized treatment facilities , challenges associated with the transport and storage of h2o2 make it an impractical solution for distributed treatment systems . electrochemical production of h2o2 from o2 is an attractive alternative means of producing h2o2 if it can be achieved without the consumption of large amounts of energy or the formation of toxic byproducts . systems in which oxygen is bubbled into a solution prior to reduction on an electrode surface consume a considerable amount of energy due to the low solubility of oxygen and the need to ensure that it reaches the electrode surface . bubbling air or pure oxygen into a solution is also an impractical approach for h2o2 production in decentralized systems because it requires pumps and controllers . furthermore , the yield of h2o2 from reduction of o2 is often quite low , which greatly increases electricity consumption . recently , gas diffusion electrodes have been used to generate h2o2 without a need to bubble air or oxygen into a solution . most of the research in this area has been focused on producing concentrated h2o2 solutions by using highly conductive solutions or organic solvents . cathodic production of h2o2 for the removal of organics has typically been used for electro - fenton treatment ; however , differences in ph needed for the optimal kinetics of the two reactions ( i.e. , production of h2o2 is most efficient at basic ph values and fenton s reaction is more effective at acidic ph values ) results in inefficient oxidation of trace organics if ph correction is not performed . to produce low concentrations of h2o2 in water immediately prior to an aop , the cathode must be capable of producing h2o2 in a low ionic strength , poorly buffered solution at circumneutral ph values . furthermore , increases in ph that occur in the cathode chamber due to consumption of protons must be compensated for by a subsequent treatment process if the water is to be sent into a water distribution system . the purpose of this study was to evaluate a system that combines in situ electrochemical production of h2o2 followed by uv irradiation and anodic ph adjustment as a cost - effective means of removing trace organic contaminants from water . this new system , which also inactivates waterborne pathogens and transforms photolabile contaminants through exposure to uv light , can be controlled by varying the production of h2o2 through adjustment of the applied current . to provide insight into the performance of the system under conditions likely to be encountered in distributed water treatment systems , three representative source waters ( i.e. , synthetic surface water , synthetic groundwater , and municipal wastewater effluent ) the performance of the system was investigated in terms of contaminant removal and energy consumption . all experiments were performed at room temperature ( 23 2 c ) with chemicals of reagent grade or higher ( sigma - aldrich , st . the composition of the waters used is summarized in table s1 , supporting information . experiments were carried out in a two - chambered parallel plate electrochemical cell consisting of two square perspex frames ( internal dimensions : 8 8 1.9 cm ) separated by a cation exchange membrane ( ultrex cmi-7000 , membranes international inc . , ringwood , nj ) . the frames were bolted together between two square perspex side plates , creating anode and cathode compartments that each had effective volumes of 122 ml ( figure s1 , supporting information ) . a solid plate was used for the anode frame , while the cathode chamber was bolted with a hollow side plate allowing for one side of the gas diffusion cathode to be exposed to air . a ti mesh electrode coated with an ir mixed - metal oxide was used as the anode ( dimensions : 7.8 7.8 cm ; 1 mm thickness ; specific surface area 1.0 m m , magneto special anodes , netherlands ) . the uv reactor consisted of a 1 l brown glass bottle ( veffective = 925 ml ) containing a low - pressure uv lamp ( arc length = 16.5 cm , optical path length = 4.3 cm ) used typically for swimming pool disinfection ( g23 odyssea pool lamp , 9w , odyssea aquarium appliance co. , ltd . , the gas diffusion cathode was created by modifying carbon fiber paper ( avcarb p75 t , 10 10 cm , fuel cell store , college station , tx ) with a conductive , hydrophobic support layer and a carbon catalyst . the air - facing side of the cathode was prepared by coating a mixture of 60 wt % ptfe and 30 wt % graphite powder ( 200 mesh , alfa aesar , ward hill , ma ) onto one side of the carbon base layer . the cathode was then air - dried at room temperature , followed by sintering at 350 c for 40 min . the liquid - facing side was prepared by applying a mixture of 3 ml of propanol with 150 mg of carbon black ( cabot black pearls 2000 , cabot , boston , ma ) and 50 mg of ptfe onto the other side of the carbon base layer . the cathode was again air - dried at room temperature , followed by sintering at 350 c for 40 min . electrolysis experiments were performed at fixed currents controlled by a multichannel potentiostat ( gamry instruments inc . , water entered the cathode compartment operating in a flow - through mode with hydraulic residence times ( hrt ) ranging from 1.5 to 5.0 min ( 12035 l d ) . cathode effluent was supplied to the uv reactor and then passed through the anode of the electrochemical cell . the applied charge density ( q , c l ) was expressed as a product of the current density ( i , a m ) , electrode surface area ( a , m ) , and the hydraulic residence time ( t , s ) normalized by the half chamber reactor volume ( v , l):1 source waters were amended with a mixture of ten test compounds each at a concentration of 10 g l. for each experiment , samples were collected prior to the electrochemical cell , after passing through the cathode chamber , after the uv reactor , and after passing through the anode . at least 3.5 l of the test solution was passed through the system prior to collection of a sample . 1.9 ml subsamples to be analyzed for trace organic compounds were mixed with 0.1 ml of methanol to quench radical reactions that could occur prior to analysis . h2o2 and ph were measured within 5 min , whereas trace contaminants were stored for a maximum of 8 h. experiments quantifying h2o2 production in the varying waters were performed with applied cathodic current densities from 0 to 30 a m under varying flow regimes ( 35120 l d ) . to assess the long - term cathode performance , 6000 l of 5 mm na2so4 in tap water ( alkalinity = 0.34 mm , [ ca ] = 0.2 mm ) was run through the cell continuously at an applied current density of 15 a m at a fixed flow rate of 120 l d. samples were collected daily and analyzed for h2o2 . the effects of dissolved oxygen concentration on h2o2 production were evaluated by sparging source water with n2 to remove o2 . h2o2 and free chlorine were measured with a shimadzu uv-2600 spectrophotometer with the titanium(iv ) sulfate method at 405 nm and the n , n - diethyl - p - phenylenediamine ( dpd ) method at 515 nm , respectively . determination of free chlorine was performed in the absence of h2o2 to eliminate the positive interference of h2o2 with dpd . the uv absorbance of the four source waters was measured with a shimadzu uv-2600 spectrophotometer . dissolved organic carbon ( doc ) and dissolved inorganic carbon ( dic ) were measured using a shimadzu toc - v analyzer . no3 , cl , and so4 were analyzed using a dionex dx-120 ion chromatograph with an as19 g column . k , na , ca , and mg were analyzed using a dionex ics-2000 ion chromatograph with a cs12a column . fluence rate values were determined by chemical actinometry using 10 m atrazine as an actinometer ( 254 = 3860 m cm , 254= 0.046 mol ei , buffered at ph = 8 using a borate buffer ; details of the calculation in the supporting information ) . conductivity was measured with an ultrameter ii 4p ( myron l company , carlsbad , ca ) . test compounds were quantified in multiple reaction monitoring ( mrm ) mode with an agilent 1200 series hplc system coupled to a 6460 triple quadrupole tandem mass spectrometer ( hplc - ms / ms ) , as described previously . analytical details and compound specific parameters are provided in the supporting information text and tables s2 and s3 , respectively . the gas diffusion electrode was polarized cathodically against a ag / agcl reference electrode ( + 0.197 v vs she ; basi , usa ) . the full cell potential between the working ( i.e. , cathode ) and counter ( i.e. , anode ) electrodes was measured in a two - electrode setup . the total system power ( ptotal , w ) is a combination of the uv lamp power ( plamp , w ) and the electrochemical cell power , which can be expressed as a product of the current density ( i , a m ) , cell potential ( vcell ) , and the electrode surface area ( a , m):2 hydrogen peroxide concentrations ( figure 1a ) and production rates ( figure s2 , supporting information ) were determined for an array of charge densities that were achieved from combinations of current densities ( 030 a m ) and cathode chamber retention times ( 1.55 min ) . a linear relationship between h2o2 production and applied charge density was observed at charge densities greater than 50 c l , independent of how the charge was obtained ( i.e. , high current density and short retention times or low current density and long retention times):3where q is the specific charge density applied in c l and [ h2o2 ] is the hydrogen peroxide concentration in mm . for all of the waters tested , the coulombic efficiency of o2 reduction to h2o2 averaged 88.8 1.8% at charge densities lower coulombic efficiencies were observed ( note the deviation from the linear fit in figure 1a at charge densities below 50 c l ) . the observed increased coulombic efficiencies at higher charge densities agreed with previously published data for electrochemical synthesis of h2o2 using a gas diffusion electrode composed of a fluorocarbon binder and activated carbon catalyst fed with conductive , alkaline solutions . production of hydrogen peroxide ( a ) and coulombic efficiency ( b ) as a function of applied charge density ( wwtp : wastewater treatment plant ) . h2o2 production was independent of the type of source water used despite the substantial variability in the composition of the matrices ( table 1 ) . this suggests that h2o2 production was not affected by ph , the presence of natural organic matter ( nom ) , dissolved ions , or conductivity over the range of applied charge densities studied . although h2o2 production was not influenced by influent water quality , the cell potential , and therefore energy consumption , was affected by the conductivity of the source waters . higher ionic strength waters exhibited lower ohmic resistances and therefore operated at lower cell potentials , thus decreasing their energy consumption . for a given applied charge , the power required decreased with increased conductivity ( synthetic surface water > synthetic groundwater > wastewater effluent electrolyte ) ( figure s3 , supporting information ) . even for low conductivity surface water , however , the energy consumption for hydrogen peroxide production at a flow rate of 120 l d was still relatively low ( 0.0180.31 kwh m for 5 < i < 30 a m ) , indicating that in situ h2o2 production required much less energy than operation of the uv lamp ( 1.8 kwh m ; calculations provided in the supporting information ) . the h2o2 production rate increased linearly with applied current density , with a maximum of between 14.4 and 14.8 mg h2o2 l min at 30 a m for all of the waters tested ( figure s2 , supporting information ) . in full - scale aop systems ( e.g. , the orange county water district s groundwater replenishment system ) , 3 mg this concentration can be obtained with the gas diffusion electrode at an applied current density of only 4.14 a m at a hydraulic residence time of 1.5 min . under these conditions , this benchtop system could process approximately 120 l of water per day while consuming approximately 1.7 wh to produce h2o2 . the removal of trace organic contaminants involved direct photolysis , reactions with ho produced by photolysis of h2o2 in the uv chamber , and direct oxidation of contaminants on the anode . at the fluence employed in the uv chamber ( f0 3000 mj cm ) , direct photolysis only removed those compounds that exhibited high quantum yields and strong light absorbance at 254 nm ( figure s4 , supporting information ) . among the compounds tested , carbamazepine exhibited the lowest tendency for direct transformation by uv light ( < 30% removal for all matrices ) , while more photoreactive compounds , such as sulfamethoxazole , propranolol , and atrazine , displayed higher removals ( 5599% ) . unlike the variability of the compounds with respect to direct photolysis , the suite of trace organics all reacted with ho at near diffusion controlled rates ( 1010 m s ; table s3 , supporting information ) . as a result of its low reactivity with uv light , carbamazepine removal in the presence of h2o2 and uv light provided useful information on the transformation of organic contaminants by ho . the extent of removal of carbamazepine varied among the different matrices ( figure 2 ) . the presence of ho scavengers explained much of the variability . in the absence of current ( and therefore h2o2 ) , the variability of carbamazepine transformation was predominately influenced by the screening of light , as accounted for by the water factor:4where z is the mixed water body depth ( m ) and is the attenuation coefficient of the water body . the water factor , which was primarily influenced by the amount of nom , followed the trend : electrolyte ( 0.998 ) > groundwater ( 0.985 ) > synthetic surface water ( 0.598 ) > wastewater effluent ( 0.508 ) . transformation of carbamazepine solely in the presence of uv light varied from 22 5% in the electrolyte solution to 15 4% in the wastewater effluent . the observed removal of carbamazepine in the surface water and wastewater effluent , however , was greater than suggested from the water factor . this may be explained by the generation of ho and dom * produced from nom sensitization by uv light , which can be significant at uv fluences employed in aops . removal of carbamazepine as a function of current density for the four types of source waters ( wwtp : wastewater treatment plant ) . the rate of transformation of trace contaminants increased with current density due to additional ho production that occurred at higher h2o2 concentrations ( tables s4s7 , supporting information ) . complete carbamazepine transformation was observed after the uv treatment chamber at an applied current density of 5 a m for the electrolyte . for the three representative source waters , carbamazepine transformation increased to 98.7 0.6% for groundwater , 93.3 1.0% for surface water , and 78.5 1.9% for wastewater effluent as the current increased to 25 a m. organic compounds that have lower reaction rate constants with ho and are not susceptible to direct photolysis will require higher current densities to achieve a similar level of treatment . the fraction of ho that reacted with the contaminants can be estimated by considering the concentrations and rate constants for reactions of different solutes with ho:5where koh , s and kho , cont are the second order reaction rate constants of scavengers and contaminants with ho , respectively , and [ s ] is the concentration of the scavenger ( e.g. , hco3 , co3 , nom , h2o2 ; tables 1 and s3 , supporting information ) . at increasing h2o2 concentrations , a trade - off exists between additional transformation of trace organics by ho produced from h2o2 photolysis and greater radical scavenging and light screening by h2o2 . therefore , despite linear increases in h2o2 production with current density , there is a diminishing benefit to the treatment . as h2o2 increased from 0.09 mm ( 4.14 a m ) to 0.54 mm ( 25 a m ) , the fraction of ho reacting with contaminants decreased by 20% , 21% , and 10% for the surface water , groundwater , and wastewater effluent , respectively . at 0.54 mm h2o2 ( 25 a m ) , there was a 4.0% , 4.7% , and 3.8% reduction in direct photolysis rates of contaminants from additional light screening by h2o2 for the surface water , groundwater , and wastewater effluent , respectively ( details of ho branching ratio and direct photolysis calculations are included in the supporting information ) . at ph 8 , approximately 6.7% , 6.5% , and 2.9% of ho reacted with the organic contaminants in the uv reactor at an initial h2o2 concentration of 3 mg l ( 0.09 mm ) for the surface water , groundwater , and wastewater effluent , respectively . at ph 10 , the fraction of ho reacting with trace organic contaminants decreased to 0.9% , 0.6% , and 0.4% for the three source waters , respectively . the significant decrease in ho reacting with the trace organic contaminants was due to scavenging by carbonate at the higher ph values . the product of this reaction , co3 , can play a significant role in the transformation of certain organic compounds ( e.g. , propranolol and sulfamethoxazole ; table s3 , supporting information ) . as a result of differences in alkalinity of the different source waters , the importance of carbonate scavenging and co3 reactions depends on the source water composition and the applied current density ( i.e. , higher applied currents result in greater ph increases in the cathode chamber ) . although nitrite is an effective scavenger of ho ( kho , no2 = 6 10 m s ) , less than 0.3% of the generated ho would be scavenged by nitrite at concentrations typically found in nitrified wastewater effluent ( i.e. , 0.1 mg l ) . the formation of halogen radicals from reactions between ho and halide ions ( chloride and bromide ) should only be significant at low ph values and therefore will have a negligible impact on contaminant transformation at the circumneutral and basic ph values observed in this system . the generation of h2o2 by the cathode consumed protons and increased the solution ph ( reaction 6 ) . in the anode , oxidation reactions produced protons and lowered the solution ph ( reaction 7):6789 for the production of 3 mg l ( 0.09 mm ) of h2o2 at a current density of 4.14 a m , approximately 0.20 mequiv l of protons should have been consumed or produced at the cathode and anode , respectively . to maintain electroneutrality , a net migration of protons occurred from the anode chamber to the cathode chamber via the cation exchange membrane . in addition to protons , cations that were present at higher concentrations ( e.g. , na , ca , mg ) also carried ionic charge through the membrane satisfying electroneutrality in the cathode chamber while creating a proton deficit in the cathode chamber . as a result of differences in buffering among matrices , the solution ph should have increased more in the cathode chamber for waters with low alkalinity . the ph in the cathode chamber increased for each of the waters as the current increased from 0 to 25 a m ( figure 3 ) . the magnitude of ph increase was most pronounced for the electrolyte and surface water ( alkalinity = 0 and 2.45 mm , respectively ) with post - cathode ph values ranging from 10 to 10.5 , while the ph never exceeded 9.9 and 9.2 in the groundwater ( alkalinity = 3.89 mm ) and the municipal wastewater effluent ( alkalinity = 4.97 mm ) , respectively . the ph increases following the cathode resulted in supersaturation with respect to calcite ( caco3(s ) ) in the groundwater ( log si > 1.31 ) , surface water ( log si > 1.58 ) , and wastewater effluent ( log si > 1.09 ) beginning at a current density of 5 a m. this reaction could result in scaling on the cathode or the ion exchange membrane that might eventually affect system performance . loss of caco3(s ) from the system could also result in an overall decrease in ph as water passed through the treatment system . for example , if the surface water solution reached equilibrium at the current density needed to produce 3 mg l ( 0.09 mm ) h2o2 , 0.74 mmol ( 74 mg ) of calcite would precipitate for each liter of water treated and the ph would have dropped from 9.82 to 7.23 . on the basis of the observed ph values , it is evident that equilibrium was not achieved . however , additional research is needed to assess the importance of calcite precipitation to scaling and ph control . ph change of the source waters prior to entering the electrochemical cell , after passing through the cathode chamber , after the uv reactor , and after the anode as a function of current density ( wwtp : wastewater treatment plant ) . to readjust the solution ph , water leaving the uv reactor was passed through the anode chamber . if no mineral precipitation occurred in the cathode and uv chambers , the final ph should have been equal to the influent ph . a slight decrease in ph was observed in all solutions to which current was applied , with greater ph decreases at higher current densities occurring in the least buffered of the three environmental matrices ( i.e. , surface water ) . under the conditions that would likely be used for treatment ( i.e. , 510 a m and short hydraulic residence times ) , due to the relatively low molar absorptivity of h2o2 at 254 nm ( 254 = 18.6 m cm ) and the limited residence times in the uv reactor ( = 660 s ) , much of the h2o2 passed through the uv chamber without undergoing photolysis . for solutions with relatively low light screening ( i.e. , electrolyte , synthetic groundwater , and synthetic surface water ) , between 40% and 50% of the h2o2 was photolyzed at current densities ranging from 5 to 25 a m ( figure 4 and figure s5 , supporting information ) . as expected , less h2o2 photolysis occurred in municipal wastewater effluent due to light screening . production of h2o2 in the cathode , residual h2o2 after the uv cell , and residual h2o2 after the anode for the four types of source waters at applied current density of 25 a m. see figure s5 , supporting information , for data on the production and removal of h2o2 over the full range of current densities ( 525 a m ) ( wwtp : wastewater treatment plant ) . in practice , many centralized treatment plants employ reducing agents ( e.g. , bisulfite ) , chlorine , or activated carbon to remove residual h2o2 before distribution . the use of activated carbon or the addition of chemicals , however , may be impractical in a distributed treatment system . partial removal of h2o2 occurred when the solution passed through the anode , especially in the electrolyte solution ( figure 4 and figure s5 , supporting information ) . anodic removal of h2o2 increased with increasing current density ( figure s5 , supporting information ) . in the nacl electrolyte solution , up to 0.12 mm h2o2 was removed at 25 a m. for the three source waters , however , the anode only removed about 25% of the amount removed in the electrolyte . removal of h2o2 in the anode was attributable to a combination of direct anodic oxidation and reactions with oxidants produced on the anode surface . for example , oxidation of chloride can result in the production of hypochlorous acid ( hocl ; pka = 7.6 ) ( reactions 8 and 9 ) . hypochlorite reacts rapidly with hydrogen peroxide under alkaline conditions with the bimolecular rate constant increasing from 196 to 7.5 10 m s from ph 6 to 9 ( see the supporting information for calculation of the ph - dependent bimolecular rate constant):10 although ti - iro2 electrodes have a high electrocatalytic activity with respect to chlorine evolution , only modest concentrations of chlorine were produced in control experiments at varying chloride concentrations due to the short hydraulic residence times and relatively low current densities applied ( table 2 ) . to separate the effects of reactive halogen species from direct electrode oxidation on the removal of h2o2 , experiments were repeated using an inert electrolyte ( i.e. , na2so4 ) ( figure s6 , supporting information ) . h2o2 removal was independent of applied current density with 37 2 m h2o2 removed from 5 to 25 a m ; a concentration equivalent to the observed h2o2 removal in the anode for the three source waters in figure s5 , supporting information . given the low chloride concentrations ( < 1 mm ) of the simulated surface water and groundwater , it is not surprising that ocl production was low and only a small quantity of h2o2 was removed in the anode . free chlorine production ( as m cl[i ] ) in the anode chamber as a function of applied current density and chloride concentration . experiments were performed with a stainless steel cathode to prevent h2o2 formation , which interferes with the chlorine measurement . despite the electrolyte and municipal wastewater effluent having roughly the same concentration of chloride , h2o2 removal was significantly higher in the electrolyte control than in the wastewater effluent , suggesting that reactive halogen species did not play an important role in h2o2 removal in the wastewater effluent matrix . nom is an effective sink of hocl / ocl ; however , under the experimental conditions used in this study , the half - life of hocl / ocl with respect to its reaction with h2o2 was much shorter than that predicted for nom ( i.e. , 1.39 and 49.3 s , respectively , as described in the supporting information ) . as a result , nom is only a minor sink for hocl / ocl in the presence of h2o2 . this was consistent with observations from experiments in which h2o2 removal decreased by less than 40% ( 58 m ) when nom was added to a solution containing a fixed concentration of chloride at a current density of 25 a m , suggesting that nom is only partially responsible for the difference in h2o2 removal between the two solutions ( figure s6 , supporting information ) . the apparent discrepancy between municipal wastewater effluent and the sodium chloride solution may be partially attributable to differences in ph values in the anode chamber . at higher ph values , like those found in the municipal wastewater effluent after anodic treatment , there is a larger driving force for oxygen evolution compared to cl2 production:1112 experiments conducted in the anode chamber at different ph values confirmed that chlorine production increased substantially as ph dropped from 9 to 7 ( figure s7 , supporting information ) . due to the presence of bicarbonate in the municipal wastewater effluent , the anode ph was considerably higher ( i.e. , 8 ) during treatment than in the unbuffered nacl electrolyte , where ph decreased to approximately 6 during anodic treatment . as a result , considerably more hocl / ocl was produced in the anode chamber when the electrolyte was treated . total hydrogen peroxide removal in the system ( i.e. , photolysis and anodic loss ) was 48 5% for the simulated groundwater , 49 3% for the simulated surface water , and 25 3% for the wastewater effluent for the array of current densities tested . at the current density required to produce 3 mg l ( 0.09 mm ) h2o2 , water leaving the treatment system effluent contained 1.52.3 mg l h2o2 . although h2o2 does not pose a health risk at these concentrations , its presence in potable water may be undesirable . in a point - of - use water treatment system , it might be possible to remove the excess h2o2 by passing it through activated carbon or a high surface area catalyst consisting of metal oxide or silver . alternatively , the efficiency of chloride oxidation in the anode chamber might be improved through the use of three - dimensional or porous electrodes that reduce mass transfer limitations or through the use of more catalytic anode materials . despite only accounting for a small fraction of the total removal observed in the treatment system , experiments conducted in the absence of uv exposure with solutions amended with 10 g l of trace organic contaminants indicated that direct anodic oxidation resulted in the removal of up to 20% of certain trace organics ( e.g. , propranolol ) at a current density of 25 a m ( figure s8 , supporting information ) . oxidation of organic compounds on the anode could be increased through the use of inactive anodes ( e.g. , boron - doped diamond , doped - sno2 , pbo2 ) . these electrodes , however , have higher capital and operating costs than ti - iro2 electrodes . although carbamazepine is relatively unreactive with hypochlorite , other compounds ( e.g. , propranolol and sulfamethoxazole ) react with hocl ( table s3 , supporting information ) . however , the presence of h2o2 reduced the importance of reactions between trace organics and chlorine species generated at the anode because chlorine preferentially reacts with h2o2 . as a result , nearly all of the observed loss of the test compounds in the anode chamber was due to direct oxidation on the anode surface . this observation was consistent with experiments comparing anodic removal of trace organics in the presence and absence of h2o2 ( figure s9 , supporting information ) . the performance of gas diffusion electrodes can decrease over time due to clogging of the pores by precipitates , fouling with nom as well as charge transfer resistance attributable to the loss of conductive graphite paste . in a long - term trial , cathode performance ( i.e. , h2o2 production at a fixed current density ) l of tap water amended with 5 mm na2so4 was passed through the system at an applied current density of 15 a m ( figure s10 , supporting information ) . calcium carbonate scaling due to the elevated ph and migration of calcium ions in the tap water into the cathode chamber was observed on the interior of the cathode . additional experiments are needed to assess the importance of scaling and the efficacy of simple descaling approaches ( i.e. , polarization reversal ) over longer time periods and more realistic operating conditions . the treatment system used electricity to produce the oxidant ( i.e. , h2o2 ) and to convert it into ho ( i.e. , the uv lamp ) . electrical energy per order ( eeo ) is a useful figure of merit for comparing the efficiency and cost of the treatment system with other aops . eeo is the electrical energy ( in kwh ) required to reduce a contaminant concentration by 1 order of magnitude in 1 m of water:13where p ( kw ) is the electrical power for the electrochemical cell and uv lamp , q ( m h ) is the system flow rate , and c0 and c ( m ) are the initial and final contaminant concentrations . without the production of h2o2 , eeo values ranged from 16.8 0.3 to 28.1 0.2 kwh m order , with larger amounts of energy needed to transform contaminants in waters that contained high concentrations of ho scavengers and chromophores ( i.e. , municipal wastewater effluent and synthetic groundwater ) ( figure 5 ) . a substantial decrease in eeo occurred when current was applied to the electrochemical cell ( i.e. , from 0 to 5 a m ) . as current density increased from 5 to 25 a m , the eeo decreased by less than 11% . aop system is much more efficient than the use of uv alone and that there is a marginal benefit associated with the production of higher h2o2 concentrations in the cathode because the reactions become less efficient at higher concentrations of h2o2 . only 814% of the energy demand was attributable to the electrochemical production of h2o2 , with the majority of the energy required for the operation of the low - pressure uv lamp . at a current density of 25 a m , an energy requirement of 1.08 0.1 to 2.84 0.1 kwh m order was observed , which was similar to results from previous studies of the transformation of trace organic compounds by uv / h2o2 in different source waters . electrical energy per order ( eeo ) for the removal of carbamazepine as a function of current density ( wwtp : wastewater treatment plant ) . the high coulombic efficiency and low current densities result in a low - cost means of h2o2 production even for treatment of water with low conductivity . for comparison , electrochemically produced h2o2 costs between 0.1 and 0.3 $ kg , while h2o2 produced by the anthraquinone process typically costs between 1 and 2 $ kg . when considering the lack of a need to transport , store , and handle h2o2 as well as modest capital and operational costs , the modular aop treatment system can be a competitive technology for point - of - use treatment at a household and community level or even for wellhead treatment of trace organic contaminants present in potable water sources . as h2o2 production was directly proportional to current density , the treatment system could be scaled up by using faster flow rates accompanied by higher applied current densities or by increasing surface area of the cathode . to obtain the equivalent contaminant removal after scale - up , optimization of the uv reactor geometry would be required . additional research is needed to assess long - term system performance under realistic operating conditions .

hydrogen peroxide ( h2o2 ) is frequently used in combination with ultraviolet ( uv ) light to treat trace organic contaminants in advanced oxidation processes ( aops ) . in small - scale applications , such as wellhead and point - of - entry water treatment systems , the need to maintain a stock solution of concentrated h2o2 increases the operational cost and complicates the operation of aops . to avoid the need for replenishing a stock solution of h2o2 , a gas diffusion electrode was used to generate low concentrations of h2o2 directly in the water prior to its exposure to uv light . following the aop , the solution was passed through an anodic chamber to lower the solution ph and remove the residual h2o2 . the effectiveness of the technology was evaluated using a suite of trace contaminants that spanned a range of reactivity with uv light and hydroxyl radical ( ho ) in three different types of source waters ( i.e. , simulated groundwater , simulated surface water , and municipal wastewater effluent ) as well as a sodium chloride solution . irrespective of the source water , the system produced enough h2o2 to treat up to 120 l water d1 . the extent of transformation of trace organic contaminants was affected by the current density and the concentrations of ho scavengers in the source water . the electrical energy per order ( eeo ) ranged from 1 to 3 kwh m3 , with the uv lamp accounting for most of the energy consumption . the gas diffusion electrode exhibited high efficiency for h2o2 production over extended periods and did not show a diminution in performance in any of the matrices .

Introduction Materials and Methods Results

distributed water treatment systems offer a potential means of exploiting alternative water sources , including municipal wastewater effluent , roof water , stormwater , and water from shallow aquifers . as a result , previous attempts to develop point - of - use treatment systems capable of removing trace organic contaminants prior to nonpotable reuse have employed electrochemical processes , but these systems suffer from limitations including the production of toxic byproducts , an inability to remove recalcitrant compounds and high cost of treatment . trace organic contaminants can be removed from water by exposure to hydroxyl radicals ( ho ) in advanced oxidation processes ( aops ) . in full - scale potable water reuse systems , trace organic contaminants are frequently removed by addition of a modest concentration of h2o2 ( e.g. , 3 mg / l ) followed by exposure to ultraviolet ( uv ) light . systems in which oxygen is bubbled into a solution prior to reduction on an electrode surface consume a considerable amount of energy due to the low solubility of oxygen and the need to ensure that it reaches the electrode surface . recently , gas diffusion electrodes have been used to generate h2o2 without a need to bubble air or oxygen into a solution . cathodic production of h2o2 for the removal of organics has typically been used for electro - fenton treatment ; however , differences in ph needed for the optimal kinetics of the two reactions ( i.e. to produce low concentrations of h2o2 in water immediately prior to an aop , the cathode must be capable of producing h2o2 in a low ionic strength , poorly buffered solution at circumneutral ph values . this new system , which also inactivates waterborne pathogens and transforms photolabile contaminants through exposure to uv light , can be controlled by varying the production of h2o2 through adjustment of the applied current . to provide insight into the performance of the system under conditions likely to be encountered in distributed water treatment systems , three representative source waters ( i.e. , synthetic surface water , synthetic groundwater , and municipal wastewater effluent ) the performance of the system was investigated in terms of contaminant removal and energy consumption . the applied charge density ( q , c l ) was expressed as a product of the current density ( i , a m ) , electrode surface area ( a , m ) , and the hydraulic residence time ( t , s ) normalized by the half chamber reactor volume ( v , l):1 source waters were amended with a mixture of ten test compounds each at a concentration of 10 g l. for each experiment , samples were collected prior to the electrochemical cell , after passing through the cathode chamber , after the uv reactor , and after passing through the anode . at least 3.5 l of the test solution was passed through the system prior to collection of a sample . the gas diffusion electrode was polarized cathodically against a ag / agcl reference electrode ( + 0.197 v vs she ; basi , usa ) . the total system power ( ptotal , w ) is a combination of the uv lamp power ( plamp , w ) and the electrochemical cell power , which can be expressed as a product of the current density ( i , a m ) , cell potential ( vcell ) , and the electrode surface area ( a , m):2 hydrogen peroxide concentrations ( figure 1a ) and production rates ( figure s2 , supporting information ) were determined for an array of charge densities that were achieved from combinations of current densities ( 030 a m ) and cathode chamber retention times ( 1.55 min ) . the observed increased coulombic efficiencies at higher charge densities agreed with previously published data for electrochemical synthesis of h2o2 using a gas diffusion electrode composed of a fluorocarbon binder and activated carbon catalyst fed with conductive , alkaline solutions . h2o2 production was independent of the type of source water used despite the substantial variability in the composition of the matrices ( table 1 ) . although h2o2 production was not influenced by influent water quality , the cell potential , and therefore energy consumption , was affected by the conductivity of the source waters . even for low conductivity surface water , however , the energy consumption for hydrogen peroxide production at a flow rate of 120 l d was still relatively low ( 0.0180.31 kwh m for 5 < i < 30 a m ) , indicating that in situ h2o2 production required much less energy than operation of the uv lamp ( 1.8 kwh m ; calculations provided in the supporting information ) . , the orange county water district s groundwater replenishment system ) , 3 mg this concentration can be obtained with the gas diffusion electrode at an applied current density of only 4.14 a m at a hydraulic residence time of 1.5 min . the removal of trace organic contaminants involved direct photolysis , reactions with ho produced by photolysis of h2o2 in the uv chamber , and direct oxidation of contaminants on the anode . unlike the variability of the compounds with respect to direct photolysis , the suite of trace organics all reacted with ho at near diffusion controlled rates ( 1010 m s ; table s3 , supporting information ) . as a result of its low reactivity with uv light , carbamazepine removal in the presence of h2o2 and uv light provided useful information on the transformation of organic contaminants by ho . in the absence of current ( and therefore h2o2 ) , the variability of carbamazepine transformation was predominately influenced by the screening of light , as accounted for by the water factor:4where z is the mixed water body depth ( m ) and is the attenuation coefficient of the water body . removal of carbamazepine as a function of current density for the four types of source waters ( wwtp : wastewater treatment plant ) . the rate of transformation of trace contaminants increased with current density due to additional ho production that occurred at higher h2o2 concentrations ( tables s4s7 , supporting information ) . for the three representative source waters , carbamazepine transformation increased to 98.7 0.6% for groundwater , 93.3 1.0% for surface water , and 78.5 1.9% for wastewater effluent as the current increased to 25 a m. organic compounds that have lower reaction rate constants with ho and are not susceptible to direct photolysis will require higher current densities to achieve a similar level of treatment . the fraction of ho that reacted with the contaminants can be estimated by considering the concentrations and rate constants for reactions of different solutes with ho:5where koh , s and kho , cont are the second order reaction rate constants of scavengers and contaminants with ho , respectively , and [ s ] is the concentration of the scavenger ( e.g. as h2o2 increased from 0.09 mm ( 4.14 a m ) to 0.54 mm ( 25 a m ) , the fraction of ho reacting with contaminants decreased by 20% , 21% , and 10% for the surface water , groundwater , and wastewater effluent , respectively . at 0.54 mm h2o2 ( 25 a m ) , there was a 4.0% , 4.7% , and 3.8% reduction in direct photolysis rates of contaminants from additional light screening by h2o2 for the surface water , groundwater , and wastewater effluent , respectively ( details of ho branching ratio and direct photolysis calculations are included in the supporting information ) . at ph 8 , approximately 6.7% , 6.5% , and 2.9% of ho reacted with the organic contaminants in the uv reactor at an initial h2o2 concentration of 3 mg l ( 0.09 mm ) for the surface water , groundwater , and wastewater effluent , respectively . at ph 10 , the fraction of ho reacting with trace organic contaminants decreased to 0.9% , 0.6% , and 0.4% for the three source waters , respectively . the significant decrease in ho reacting with the trace organic contaminants was due to scavenging by carbonate at the higher ph values . as a result of differences in alkalinity of the different source waters , the importance of carbonate scavenging and co3 reactions depends on the source water composition and the applied current density ( i.e. although nitrite is an effective scavenger of ho ( kho , no2 = 6 10 m s ) , less than 0.3% of the generated ho would be scavenged by nitrite at concentrations typically found in nitrified wastewater effluent ( i.e. in the anode , oxidation reactions produced protons and lowered the solution ph ( reaction 7):6789 for the production of 3 mg l ( 0.09 mm ) of h2o2 at a current density of 4.14 a m , approximately 0.20 mequiv l of protons should have been consumed or produced at the cathode and anode , respectively . as a result of differences in buffering among matrices , the solution ph should have increased more in the cathode chamber for waters with low alkalinity . the magnitude of ph increase was most pronounced for the electrolyte and surface water ( alkalinity = 0 and 2.45 mm , respectively ) with post - cathode ph values ranging from 10 to 10.5 , while the ph never exceeded 9.9 and 9.2 in the groundwater ( alkalinity = 3.89 mm ) and the municipal wastewater effluent ( alkalinity = 4.97 mm ) , respectively . the ph increases following the cathode resulted in supersaturation with respect to calcite ( caco3(s ) ) in the groundwater ( log si > 1.31 ) , surface water ( log si > 1.58 ) , and wastewater effluent ( log si > 1.09 ) beginning at a current density of 5 a m. this reaction could result in scaling on the cathode or the ion exchange membrane that might eventually affect system performance . for example , if the surface water solution reached equilibrium at the current density needed to produce 3 mg l ( 0.09 mm ) h2o2 , 0.74 mmol ( 74 mg ) of calcite would precipitate for each liter of water treated and the ph would have dropped from 9.82 to 7.23 . ph change of the source waters prior to entering the electrochemical cell , after passing through the cathode chamber , after the uv reactor , and after the anode as a function of current density ( wwtp : wastewater treatment plant ) . to readjust the solution ph , water leaving the uv reactor was passed through the anode chamber . a slight decrease in ph was observed in all solutions to which current was applied , with greater ph decreases at higher current densities occurring in the least buffered of the three environmental matrices ( i.e. , 510 a m and short hydraulic residence times ) , due to the relatively low molar absorptivity of h2o2 at 254 nm ( 254 = 18.6 m cm ) and the limited residence times in the uv reactor ( = 660 s ) , much of the h2o2 passed through the uv chamber without undergoing photolysis . , electrolyte , synthetic groundwater , and synthetic surface water ) , between 40% and 50% of the h2o2 was photolyzed at current densities ranging from 5 to 25 a m ( figure 4 and figure s5 , supporting information ) . production of h2o2 in the cathode , residual h2o2 after the uv cell , and residual h2o2 after the anode for the four types of source waters at applied current density of 25 a m. see figure s5 , supporting information , for data on the production and removal of h2o2 over the full range of current densities ( 525 a m ) ( wwtp : wastewater treatment plant ) . partial removal of h2o2 occurred when the solution passed through the anode , especially in the electrolyte solution ( figure 4 and figure s5 , supporting information ) . in the nacl electrolyte solution , up to 0.12 mm h2o2 was removed at 25 a m. for the three source waters , however , the anode only removed about 25% of the amount removed in the electrolyte . hypochlorite reacts rapidly with hydrogen peroxide under alkaline conditions with the bimolecular rate constant increasing from 196 to 7.5 10 m s from ph 6 to 9 ( see the supporting information for calculation of the ph - dependent bimolecular rate constant):10 although ti - iro2 electrodes have a high electrocatalytic activity with respect to chlorine evolution , only modest concentrations of chlorine were produced in control experiments at varying chloride concentrations due to the short hydraulic residence times and relatively low current densities applied ( table 2 ) . to separate the effects of reactive halogen species from direct electrode oxidation on the removal of h2o2 , experiments were repeated using an inert electrolyte ( i.e. given the low chloride concentrations ( < 1 mm ) of the simulated surface water and groundwater , it is not surprising that ocl production was low and only a small quantity of h2o2 was removed in the anode . free chlorine production ( as m cl[i ] ) in the anode chamber as a function of applied current density and chloride concentration . despite the electrolyte and municipal wastewater effluent having roughly the same concentration of chloride , h2o2 removal was significantly higher in the electrolyte control than in the wastewater effluent , suggesting that reactive halogen species did not play an important role in h2o2 removal in the wastewater effluent matrix . nom is an effective sink of hocl / ocl ; however , under the experimental conditions used in this study , the half - life of hocl / ocl with respect to its reaction with h2o2 was much shorter than that predicted for nom ( i.e. the apparent discrepancy between municipal wastewater effluent and the sodium chloride solution may be partially attributable to differences in ph values in the anode chamber . due to the presence of bicarbonate in the municipal wastewater effluent , the anode ph was considerably higher ( i.e. total hydrogen peroxide removal in the system ( i.e. , photolysis and anodic loss ) was 48 5% for the simulated groundwater , 49 3% for the simulated surface water , and 25 3% for the wastewater effluent for the array of current densities tested . in a point - of - use water treatment system , it might be possible to remove the excess h2o2 by passing it through activated carbon or a high surface area catalyst consisting of metal oxide or silver . despite only accounting for a small fraction of the total removal observed in the treatment system , experiments conducted in the absence of uv exposure with solutions amended with 10 g l of trace organic contaminants indicated that direct anodic oxidation resulted in the removal of up to 20% of certain trace organics ( e.g. the performance of gas diffusion electrodes can decrease over time due to clogging of the pores by precipitates , fouling with nom as well as charge transfer resistance attributable to the loss of conductive graphite paste . , h2o2 production at a fixed current density ) l of tap water amended with 5 mm na2so4 was passed through the system at an applied current density of 15 a m ( figure s10 , supporting information ) . electrical energy per order ( eeo ) is a useful figure of merit for comparing the efficiency and cost of the treatment system with other aops . eeo is the electrical energy ( in kwh ) required to reduce a contaminant concentration by 1 order of magnitude in 1 m of water:13where p ( kw ) is the electrical power for the electrochemical cell and uv lamp , q ( m h ) is the system flow rate , and c0 and c ( m ) are the initial and final contaminant concentrations . without the production of h2o2 , eeo values ranged from 16.8 0.3 to 28.1 0.2 kwh m order , with larger amounts of energy needed to transform contaminants in waters that contained high concentrations of ho scavengers and chromophores ( i.e. aop system is much more efficient than the use of uv alone and that there is a marginal benefit associated with the production of higher h2o2 concentrations in the cathode because the reactions become less efficient at higher concentrations of h2o2 . only 814% of the energy demand was attributable to the electrochemical production of h2o2 , with the majority of the energy required for the operation of the low - pressure uv lamp . at a current density of 25 a m , an energy requirement of 1.08 0.1 to 2.84 0.1 kwh m order was observed , which was similar to results from previous studies of the transformation of trace organic compounds by uv / h2o2 in different source waters . electrical energy per order ( eeo ) for the removal of carbamazepine as a function of current density ( wwtp : wastewater treatment plant ) . when considering the lack of a need to transport , store , and handle h2o2 as well as modest capital and operational costs , the modular aop treatment system can be a competitive technology for point - of - use treatment at a household and community level or even for wellhead treatment of trace organic contaminants present in potable water sources .

this retrospective study included a total of 25 eyes of 14 patients with ages ranging between 52 and 77 years old . all these eyes underwent cataract surgery with implantation of the accommodating iol crystalens hd ( bausch and lomb ) . the inclusion criteria of this study were patients with visually significant cataract or presbyopic / pre presbyopic patients suitable for refractive lens exchange and demanding complete spectacle - independence . the exclusion criteria were patients with active ocular diseases , ruptured posterior capsule , zonulodialysis , scotopic pupil size of more than 6.0 mm , illiteracy and topographic astigmatisms higher than 1.25 d. all volunteers were adequately informed and signed a consent form . the study adhered to the tenets of the declaration of helsinki and was approved by the local ethical committee . the accommodating iol used in this study was the crystalens hd ( bausch and lomb ) , which has a biconvex single - optic design . the iol is of a biocompatible third - generation silicone ( biosil ) with a refractive index of 1.428 . it has a central bi - aspheric modification ( around 1.5-mm diameter ) to increase depth of focus and thus provide better intermediate and near foci . two sizes are available depending on the required power , the 12.0 mm model ( hd520 ) for powers between 10.00 and 16.50 d , and the 11.5 mm model ( hd500 ) for powers between 17.00 and 33.00 d. according to the manufacturer , the iol has a double mechanism to improve the near visual function : axial movement of the optic as a consequence of the ciliary muscle changes and variation of the radius of curvature of the anterior iol surface ( arching optic ) . in the current study , the srk / t formula and the iol master software ( carl zeiss meditec , jena , germany ) were used in all cases for the iol power calculation , with an a - constant value of 118.8 . all surgeries were performed by one of the experienced surgeons ( mlr ) using a standard technique of phacoemulsification . in all cases , topical anesthesia was administered , and pupillary dilation was induced with a combination of tropicamide and phenylephrine 10% every 15 min h prior to the procedure . a 2.75-mm clear incision was made with a diamond knife on the steepest meridian to minimize post - surgical astigmatism . a paracentesis was made 6090 clockwise from the main incision and the anterior chamber was filled with viscoelastic material . after the crystalline lens removal , the iols were implanted through the incision into the capsular bag using a specific injector developed by the manufacturer for such purpose . finally , the surgeon proceeded to retrieve the viscoelastic material using the irrigation - aspiration system . a combination of topical steroid and antibiotic ( tobradex , alcon , fort worth , tx , usa ) as well as a nonsteroidal anti - inflammatory drops ( dicloabak , laboratorios thea , barcelona , spain ) were prescribed to be applied four times daily for a week after the surgery and 3 times daily the second postoperative week . in addition , the nonsteroidal anti - inflammatory drops were also prescribed to be applying three times daily during 2 weeks more after surgery . almost all theoretical formulas for iol power calculation are based on the use of a simplified eye model , with a thin cornea and crystalline lens model . according to such approach , the power of the iol ( piol ) can be easily calculated using the gauss equation in paraxial optics : where pc is the total corneal power , elp the effective lens plane , al the axial length ( al ) , nha the aqueous humor refractive index , nhv the vitreous humor refractive index and rdes is the postoperative desired refraction calculated at corneal vertex . our research group has recently proposed the use of a variable keratometric index ( nkadj ) depending on the radius of the anterior corneal surface ( r1c ) expressed in millimeters for minimizing the error associated to the keratometric approach for corneal power calculation . specifically , the following expression was defined according to the gullstrand eye model : using this algorithm , a new keratometric corneal power , named adjusted keratometric corneal power ( pkadj ) , can be calculated using the classical keratometric corneal power formula . in the current study , the adjusted iol power ( pioladj ) was calculated , defined as the iol power calculated from the equation 1 using the nkadj value for the estimation of the corneal power ( pkadj ) , the nha and nhv values corresponding to the gullstrand eye model ( 1.336 for both index ) . in such calculation , the postoperative spherical equivalent ( se ) at corneal vertex was considered as the desired refraction ( rdes = sepost ) . afterward , this iol power ( pioladj ) was compared with the real power of the iol implanted ( piolreal ) . the pioladj calculation was performed after estimating the elp using two different approaches : elp calculation following the srk / t formula guidelines ( named pioladjsrk / t ) and elp calculation using a mathematical expression obtained by multiple regression analysis ( named pioladj ) , as explained carefully in the next section . furthermore , the piol was also calculated using three conventional formulae ( haigis , hofferq and holladay i ) considering the elp defined for each formula and that rdes = sepost . a comparative analysis was done between these values of piol and pioladj . considering equation 1 , piolreal , pkadj and a multiple regression analysis was performed with the aim of obtaining a mathematic expression for predicting the elpadj from different anatomical and clinical parameters . preoperatively , all patients had a full ophthalmologic examination including the evaluation of the refractive status , the distance and near visual acuities , slit lamp examination , optical biometry ( iol - master , carl zeiss meditec , jena , germany ) , goldman tonometry and funduscopy . distance ( 4 m ) and near ( 40 cm ) visual acuities were evaluated with etdrs charts . postoperatively , patients were evaluated at 1-day , 1-week , 1-month , and 3 months after surgery . at all visits , visual acuity , refraction and the integrity of the anterior segment the statistical analysis was performed using the spss statistics software package version 19.0 for windows ( ibm , armonk , ny , usa ) . normality of data samples was evaluated by means of the kolmogorov - smirnov test . when parametric analysis was possible , the student 's t - test for paired data was used for comparing the different approaches for piol calculation and also for comparing preoperative and postoperative data . when parametric analysis was not possible , the wilcoxon rank sum test was applied to assess the significance of such comparisons . correlation coefficients ( pearson or spearman depending if normality condition could be assumed ) were used to assess the correlation between different variables . regarding the interchangeability between pairs of methods used for obtaining piol , this is a graphical method for assessing if there is an agreement between two clinical procedures . specifically , bland - altman plots show the differences between the methods plotted against the mean of the 2 methods . the limits of agreement ( loa ) are defined as the mean 1.96 standard deviation ( sd ) of the differences . if the limits are clinically relevant , the 2 methods can not be used interchangeably . in the current study , differences in iol power between the different formulas evaluated was considered as clinically relevant for values of more than 0.5 d because this value is the iol power step provided currently by most of manufacturers and has been shown to be the optical neutralization tolerance since many years ago . a multiple regression analysis was used for predicting the elpadj from different preoperative anatomical and clinical parameters . model assumptions were evaluated by analyzing residuals , the normality of nonstandardized residuals ( homoscedasticity ) , and the cook distance to detect influential points or outliers . in addition , the lack of correlation between errors and multicollinearity was assessed using the durbin watson test , the calculation of the colinearity tolerance , and the variance inflation factor . this retrospective study included a total of 25 eyes of 14 patients with ages ranging between 52 and 77 years old . all these eyes underwent cataract surgery with implantation of the accommodating iol crystalens hd ( bausch and lomb ) . the inclusion criteria of this study were patients with visually significant cataract or presbyopic / pre presbyopic patients suitable for refractive lens exchange and demanding complete spectacle - independence . the exclusion criteria were patients with active ocular diseases , ruptured posterior capsule , zonulodialysis , scotopic pupil size of more than 6.0 mm , illiteracy and topographic astigmatisms higher than 1.25 d. all volunteers were adequately informed and signed a consent form . the study adhered to the tenets of the declaration of helsinki and was approved by the local ethical committee . the accommodating iol used in this study was the crystalens hd ( bausch and lomb ) , which has a biconvex single - optic design . the iol is of a biocompatible third - generation silicone ( biosil ) with a refractive index of 1.428 . it has a central bi - aspheric modification ( around 1.5-mm diameter ) to increase depth of focus and thus provide better intermediate and near foci . two sizes are available depending on the required power , the 12.0 mm model ( hd520 ) for powers between 10.00 and 16.50 d , and the 11.5 mm model ( hd500 ) for powers between 17.00 and 33.00 d. according to the manufacturer , the iol has a double mechanism to improve the near visual function : axial movement of the optic as a consequence of the ciliary muscle changes and variation of the radius of curvature of the anterior iol surface ( arching optic ) . in the current study , the srk / t formula and the iol master software ( carl zeiss meditec , jena , germany ) were used in all cases for the iol power calculation , with an a - constant value of 118.8 . all surgeries were performed by one of the experienced surgeons ( mlr ) using a standard technique of phacoemulsification . in all cases , topical anesthesia was administered , and pupillary dilation was induced with a combination of tropicamide and phenylephrine 10% every 15 min h prior to the procedure . a 2.75-mm clear incision was made with a diamond knife on the steepest meridian to minimize post - surgical astigmatism . a paracentesis was made 6090 clockwise from the main incision and the anterior chamber was filled with viscoelastic material . a continuous curvilinear capsulorhexis between 5.5 and 6.0 mm was performed . after the crystalline lens removal , the iols were implanted through the incision into the capsular bag using a specific injector developed by the manufacturer for such purpose . finally , the surgeon proceeded to retrieve the viscoelastic material using the irrigation - aspiration system . a combination of topical steroid and antibiotic ( tobradex , alcon , fort worth , tx , usa ) as well as a nonsteroidal anti - inflammatory drops ( dicloabak , laboratorios thea , barcelona , spain ) were prescribed to be applied four times daily for a week after the surgery and 3 times daily the second postoperative week . in addition , the nonsteroidal anti - inflammatory drops were also prescribed to be applying three times daily during 2 weeks more after surgery . almost all theoretical formulas for iol power calculation are based on the use of a simplified eye model , with a thin cornea and crystalline lens model . according to such approach , the power of the iol ( piol ) can be easily calculated using the gauss equation in paraxial optics : where pc is the total corneal power , elp the effective lens plane , al the axial length ( al ) , nha the aqueous humor refractive index , nhv the vitreous humor refractive index and rdes is the postoperative desired refraction calculated at corneal vertex . our research group has recently proposed the use of a variable keratometric index ( nkadj ) depending on the radius of the anterior corneal surface ( r1c ) expressed in millimeters for minimizing the error associated to the keratometric approach for corneal power calculation . specifically , the following expression was defined according to the gullstrand eye model : using this algorithm , a new keratometric corneal power , named adjusted keratometric corneal power ( pkadj ) , can be calculated using the classical keratometric corneal power formula . in the current study , the adjusted iol power ( pioladj ) was calculated , defined as the iol power calculated from the equation 1 using the nkadj value for the estimation of the corneal power ( pkadj ) , the nha and nhv values corresponding to the gullstrand eye model ( 1.336 for both index ) . in such calculation , the postoperative spherical equivalent ( se ) at corneal vertex was considered as the desired refraction ( rdes = sepost ) . afterward , this iol power ( pioladj ) was compared with the real power of the iol implanted ( piolreal ) . the pioladj calculation was performed after estimating the elp using two different approaches : elp calculation following the srk / t formula guidelines ( named pioladjsrk / t ) and elp calculation using a mathematical expression obtained by multiple regression analysis ( named pioladj ) , as explained carefully in the next section . furthermore , the piol was also calculated using three conventional formulae ( haigis , hofferq and holladay i ) considering the elp defined for each formula and that rdes = sepost . considering equation 1 , piolreal , pkadj and rdes = sepost in each case , elp was obtained and named adjusted elpadj . a multiple regression analysis was performed with the aim of obtaining a mathematic expression for predicting the elpadj from different anatomical and clinical parameters . preoperatively , all patients had a full ophthalmologic examination including the evaluation of the refractive status , the distance and near visual acuities , slit lamp examination , optical biometry ( iol - master , carl zeiss meditec , jena , germany ) , goldman tonometry and funduscopy . distance ( 4 m ) and near ( 40 cm ) visual acuities were evaluated with etdrs charts . postoperatively , patients were evaluated at 1-day , 1-week , 1-month , and 3 months after surgery . at all visits , visual acuity , refraction and the integrity of the anterior segment the statistical analysis was performed using the spss statistics software package version 19.0 for windows ( ibm , armonk , ny , usa ) . when parametric analysis was possible , the student 's t - test for paired data was used for comparing the different approaches for piol calculation and also for comparing preoperative and postoperative data . when parametric analysis was not possible , correlation coefficients ( pearson or spearman depending if normality condition could be assumed ) were used to assess the correlation between different variables . regarding the interchangeability between pairs of methods used for obtaining piol , this is a graphical method for assessing if there is an agreement between two clinical procedures . specifically , bland - altman plots show the differences between the methods plotted against the mean of the 2 methods . the limits of agreement ( loa ) are defined as the mean 1.96 standard deviation ( sd ) of the differences . if the limits are clinically relevant , the 2 methods can not be used interchangeably . in the current study , differences in iol power between the different formulas evaluated was considered as clinically relevant for values of more than 0.5 d because this value is the iol power step provided currently by most of manufacturers and has been shown to be the optical neutralization tolerance since many years ago . a multiple regression analysis was used for predicting the elpadj from different preoperative anatomical and clinical parameters . model assumptions were evaluated by analyzing residuals , the normality of nonstandardized residuals ( homoscedasticity ) , and the cook distance to detect influential points or outliers . in addition , the lack of correlation between errors and multicollinearity was assessed using the durbin watson test , the calculation of the colinearity tolerance , and the variance inflation factor . this study evaluated 25 eyes of 14 patients ( 16 men [ 64% ] ) , with a mean age of 65.9 years 8.9 ( sd ) ( range , 5279 years ) . mean preoperative keratometry , al and anterior chamber depth ( acd ) were 43.29 d 1.45 ( range , 40.9145.89 d ) , 23.21 mm 0.89 mm ( range , 21.6525.04 mm ) , and 3.27 mm 0.30 mm ( range , 2.633.84 mm ) , respectively . according to all these data and using the srk - t formula , mean iol power implanted was 22.53 d 2.70 ( sd ) ( range , 1628 d ) . table 1 summarizes the preoperative and postoperative visual and refractive data , and table 2 displays the biometric and iol power calculation data of the eyes evaluated . the corresponding p values for the comparison between the preoperative and postoperative data are shown for each parameter evaluated mean biometric and iol power calculation data statistically significant differences were found between pioladjsrk / t and piolreal when elp was calculated with the srk / t formula guidelines and rdes = eepost ( p < 0.01 , paired student 's t - test ) . a very strong and statistically significant correlation was found between pioladj - srk / t and piolreal ( r = 0.960 , p < 0.01 ) [ fig . 1 ] . according to the bland and altman method , the pioladj - srk / t was higher than piolreal ( mean of differences 1.97 d ) , with clinically relevant loa ( 3.39 and 0.36 d ) . scattergram showing the relationship between the adjusted intraocular lenses ( iol ) power using the effective lens position estimated using the srk - t formula guidelines ( pioladj - srk / t ) and the real power of the iol implanted ( piolreal ) bland altman plots for the comparison between the adjusted intraocular lenses ( iol ) power using the effective lens position estimated using the srk - t formula guidelines ( pioladj - srk / t ) and the real power of the iol implanted ( piolreal ) . the dotted lines show the limits of agreement ( 1.96 sd ) the multiple regression analysis revealed that the elpadj was significantly correlated with al , acd , pkadj and age ( p < 0.001 ) : the homoscedasticity of the model was confirmed by the normality of the nonstandardized residuals distribution ( p = 0.20 ) and the absence of influential points or outliers ( mean cook 's distance : 0.049 0.081 ) . with this model , the poor correlation between residuals ( durbin - watson test : 2.165 ) and the lack of multicollinearity ( tolerance 0.4860.992 ; variance inflation factors 2.0561.008 ) was also confirmed . a statistically significant difference was found between elp calculated with the srk / t formula guidelines and the elpadj ( p < 0.01 , paired student 's t - test ) , with the lowest value for the adjusted calculation [ table 1 ] . no statistically significant differences were found between pioladj and piolreal when elpadj was used and rdes = sepost were considered for pioladj calculation ( p = 0.10 , paired student 's t - test ) . a very strong and statistically significant correlation was found between pioladj and piolreal ( r = 0.97 , p < 0.01 ) [ fig . 3 ] . according to the bland and altman method , the mean difference between both pioladj and piolreal was 0.002 d , with loa of 1.229 and 1.225 d. fig . scattergram showing the relationship between the adjusted intraocular lenses ( iol ) power using the regression analysis adjusted effective lens position ( pioladj ) and the real power of the iol implanted ( piolreal ) bland altman plots for the comparison between the adjusted intraocular lenses ( iol ) power using the regression analysis adjusted effective lens position ( pioladj ) and the real power of the iol implanted ( piolreal ) . the dotted lines show the limits of agreement ( 1.96 sd ) statistically significant differences were found between pioladj and each of the formulas studied ( p < 0.01 , paired student 's t - test ) . a very strong and statistically significant correlation was found between piolhaigis and pioladj ( r = 0.983 , p < 0.01 ) , between piolhofferq and pioladj ( r = 0.992 , p < 0.01 ) and between piolholladay and pioladj ( r = 0.987 , p < 0.01 ) . table 3 shows the bland and altman analysis outcomes corresponding to all comparisons done . furthermore , the elpadj ( mean sd : 4.18 0.27 mm , range 3.704.83 mm ) was significantly lower than the elp obtained following the guidelines proposed by each of the formulas used ( paired student 's t - test , p < 0.01 ) [ table 1 ] . bland and altman analysis outcomes of the comparison between pioladj and the iol power obtained with other commonly used formulas statistically significant differences were found between pioladjsrk / t and piolreal when elp was calculated with the srk / t formula guidelines and rdes = eepost ( p < 0.01 , paired student 's t - test ) . a very strong and statistically significant correlation was found between pioladj - srk / t and piolreal ( r = 0.960 , p < 0.01 ) [ fig . 1 ] . according to the bland and altman method , the pioladj - srk / t was higher than piolreal ( mean of differences 1.97 d ) , with clinically relevant loa ( 3.39 and 0.36 d ) . scattergram showing the relationship between the adjusted intraocular lenses ( iol ) power using the effective lens position estimated using the srk - t formula guidelines ( pioladj - srk / t ) and the real power of the iol implanted ( piolreal ) bland altman plots for the comparison between the adjusted intraocular lenses ( iol ) power using the effective lens position estimated using the srk - t formula guidelines ( pioladj - srk / t ) and the real power of the iol implanted ( piolreal ) . the multiple regression analysis revealed that the elpadj was significantly correlated with al , acd , pkadj and age ( p < 0.001 ) : the homoscedasticity of the model was confirmed by the normality of the nonstandardized residuals distribution ( p = 0.20 ) and the absence of influential points or outliers ( mean cook 's distance : 0.049 0.081 ) . with this model , the poor correlation between residuals ( durbin - watson test : 2.165 ) and the lack of multicollinearity ( tolerance 0.4860.992 ; variance inflation factors 2.0561.008 ) was also confirmed . a statistically significant difference was found between elp calculated with the srk / t formula guidelines and the elpadj ( p < 0.01 , paired student 's t - test ) , with the lowest value for the adjusted calculation [ table 1 ] . no statistically significant differences were found between pioladj and piolreal when elpadj was used and rdes = sepost were considered for pioladj calculation ( p = 0.10 , paired student 's t - test ) . a very strong and statistically significant correlation was found between pioladj and piolreal ( r = 0.97 , p < 0.01 ) [ fig . 3 ] . according to the bland and altman method , the mean difference between both pioladj and piolreal was 0.002 d , with loa of 1.229 and 1.225 d. fig . scattergram showing the relationship between the adjusted intraocular lenses ( iol ) power using the regression analysis adjusted effective lens position ( pioladj ) and the real power of the iol implanted ( piolreal ) bland altman plots for the comparison between the adjusted intraocular lenses ( iol ) power using the regression analysis adjusted effective lens position ( pioladj ) and the real power of the iol implanted ( piolreal ) . statistically significant differences were found between pioladj and each of the formulas studied ( p < 0.01 , paired student 's t - test ) . a very strong and statistically significant correlation was found between piolhaigis and pioladj ( r = 0.983 , p < 0.01 ) , between piolhofferq and pioladj ( r = 0.992 , p < 0.01 ) and between piolholladay and pioladj ( r = 0.987 , p < 0.01 ) . table 3 shows the bland and altman analysis outcomes corresponding to all comparisons done . furthermore , the elpadj ( mean sd : 4.18 0.27 mm , range 3.704.83 mm ) was significantly lower than the elp obtained following the guidelines proposed by each of the formulas used ( paired student 's t - test , p < 0.01 ) [ table 1 ] . bland and altman analysis outcomes of the comparison between pioladj and the iol power obtained with other commonly used formulas currently , a great variety of options are available for the correction of presbyopia , such as the replacement of the transparent crystalline lens by an accommodating iol that theoretically provide a restoration of the visual function not only at distance , but also at intermediate and near . however , the various preliminary models of accommodating iols were found to provide limited near visual outcomes and the results with the new generation of accommodating iols are not completely successful . beiko concluded from a comparative study that the single - optic accommodating iols , such as crystalens hd and tetraflex , did not offer a significant advantage in near visual acuity over mini - monovision with a monofocal iol . zamora - alejo et al . concluded in another comparative study that the crystalens hd was able to provide some benefit for intermediate visual function compared to a monofocal iol . likewise , ali et al . compared this iol with a standard monofocal iol and concluded that the intraocular optical quality achieved with this iol was similar to that obtained with a conventional monofocal iol . however , the refractive predictability was observed to be limited in some cases showing an unexpected postoperative myopic or hyperopic postoperative refractive error . in our study , the postoperative se ranged from 3.13 to + 1.14 d , which confirms the presence of a significant variability with a trend to postoperative myopia . according to all this evidence , some optimizations seem to be necessary in the calculation of the power required to be implanted with this accommodating iol . possible sources of error in the calculation of this accommodation iol might be the bias introduced by considering the corneal power assuming the keratometric error , errors in the determination of the al or inaccuracy in the estimation of the elp for this specific iol . first , the potential impact of the keratometric error was analyzed by calculating the corneal power using an adjusted keratometric index aimed at minimizing the clinical error in the estimation of the corneal power . however , we still obtained statistically significant and clinically relevant differences between the adjusted calculation , and the real power of the iol implanted that was selected according to the srk - t formula outcomes . as the accuracy of the iol - master for obtaining al measurements has been widely demonstrated , the elp was thought to be a critical factor for the presence of a relatively limited predictability with the accommodating iol evaluated . for such purpose , an expression for estimating an optimized elp according to some preoperative parameters , designated as adjusted elp , the iol power calculation was performed considering this adjusted elp and the results were compared to those obtained with other predicting algorithms of elp . this analysis revealed that the elpadj was significantly lower compared to the values estimated with the commonly used formulas . one of the main factors that may account for this finding is the potentially more anterior position of the optic of the evaluated accommodating iol due to the flexible haptics . indeed , considering equation 1 , a longer elp would lead to the calculation of a higher value of iol power that may potentially lead to the presence of postoperative myopia . indeed , when the calculation of iol power was done correcting the keratometric power and also assuming the elpadj value , no statistically significant differences were found between the implanted and the estimated iol power . in contrast , significant differences in iol power were observed with the other commonly used formulas , haigis , hofferq and holladay , which used significantly higher values of elp . regarding the clinical interchangeability of piolreal and pioladj , a range of agreement of 1.23 d was found which is limited considering that the evaluated iol is available in half diopter steps . this confirms that although a potential more anterior position of the iol may contribute to elp errors with the accommodating iol evaluated , some positional instability of this iol within the capsular bag could also influence on them . this is consistent with the results of some ultrasonographic studies revealing the presence of unexpected positions with this type of accommodating iol . finally , elpadj was found to be related to some factors , such as the al , the adjusted keratometric corneal power ( pkadj ) , the acd and age . specifically , the longer the eye , the higher was the elpadj . this is consistent with previous outcomes reported by other authors such as olsen et al . who found that short eyes tended to have a shallow anterior chamber postoperatively and vice versa . these authors also found that myopic eyes with a large capsular bag showed less iol movement postoperatively . however , not only anatomical parameters influenced on elp ; age was also found to be an influencing factor . similarly , other authors have reported a similar finding for another model of accommodating iol . the interaction between capsular bag fusion and the fibrotic reaction following iol implantation that leads to capsular bag shrinkage seems to be the main factor accounting for this . there are several limitations in the current research , such as the limited sample size or the short follow - up . it should be considered that , although rare , changes in iol position has been described more than 3 months after surgery , especially after nd : yag capsulotomy . as previously mentioned , the crystalens hd iol has a central bi - aspheric optical modification generating theoretically some level of negative spherical aberration and therefore contributing to the increase of the depth of focus . this may have led to some degree of myopia under pupillary constriction and therefore to some bias in the estimation of the refraction . however , it should be considered that small levels of intraocular primary spherical aberration have been reported with this accommodating iol , and of positive sign in some cases . residual myopic refractive errors of more than 0.50 d can not be attributed to these limited levels of spherical aberration . furthermore , there were several cases with clinically significant hyperopic residual refractive errors , not only myopic . another factor that may have contributed to some variability and bias in the estimation of refraction would be the presence of iol tilts or decentration leading to a degradation of the visual quality and therefore limiting the accuracy of manifest refraction . some authors have reported cases of misalignment , tilting or bad positioning with previous models of the evaluated accommodating iol leading to significant levels of visual deterioration . in our sample , iol misalignment and tilt were not observed in the slitlamp examination , but a more detailed analysis with advanced imaging techniques , such as optical coherence tomography or ultrasonography would be recommendable . future studies should be conducted to evaluate the position adopted by this iol into the capsular bag and how it is relation to limitation in the precision of the refractive correction . finally , it must be acknowledged as an additional limitation that intermediate visual acuity was not recorded in the current series . refractive outcomes after cataract surgery with implantation of the accommodating iol crystalens hd can be optimized by minimizing the keratometric error using a variable keratometric index for corneal power estimation and by estimating elp using a mathematical expression dependent on anatomical factors and age . the correction only of the error associated with the keratometric estimation of the corneal power using a variable refractive index does not improve significantly the refractive precision achieved with the accommodating iol evaluated . future studies should be performed to validate this model of iol power calculation for the crystalens hd iol with larger sample of sizes including more extreme cases ( long and short ) .

purpose : to evaluate the predictability of the refractive correction achieved with a positional accommodating intraocular lenses ( iol ) and to develop a potential optimization of it by minimizing the error associated with the keratometric estimation of the corneal power and by developing a predictive formula for the effective lens position ( elp).materials and methods : clinical data from 25 eyes of 14 patients ( age range , 5277 years ) and undergoing cataract surgery with implantation of the accommodating iol crystalens hd ( bausch and lomb ) were retrospectively reviewed . in all cases , the calculation of an adjusted iol power ( pioladj ) based on gaussian optics considering the residual refractive error was done using a variable keratometric index value ( nkadj ) for corneal power estimation with and without using an estimation algorithm for elp obtained by multiple regression analysis ( elpadj ) . pioladj was compared to the real iol power implanted ( piolreal , calculated with the srk - t formula ) and also to the values estimated by the haigis , hofferq , and holladay i formulas.results:no statistically significant differences were found between piolreal and pioladj when elpadj was used ( p = 0.10 ) , with a range of agreement between calculations of 1.23 d. in contrast , piolreal was significantly higher when compared to pioladj without using elpadj and also compared to the values estimated by the other formulas.conclusions:predictable refractive outcomes can be obtained with the accommodating iol crystalens hd using a variable keratometric index for corneal power estimation and by estimating elp with an algorithm dependent on anatomical factors and age .

Materials and Methods Patients Intraocular lens Surgical technique Calculation of the adjusted IOL power to minimize the keratometric error Estimation of adjusted ELP Preoperative and postoperative examinations Statistical analysis Results Agreement of P Estimation of ELP Agreement between P Agreement of P Discussion Conclusion

this retrospective study included a total of 25 eyes of 14 patients with ages ranging between 52 and 77 years old . all these eyes underwent cataract surgery with implantation of the accommodating iol crystalens hd ( bausch and lomb ) . the accommodating iol used in this study was the crystalens hd ( bausch and lomb ) , which has a biconvex single - optic design . two sizes are available depending on the required power , the 12.0 mm model ( hd520 ) for powers between 10.00 and 16.50 d , and the 11.5 mm model ( hd500 ) for powers between 17.00 and 33.00 d. according to the manufacturer , the iol has a double mechanism to improve the near visual function : axial movement of the optic as a consequence of the ciliary muscle changes and variation of the radius of curvature of the anterior iol surface ( arching optic ) . in the current study , the srk / t formula and the iol master software ( carl zeiss meditec , jena , germany ) were used in all cases for the iol power calculation , with an a - constant value of 118.8 . in all cases , topical anesthesia was administered , and pupillary dilation was induced with a combination of tropicamide and phenylephrine 10% every 15 min h prior to the procedure . almost all theoretical formulas for iol power calculation are based on the use of a simplified eye model , with a thin cornea and crystalline lens model . according to such approach , the power of the iol ( piol ) can be easily calculated using the gauss equation in paraxial optics : where pc is the total corneal power , elp the effective lens plane , al the axial length ( al ) , nha the aqueous humor refractive index , nhv the vitreous humor refractive index and rdes is the postoperative desired refraction calculated at corneal vertex . our research group has recently proposed the use of a variable keratometric index ( nkadj ) depending on the radius of the anterior corneal surface ( r1c ) expressed in millimeters for minimizing the error associated to the keratometric approach for corneal power calculation . specifically , the following expression was defined according to the gullstrand eye model : using this algorithm , a new keratometric corneal power , named adjusted keratometric corneal power ( pkadj ) , can be calculated using the classical keratometric corneal power formula . in the current study , the adjusted iol power ( pioladj ) was calculated , defined as the iol power calculated from the equation 1 using the nkadj value for the estimation of the corneal power ( pkadj ) , the nha and nhv values corresponding to the gullstrand eye model ( 1.336 for both index ) . afterward , this iol power ( pioladj ) was compared with the real power of the iol implanted ( piolreal ) . the pioladj calculation was performed after estimating the elp using two different approaches : elp calculation following the srk / t formula guidelines ( named pioladjsrk / t ) and elp calculation using a mathematical expression obtained by multiple regression analysis ( named pioladj ) , as explained carefully in the next section . furthermore , the piol was also calculated using three conventional formulae ( haigis , hofferq and holladay i ) considering the elp defined for each formula and that rdes = sepost . considering equation 1 , piolreal , pkadj and a multiple regression analysis was performed with the aim of obtaining a mathematic expression for predicting the elpadj from different anatomical and clinical parameters . preoperatively , all patients had a full ophthalmologic examination including the evaluation of the refractive status , the distance and near visual acuities , slit lamp examination , optical biometry ( iol - master , carl zeiss meditec , jena , germany ) , goldman tonometry and funduscopy . in addition , the lack of correlation between errors and multicollinearity was assessed using the durbin watson test , the calculation of the colinearity tolerance , and the variance inflation factor . this retrospective study included a total of 25 eyes of 14 patients with ages ranging between 52 and 77 years old . all these eyes underwent cataract surgery with implantation of the accommodating iol crystalens hd ( bausch and lomb ) . the accommodating iol used in this study was the crystalens hd ( bausch and lomb ) , which has a biconvex single - optic design . two sizes are available depending on the required power , the 12.0 mm model ( hd520 ) for powers between 10.00 and 16.50 d , and the 11.5 mm model ( hd500 ) for powers between 17.00 and 33.00 d. according to the manufacturer , the iol has a double mechanism to improve the near visual function : axial movement of the optic as a consequence of the ciliary muscle changes and variation of the radius of curvature of the anterior iol surface ( arching optic ) . in the current study , the srk / t formula and the iol master software ( carl zeiss meditec , jena , germany ) were used in all cases for the iol power calculation , with an a - constant value of 118.8 . in all cases , topical anesthesia was administered , and pupillary dilation was induced with a combination of tropicamide and phenylephrine 10% every 15 min h prior to the procedure . according to such approach , the power of the iol ( piol ) can be easily calculated using the gauss equation in paraxial optics : where pc is the total corneal power , elp the effective lens plane , al the axial length ( al ) , nha the aqueous humor refractive index , nhv the vitreous humor refractive index and rdes is the postoperative desired refraction calculated at corneal vertex . our research group has recently proposed the use of a variable keratometric index ( nkadj ) depending on the radius of the anterior corneal surface ( r1c ) expressed in millimeters for minimizing the error associated to the keratometric approach for corneal power calculation . specifically , the following expression was defined according to the gullstrand eye model : using this algorithm , a new keratometric corneal power , named adjusted keratometric corneal power ( pkadj ) , can be calculated using the classical keratometric corneal power formula . in the current study , the adjusted iol power ( pioladj ) was calculated , defined as the iol power calculated from the equation 1 using the nkadj value for the estimation of the corneal power ( pkadj ) , the nha and nhv values corresponding to the gullstrand eye model ( 1.336 for both index ) . afterward , this iol power ( pioladj ) was compared with the real power of the iol implanted ( piolreal ) . the pioladj calculation was performed after estimating the elp using two different approaches : elp calculation following the srk / t formula guidelines ( named pioladjsrk / t ) and elp calculation using a mathematical expression obtained by multiple regression analysis ( named pioladj ) , as explained carefully in the next section . furthermore , the piol was also calculated using three conventional formulae ( haigis , hofferq and holladay i ) considering the elp defined for each formula and that rdes = sepost . preoperatively , all patients had a full ophthalmologic examination including the evaluation of the refractive status , the distance and near visual acuities , slit lamp examination , optical biometry ( iol - master , carl zeiss meditec , jena , germany ) , goldman tonometry and funduscopy . in addition , the lack of correlation between errors and multicollinearity was assessed using the durbin watson test , the calculation of the colinearity tolerance , and the variance inflation factor . this study evaluated 25 eyes of 14 patients ( 16 men [ 64% ] ) , with a mean age of 65.9 years 8.9 ( sd ) ( range , 5279 years ) . according to all these data and using the srk - t formula , mean iol power implanted was 22.53 d 2.70 ( sd ) ( range , 1628 d ) . the corresponding p values for the comparison between the preoperative and postoperative data are shown for each parameter evaluated mean biometric and iol power calculation data statistically significant differences were found between pioladjsrk / t and piolreal when elp was calculated with the srk / t formula guidelines and rdes = eepost ( p < 0.01 , paired student 's t - test ) . according to the bland and altman method , the pioladj - srk / t was higher than piolreal ( mean of differences 1.97 d ) , with clinically relevant loa ( 3.39 and 0.36 d ) . scattergram showing the relationship between the adjusted intraocular lenses ( iol ) power using the effective lens position estimated using the srk - t formula guidelines ( pioladj - srk / t ) and the real power of the iol implanted ( piolreal ) bland altman plots for the comparison between the adjusted intraocular lenses ( iol ) power using the effective lens position estimated using the srk - t formula guidelines ( pioladj - srk / t ) and the real power of the iol implanted ( piolreal ) . the dotted lines show the limits of agreement ( 1.96 sd ) the multiple regression analysis revealed that the elpadj was significantly correlated with al , acd , pkadj and age ( p < 0.001 ) : the homoscedasticity of the model was confirmed by the normality of the nonstandardized residuals distribution ( p = 0.20 ) and the absence of influential points or outliers ( mean cook 's distance : 0.049 0.081 ) . a statistically significant difference was found between elp calculated with the srk / t formula guidelines and the elpadj ( p < 0.01 , paired student 's t - test ) , with the lowest value for the adjusted calculation [ table 1 ] . no statistically significant differences were found between pioladj and piolreal when elpadj was used and rdes = sepost were considered for pioladj calculation ( p = 0.10 , paired student 's t - test ) . according to the bland and altman method , the mean difference between both pioladj and piolreal was 0.002 d , with loa of 1.229 and 1.225 d. fig . scattergram showing the relationship between the adjusted intraocular lenses ( iol ) power using the regression analysis adjusted effective lens position ( pioladj ) and the real power of the iol implanted ( piolreal ) bland altman plots for the comparison between the adjusted intraocular lenses ( iol ) power using the regression analysis adjusted effective lens position ( pioladj ) and the real power of the iol implanted ( piolreal ) . the dotted lines show the limits of agreement ( 1.96 sd ) statistically significant differences were found between pioladj and each of the formulas studied ( p < 0.01 , paired student 's t - test ) . a very strong and statistically significant correlation was found between piolhaigis and pioladj ( r = 0.983 , p < 0.01 ) , between piolhofferq and pioladj ( r = 0.992 , p < 0.01 ) and between piolholladay and pioladj ( r = 0.987 , p < 0.01 ) . furthermore , the elpadj ( mean sd : 4.18 0.27 mm , range 3.704.83 mm ) was significantly lower than the elp obtained following the guidelines proposed by each of the formulas used ( paired student 's t - test , p < 0.01 ) [ table 1 ] . bland and altman analysis outcomes of the comparison between pioladj and the iol power obtained with other commonly used formulas statistically significant differences were found between pioladjsrk / t and piolreal when elp was calculated with the srk / t formula guidelines and rdes = eepost ( p < 0.01 , paired student 's t - test ) . scattergram showing the relationship between the adjusted intraocular lenses ( iol ) power using the effective lens position estimated using the srk - t formula guidelines ( pioladj - srk / t ) and the real power of the iol implanted ( piolreal ) bland altman plots for the comparison between the adjusted intraocular lenses ( iol ) power using the effective lens position estimated using the srk - t formula guidelines ( pioladj - srk / t ) and the real power of the iol implanted ( piolreal ) . the multiple regression analysis revealed that the elpadj was significantly correlated with al , acd , pkadj and age ( p < 0.001 ) : the homoscedasticity of the model was confirmed by the normality of the nonstandardized residuals distribution ( p = 0.20 ) and the absence of influential points or outliers ( mean cook 's distance : 0.049 0.081 ) . a statistically significant difference was found between elp calculated with the srk / t formula guidelines and the elpadj ( p < 0.01 , paired student 's t - test ) , with the lowest value for the adjusted calculation [ table 1 ] . no statistically significant differences were found between pioladj and piolreal when elpadj was used and rdes = sepost were considered for pioladj calculation ( p = 0.10 , paired student 's t - test ) . according to the bland and altman method , the mean difference between both pioladj and piolreal was 0.002 d , with loa of 1.229 and 1.225 d. fig . scattergram showing the relationship between the adjusted intraocular lenses ( iol ) power using the regression analysis adjusted effective lens position ( pioladj ) and the real power of the iol implanted ( piolreal ) bland altman plots for the comparison between the adjusted intraocular lenses ( iol ) power using the regression analysis adjusted effective lens position ( pioladj ) and the real power of the iol implanted ( piolreal ) . statistically significant differences were found between pioladj and each of the formulas studied ( p < 0.01 , paired student 's t - test ) . a very strong and statistically significant correlation was found between piolhaigis and pioladj ( r = 0.983 , p < 0.01 ) , between piolhofferq and pioladj ( r = 0.992 , p < 0.01 ) and between piolholladay and pioladj ( r = 0.987 , p < 0.01 ) . furthermore , the elpadj ( mean sd : 4.18 0.27 mm , range 3.704.83 mm ) was significantly lower than the elp obtained following the guidelines proposed by each of the formulas used ( paired student 's t - test , p < 0.01 ) [ table 1 ] . bland and altman analysis outcomes of the comparison between pioladj and the iol power obtained with other commonly used formulas currently , a great variety of options are available for the correction of presbyopia , such as the replacement of the transparent crystalline lens by an accommodating iol that theoretically provide a restoration of the visual function not only at distance , but also at intermediate and near . according to all this evidence , some optimizations seem to be necessary in the calculation of the power required to be implanted with this accommodating iol . possible sources of error in the calculation of this accommodation iol might be the bias introduced by considering the corneal power assuming the keratometric error , errors in the determination of the al or inaccuracy in the estimation of the elp for this specific iol . first , the potential impact of the keratometric error was analyzed by calculating the corneal power using an adjusted keratometric index aimed at minimizing the clinical error in the estimation of the corneal power . however , we still obtained statistically significant and clinically relevant differences between the adjusted calculation , and the real power of the iol implanted that was selected according to the srk - t formula outcomes . as the accuracy of the iol - master for obtaining al measurements has been widely demonstrated , the elp was thought to be a critical factor for the presence of a relatively limited predictability with the accommodating iol evaluated . for such purpose , an expression for estimating an optimized elp according to some preoperative parameters , designated as adjusted elp , the iol power calculation was performed considering this adjusted elp and the results were compared to those obtained with other predicting algorithms of elp . this analysis revealed that the elpadj was significantly lower compared to the values estimated with the commonly used formulas . indeed , considering equation 1 , a longer elp would lead to the calculation of a higher value of iol power that may potentially lead to the presence of postoperative myopia . indeed , when the calculation of iol power was done correcting the keratometric power and also assuming the elpadj value , no statistically significant differences were found between the implanted and the estimated iol power . in contrast , significant differences in iol power were observed with the other commonly used formulas , haigis , hofferq and holladay , which used significantly higher values of elp . regarding the clinical interchangeability of piolreal and pioladj , a range of agreement of 1.23 d was found which is limited considering that the evaluated iol is available in half diopter steps . this confirms that although a potential more anterior position of the iol may contribute to elp errors with the accommodating iol evaluated , some positional instability of this iol within the capsular bag could also influence on them . finally , elpadj was found to be related to some factors , such as the al , the adjusted keratometric corneal power ( pkadj ) , the acd and age . as previously mentioned , the crystalens hd iol has a central bi - aspheric optical modification generating theoretically some level of negative spherical aberration and therefore contributing to the increase of the depth of focus . future studies should be conducted to evaluate the position adopted by this iol into the capsular bag and how it is relation to limitation in the precision of the refractive correction . refractive outcomes after cataract surgery with implantation of the accommodating iol crystalens hd can be optimized by minimizing the keratometric error using a variable keratometric index for corneal power estimation and by estimating elp using a mathematical expression dependent on anatomical factors and age . the correction only of the error associated with the keratometric estimation of the corneal power using a variable refractive index does not improve significantly the refractive precision achieved with the accommodating iol evaluated .

radiocarbon dating is a chronological method which uses radioisotope carbon-14 ( c ) to investigate the ages of carbonaceous samples [ 1 , 2 ] . c atom forms in the nuclear reaction:\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ n + { } _ { 7}^{14 } { \text{n}}^{1 + } \to { } _ { 6}^{14 } { \text{c } } + p $ $ \end{document}where p is the proton , n is the atom of nitrogen , n is the neutron resulting from the cosmic ray . then c spreads throughout the atmosphere and forms carbon dioxide ( co2 ) when it reacts with oxygen . when sampled , the carbonaceous materials become clocks due to the decaying c in them . since the half - life of c is moderate , i.e. 5568 30 or 5730 40 years , grosse and libby at the university of chicago developed the radiocarbon dating method . to achieve high accuracy calendar dates by this method , scientists had advanced several calibration methods , including tree rings , annually laminated sediments , corals [ 7 , 8 ] , intcal98 , intcal04 , and intcal09 . the calculations of such calibrations are based on the conventional formula , which was given by stuiver and polach in 1977 . in this formula , the correction of the isotopic fractionation plays a significant role . the correction is that the difference in c , normalized to the postulated mean value , i.e. 25 per mil with respect to pdb , of terrestrial wood , simply indicates a double change of that in c within the carbonaceous matters . however , neither might this quantitative relation always be true for specific processes such as chemical processes in which the factor is 1.9 [ 11 , 12 ] , nor does the conventional formula itself include the effect of temperature , at which the sample formed , on the fractionation correction . the following section tries to relate the equilibrium isotopic fractionation correction , depending on the temperature , to the radiocarbon dating method . and the derivations in present paper are based on the postulate : samples are well preserved without chemical exchange after they formed . does not use libby s first assumption because published works shows the variability of c in the atmosphere [ 1117 ] . this variability is due to the varying intensities of cosmic radiation with time , and the release of c and c into the atmosphere by , for example , burning of fossil ( known as suess effect [ 1921 ] ) and explosion of nuclear bombs , respectively . as mentioned above , co2 in the atmosphere is the significant medium to preserve c into the carbonaceous matter , xcn , where x stands for the stoichiometry of all atoms except carbon ( c ) in the sample , and n for the number of c in it . this process involves an isotope reaction [ 23 , 24]:1\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { } ^{14}{\text{co}}_{2 } + \frac{1}{n}x{}^{12}{\text{c}}_{n } \overset k \rightleftharpoons { } ^{12}{\text{co}}_{2 } + \frac{1}{n}x{}^{14}{\text{c}}_{n } $ $ \end{document}where k is the equilibrium constant , depending at most on the temperature t . incorporation of c into the carbonaceous matter xcn , assuming no further exchange , starts the radiocarbon clock . the number of c in xcn at t = 0 , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ n_{{x{\text{c}}_{n } , 0}}^{{{}^{14}{\text{c } } } } $ $ \end{document } , and that at present t = t , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ n_{{x{\text{c}}_{n } , t}}^{{{}^{14}{\text{c } } } } $ $ \end{document } , follow an exponential decay:2\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ n_{{x{\text{c}}_{n } , t}}^{{{}^{14}{\text{c } } } } = n_{{x{\text{c}}_{n } , 0}}^{{{}^{14}{\text{c } } } } \begin{array}{*{20}c } { { \text{e}}^ { - \lambda t } } \\ \end{array } $ $ \end{document}where 1/ = 8033 year ( libby value ) is the mean life of c. now let \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ n_{{x{\text{c}}_{n}}}^{{{}^{12}{\text{c } } } } $ $ \end{document } denote the number of c in xcn . division of eq . 2 by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ n_{{x{\text{c}}_{n}}}^{{{}^{12}{\text{c } } } } $ $ \end{document } gives3\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \frac{{n_{{x{\text{c}}_{n } , t}}^{{{}^{14}{\text{c } } } } } } { { n_{{x{\text{c}}_{n } } } ^{{{}^{12}{\text{c } } } } } } = \frac{{n_{{x{\text{c}}_{n } , 0}}^{{{}^{14}{\text{c } } } } } } { { n_{{x{\text{c}}_{n } } } ^{{{}^{12}{\text{c } } } } } } { \text{e}}^ { - \lambda t } $ $ \end{document}since \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ n_{{x{\text{c}}_{n } } } ^{{^{12 } { \text{c } } } } $ $ \end{document } was not altered after the sample formed . in this equation , the left side and the first term of the right side represent the ratio of c / c in the sample at t = t and t = 0 , respectively . the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { { n_{{x{\text{c}}_{n } , 0}}^{{{}^{14}{\text{c } } } } } \mathord{\left/ { \vphantom { { n_{{x{\text{c}}_{n } , 0}}^{{{}^{14}{\text{c } } } } } { n_{{x{\text{c}}_{n } } } ^{{{}^{12}{\text{c } } } } } } } \right . \kern-\nulldelimiterspace } { n_{{x{\text{c}}_{n } } } ^{{{}^{12}{\text{c } } } } } } $ $ \end{document } ratio in eq . 3 can be evaluated by studying the equilibrium constant k of reaction 1 at t = 0 . for reaction 1 , the molality - scale equilibrium constant is 4\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ k = \frac{{\left [ { x{}^{14}{\text{c}}_{n } } \right]^{\frac{1}{n } } \left [ { { } ^{12}{\text{co}}_{2 } } \right]}}{{\left [ { x{}^{12}{\text{c}}_{n } } \right]^{\frac{1}{n } } \left [ { { } ^{14}{\text{co}}_{2 } } \right ] } } = \frac{{\left ( { \frac{{\left [ { x{}^{14}{\text{c}}_{n } } \right]}}{{\left [ { x{}^{12}{\text{c}}_{n } } \right ] } } } \right)^{\frac{1}{n } } } } { { \frac{{\left [ { { } ^{14}{\text{co}}_{2 } } \right]}}{{\left [ { { } ^{12}{\text{co}}_{2 } } \right ] } } } } = \frac{{\frac{{n_{{xc_{n } , 0}}^{{{}^{14}{\text{c } } } } } } { { n_{{x{\text{c}}_{n } } } ^{{{}^{12}{\text{c } } } } } } } } { { \frac{{n_{{{\text{co}}_{2 } , 0}}^{{{}^{14}{\text{c } } } } } } { { n_{{{\text{co}}_{2 } } } ^{{{}^{12}{\text{c } } } } } } } } $ $ \end{document}with the last term expressing the concentration by the number of c and c in those two chemicals : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ n_{{{\text{co}}_{2 } , 0}}^{{{}^{14}{\text{c } } } } $ $ \end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ n_{{{\text{co}}_{2 } } } ^{{{}^{12}{\text{c } } } } $ $ \end{document } are the numbers of c and c in co2 in the atmosphere at t = 0 , respectively . this term is the c / c ratio in xcn divided by that in co2 , and it is just the expression of the fractionation factor , , defined by experimental chemists , giving k = . for theoretical chemists , is the ratio of reduced partition function ratios ( rpfrs ) between those two substances [ 23 , 24 ] . since the factor is unique for a specific process , we have5\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \frac{{\frac{{n_{{x{\text{c}}_{n } , 0}}^{{{}^{14}{\text{c } } } } } } { { n_{{x{\text{c}}_{n } } } ^{{{}^{12}{\text{c } } } } } } } } { { \frac{{n_{{{\text{co}}_{2 } , 0}}^{{{}^{14}{\text{c } } } } } } { { n_{{{\text{co}}_{2 } } } ^{{{}^{12}{\text{c } } } } } } } } = \alpha = \frac{{{\text{rpfr}}_{{x{}^{14/12}{\text{c}}_{n } } } } } { { { \text{rpfr}}_{{{}^{14/12}{\text{co}}_{2 } } } } } $ $ \end{document}or6\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \frac{{n_{{x{\text{c}}_{n } , 0}}^{{{}^{14}{\text{c } } } } } } { { n_{{x{\text{c}}_{n } } } ^{{{}^{12}{\text{c } } } } } } = \frac{{{\text{rpfr}}_{{x{}^{14/12}{\text{c}}_{n } } } } } { { { \text{rpfr}}_{{{}^{14/12}{\text{co}}_{2 } } } } } \frac{{n_{{{\text{co}}_{2 } , 0}}^{{{}^{14}{\text{c } } } } } } { { n_{{{\text{co}}_{2 } } } ^{{{}^{12}{\text{c } } } } } } $ $ \end{document}where7\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \text{rpfr } } = \left ( { \frac{{\sigma^{{{}^{12}{\text{c } } } } } } { { \sigma^{{{}^{14}{\text{c } } } } } } \prod\limits_{k}^{3n - 6(5 ) } { \frac{{u_{k}^{{{}^{14}{\text{c } } } } } } { { u_{k}^{{{}^{12}{\text{c } } } } } } } \frac{{\exp \left ( { - u_{k}^{{{}^{14}{\text{c } } } } /2 } \right)}}{{\exp \left ( { - u_{k}^{{{}^{12}{\text{c } } } } /2 } \right)}}\frac{{1 - \exp \left ( { - u_{k}^{{{}^{12}{\text{c } } } } } \right)}}{{1 - \exp \left ( { - u_{k}^{{{}^{14}{\text{c } } } } } \right ) } } } \right)^{{{1 \mathord{\left/ { \vphantom { 1 n } } \right . \kern-\nulldelimiterspace } n } } } $ $ \end{document}in which is the symmetry number , u = hc/kbt , h is the planck s constant , c is the speed of light , is the frequency in cm , kb is the boltzmann constant , t is the temperature . substitution of eq . 6 into eq . 3 gives8\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \frac{{n_{{x{\text{c}}_{n } , t}}^{{{}^{14}{\text{c } } } } } } { { n_{{x{\text{c}}_{n } } } ^{{{}^{12}{\text{c } } } } } } { \text{e}}^{\lambda t } = \frac{{{\text{rpfr}}_{{x{}^{14/12}{\text{c}}_{n } } } } } { { { \text{rpfr}}_{{{}^{14/12}{\text{co}}_{2 } } } } } \frac{{n_{{{\text{co}}_{2 } , 0}}^{{{}^{14}{\text{c } } } } } } { { n_{{{\text{co}}_{2 } } } ^{{{}^{12}{\text{c } } } } } } $ $ \end{document}use of the natural logarithm to eq . 8 leads to9\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ t = \frac{1}{\lambda } \left ( { \ln \left ( { { \text{rpfr}}_{{x{}^{14/12}{\text{c}}_{n } } } } \right ) - \ln \left ( { { \text{rpfr}}_{{{}^{14/12}{\text{co}}_{2 } } } } \right ) - \ln \left ( { \frac{{n_{{x{\text{c}}_{n } , t}}^{{{}^{14}{\text{c } } } } } } { { n_{{x{\text{c}}_{n } } } ^{{{}^{12}{\text{c } } } } } } } \right ) + \ln \left ( { \frac{{n_{{{\text{co}}_{2 } , 0}}^{{{}^{14}{\text{c } } } } } } { { n_{{{\text{co}}_{2 } } } ^{{{}^{12}{\text{c } } } } } } } \right ) } \right ) $ $ \end{document } this equation shows that the time t the clock recorded is a function of , t , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { { n_{{x{\text{c}}_{n } , t}}^{{{}^{14}{\text{c } } } } } \mathord{\left/ { \vphantom { { n_{{x{\text{c}}_{n } , t}}^{{{}^{14}{\text{c } } } } } { n_{{x{\text{c}}_{n } } } ^{{{}^{12}{\text{c } } } } } } } \right . \kern-\nulldelimiterspace } { n_{{x{\text{c}}_{n } } } ^{{{}^{12}{\text{c } } } } } } $ $ \end{document } , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { { n_{{{\text{co}}_{2 } , 0}}^{{{}^{14}{\text{c } } } } } \mathord{\left/ { \vphantom { { n_{{{\text{co}}_{2 } , 0}}^{{{}^{14}{\text{c } } } } } { n_{{{\text{co}}_{2 } } } ^{{{}^{12}{\text{c } } } } } } } \right . \kern-\nulldelimiterspace } { n_{{{\text{co}}_{2 } } } ^{{{}^{12}{\text{c } } } } } } $ $ \end{document } and other physical constants . the first two terms in the right side of above equation is called the isotopic fractionation correction to the radiocarbon dating method . for a specific sample , the accurate value of and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { { n_{{x{\text{c}}_{n } , t}}^{{{}^{14}{\text{c } } } } } \mathord{\left/ { \vphantom { { n_{{x{\text{c}}_{n } , t}}^{{{}^{14}{\text{c } } } } } { n_{{x{\text{c}}_{n } } } ^{{{}^{12}{\text{c } } } } } } } \right . \kern-\nulldelimiterspace } { n_{{x{\text{c}}_{n } } } ^{{{}^{12}{\text{c } } } } } } $ $ \end{document } in eq . 9 can be directly evaluated by ab initio quantum calculations [ 23 , 24 , 2631 ] and accelerator mass spectrometry ( ams ) technique [ 3242 ] , respectively . the methods to evaluate t and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { { n_{{{\text{co}}_{2 } , 0}}^{{{}^{14}{\text{c } } } } } \mathord{\left/ { \vphantom { { n_{{{\text{co}}_{2 } , 0}}^{{{}^{14}{\text{c } } } } } { n_{{{\text{co}}_{2 } } } ^{{{}^{12}{\text{c } } } } } } } \right . \kern-\nulldelimiterspace } { n_{{{\text{co}}_{2 } } } ^{{{}^{12}{\text{c } } } } } } $ $ \end{document } are illustrated below . 9 is the unique temperature at which the sample formed and recorded . according to eq . 5 , is a function of temperature , and this allow us to find the inverse function of to evaluate t : that is , if = f ( t ) , then we have t = f( ) which gives t if is known . if is , however , given by the theoretical calculations , t locates in an interval of temperatures at which xcn can form [ 2629 ] rather than at a unique temperature the specific sample formed . so we have to experimentally measure the isotope ratios kept in the sample , and then get the unique and , therefore , the unique t. a good example of such case is to determine t by studying the clumped - isotopes in calcite [ 43 , 44 ] . the equilibrium constant k3866 ( or 47 , see definitions and details of experiments in refs . [ 43 , 44 ] ) of clumped - isotopes in calcite can be given as a function of t from 273.15 to 1273.15 k by theoretical predictions , while a specific sample such as calcite ha4 in ref . only formed at 323.15 2(1 ) k with k3866 = 1.00053 0.011(1 ) . therefore , as ref . did , one might use the experimental results of clumped - isotope ratios in the calcite rings of the stalagmite to determine k3866 , and then compare this value to the experimental fitted polynomial , t = f(k3866 ) , to get t . since samples ( formed in reaction 1 ) such as stalagmites and trees are in a single phase , ts recorded by them are suggested to be experimentally given by studying the clumped - isotopes [ 4550 ] kept in them . \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { { n_{{{\text{co}}_{2 } , 0}}^{{{}^{14}{\text{c } } } } } \mathord{\left/ { \vphantom { { n_{{{\text{co}}_{2 } , 0}}^{{{}^{14}{\text{c } } } } } { n_{{{\text{co}}_{2 } } } ^{{{}^{12}{\text{c } } } } } } } \right . \kern-\nulldelimiterspace } { n_{{{\text{co}}_{2 } } } ^{{{}^{12}{\text{c } } } } } } $ $ \end{document } in eq . rewriting eq . 8 , we have10\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \frac{{n_{{{\text{co}}_{2 } , 0}}^{{{}^{14}{\text{c } } } } } } { { n_{{{\text{co}}_{2 } } } ^{{{}^{12}{\text{c } } } } } } = \frac{{{\text{rpfr}}_{{{}^{14/12}{\text{co}}_{2 } } } } } { { { \text{rpfr}}_{{x{}^{14/12}{\text{c}}_{n } } } } } \frac{{n_{{x{\text{c}}_{n } , t}}^{{{}^{14}{\text{c } } } } } } { { n_{{x{\text{c}}_{n } } } ^{{{}^{12}{\text{c } } } } } } { \text{e}}^{\lambda t } $ $ \end{document } in this equation , the left side is the c / c ratio in the atmosphere and the right side shows information from a sample : the isotope fractionation factor of c between co2 and the sample , the ams results of c / c ratio in it , and the time t which can be directly given by examining the number of the growth rings of , for example , a tree . with such information on the samples , eq . 10 gives the ratio of c / c in the atmosphere where the sample formed in the geological time . if the c / c ratio in the past atmosphere were generated in future literature , we shall obtain a new relationship between this c / c ratio and t. then we expect a reasonable reference value ( which plays the role as absolute international standard activity aabs does in the conventional formula ) of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \left ( { { { n_{{{\text{co}}_{2 } , 0}}^{{{}^{14}{\text{c } } } } } \mathord{\left/ { \vphantom { { n_{{{\text{co}}_{2 } , 0}}^{{{}^{14}{\text{c } } } } } { n_{{{\text{co}}_{2 } } } ^{{{}^{12}{\text{c } } } } } } } \right . \kern-\nulldelimiterspace } { n_{{{\text{co}}_{2 } } } ^{{{}^{12}{\text{c } } } } } } } \right)_{\text{ref } } $ $ \end{document } that makes eq this is beyond the ability , and thus a limitation , of this theoretical paper : we can not set the last term in eq . 9 down now . from the above derivations , the conclusions can be drawn as follows : this work introduces us a new dimension , the temperature t , to study radiocarbon dating.the mechanism of isotopic fractionation correction to radiocarbon dating method is clear . this correction , shown in eq . 9 , is a function of and t resulting from studying the equilibrium isotopic reaction 1 . since the fractionation caused by reaction 1 can not happen in the classical mechanism and it can be derived only from quantum mechanism [ 23 , 24 ] , the correction is a quantum - mechanical equilibrium isotopic fractionation correction to radiocarbon dating.one application of this work , as eq . 9 shows , is to explain why so many calibrations , shown in the introduction , are needed for radiocarbon dating . the reasons for the need are : the first comes from the difference of carbonaceous matters . 9 when we suppose a constant t and a fixed c / c ratio in the last term . for example , carbonaceous matters xcn are fiber and calcite in tree rings and corals [ 5 , 51 ] , respectively . these chemicals have different vibrations and therefore different rpfrs at the same t;the second is from the temperature . since between , for example , calcite and carbon dioxide in eq . 9 is generally not a linear function of t [ 2628 ] , t affects t in a non - linear way if the last two terms in eq , the temperature of one ring of calcite differs from that of another;the third is due to the variation of c / c ratio in the atmosphere . if the first three terms in eq . 9 are fixed , then t is a function of the variation of c / c ratio . as published works showed [ 1114 ] , there is no linear function to describe this ratio as a function of t : this ratio affects t in some non - linear way . briefly , these three parameters separately have a non - linear relationship with t.in the nature , the relationship between t and the three parameters is more complex . when reaction 1 happened , these three influence factors might change together with the latitude and the altitude or the depth and in seasonable scale or even hundred - year scale [ 1114 , 5254 ] . consequently , the years given by conventional formula deviate from a straight or linear line and need to be calibrated when high accuracies are required . 9 itself contains more information than the conventional one and , if eq . 9 were used to determine the years of samples , the deviation would be significant small.another application of present work , as eq . 10 shows , is to generate the c / c ratio in the atmosphere in the geological history by studying the examples of which the growth years can be directly examined . this work introduces us a new dimension , the temperature t , to study radiocarbon dating . 9 , is a function of and t resulting from studying the equilibrium isotopic reaction 1 . since the fractionation caused by reaction 1 can not happen in the classical mechanism and it can be derived only from quantum mechanism [ 23 , 24 ] , the correction is a quantum - mechanical equilibrium isotopic fractionation correction to radiocarbon dating . 9 shows , is to explain why so many calibrations , shown in the introduction , are needed for radiocarbon dating . the reasons for the need are : the first comes from the difference of carbonaceous matters . 9 when we suppose a constant t and a fixed c / c ratio in the last term . for example , carbonaceous matters xcn are fiber and calcite in tree rings and corals [ 5 , 51 ] , respectively . these chemicals have different vibrations and therefore different rpfrs at the same t;the second is from the temperature . since between , for example , calcite and carbon dioxide in eq . 9 is generally not a linear function of t [ 2628 ] , t affects t in a non - linear way if the last two terms in eq , the temperature of one ring of calcite differs from that of another;the third is due to the variation of c / c ratio in the atmosphere . if the first three terms in eq . 9 are fixed , then t is a function of the variation of c / c ratio . as published works showed [ 1114 ] , there is no linear function to describe this ratio as a function of t : this ratio affects t in some non - linear way . briefly , these three parameters separately have a non - linear relationship with t. the first comes from the difference of carbonaceous matters . 9 when we suppose a constant t and a fixed c / c ratio in the last term . for example , carbonaceous matters xcn are fiber and calcite in tree rings and corals [ 5 , 51 ] , respectively . these chemicals have different vibrations and therefore different rpfrs at the same t ; the second is from the temperature . since between , for example , calcite and carbon dioxide in eq . 9 is generally not a linear function of t [ 2628 ] , t affects t in a non - linear way if the last two terms in eq . , the temperature of one ring of calcite differs from that of another ; the third is due to the variation of c / c ratio in the atmosphere . if the first three terms in eq . 9 are fixed , then t is a function of the variation of c / c ratio . as published works showed [ 1114 ] , there is no linear function to describe this ratio as a function of t : this ratio affects t in some non - linear way . briefly , these three parameters separately have a non - linear relationship with t. in the nature , the relationship between t and the three parameters is more complex . when reaction 1 happened , these three influence factors might change together with the latitude and the altitude or the depth and in seasonable scale or even hundred - year scale [ 1114 , 5254 ] . consequently , the years given by conventional formula deviate from a straight or linear line and need to be calibrated when high accuracies are required . 9 itself contains more information than the conventional one and , if eq . 9 were used to determine the years of samples , the deviation would be significant small . 10 shows , is to generate the c / c ratio in the atmosphere in the geological history by studying the examples of which the growth years can be directly examined .

this paper relates the quantum mechanical equilibrium isotopic fractionation correction to the radiocarbon dating method by eq . 9 , and also shows the significant influence of temperature on the method . it is suggested that the correction is a function of the frequencies and temperature of a specific sample and these two variables can be evaluated theoretically by the ab initio quantum calculations and experimentally by analyzing the clumped - isotope ratios in it , respectively . this paper also suggests that the 14c/12c ratio in the atmosphere in geological time can be calculated by eq . 10 .

Introduction Assumptions and derivations Conclusions

radiocarbon dating is a chronological method which uses radioisotope carbon-14 ( c ) to investigate the ages of carbonaceous samples [ 1 , 2 ] . 5568 30 or 5730 40 years , grosse and libby at the university of chicago developed the radiocarbon dating method . in this formula , the correction of the isotopic fractionation plays a significant role . the correction is that the difference in c , normalized to the postulated mean value , i.e. however , neither might this quantitative relation always be true for specific processes such as chemical processes in which the factor is 1.9 [ 11 , 12 ] , nor does the conventional formula itself include the effect of temperature , at which the sample formed , on the fractionation correction . the following section tries to relate the equilibrium isotopic fractionation correction , depending on the temperature , to the radiocarbon dating method . does not use libby s first assumption because published works shows the variability of c in the atmosphere [ 1117 ] . this variability is due to the varying intensities of cosmic radiation with time , and the release of c and c into the atmosphere by , for example , burning of fossil ( known as suess effect [ 1921 ] ) and explosion of nuclear bombs , respectively . as mentioned above , co2 in the atmosphere is the significant medium to preserve c into the carbonaceous matter , xcn , where x stands for the stoichiometry of all atoms except carbon ( c ) in the sample , and n for the number of c in it . in this equation , the left side and the first term of the right side represent the ratio of c / c in the sample at t = t and t = 0 , respectively . 3 can be evaluated by studying the equilibrium constant k of reaction 1 at t = 0 . for reaction 1 , the molality - scale equilibrium constant is 4\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ k = \frac{{\left [ { x{}^{14}{\text{c}}_{n } } \right]^{\frac{1}{n } } \left [ { { } ^{12}{\text{co}}_{2 } } \right]}}{{\left [ { x{}^{12}{\text{c}}_{n } } \right]^{\frac{1}{n } } \left [ { { } ^{14}{\text{co}}_{2 } } \right ] } } = \frac{{\left ( { \frac{{\left [ { x{}^{14}{\text{c}}_{n } } \right]}}{{\left [ { x{}^{12}{\text{c}}_{n } } \right ] } } } \right)^{\frac{1}{n } } } } { { \frac{{\left [ { { } ^{14}{\text{co}}_{2 } } \right]}}{{\left [ { { } ^{12}{\text{co}}_{2 } } \right ] } } } } = \frac{{\frac{{n_{{xc_{n } , 0}}^{{{}^{14}{\text{c } } } } } } { { n_{{x{\text{c}}_{n } } } ^{{{}^{12}{\text{c } } } } } } } } { { \frac{{n_{{{\text{co}}_{2 } , 0}}^{{{}^{14}{\text{c } } } } } } { { n_{{{\text{co}}_{2 } } } ^{{{}^{12}{\text{c } } } } } } } } $ $ \end{document}with the last term expressing the concentration by the number of c and c in those two chemicals : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ n_{{{\text{co}}_{2 } , 0}}^{{{}^{14}{\text{c } } } } $ $ \end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ n_{{{\text{co}}_{2 } } } ^{{{}^{12}{\text{c } } } } $ $ \end{document } are the numbers of c and c in co2 in the atmosphere at t = 0 , respectively . this term is the c / c ratio in xcn divided by that in co2 , and it is just the expression of the fractionation factor , , defined by experimental chemists , giving k = . 8 leads to9\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ t = \frac{1}{\lambda } \left ( { \ln \left ( { { \text{rpfr}}_{{x{}^{14/12}{\text{c}}_{n } } } } \right ) - \ln \left ( { { \text{rpfr}}_{{{}^{14/12}{\text{co}}_{2 } } } } \right ) - \ln \left ( { \frac{{n_{{x{\text{c}}_{n } , t}}^{{{}^{14}{\text{c } } } } } } { { n_{{x{\text{c}}_{n } } } ^{{{}^{12}{\text{c } } } } } } } \right ) + \ln \left ( { \frac{{n_{{{\text{co}}_{2 } , 0}}^{{{}^{14}{\text{c } } } } } } { { n_{{{\text{co}}_{2 } } } ^{{{}^{12}{\text{c } } } } } } } \right ) } \right ) $ $ \end{document } this equation shows that the time t the clock recorded is a function of , t , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { { n_{{x{\text{c}}_{n } , t}}^{{{}^{14}{\text{c } } } } } \mathord{\left/ { \vphantom { { n_{{x{\text{c}}_{n } , t}}^{{{}^{14}{\text{c } } } } } { n_{{x{\text{c}}_{n } } } ^{{{}^{12}{\text{c } } } } } } } \right . the first two terms in the right side of above equation is called the isotopic fractionation correction to the radiocarbon dating method . for a specific sample , the accurate value of and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { { n_{{x{\text{c}}_{n } , t}}^{{{}^{14}{\text{c } } } } } \mathord{\left/ { \vphantom { { n_{{x{\text{c}}_{n } , t}}^{{{}^{14}{\text{c } } } } } { n_{{x{\text{c}}_{n } } } ^{{{}^{12}{\text{c } } } } } } } \right . 9 can be directly evaluated by ab initio quantum calculations [ 23 , 24 , 2631 ] and accelerator mass spectrometry ( ams ) technique [ 3242 ] , respectively . 5 , is a function of temperature , and this allow us to find the inverse function of to evaluate t : that is , if = f ( t ) , then we have t = f( ) which gives t if is known . if is , however , given by the theoretical calculations , t locates in an interval of temperatures at which xcn can form [ 2629 ] rather than at a unique temperature the specific sample formed . so we have to experimentally measure the isotope ratios kept in the sample , and then get the unique and , therefore , the unique t. a good example of such case is to determine t by studying the clumped - isotopes in calcite [ 43 , 44 ] . [ 43 , 44 ] ) of clumped - isotopes in calcite can be given as a function of t from 273.15 to 1273.15 k by theoretical predictions , while a specific sample such as calcite ha4 in ref . did , one might use the experimental results of clumped - isotope ratios in the calcite rings of the stalagmite to determine k3866 , and then compare this value to the experimental fitted polynomial , t = f(k3866 ) , to get t . since samples ( formed in reaction 1 ) such as stalagmites and trees are in a single phase , ts recorded by them are suggested to be experimentally given by studying the clumped - isotopes [ 4550 ] kept in them . 8 , we have10\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \frac{{n_{{{\text{co}}_{2 } , 0}}^{{{}^{14}{\text{c } } } } } } { { n_{{{\text{co}}_{2 } } } ^{{{}^{12}{\text{c } } } } } } = \frac{{{\text{rpfr}}_{{{}^{14/12}{\text{co}}_{2 } } } } } { { { \text{rpfr}}_{{x{}^{14/12}{\text{c}}_{n } } } } } \frac{{n_{{x{\text{c}}_{n } , t}}^{{{}^{14}{\text{c } } } } } } { { n_{{x{\text{c}}_{n } } } ^{{{}^{12}{\text{c } } } } } } { \text{e}}^{\lambda t } $ $ \end{document } in this equation , the left side is the c / c ratio in the atmosphere and the right side shows information from a sample : the isotope fractionation factor of c between co2 and the sample , the ams results of c / c ratio in it , and the time t which can be directly given by examining the number of the growth rings of , for example , a tree . 10 gives the ratio of c / c in the atmosphere where the sample formed in the geological time . if the c / c ratio in the past atmosphere were generated in future literature , we shall obtain a new relationship between this c / c ratio and t. then we expect a reasonable reference value ( which plays the role as absolute international standard activity aabs does in the conventional formula ) of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \left ( { { { n_{{{\text{co}}_{2 } , 0}}^{{{}^{14}{\text{c } } } } } \mathord{\left/ { \vphantom { { n_{{{\text{co}}_{2 } , 0}}^{{{}^{14}{\text{c } } } } } { n_{{{\text{co}}_{2 } } } ^{{{}^{12}{\text{c } } } } } } } \right . from the above derivations , the conclusions can be drawn as follows : this work introduces us a new dimension , the temperature t , to study radiocarbon dating.the mechanism of isotopic fractionation correction to radiocarbon dating method is clear . 9 , is a function of and t resulting from studying the equilibrium isotopic reaction 1 . since the fractionation caused by reaction 1 can not happen in the classical mechanism and it can be derived only from quantum mechanism [ 23 , 24 ] , the correction is a quantum - mechanical equilibrium isotopic fractionation correction to radiocarbon dating.one application of this work , as eq . 9 shows , is to explain why so many calibrations , shown in the introduction , are needed for radiocarbon dating . 9 when we suppose a constant t and a fixed c / c ratio in the last term . 9 is generally not a linear function of t [ 2628 ] , t affects t in a non - linear way if the last two terms in eq , the temperature of one ring of calcite differs from that of another;the third is due to the variation of c / c ratio in the atmosphere . 9 are fixed , then t is a function of the variation of c / c ratio . as published works showed [ 1114 ] , there is no linear function to describe this ratio as a function of t : this ratio affects t in some non - linear way . 10 shows , is to generate the c / c ratio in the atmosphere in the geological history by studying the examples of which the growth years can be directly examined . 9 , is a function of and t resulting from studying the equilibrium isotopic reaction 1 . since the fractionation caused by reaction 1 can not happen in the classical mechanism and it can be derived only from quantum mechanism [ 23 , 24 ] , the correction is a quantum - mechanical equilibrium isotopic fractionation correction to radiocarbon dating . 9 shows , is to explain why so many calibrations , shown in the introduction , are needed for radiocarbon dating . 9 is generally not a linear function of t [ 2628 ] , t affects t in a non - linear way if the last two terms in eq , the temperature of one ring of calcite differs from that of another;the third is due to the variation of c / c ratio in the atmosphere . 9 are fixed , then t is a function of the variation of c / c ratio . as published works showed [ 1114 ] , there is no linear function to describe this ratio as a function of t : this ratio affects t in some non - linear way . , the temperature of one ring of calcite differs from that of another ; the third is due to the variation of c / c ratio in the atmosphere . 9 are fixed , then t is a function of the variation of c / c ratio . as published works showed [ 1114 ] , there is no linear function to describe this ratio as a function of t : this ratio affects t in some non - linear way . 10 shows , is to generate the c / c ratio in the atmosphere in the geological history by studying the examples of which the growth years can be directly examined .

acute liver failure , frequently results from hepatitis virus infection , induction of drugs and toxins , or hepatic ischemia - reperfusion injury , is an important dramatic clinical syndrome . liver oxidative stress and inflammation are significant determinants in the physiopathology of acute liver failure . pro - inflammatory cytokines and reactive oxygen species have key roles in the mentioned liver failure ( 1 ) . additionally , cytokines are one of the most important regulators of host responses to infection , immune responses , and inflammation . some cytokines make the disease worse ( pro - inflammatory ) , while others act to reduce inflammation ( anti - inflammatory ) . the well - known examples of pro - inflammatory cytokines are interleukin-6 ( il-6 ) and tumor necrosis factor ( tnf ) ( 2 ) . on the other hand , oxidative stress is described as a disturbance in the balance between the production of reactive oxygen species and antioxidant defenses . in current literature , most claim that a close relationship exists between oxidative stress and inflammation ( 3 , 4 ) . intestinal bacteria and their metabolites cause over - activation of the immune system when liver function is severely damaged , which leads to hepatocyte necrosis ( 5 ) . exposure to large amounts or high doses of lps under certain conditions may contribute to sepsis associated with fever , circulatory shock , and injury to several organs , including the liver ( 6 ) . the commonly accepted strategy used for prevention of lps - based damage is the reduction of reactive metabolites and the use of antioxidants ( 7 ) . epigallocatechin-3-gallate ( egcg ) , a major catechin isolated from green tea , is known to be a potential antioxidant . it has been used to treat a variety of diseases such as diabetes , cancer , cardiovascular disease , and nephrotoxicity ( 8 - 10 ) . egcg suppresses the migration and adhesion of multiple cell types , and it possesses excellent chemopreventive properties . egcg plays anti - inflammatory and anti - oxidant roles in the elimination of cellular damage ( 11 ) . more recently , reports have demonstrated the potential of egcg to generate oxidative stress in vitro ( 12 ) . therefore , the aim of this study was to investigate the alternative protective effects of egcg on lps - induced hepatotoxicity by using hep3b human hepatoma cells . specifically , the study examines the role of some proinflammatory markers and oxidative damage as possible mechanisms of lps - associated cytotoxicity . consequently , the hepatocellular carcinoma cell line hep3b was chosen as a model for investigation of lps toxicity and the effect of egcg on lps - exposed cells . the human hepatoma - derived cell line hep3b , was purchased from the american type culture collection ( atcc , usa ) , was cultured in dulbecco s modified eagle s medium ( dmem ) supplemented with a 10% fetal bovine serum ( fbs ) , 1% penicillin - streptomycin solution ( gibco , pittsburgh , usa ) at 37c in a humidified atmosphere of 5% co2 . subconfluent cultures were detached with trypsin / edta ( gibco , pittsburgh , usa ) and counted with trypan blue ( sigma - aldrich , usa ) counting method , and cells were seeded in six - well plates in 2 ml of growth medium . next , epigallocatechin gallate ( egcg ) ( santa cruz biotechnology , ca , usa ) and a single dose application ( 100 ng / ml ) of lps ( sigma - aldrich , usa ) were conducted on related groups in the appropriate doses . the egcg doses were 400 m , 200 m , 100 m , and 50 m , respectively . the cells were then washed with dulbecco s phosphate - buffered saline ( dpbs ) ( sigma - aldrich , usa ) , and a(3-([4 , 5-dimethylthiazol-2-yl)]-2,5-diphenyltetrazolium bromide ( mtt ) cell proliferation assay ( sigma - aldrich , usa ) was performed . optimal doses of egcg and lps were determined by the mtt assay , cells were seeded in 96-well plates in 100 l of growth medium , and the optimum doses were applied . hep3b hepatoma cells were seeded into 96-well plates at 2 x 10 cells per well and treated with egcg and lps . next , 10 l of mtt was added to each well and incubated for 4 hr at 37 c . the medium was then removed , and 150 l of dimethyl sulfoxide ( dmso ) was added to solubilize the mtt formazan . the absorbance of converted dye by living cells was measured at the wavelength of 570 nm . the total rna from hep3b cells was isolated using a qiagen rna isolation kit with an automated device ( qiacube , qiagen , germany ) according to the manufacturer s protocol . the cdna synthesis was performed by using the first strand synthesis kit ( qiagen , germany ) . real - time pcr was done using tnf - alpha , il-6 , and human beta - actin ( actb ) reference gene hybrid probes with roche cobas z480 ( roche , basel , switzerland ) . the values for specific genes were normalized by using beta - actin reference gene . in each pcr run , the cdna samples were amplified in triplicate . pcr primer sequences of tnf - alpha , il-6 and -actin cells were incubated , the culture supernatant was collected , and the concentrations of aspartate aminotransferase ( ast ) and alanine aminotra- nsferase ( alt ) were determined for each culture using an automatic biochemical analyzer . the cells were centrifuged at 600 g for 10 min at 4 c to remove nuclear fractions , and the remaining separated supernatant was recentrifuged at 10,000 g for 20 min at 4 c to collect the mitochondrial fraction ( pellet ) including peroxisomes for a catalase activity ( cat ) assay . superoxide dismutase ( sod ) activity was detected and inhibition of the formation of nicotinamide adenine dinucleotide phosphate ( nadph)-phenazine methosulphate nitroblue tetrazolium formazan was measured spectropho- tometrically at 560 nm . the sod and cat activities in the culture media were assayed with sod and cat assay kits ( enzo , telluride , usa ) according to the manufacturers instructions . the activity of glutathione peroxidase ( gsh - px ) in the culture medium was determined with human gsh - px elisa kit ( elabscience biotechnology co. , ltd , usa ) according to the manufacturers instructions . gsh - px activity was assayed using a method based on the reaction between glutathione remaining after the action of gsh - px and 5,5-dithiobid-2-nitrobenzoic acid to form a complex that absorbs maximally at 412 nm . all parameters recorded during sampling and laboratory findings were entered and stored in microsoft - excel . the data were analyzed using the ibm statistical package for social sciences ( spss ) version 20.0 statistical software ( ibm , ny , usa ) . statistical analysis was performed using one - way analysis of variance ( anova ) and duncan s multiple range tests ( dmrt ) . all p - values were based on a two - sided test of statistical significance and significance was accepted at the level of p < 0.05 . the human hepatoma - derived cell line hep3b , was purchased from the american type culture collection ( atcc , usa ) , was cultured in dulbecco s modified eagle s medium ( dmem ) supplemented with a 10% fetal bovine serum ( fbs ) , 1% penicillin - streptomycin solution ( gibco , pittsburgh , usa ) at 37c in a humidified atmosphere of 5% co2 . subconfluent cultures were detached with trypsin / edta ( gibco , pittsburgh , usa ) and counted with trypan blue ( sigma - aldrich , usa ) counting method , and cells were seeded in six - well plates in 2 ml of growth medium . next , epigallocatechin gallate ( egcg ) ( santa cruz biotechnology , ca , usa ) and a single dose application ( 100 ng / ml ) of lps ( sigma - aldrich , usa ) were conducted on related groups in the appropriate doses . the egcg doses were 400 m , 200 m , 100 m , and 50 m , respectively . the cells were then washed with dulbecco s phosphate - buffered saline ( dpbs ) ( sigma - aldrich , usa ) , and a(3-([4 , 5-dimethylthiazol-2-yl)]-2,5-diphenyltetrazolium bromide ( mtt ) cell proliferation assay ( sigma - aldrich , usa ) was performed . optimal doses of egcg and lps were determined by the mtt assay , cells were seeded in 96-well plates in 100 l of growth medium , and the optimum doses were applied . hep3b hepatoma cells were seeded into 96-well plates at 2 x 10 cells per well and treated with egcg and lps . next , 10 l of mtt was added to each well and incubated for 4 hr at 37 c . the medium was then removed , and 150 l of dimethyl sulfoxide ( dmso ) was added to solubilize the mtt formazan . the absorbance of converted dye by living cells was measured at the wavelength of 570 nm . the total rna from hep3b cells was isolated using a qiagen rna isolation kit with an automated device ( qiacube , qiagen , germany ) according to the manufacturer s protocol . the cdna synthesis was performed by using the first strand synthesis kit ( qiagen , germany ) . real - time pcr was done using tnf - alpha , il-6 , and human beta - actin ( actb ) reference gene hybrid probes with roche cobas z480 ( roche , basel , switzerland ) . the values for specific genes were normalized by using beta - actin reference gene . in each pcr run cells were incubated , the culture supernatant was collected , and the concentrations of aspartate aminotransferase ( ast ) and alanine aminotra- nsferase ( alt ) were determined for each culture using an automatic biochemical analyzer . the cells were centrifuged at 600 g for 10 min at 4 c to remove nuclear fractions , and the remaining separated supernatant was recentrifuged at 10,000 g for 20 min at 4 c to collect the mitochondrial fraction ( pellet ) including peroxisomes for a catalase activity ( cat ) assay . superoxide dismutase ( sod ) activity was detected and inhibition of the formation of nicotinamide adenine dinucleotide phosphate ( nadph)-phenazine methosulphate nitroblue tetrazolium formazan was measured spectropho- tometrically at 560 nm . the sod and cat activities in the culture media were assayed with sod and cat assay kits ( enzo , telluride , usa ) according to the manufacturers instructions . the activity of glutathione peroxidase ( gsh - px ) in the culture medium was determined with human gsh - px elisa kit ( elabscience biotechnology co. , ltd , usa ) according to the manufacturers instructions . gsh - px activity was assayed using a method based on the reaction between glutathione remaining after the action of gsh - px and 5,5-dithiobid-2-nitrobenzoic acid to form a complex that absorbs maximally at 412 nm . all parameters recorded during sampling and laboratory findings were entered and stored in microsoft - excel . the data were analyzed using the ibm statistical package for social sciences ( spss ) version 20.0 statistical software ( ibm , ny , usa ) . statistical analysis was performed using one - way analysis of variance ( anova ) and duncan s multiple range tests ( dmrt ) . all p - values were based on a two - sided test of statistical significance and significance was accepted at the level of p < 0.05 . the cytotoxic effects of chemicals ( egcg and lps ) , ast and alt levels , sod and cat activities , gsh - px , tnf - alpha , and il-6 levels were determined by using different biochemical and molecular methods . the mtt assay shows cell viability . according to the mtt assay results of both 24 and 48 hr applications , , there was a significant difference between the lps application group and all other experimental groups in terms of cell viability ( p<0.05 ) . however , all doses of egcg led to an increase in cell viability at different levels in the lps - induced hepatotoxicity groups ( figure 1 ) . cell viability of all experimental groups detected by mtt assay a and b show the significant differences among 24 hr treated experimental groups ( p<0.05 ) 15 show the significant differences among 48 hr treated experimental groups ( p<0.05 ) in this study , the liver enzyme levels ( ast and alt ) were checked to determine whether or not lps lead to hepatotoxicity . both alt and ast enzyme levels were highest in the lps and lps+egcg400 m groups . there were significant differences between the control group , lps , and lps+egcg400 m groups ( p<0.05 ) . normalization of ast and alt levels was detected in egcg 200 , 100 , and 50 m treatment groups . the effects of lps and egcg treatment on ast and alt enzyme levels a e show the significant differences among experimental groups in ast enzyme levels ( p<0.05 ) 1 and 2 show the significant differences among experimental groups in alt enzyme levels ( p<0.05 ) some anti - oxidant system parameters such as sod , cat , and gsh - px were detected by biochemical methods as shown in figures 3 , 4 , and 5 . the highest treatment dose , 400 m of egcg , lead to a dramatic decrease in sod level of hep3b cells , and there were significant differences in the control group versus the egcg400 and control vs. lps+egcg400 groups ( p<0.05 ) . however , other treatment doses of egcg normalized the sod level as seen in figure 3 . on the other hand , catalase is also an antioxidant enzyme which catalyzes the conversion of h2o2 to water and oxygen . the results were nearly the same as sod activity results . when the highest dose of egcg was applied to lps - induced hep3b cells , however , other doses of egcg showed a possible treatment effect on lps - induced hepatotoxicity . they reduced the cat activity level in contrast to egcg400 m application ( figure 4 ) . finally , gsh - px enzymes catalyzed the reduction of hydroperoxides to water and the respective alcohols by oxidizing gsh to oxidized glutathione ( gssg ) ( 13 , 14 ) . the effects of lps and egcg treatment on sod enzyme activity in hep3b cells a c show the significant differences among experimental groups in sod enzyme activity ( p<0.05 ) the effects of lps and egcg treatment on cat enzyme activity in hep3b cells a f show the significant differences among experimental groups in cat enzyme activity ( p<0.05 ) the effects of lps and egcg treatment on gsh - px enzyme activity in hep3b cells a d show the significant differences among experimental groups in gsh - px enzyme activity ( p<0.05 ) when gsh - px levels were evaluated in the present study , there were statistical differences ( p<0.05 ) between the control and other experimental groups . as with sod and cat activity levels , the highest dose of egcg led to a significant increase in lps - induced hepatotoxicity ( figure 5 ) . initially , it was seen that lps caused an increase in both tnf - alpha and il-6 levels and led to inflammation . at that point , attempts were made to treat and normalize these levels with various doses of egcg . however , the highest dose of egcg also led to a dramatic increase in these cytokine levels ( figure 6 ) . tnf - alpha and il-6 levels increased 5-fold and 2-fold in the lps administration group , respectively . it was understood that high dose egcg treatment caused serious changes in some crucial parameters . tnf - alpha and il-6 levels increased 36-fold and 14-fold in the high dose egcg+lps group , respectively . other treatment doses of egcg were found to be beneficial doses because they normalized these cytokine levels after lps - induced hepatotoxicity . tnf - alpha and il-6 mrna expression levels of all experimental groups a g show the significant differences among experimental groups in tnf - alpha mrna expression levels ( p<0.05 ) 17 show the significant differences among experimental groups in il-6 mrna expression levels ( p<0.05 ) infection , especially gram - negative bacteria , is the most common cause of sepsis , and lps found in such bacteria is an endotoxin that has been implicated in the pathogenesis of infection and as a cause of septic shock . lps - induced hepatotoxicity is characterized by disturbed intracellular redox balance and excessive reactive oxygen species ( ros ) accumulation , leading to liver injury ( 15 ) . furthermore , human hepatoblastoma hep3b cells have been used in many toxicity studies to screen for hepatotoxic compounds ( 16 ) . these cells have low levels of phase i cytochrome p450 enzymes when compared with primary hepatocytes , but they have normal levels of phase ii enzymes ( 17 , 18 ) . some studies have indicated that hepatocellular carcinoma cells present a valuable in vitro model for hepatotoxicity studies ( 18 - 20 ) . this study evaluated the possible role of egcg in lps - induced hepatotoxicity using hep3b cells . for this reason , mtt assay , ast and alt liver enzyme levels , gsh level , sod and cat activities , tnf - alpha and il-6 levels were investigated by different methods in this study . in current literature , there are few published studies about the anti - inflammatory effects of egcg on various cells and organs in either animal or in vitro models . however , limited studies have examined the relationship between egcg and lps - stimulated hepatotoxicity . the goal of our study was to investigate both the anti - oxidant and anti - inflammatory effects of egcg on lps - induced hepatotoxicity under in vitro conditions on hep3b cells . initially , it was seen that a high dose egcg is also a toxic agent for hepatocytes as it negatively affects all parameters in hep3b cells . a 400 m egcg treatment leads to an increase in the levels of cat , gsh - px , ast , alt , tnf - alpha , and il-6 , while it decreases the level of sod . however , other doses of egcg treatment may be as effective , especially doses of 100 and 200 m doses . when mtt results were considered , it was seen that lps and a high dose of egcg caused a serious reduction in the level of cell viability . most in vitro studies have shown that lps is a serious cytotoxic agent especially on hepatocytes ( 18 ) . however , a study confirms our hypothesis that a high dose of egcg ( 400 m ) is cytotoxic on cell viability ( 21 ) . the other two doses of egcg ( 200 and 100 m ) were the most effective doses of egcg treatment used in this study . these doses normalized nearly all the parameters and cell viability . as in this study , kim et al showed that the protection by egcg is most effective when cells are exposed to egcg for short periods at 100 or 200 m concentrations ( 22 ) . evaluation of several biochemical parameters , including enzymes , can be helpful in identifying damage to target tissues , as well as identifying the health status of the host . in addition , ast and alt are the most important enzymes acting as transaminases involved in amino acid metabolism ( 23 ) . for this reason , liver ast and alt enzymes are considered relevant stress indicators , and they were used to determine liver toxicity in this study . as seen in figure 2 , lps causes an increase in both ast and alt levels in hep3b cells . additionally , a high dose of egcg exacerbated the increase of liver enzymes . previous studies have shown that lps can lead to a dramatic increase in ast , alp , and alt levels ( 5 , 24 , 25 ) . oxidative stress is a system made up of a group of antioxidant enzymes and non - enzymatic antioxidant substances capable of neutralizing free radicals and preventing an excess production of ros ( 26 ) . sod , gsh - px , and catalase enzymes are the first line of cellular defense against oxidative stress . in our study , these increases are an expected result when the toxicity potential of lps and high dose egcg are suspected . however , as expected , effective doses of egcg treatment normalized these enzyme levels . study showed that non - lethal toxic doses of egcg treatment markedly decrease the levels of sod , catalase , and glutathione peroxidase . it also showed that egcg also significantly suppresses mrna levels of hepatic sod and cat levels ( 27 ) . moreover , most scientific studies have proven that lps leads to an increase in oxidative stress parameters ( 18 ) . oxidative stress mediated tissue damage can be reversed by the sod enzyme and glutathione ( gsh ) . the action of these two antioxidant parameters prevents the cytotoxic effects of toxic free radicals . cat is also an anti - oxidant enzyme that reacts with hydrogen peroxide to produce water and molecular oxygen . the currently accepted knowledge supports our anti - oxidant data that egcg can affect the antioxidant enzyme levels and prevent oxidative damage . in addition , cytokines play an important role in many cellular processes such as immunity , inflammation , cell proliferation , differentiation , and cell death . among the inflammatory networks , tnf- , il-1 , and il-6 are known to be the most important inflammatory mediators involved in the initiation of acute inflammation . tnf - alpha has been linked to increased oxidative stress and is known to activate other inflammatory cells ( 28 , 29 ) . also , it is the earliest and primary endogenous mediator for the process of inflammatory responses and has been linked to increased oxidative stress ( 28 ) . according to literature , it has been shown that il-6 triggers some cellular signaling pathways and induces some pro - apoptotic proteins such as b - cell cll / lymphoma-2 ( bcl-2 ) ( 30 ) . in the present study , only lps administration , only high dose egcg treatment , and lps+egcg400 administration led to a serious increase in both the tnf - alpha and il-6 levels and also caused acute inflammation . however , effective doses of egcg were also used in an attempt to normalize and reduced cytokine levels . a previous study has concluded that the anti - inflammatory mechanism of green tea polyphenols contributes to down - regulation of tnf - alpha gene expression ( 5 ) . increases in oxidative stress can increase the production of inflammatory cytokines and in turn , an increase in inflammatory cytokines can induce the production of radicals . for example , chen et al examined the possible protective effects of egcg in mastitis rats . it was found that egcg inhibits the lps - induced inflammatory response and normalizes anti - oxidant enzyme levels ( 29 ) . another study ( 31 ) claimed that egcg could inhibit il-6 and nuclear factor - kappa - light - chain - enhancer of activated b cell ( nf - kb ) activities in human gingival fibroblasts . a similar study ( 32 ) showed that some egcg derivates suppress the lps - induced production of nitric oxide and pro - inflammatory cytokines in macrophages . another study , performed in gingival epithelial cells , showed that egcg inhibited inflammatory cytokine secretion and also suggested that it may be a novel adjunctive therapeutic agent ( 33 ) . liu et al ( 5 ) used lps - induced l0 hepatocytes in their study to examine the effects of egcg on liver cells . they showed that pre - treatment of egcg reduces the production of tnf - alpha and nitric oxide production in a dose - dependent manner . in conclusion , lps caused hepatotoxicity , but interestingly , a high dose of egcg was found to be a cytotoxic agent in this study . however , other two doses of egcg led to a decrease in both inflammatory cytokine levels and antioxidant enzyme levels in lps - induced hep3b cells . to better understand the molecular mechanisms of the anti - inflammatory and anti - oxidant effects of egcg , further studies should examine the effect of egcg on secondary cellular signaling pathways such as mapk in lps - stimulated hepatocytes because the erk1/2 and p38 are the major members of mapk family and are associated with cellular oxidative stress , inflammation , proliferation , and migration . finally , the protective effect of egcg in lps - enhanced hep3b cells is associated with the inhibition of inflammatory mediators , including tnf - alpha and il-6 , and may affect the antioxidant enzyme levels . taking everything into account , egcg may be seen as a promising agent for the treatment of hepatic over - active inflammatory response .

objective(s):in the present study , our aim was to investigate the possible protective effects of epigallocatechin gallate ( egcg ) on lipopolysaccharide ( lps)-induced hepatotoxicity by using hep3b human hepatoma cells . specifically , the study examines the role of some proinflammatory markers and oxidative damage as possible mechanisms of lps - associated cytotoxicity . consequently , the hepatocellular carcinoma cell line hep3b was chosen as a model for investigation of lps toxicity and the effect of egcg on lps - exposed cells.materials and methods : the hep3b human hepatoma cells were used for this study . the cytotoxic effects of chemicals ( egcg and lps ) , ast and alt levels , sod and cat activities , gsh - px level and tnf - alpha and il-6 levels were detected by using different biochemical and molecular methods . lps and egcg were applied to cells at various times and doses.results:the highest treatment dose of egcg ( 400 m ) led to a dramatic decrease in sod level and increase in cat and gsh levels . additionally , the highest dose of egcg also led to a dramatic increase in tnf - alpha and il-6 levels . on the other hand , effective doses of egcg ( 200 and 100 m ) normalized all related parameters levels.conclusion:lps caused hepatotoxicity , but interestingly , a high dose of egcg was found to be a cytotoxic agent in this study . however , other two doses of egcg led to a decrease in both inflammatory cytokine levels and antioxidant enzyme levels . further studies should examine the effect of egcg on secondary cellular signaling pathways .

Introduction Material and Methods Cell Culture and Treatment MTT assay Quantitative real-time PCR (Q-PCR) analysis Biochemical Examination GSH-Px activity detection by enzyme-linked immunosorbent assay (ELISA) Statistical analysis Results Discussion Conclusion Conflict of interest

on the other hand , oxidative stress is described as a disturbance in the balance between the production of reactive oxygen species and antioxidant defenses . exposure to large amounts or high doses of lps under certain conditions may contribute to sepsis associated with fever , circulatory shock , and injury to several organs , including the liver ( 6 ) . the commonly accepted strategy used for prevention of lps - based damage is the reduction of reactive metabolites and the use of antioxidants ( 7 ) . epigallocatechin-3-gallate ( egcg ) , a major catechin isolated from green tea , is known to be a potential antioxidant . therefore , the aim of this study was to investigate the alternative protective effects of egcg on lps - induced hepatotoxicity by using hep3b human hepatoma cells . specifically , the study examines the role of some proinflammatory markers and oxidative damage as possible mechanisms of lps - associated cytotoxicity . consequently , the hepatocellular carcinoma cell line hep3b was chosen as a model for investigation of lps toxicity and the effect of egcg on lps - exposed cells . the human hepatoma - derived cell line hep3b , was purchased from the american type culture collection ( atcc , usa ) , was cultured in dulbecco s modified eagle s medium ( dmem ) supplemented with a 10% fetal bovine serum ( fbs ) , 1% penicillin - streptomycin solution ( gibco , pittsburgh , usa ) at 37c in a humidified atmosphere of 5% co2 . subconfluent cultures were detached with trypsin / edta ( gibco , pittsburgh , usa ) and counted with trypan blue ( sigma - aldrich , usa ) counting method , and cells were seeded in six - well plates in 2 ml of growth medium . next , epigallocatechin gallate ( egcg ) ( santa cruz biotechnology , ca , usa ) and a single dose application ( 100 ng / ml ) of lps ( sigma - aldrich , usa ) were conducted on related groups in the appropriate doses . the egcg doses were 400 m , 200 m , 100 m , and 50 m , respectively . the cells were then washed with dulbecco s phosphate - buffered saline ( dpbs ) ( sigma - aldrich , usa ) , and a(3-([4 , 5-dimethylthiazol-2-yl)]-2,5-diphenyltetrazolium bromide ( mtt ) cell proliferation assay ( sigma - aldrich , usa ) was performed . optimal doses of egcg and lps were determined by the mtt assay , cells were seeded in 96-well plates in 100 l of growth medium , and the optimum doses were applied . hep3b hepatoma cells were seeded into 96-well plates at 2 x 10 cells per well and treated with egcg and lps . real - time pcr was done using tnf - alpha , il-6 , and human beta - actin ( actb ) reference gene hybrid probes with roche cobas z480 ( roche , basel , switzerland ) . pcr primer sequences of tnf - alpha , il-6 and -actin cells were incubated , the culture supernatant was collected , and the concentrations of aspartate aminotransferase ( ast ) and alanine aminotra- nsferase ( alt ) were determined for each culture using an automatic biochemical analyzer . the cells were centrifuged at 600 g for 10 min at 4 c to remove nuclear fractions , and the remaining separated supernatant was recentrifuged at 10,000 g for 20 min at 4 c to collect the mitochondrial fraction ( pellet ) including peroxisomes for a catalase activity ( cat ) assay . the sod and cat activities in the culture media were assayed with sod and cat assay kits ( enzo , telluride , usa ) according to the manufacturers instructions . the activity of glutathione peroxidase ( gsh - px ) in the culture medium was determined with human gsh - px elisa kit ( elabscience biotechnology co. , ltd , usa ) according to the manufacturers instructions . gsh - px activity was assayed using a method based on the reaction between glutathione remaining after the action of gsh - px and 5,5-dithiobid-2-nitrobenzoic acid to form a complex that absorbs maximally at 412 nm . the human hepatoma - derived cell line hep3b , was purchased from the american type culture collection ( atcc , usa ) , was cultured in dulbecco s modified eagle s medium ( dmem ) supplemented with a 10% fetal bovine serum ( fbs ) , 1% penicillin - streptomycin solution ( gibco , pittsburgh , usa ) at 37c in a humidified atmosphere of 5% co2 . subconfluent cultures were detached with trypsin / edta ( gibco , pittsburgh , usa ) and counted with trypan blue ( sigma - aldrich , usa ) counting method , and cells were seeded in six - well plates in 2 ml of growth medium . next , epigallocatechin gallate ( egcg ) ( santa cruz biotechnology , ca , usa ) and a single dose application ( 100 ng / ml ) of lps ( sigma - aldrich , usa ) were conducted on related groups in the appropriate doses . the egcg doses were 400 m , 200 m , 100 m , and 50 m , respectively . the cells were then washed with dulbecco s phosphate - buffered saline ( dpbs ) ( sigma - aldrich , usa ) , and a(3-([4 , 5-dimethylthiazol-2-yl)]-2,5-diphenyltetrazolium bromide ( mtt ) cell proliferation assay ( sigma - aldrich , usa ) was performed . optimal doses of egcg and lps were determined by the mtt assay , cells were seeded in 96-well plates in 100 l of growth medium , and the optimum doses were applied . hep3b hepatoma cells were seeded into 96-well plates at 2 x 10 cells per well and treated with egcg and lps . real - time pcr was done using tnf - alpha , il-6 , and human beta - actin ( actb ) reference gene hybrid probes with roche cobas z480 ( roche , basel , switzerland ) . the values for specific genes were normalized by using beta - actin reference gene . in each pcr run cells were incubated , the culture supernatant was collected , and the concentrations of aspartate aminotransferase ( ast ) and alanine aminotra- nsferase ( alt ) were determined for each culture using an automatic biochemical analyzer . the cells were centrifuged at 600 g for 10 min at 4 c to remove nuclear fractions , and the remaining separated supernatant was recentrifuged at 10,000 g for 20 min at 4 c to collect the mitochondrial fraction ( pellet ) including peroxisomes for a catalase activity ( cat ) assay . the sod and cat activities in the culture media were assayed with sod and cat assay kits ( enzo , telluride , usa ) according to the manufacturers instructions . the activity of glutathione peroxidase ( gsh - px ) in the culture medium was determined with human gsh - px elisa kit ( elabscience biotechnology co. , ltd , usa ) according to the manufacturers instructions . gsh - px activity was assayed using a method based on the reaction between glutathione remaining after the action of gsh - px and 5,5-dithiobid-2-nitrobenzoic acid to form a complex that absorbs maximally at 412 nm . the cytotoxic effects of chemicals ( egcg and lps ) , ast and alt levels , sod and cat activities , gsh - px , tnf - alpha , and il-6 levels were determined by using different biochemical and molecular methods . however , all doses of egcg led to an increase in cell viability at different levels in the lps - induced hepatotoxicity groups ( figure 1 ) . cell viability of all experimental groups detected by mtt assay a and b show the significant differences among 24 hr treated experimental groups ( p<0.05 ) 15 show the significant differences among 48 hr treated experimental groups ( p<0.05 ) in this study , the liver enzyme levels ( ast and alt ) were checked to determine whether or not lps lead to hepatotoxicity . both alt and ast enzyme levels were highest in the lps and lps+egcg400 m groups . normalization of ast and alt levels was detected in egcg 200 , 100 , and 50 m treatment groups . the effects of lps and egcg treatment on ast and alt enzyme levels a e show the significant differences among experimental groups in ast enzyme levels ( p<0.05 ) 1 and 2 show the significant differences among experimental groups in alt enzyme levels ( p<0.05 ) some anti - oxidant system parameters such as sod , cat , and gsh - px were detected by biochemical methods as shown in figures 3 , 4 , and 5 . the highest treatment dose , 400 m of egcg , lead to a dramatic decrease in sod level of hep3b cells , and there were significant differences in the control group versus the egcg400 and control vs. lps+egcg400 groups ( p<0.05 ) . however , other treatment doses of egcg normalized the sod level as seen in figure 3 . on the other hand , catalase is also an antioxidant enzyme which catalyzes the conversion of h2o2 to water and oxygen . when the highest dose of egcg was applied to lps - induced hep3b cells , however , other doses of egcg showed a possible treatment effect on lps - induced hepatotoxicity . finally , gsh - px enzymes catalyzed the reduction of hydroperoxides to water and the respective alcohols by oxidizing gsh to oxidized glutathione ( gssg ) ( 13 , 14 ) . the effects of lps and egcg treatment on sod enzyme activity in hep3b cells a c show the significant differences among experimental groups in sod enzyme activity ( p<0.05 ) the effects of lps and egcg treatment on cat enzyme activity in hep3b cells a f show the significant differences among experimental groups in cat enzyme activity ( p<0.05 ) the effects of lps and egcg treatment on gsh - px enzyme activity in hep3b cells a d show the significant differences among experimental groups in gsh - px enzyme activity ( p<0.05 ) when gsh - px levels were evaluated in the present study , there were statistical differences ( p<0.05 ) between the control and other experimental groups . as with sod and cat activity levels , the highest dose of egcg led to a significant increase in lps - induced hepatotoxicity ( figure 5 ) . initially , it was seen that lps caused an increase in both tnf - alpha and il-6 levels and led to inflammation . at that point , attempts were made to treat and normalize these levels with various doses of egcg . however , the highest dose of egcg also led to a dramatic increase in these cytokine levels ( figure 6 ) . tnf - alpha and il-6 levels increased 5-fold and 2-fold in the lps administration group , respectively . tnf - alpha and il-6 levels increased 36-fold and 14-fold in the high dose egcg+lps group , respectively . other treatment doses of egcg were found to be beneficial doses because they normalized these cytokine levels after lps - induced hepatotoxicity . tnf - alpha and il-6 mrna expression levels of all experimental groups a g show the significant differences among experimental groups in tnf - alpha mrna expression levels ( p<0.05 ) 17 show the significant differences among experimental groups in il-6 mrna expression levels ( p<0.05 ) infection , especially gram - negative bacteria , is the most common cause of sepsis , and lps found in such bacteria is an endotoxin that has been implicated in the pathogenesis of infection and as a cause of septic shock . these cells have low levels of phase i cytochrome p450 enzymes when compared with primary hepatocytes , but they have normal levels of phase ii enzymes ( 17 , 18 ) . some studies have indicated that hepatocellular carcinoma cells present a valuable in vitro model for hepatotoxicity studies ( 18 - 20 ) . this study evaluated the possible role of egcg in lps - induced hepatotoxicity using hep3b cells . for this reason , mtt assay , ast and alt liver enzyme levels , gsh level , sod and cat activities , tnf - alpha and il-6 levels were investigated by different methods in this study . in current literature , there are few published studies about the anti - inflammatory effects of egcg on various cells and organs in either animal or in vitro models . however , limited studies have examined the relationship between egcg and lps - stimulated hepatotoxicity . the goal of our study was to investigate both the anti - oxidant and anti - inflammatory effects of egcg on lps - induced hepatotoxicity under in vitro conditions on hep3b cells . initially , it was seen that a high dose egcg is also a toxic agent for hepatocytes as it negatively affects all parameters in hep3b cells . a 400 m egcg treatment leads to an increase in the levels of cat , gsh - px , ast , alt , tnf - alpha , and il-6 , while it decreases the level of sod . however , other doses of egcg treatment may be as effective , especially doses of 100 and 200 m doses . when mtt results were considered , it was seen that lps and a high dose of egcg caused a serious reduction in the level of cell viability . however , a study confirms our hypothesis that a high dose of egcg ( 400 m ) is cytotoxic on cell viability ( 21 ) . the other two doses of egcg ( 200 and 100 m ) were the most effective doses of egcg treatment used in this study . as in this study , kim et al showed that the protection by egcg is most effective when cells are exposed to egcg for short periods at 100 or 200 m concentrations ( 22 ) . in addition , ast and alt are the most important enzymes acting as transaminases involved in amino acid metabolism ( 23 ) . for this reason , liver ast and alt enzymes are considered relevant stress indicators , and they were used to determine liver toxicity in this study . as seen in figure 2 , lps causes an increase in both ast and alt levels in hep3b cells . additionally , a high dose of egcg exacerbated the increase of liver enzymes . previous studies have shown that lps can lead to a dramatic increase in ast , alp , and alt levels ( 5 , 24 , 25 ) . sod , gsh - px , and catalase enzymes are the first line of cellular defense against oxidative stress . in our study , these increases are an expected result when the toxicity potential of lps and high dose egcg are suspected . however , as expected , effective doses of egcg treatment normalized these enzyme levels . study showed that non - lethal toxic doses of egcg treatment markedly decrease the levels of sod , catalase , and glutathione peroxidase . it also showed that egcg also significantly suppresses mrna levels of hepatic sod and cat levels ( 27 ) . the action of these two antioxidant parameters prevents the cytotoxic effects of toxic free radicals . cat is also an anti - oxidant enzyme that reacts with hydrogen peroxide to produce water and molecular oxygen . the currently accepted knowledge supports our anti - oxidant data that egcg can affect the antioxidant enzyme levels and prevent oxidative damage . among the inflammatory networks , tnf- , il-1 , and il-6 are known to be the most important inflammatory mediators involved in the initiation of acute inflammation . tnf - alpha has been linked to increased oxidative stress and is known to activate other inflammatory cells ( 28 , 29 ) . according to literature , it has been shown that il-6 triggers some cellular signaling pathways and induces some pro - apoptotic proteins such as b - cell cll / lymphoma-2 ( bcl-2 ) ( 30 ) . in the present study , only lps administration , only high dose egcg treatment , and lps+egcg400 administration led to a serious increase in both the tnf - alpha and il-6 levels and also caused acute inflammation . however , effective doses of egcg were also used in an attempt to normalize and reduced cytokine levels . a previous study has concluded that the anti - inflammatory mechanism of green tea polyphenols contributes to down - regulation of tnf - alpha gene expression ( 5 ) . for example , chen et al examined the possible protective effects of egcg in mastitis rats . it was found that egcg inhibits the lps - induced inflammatory response and normalizes anti - oxidant enzyme levels ( 29 ) . a similar study ( 32 ) showed that some egcg derivates suppress the lps - induced production of nitric oxide and pro - inflammatory cytokines in macrophages . another study , performed in gingival epithelial cells , showed that egcg inhibited inflammatory cytokine secretion and also suggested that it may be a novel adjunctive therapeutic agent ( 33 ) . liu et al ( 5 ) used lps - induced l0 hepatocytes in their study to examine the effects of egcg on liver cells . they showed that pre - treatment of egcg reduces the production of tnf - alpha and nitric oxide production in a dose - dependent manner . in conclusion , lps caused hepatotoxicity , but interestingly , a high dose of egcg was found to be a cytotoxic agent in this study . however , other two doses of egcg led to a decrease in both inflammatory cytokine levels and antioxidant enzyme levels in lps - induced hep3b cells . to better understand the molecular mechanisms of the anti - inflammatory and anti - oxidant effects of egcg , further studies should examine the effect of egcg on secondary cellular signaling pathways such as mapk in lps - stimulated hepatocytes because the erk1/2 and p38 are the major members of mapk family and are associated with cellular oxidative stress , inflammation , proliferation , and migration . finally , the protective effect of egcg in lps - enhanced hep3b cells is associated with the inhibition of inflammatory mediators , including tnf - alpha and il-6 , and may affect the antioxidant enzyme levels . taking everything into account , egcg may be seen as a promising agent for the treatment of hepatic over - active inflammatory response .

since the first studies on the prognosis of endodontic therapy , the quality of root canal filling has been considered essential for treatment success . the presence of voids in the root canal filling facilitates bacterial proliferation and the perpetuation of periapical lesions . among the several factors that can interfere in the root canal filling these materials should present biocompatibility and suitable antimicrobialand physiochemical properties . in this context , it is important to study the properties of the filling materials in order to establish the appropriate parameters for the development of new products , as well as to evaluate those already available on the market . root canal sealers can be classified according to the chemical composition in zinc oxide - eugenol - based , calcium hydroxide - containing , glass - ionomer - based , epoxy - resin - based and methacrylate - resin - based sealers . the zinc oxide - eugenol - based sealers were introduced in endodontics by grossman , in 1936 , to be used in conjunction with the gutta - percha or silver cones in root canal fillings . endofill ( dentsply - mallefer , dentsply indstria e comrcio ltda . , petrpolis , rj , brazil ) and endomthasone ( spcialities septodont , saint - maurdes - fosss , cedex , france ) are commonly used zinc oxide - eugenol - based sealers , both available in a powder - liquid presentation . sealers that contain calcium hydroxide were idealized with the purpose of improving the biological properties and ensuring a good seal of root canal system . among these products , apexit plus ( ivoclar , vivadent , frstentum , schaan , liechtenstein ) and sealapex ( sybron - endo , glendora , ca , usa ) are available in the market in a paste - paste presentation . the first resin - based sealer was proposed by schreder , in 1954 , and contained epoxy resin and bisphenol . since them , studies have contributed to the improvement of sealers ' quality , leading to the development of an epoxy resin - based sealer with good physicochemical properties , ah plus ( dentsply , detrey gmbh , konstanz , germany ) . much research has been done to develop products derived from biomaterials , in order to increase the compatibility to biological tissues . in the 1980 's , an experimental sealer , called polifil , was developed at the araraquara school of dentistry ( sp , brazil ) . this sealer is based on a polyurethane vegetable resin , ricinus communis extract , and has been demonstrating good flow , efficient sealing capacity and some antimicrobial activity . in 1983 , the american national standards institute / american dental association ( ansi / ada ) released a series of norms and tests ( named specification 57 ) to evaluate the physiochemical properties of endodontic sealers , aiming at standardizing the tests and promote larger scientific quality in the researches . this specification was revised in 2000 and it includes the following tests : film thickness , setting time , flow , radiopacity , solubility and dimensional change following setting . due to the diversity of composition of the available sealers and considering the ansi / ada standards , the objective of this study was to evaluate in vitro the physiochemical properties of sealers of different bases : endofill and endomthasone ( zinc oxide and eugenol ) , apexit plus and sealapex ( calcium hydroxide ) , polifil ( ricinus communis polymer ) and ah plus ( epoxy resin ) , in order to contribute to the selection of the appropriate material for clinical practice . setting time , flow , radiopacity , solubility , and dimensional change after setting for ah plus , polifil , apexit plus , sealapex , endomthasone and endofill sealers were measured according to ansi / ada standards for dental root canal sealing materials by one examiner blinded to the identification of the materials . details of the endodontic sealers used in the study all tested materials were prepared in accordance to the manufacturers ' instructions . ah plus , iii and iv are presented as two pastes and , for each tested sample , 15 mm of the sealer ( 1:1 ) was dispensed onto a mixing pad and mixed until obtaining a homogeneous mixture . in group ii ( polifil ) , 0.18 ml of the liquid was mixed to 0.44 ml of the paste , measure through a graduate syringe and the mixture was mixed on a glass plate . for endomthasone and endofill , 3 drops of the liquid and a portion of the powder were released in glass plate and mixed in a gradual way until getting the ideal consistence , characterized by a cement thread of approximately 2 cm , without falling during 15 s. for the physicochemical tests , the arithmetic mean of five replicates for each sealer was recorded and considered as the result of the test . five plaster of cast rings having an internal diameter of 10 mm and a thickness of 2 mm were prepared for each group . the external borders of the moulds were fixed with wax on a glass plate ( 75x25x1 mm ) . the moulds were filled with the material and transferred to a chamber with 95% relative humidity and a temperature of 37c . after 15010 s from the onset of mixing , a gilmore - type needle with a mass of 1000.5 g having a flat end of 2.00.1 mm in diameter was carefully lowered vertically onto the horizontal surface of the testing sample . the needle tip was cleaned and the probing was repeated until indentations ceased to be visible . the time used from the start of mixing to this point was recorded . if the results differed by more than 5% , the test was repeated . the amount of 0.5 ml of each sealer tested was placed on a glass plate ( 10x10x3 mm ) using a graduated disposable 3-ml syringe . at 1805 seconds after the onset of mixing , another plate with a mass of 20 + 2 g and a load of 100 g were carefully applied on top of the material . ten min after mixing the cement , the load was removed and the major and minor diameters of the compressed discs were measured using a digital calliper with a resolution of 0.01 mm ( mitutoyo mti corporation , tokyo , japan ) . if both measurements were consistent to within 1 mm , the results were recorded . if the major and minor diameter discs were not uniformly circular or did not match within 1 mm , the test was repeated . five acrylic plates ( 2.2 cm x 4.5 cm x 1 mm ) , containing four wells measuring 1 mm in depth and 5 mm in diameter , were prepared and placed over a glass plate covered by cellophane sheet . in group i , the freshly mixed sealer was introduced into the wells using a syringe , to avoid the formation of bubbles . in groups ii , iii the respective material applicators were used to fill the wells . another glass plate covered with cellophane each plate was kept in an incubator ( 37c , 95% relative humidity ) for a period corresponding to three times the setting time . each well was filled with one of the sealers , following a sequence according to the setting time of the material , from the longest to the shortest , so that the samples would be ready for radiographic evaluation after the final setting of all materials . each one of the acrylic plates containing the root filling materials was positioned , at the time of the radiographic exposure , alongside to another acrylic plate ( 1.3 cm x 4.5 cm x 1 mm ) , containing an aluminum stepwedge , made of 1100 alloy , with the thickness varying from 1 to 10 mm , in uniform steps of 1 mm each . this set of acrylic plates was placed in front of this phosphor plate and a digital radiograph was taken ( digora system , soredex orion corporation , helsinki , finland ) . care was taken to place the samples next to the aluminum step wedge and in the middle of the phosphor plate . radiographic images were obtained using the spectro 70x x - ray machine ( dabi atlante , ribeiro preto , sp , brazil ) , at 70 kvp and 8 ma . the object - to - focus distance was 30 cm and the exposure time was 0.2 s. exposed imaging plates of the test samples were immediately scanned after exposure ( digora scanner ) and analyzed using digora for windows 5.1 software . five teflon moulds , prepared for the production of 3.58-mm high cylindrical test bodies measuring 3 mm in diameter , were placed on a glass plate wrapped with a fine cellophane sheet . the moulds were filled with a slight excess of freshly mixed sealers and a microscope slide , also wrapped in cellophane , was pressed onto the upper surface of the mould . the assembled group was kept firmly joined with a c - shaped clamp and transferred to an incubator ( 37c , 95% relative humidity ) left to stand for a period corresponding to three times the setting time . after this period , the flat ends of the moulds , containing the samples , were grinded with 600 grit wet sandpaper . the samples were removed from the mould , measured with a digital caliper , stored in a 50-ml vessel containing 2.24 ml of distilled and deionized water , and kept in an incubator ( 37c , 95% relative humidity ) for 30 days . the sample was then removed from the container , blotted dry on absorbent paper , and measured again for length . the percentage of the dimensional alterations was calculated using the equation : l30ll100 where l30 is the length of the sample after 30 days of storage and l is the initial length of the sample . a 1.5-mm - thick cylindrical teflon ( polytetrafluoroethylene , dupont , habia , knivsta , sweden ) mould measuring 7.75 mm in inner diameter was filled with freshly mixed sealers . the mould was supported by a larger glass plate and covered with a cellophane sheet . an impermeable nylon thread was placed inside the material and another glass plate , also covered with cellophane film , was positioned on the mould and pressed manually in such a way that the plates touched the entire mould in a uniform manner . the assembly was placed in an incubator ( 37c , 95% relative humidity ) and left to stand for a period corresponding to three times the setting time . as soon as the samples were removed from the mould , they were weighed three times each in an analytical balance ( hm-200 ; a&d engineering inc . , the samples were suspended by nylon thread and placed two - by - two inside a plastic vessel with a wide opening containing 7.5 ml of distilled and deionized water , taking care to avoid any contact between them and the inner surface of the container . the containers were sealed and left for 7 days in an incubator ( 37c , 95% relative humidity ) . after this period , the samples were removed from the containers , rinsed with distilled and deionized water , blotted dry with absorbent paper , and placed in a dehumidifier for 24 h. afterwards , they were weighed again . the weight loss of each sample , expressed as percentage of the original mass ( m%=mi - mf ) , was taken as the solubility of the sealer . the solution in which the specimens were immersed was analyzed by atomic absorption spectrometry ( perkin elmer instruments gmbh , berlingen , germany ) to quantify the levels of na , k , ni and zn ions released in the solution . merck solutions with 1000 mg / l concentration ( merck , darmstadt , germany ) were used to prepare the standard solution of the different metals . metals ' analytical curves were obtained from appropriate dilutions of each respective stock solution . this spectrophotometer is supplied with hollow cathode lamps with different light spectrums exclusively for measuring metallic ions . the obtained solutions were sprayed into the atomic absorption spectrophotometer for ions quantification . the arithmetic mean was done and considered as the results of the concentration of na , k , zn and ni , expressed as g / ml . the kolmogorov - smirnov showed that the results were consistent with a normal distribution curve . the parametric statistical analysis was performed by one - way anova and tukey - kramer post - hoc test ) and the significance level was set as 5% ( graphpad instat , graphpad software inc . , san diego , ca , usa ) . five plaster of cast rings having an internal diameter of 10 mm and a thickness of 2 mm were prepared for each group . the external borders of the moulds were fixed with wax on a glass plate ( 75x25x1 mm ) . the moulds were filled with the material and transferred to a chamber with 95% relative humidity and a temperature of 37c . after 15010 s from the onset of mixing , a gilmore - type needle with a mass of 1000.5 g having a flat end of 2.00.1 mm in diameter was carefully lowered vertically onto the horizontal surface of the testing sample . the needle tip was cleaned and the probing was repeated until indentations ceased to be visible . the time used from the start of mixing to this point was recorded . if the results differed by more than 5% , the test was repeated . the amount of 0.5 ml of each sealer tested was placed on a glass plate ( 10x10x3 mm ) using a graduated disposable 3-ml syringe . at 1805 seconds after the onset of mixing , another plate with a mass of 20 + 2 g and a load of 100 g ten min after mixing the cement , the load was removed and the major and minor diameters of the compressed discs were measured using a digital calliper with a resolution of 0.01 mm ( mitutoyo mti corporation , tokyo , japan ) . if both measurements were consistent to within 1 mm , the results were recorded . if the major and minor diameter discs were not uniformly circular or did not match within 1 mm , the test was repeated . five acrylic plates ( 2.2 cm x 4.5 cm x 1 mm ) , containing four wells measuring 1 mm in depth and 5 mm in diameter , were prepared and placed over a glass plate covered by cellophane sheet . in group i , the freshly mixed sealer was introduced into the wells using a syringe , to avoid the formation of bubbles . in groups ii , iii the respective material applicators were used to fill the wells . another glass plate covered with cellophane each plate was kept in an incubator ( 37c , 95% relative humidity ) for a period corresponding to three times the setting time . each well was filled with one of the sealers , following a sequence according to the setting time of the material , from the longest to the shortest , so that the samples would be ready for radiographic evaluation after the final setting of all materials . each one of the acrylic plates containing the root filling materials was positioned , at the time of the radiographic exposure , alongside to another acrylic plate ( 1.3 cm x 4.5 cm x 1 mm ) , containing an aluminum stepwedge , made of 1100 alloy , with the thickness varying from 1 to 10 mm , in uniform steps of 1 mm each . this set of acrylic plates was placed in front of this phosphor plate and a digital radiograph was taken ( digora system , soredex orion corporation , helsinki , finland ) . care was taken to place the samples next to the aluminum step wedge and in the middle of the phosphor plate . radiographic images were obtained using the spectro 70x x - ray machine ( dabi atlante , ribeiro preto , sp , brazil ) , at 70 kvp and 8 ma . the object - to - focus distance was 30 cm and the exposure time was 0.2 s. exposed imaging plates of the test samples were immediately scanned after exposure ( digora scanner ) and analyzed using digora for windows 5.1 software . five teflon moulds , prepared for the production of 3.58-mm high cylindrical test bodies measuring 3 mm in diameter , were placed on a glass plate wrapped with a fine cellophane sheet . the moulds were filled with a slight excess of freshly mixed sealers and a microscope slide , also wrapped in cellophane , was pressed onto the upper surface of the mould . the assembled group was kept firmly joined with a c - shaped clamp and transferred to an incubator ( 37c , 95% relative humidity ) left to stand for a period corresponding to three times the setting time . after this period , the flat ends of the moulds , containing the samples , were grinded with 600 grit wet sandpaper . the samples were removed from the mould , measured with a digital caliper , stored in a 50-ml vessel containing 2.24 ml of distilled and deionized water , and kept in an incubator ( 37c , 95% relative humidity ) for 30 days . the sample was then removed from the container , blotted dry on absorbent paper , and measured again for length . the percentage of the dimensional alterations was calculated using the equation : l30ll100 where l30 is the length of the sample after 30 days of storage and l is the initial length of the sample . a 1.5-mm - thick cylindrical teflon ( polytetrafluoroethylene , dupont , habia , knivsta , sweden ) mould measuring 7.75 mm in inner diameter was filled with freshly mixed sealers . the mould was supported by a larger glass plate and covered with a cellophane sheet . an impermeable nylon thread was placed inside the material and another glass plate , also covered with cellophane film , was positioned on the mould and pressed manually in such a way that the plates touched the entire mould in a uniform manner . the assembly was placed in an incubator ( 37c , 95% relative humidity ) and left to stand for a period corresponding to three times the setting time . as soon as the samples were removed from the mould , they were weighed three times each in an analytical balance ( hm-200 ; a&d engineering inc . , the samples were suspended by nylon thread and placed two - by - two inside a plastic vessel with a wide opening containing 7.5 ml of distilled and deionized water , taking care to avoid any contact between them and the inner surface of the container . the containers were sealed and left for 7 days in an incubator ( 37c , 95% relative humidity ) . after this period , the samples were removed from the containers , rinsed with distilled and deionized water , blotted dry with absorbent paper , and placed in a dehumidifier for 24 h. afterwards , they were weighed again . the weight loss of each sample , expressed as percentage of the original mass ( m%=mi - mf ) , was taken as the solubility of the sealer . the solution in which the specimens were immersed was analyzed by atomic absorption spectrometry ( perkin elmer instruments gmbh , berlingen , germany ) to quantify the levels of na , k , ni and zn ions released in the solution . merck solutions with 1000 mg / l concentration ( merck , darmstadt , germany ) were used to prepare the standard solution of the different metals . metals ' analytical curves were obtained from appropriate dilutions of each respective stock solution . this spectrophotometer is supplied with hollow cathode lamps with different light spectrums exclusively for measuring metallic ions . the obtained solutions were sprayed into the atomic absorption spectrophotometer for ions quantification . the arithmetic mean was done and considered as the results of the concentration of na , k , zn and ni , expressed as g / ml . five specimens from each group were tested and the means were statistically compared . the kolmogorov - smirnov showed that the results were consistent with a normal distribution curve . the parametric statistical analysis was performed by one - way anova and tukey - kramer post - hoc test ) and the significance level was set as 5% ( graphpad instat , graphpad software inc . , san diego , ca , usa ) . table 1 shows the mean values and standard deviations for the physicochemical properties ( setting time , flow , radiopacity , dimensional change following setting and solubility ) of the tested sealers . mean values and standard deviation of the physiochemical properties for each sealer same letters indicate statistical similarity ( p>0.05 ) nh : not set ; np : not performed the ansi / ada specification requires that the setting time of a sealer shall be within 10% of that stated by the manufacturers . in this study , ah plus and apexit plus the manufacturers of endomthasone and endofill do not mention the setting time of these sealers and polifil is still in experimental phase . statistical analysis demonstrated that all sealers displayed mean values statistically different among themselves ( p<0.05 ) and endomthasone and endofill presented , respectively , the longest and the shortest setting time . the sealapex did not set after 168 h and , for this reason , it was not subjected to the other tests . the ansi / ada specification requires that a sealer shall have a diameter of no less than 20 mm and all groups of this study conformed to the standards . statistical analysis showed that endofill and apexit plus presented the highest mean values ( p<0.05 ) and were statistically similar between themselves . ah plus and polifil presented intermediate values and were statistically similar between themselves ( p>0.05 ) . all materials showed radiopacity above the 3 mm aluminum recommended by ansi / ada specification 57 ( 2000 ) . ah plus presented higher values statistically similar to endomthasone ( p>0.05 ) and significantly different from the other groups ( p<0.05 ) , . endomthasone showed intermediate values , statistically similar to the other sealers ( p>0.05 ) . ansi / ada states that the maximum limit is 1% for linear shrinkage and 0.1% for expansion . the dimensional change of all sealers was greater than the values considered acceptable by ansi / ada . ah plus , polifil and endomthasone were statistically similar among themselves ( p>0.05 ) and were different from the other materials ( p<0.05 ) . apexit plus and endofill presented the lowest mean values and were statistically similar among themselves ( p>0.05 ) . a root canal sealer should not exceed 3% by mass when the solubility of the set material is tested . all sealers are in agreement with ansi / ada standards . statistical analysis showed that sealers were statistically similar among themselves ( p>0.05 ) . the distilled and deionized water used for the solubility test was analyzed by atomic absorption spectrometry . the amount of ions released in the immersion solution , in each group , is displayed in table 2 . the concentration of na ions was higher for apexit plus , followed by ah plus , endomthasone and endofill . the concentration of na ions was not determined for polifil sealer because , even after successive dilutions , the concentration of this ion in the immersion solution was much higher than the concentration of the most concentrated pattern of the analytical curve . for k ions , the release was higher in endomthasone , followed by apexit plus , ah plus , polifil and endofill . regarding zn , the highest release was verified for endofill , followed by endomthasone and polifil . for niions , all groups presented concentration inferior to 0.6 mg / l . metallic ions concentration ( mg / l ) found in the immersion liquid of the solubility test the ansi / ada specification requires that the setting time of a sealer shall be within 10% of that stated by the manufacturers . in this study , ah plus and apexit plus the manufacturers of endomthasone and endofill do not mention the setting time of these sealers and polifil is still in experimental phase . statistical analysis demonstrated that all sealers displayed mean values statistically different among themselves ( p<0.05 ) and endomthasone and endofill presented , respectively , the longest and the shortest setting time . the sealapex did not set after 168 h and , for this reason , it was not subjected to the other tests . the ansi / ada specification requires that a sealer shall have a diameter of no less than 20 mm and all groups of this study conformed to the standards . statistical analysis showed that endofill and apexit plus presented the highest mean values ( p<0.05 ) and were statistically similar between themselves . ah plus and polifil presented intermediate values and were statistically similar between themselves ( p>0.05 ) . all materials showed radiopacity above the 3 mm aluminum recommended by ansi / ada specification 57 ( 2000 ) . ah plus presented higher values statistically similar to endomthasone ( p>0.05 ) and significantly different from the other groups ( p<0.05 ) , . endomthasone showed intermediate values , statistically similar to the other sealers ( p>0.05 ) . ansi / ada states that the maximum limit is 1% for linear shrinkage and 0.1% for expansion . the dimensional change of all sealers was greater than the values considered acceptable by ansi / ada . ah plus , polifil and endomthasone were statistically similar among themselves ( p>0.05 ) and were different from the other materials ( p<0.05 ) . apexit plus and endofill presented the lowest mean values and were statistically similar among themselves ( p>0.05 ) . a root canal sealer should not exceed 3% by mass when the solubility of the set material is tested . all sealers are in agreement with ansi / ada standards . statistical analysis showed that sealers were statistically similar among themselves ( p>0.05 ) . the distilled and deionized water used for the solubility test was analyzed by atomic absorption spectrometry . the amount of ions released in the immersion solution , in each group , is displayed in table 2 . the concentration of na ions was higher for apexit plus , followed by ah plus , endomthasone and endofill . the concentration of na ions was not determined for polifil sealer because , even after successive dilutions , the concentration of this ion in the immersion solution was much higher than the concentration of the most concentrated pattern of the analytical curve . for k ions , the release was higher in endomthasone , followed by apexit plus , ah plus , polifil and endofill . regarding zn , the highest release was verified for endofill , followed by endomthasone and polifil . for niions , all groups presented concentration inferior to 0.6 mg / l . metallic ions concentration ( mg / l ) found in the immersion liquid of the solubility test the properties of root canal sealers can be divided into the following categories : physicochemical , antimicrobial , and biological . when studying the ideal properties of a filling material , it is possible to establish research parameters for the development of new products and to evaluate those already existing on the market , thus achieving better clinical results in clinical practice . the physiochemical tests conducted in this study were based on the specification 57 of ansi / ada , following the modifications proposed by carvalho - jnior , et al . ( 2007 ) , who suggested a reduction of 80% in volume of the test samples dimensions , aiming to contribute to the rational use of endodontic materials without affecting the results . the images were obtained using the digora digital system ( digora software for windows ) , which requires a shorter exposure time and the operating system captures , processes , stores and measures the images . it also presents a scale that achieves 256 gray levels . according to the ansi / ada , the setting time of a sealer should vary only 10% in relation to the established by the manufacturer . in the present study , ah plus and apexit plus are in agreement with ansi / ada standards . the manufacturers of endomthasone and endofill do not mention the setting time of these sealers and polifil is still in experimental phase , thus , no setting time has been defined yet . the setting time is primarily a control test on the stable behavior of a product and is dependent on the sealer components , particle size , room temperature , and relative humidity . the chemical agents used to promote the radiopacity of endomthasone ( lead oxide and bismuth subnitrate ) and ah plus ( zirconium oxide and calcium tungstate ) can be responsible for the longer setting time , since these radiopacifying agents have low solubility in water . apexit plus sealer has calcium oxide in its composition , which is converted into calcium hydroxide when in contact with water , which delay the setting process due to the inclusion of another chemical reaction . sealapex did not set in the experimental phase of this study ( 1 week - period ) , which is in agreement with previous reports . the short setting time was reached by endofill , probably due to the absence of calcium hydroxide and the presence of a single radiopacifying agent on its composition . ansi / ada establishes as 20 mm the acceptable minimum value for the diameter of the disc formed by the sealer . in this study it is possible to attribute endofill 's higher values in the flow test to the presence of hydrogenated resin on its formula . for apexit plus , it is important to highlight the presence of epoxy resin , probably responsible for the increase of sealer viscosity . for polifil , it can be speculated that the physiochemical characteristics of the vegetal polymer favored the flow property . the lower values achieved by endomthasone could be attributed to the absence of hydrogenated resin or epoxy resin on its composition . root canal sealers should also have sufficient radiopacity to allow for a clear distinction between the materials and surrounding anatomic structures and to facilitate the evaluation of the quality of the root fillings , which can be undertaken only through radiographic examination . ansi / ada establishes that the radiopacity should be larger or equal of 3 mm of aluminum . all materials of the present study were in accordance with the ansi / ada specification . the difference in the results can be attributed to the radiopacifying agents present in the sealers . dimensional change demonstrates , in percentage , the shrinkage or expansion of the material following setting . ansi / ada states that the maximum limit is 1% for linear shrinkage and 0.1% for expansion . the dimensional change of all tested sealers was greater than the values considered acceptable by ansi / ada . ah plus , endomthasone and polifil presented expansion , while apexit plus and endofill presented contraction . in other literature reports , expansion was also verified for ah plusand endofill . polifil also presented expansion possibly due to water sorption by its components ( zinc oxide and calcium carbonate ) after polymerization . however , this is an experimental sealer and , consequently , the mechanism related to water sorption and diffusion in the matrix is still not completely elucidated . the contraction presented by apexit plus can be attributed to its high solubility , which affected the dimensional stability of the sealer . endofill also presented contraction and it lost an equivalent amount of zinc as that of endomthasone . the zinc found in the immersion solution can be related to the continuous loss of eugenol from the matrix . ansi / ada establishes that solubility of sealers should not exceed 3% by mass . solubility results of all groups were within ansi / ada recommendations . ah plus , apexit plus and endofill had solubilization with mass loss . previous studies of solubility only found material loss by solubilization of the sealer structure . in this study , the sealers were statistically similar regarding solubility . however , ah plus presented the lowest mean values , probably due to the presence of hema in its composition . the highest solubility was presented by endofill due to the continuous loss of eugenol from the sealer matrix . the analysis of the immersion solutions revealed that overall the sealers presented low levels of metals concentration . the amount ni ions found was below the level required for quantification , which is a good result . zn levels were higher for endomthasone and endofill , which is an expected result since they are zinc oxide - eugenol - based sealers . further studies should aim for better understanding of physical , mechanical and chemical properties of the endodontic sealers , supporting researchers and clinicians to determine their ideal applicability in specific clinical procedures . based on the results of this laboratory research , it may be concluded that : ah plus , apexit plus and endofill sealers are in accordance with ansi / ada standards . endomthasone 's manufacturer did not mention the setting time , polifil is an experimental sealer and sealapex did not set ; all tested sealers were in accordance with ansi / ada for flow , radiopacity and solubility ; the dimensional change of all sealers did not fulfill the ansi / ada requirements .

objectiveto assess the setting time ( st ) , flow ( fl ) , radiopacity ( rd ) , solubility ( sb ) and dimensional change following setting ( dc ) of different sealers ( ah plus , polifil , apexit plus , sealapex , endomthasone and endofill according to american national standards institute / american dental association ( ansi / ada ) specification 57 . material and methodsfive samples of each material were used for each test . for st , cast rings were filled with sealers and tested with a gilmore needle . for fl , the sealer was placed on a glass plate . after 180 s , another plate with 20 g and a load of 100 g were applied on the material , and the diameters of the discs formed were measured . in rd , circular molds were filled with the sealers , radiographed and analyzed using digora software . for sb , circular molds were filled with the sealers , a nylon thread was placed inside the material and another glass plate was positioned on the set , pressed and stored at 37c . samples were weighed , placed in water , dried and reweighed . the water used for sb was analyzed by atomic absorption spectrometry . for dc , circular molds were filled with the sealers , covered by glass plates and stored at 37c . samples were measured and stored in water for 30 days . after this period , they were dryed and measured again . resultsregarding st , ah plus , apexit and endofil sealers are in accordance with ansi / ada standards . endomthasone 's manufacturer did not mention the st ; polifil is an experimental sealer and sealapex did not set . considering rd , sb and dc , all sealers were in accordance with ansi / ada . the spectrometric analysis showed that a significant amount of k+ and zn2 + ions was released from apexit plus and endofill , respectively . conclusionexcept for dc , all other physicochemical properties of the tested sealers conformed to ansi / ada requirements .

INTRODUCTION MATERIAL AND METHODS Setting time Flow test Radiopacity test Dimensional change following setting Solubility Statistical analysis RESULTS Setting time Flow test Radiopacity test Dimensional change after setting Solubility DISCUSSION CONCLUSIONS

among these products , apexit plus ( ivoclar , vivadent , frstentum , schaan , liechtenstein ) and sealapex ( sybron - endo , glendora , ca , usa ) are available in the market in a paste - paste presentation . in 1983 , the american national standards institute / american dental association ( ansi / ada ) released a series of norms and tests ( named specification 57 ) to evaluate the physiochemical properties of endodontic sealers , aiming at standardizing the tests and promote larger scientific quality in the researches . this specification was revised in 2000 and it includes the following tests : film thickness , setting time , flow , radiopacity , solubility and dimensional change following setting . due to the diversity of composition of the available sealers and considering the ansi / ada standards , the objective of this study was to evaluate in vitro the physiochemical properties of sealers of different bases : endofill and endomthasone ( zinc oxide and eugenol ) , apexit plus and sealapex ( calcium hydroxide ) , polifil ( ricinus communis polymer ) and ah plus ( epoxy resin ) , in order to contribute to the selection of the appropriate material for clinical practice . setting time , flow , radiopacity , solubility , and dimensional change after setting for ah plus , polifil , apexit plus , sealapex , endomthasone and endofill sealers were measured according to ansi / ada standards for dental root canal sealing materials by one examiner blinded to the identification of the materials . ah plus , iii and iv are presented as two pastes and , for each tested sample , 15 mm of the sealer ( 1:1 ) was dispensed onto a mixing pad and mixed until obtaining a homogeneous mixture . in group ii ( polifil ) , 0.18 ml of the liquid was mixed to 0.44 ml of the paste , measure through a graduate syringe and the mixture was mixed on a glass plate . for endomthasone and endofill , 3 drops of the liquid and a portion of the powder were released in glass plate and mixed in a gradual way until getting the ideal consistence , characterized by a cement thread of approximately 2 cm , without falling during 15 s. for the physicochemical tests , the arithmetic mean of five replicates for each sealer was recorded and considered as the result of the test . the external borders of the moulds were fixed with wax on a glass plate ( 75x25x1 mm ) . the moulds were filled with the material and transferred to a chamber with 95% relative humidity and a temperature of 37c . after 15010 s from the onset of mixing , a gilmore - type needle with a mass of 1000.5 g having a flat end of 2.00.1 mm in diameter was carefully lowered vertically onto the horizontal surface of the testing sample . the amount of 0.5 ml of each sealer tested was placed on a glass plate ( 10x10x3 mm ) using a graduated disposable 3-ml syringe . at 1805 seconds after the onset of mixing , another plate with a mass of 20 + 2 g and a load of 100 g were carefully applied on top of the material . ten min after mixing the cement , the load was removed and the major and minor diameters of the compressed discs were measured using a digital calliper with a resolution of 0.01 mm ( mitutoyo mti corporation , tokyo , japan ) . five acrylic plates ( 2.2 cm x 4.5 cm x 1 mm ) , containing four wells measuring 1 mm in depth and 5 mm in diameter , were prepared and placed over a glass plate covered by cellophane sheet . another glass plate covered with cellophane each plate was kept in an incubator ( 37c , 95% relative humidity ) for a period corresponding to three times the setting time . each well was filled with one of the sealers , following a sequence according to the setting time of the material , from the longest to the shortest , so that the samples would be ready for radiographic evaluation after the final setting of all materials . each one of the acrylic plates containing the root filling materials was positioned , at the time of the radiographic exposure , alongside to another acrylic plate ( 1.3 cm x 4.5 cm x 1 mm ) , containing an aluminum stepwedge , made of 1100 alloy , with the thickness varying from 1 to 10 mm , in uniform steps of 1 mm each . the object - to - focus distance was 30 cm and the exposure time was 0.2 s. exposed imaging plates of the test samples were immediately scanned after exposure ( digora scanner ) and analyzed using digora for windows 5.1 software . five teflon moulds , prepared for the production of 3.58-mm high cylindrical test bodies measuring 3 mm in diameter , were placed on a glass plate wrapped with a fine cellophane sheet . the moulds were filled with a slight excess of freshly mixed sealers and a microscope slide , also wrapped in cellophane , was pressed onto the upper surface of the mould . after this period , the flat ends of the moulds , containing the samples , were grinded with 600 grit wet sandpaper . the samples were removed from the mould , measured with a digital caliper , stored in a 50-ml vessel containing 2.24 ml of distilled and deionized water , and kept in an incubator ( 37c , 95% relative humidity ) for 30 days . an impermeable nylon thread was placed inside the material and another glass plate , also covered with cellophane film , was positioned on the mould and pressed manually in such a way that the plates touched the entire mould in a uniform manner . the assembly was placed in an incubator ( 37c , 95% relative humidity ) and left to stand for a period corresponding to three times the setting time . as soon as the samples were removed from the mould , they were weighed three times each in an analytical balance ( hm-200 ; a&d engineering inc . , the samples were suspended by nylon thread and placed two - by - two inside a plastic vessel with a wide opening containing 7.5 ml of distilled and deionized water , taking care to avoid any contact between them and the inner surface of the container . after this period , the samples were removed from the containers , rinsed with distilled and deionized water , blotted dry with absorbent paper , and placed in a dehumidifier for 24 h. afterwards , they were weighed again . the weight loss of each sample , expressed as percentage of the original mass ( m%=mi - mf ) , was taken as the solubility of the sealer . the solution in which the specimens were immersed was analyzed by atomic absorption spectrometry ( perkin elmer instruments gmbh , berlingen , germany ) to quantify the levels of na , k , ni and zn ions released in the solution . the external borders of the moulds were fixed with wax on a glass plate ( 75x25x1 mm ) . the moulds were filled with the material and transferred to a chamber with 95% relative humidity and a temperature of 37c . after 15010 s from the onset of mixing , a gilmore - type needle with a mass of 1000.5 g having a flat end of 2.00.1 mm in diameter was carefully lowered vertically onto the horizontal surface of the testing sample . the amount of 0.5 ml of each sealer tested was placed on a glass plate ( 10x10x3 mm ) using a graduated disposable 3-ml syringe . at 1805 seconds after the onset of mixing , another plate with a mass of 20 + 2 g and a load of 100 g ten min after mixing the cement , the load was removed and the major and minor diameters of the compressed discs were measured using a digital calliper with a resolution of 0.01 mm ( mitutoyo mti corporation , tokyo , japan ) . five acrylic plates ( 2.2 cm x 4.5 cm x 1 mm ) , containing four wells measuring 1 mm in depth and 5 mm in diameter , were prepared and placed over a glass plate covered by cellophane sheet . another glass plate covered with cellophane each plate was kept in an incubator ( 37c , 95% relative humidity ) for a period corresponding to three times the setting time . each well was filled with one of the sealers , following a sequence according to the setting time of the material , from the longest to the shortest , so that the samples would be ready for radiographic evaluation after the final setting of all materials . each one of the acrylic plates containing the root filling materials was positioned , at the time of the radiographic exposure , alongside to another acrylic plate ( 1.3 cm x 4.5 cm x 1 mm ) , containing an aluminum stepwedge , made of 1100 alloy , with the thickness varying from 1 to 10 mm , in uniform steps of 1 mm each . the object - to - focus distance was 30 cm and the exposure time was 0.2 s. exposed imaging plates of the test samples were immediately scanned after exposure ( digora scanner ) and analyzed using digora for windows 5.1 software . five teflon moulds , prepared for the production of 3.58-mm high cylindrical test bodies measuring 3 mm in diameter , were placed on a glass plate wrapped with a fine cellophane sheet . the moulds were filled with a slight excess of freshly mixed sealers and a microscope slide , also wrapped in cellophane , was pressed onto the upper surface of the mould . after this period , the flat ends of the moulds , containing the samples , were grinded with 600 grit wet sandpaper . the samples were removed from the mould , measured with a digital caliper , stored in a 50-ml vessel containing 2.24 ml of distilled and deionized water , and kept in an incubator ( 37c , 95% relative humidity ) for 30 days . an impermeable nylon thread was placed inside the material and another glass plate , also covered with cellophane film , was positioned on the mould and pressed manually in such a way that the plates touched the entire mould in a uniform manner . the assembly was placed in an incubator ( 37c , 95% relative humidity ) and left to stand for a period corresponding to three times the setting time . as soon as the samples were removed from the mould , they were weighed three times each in an analytical balance ( hm-200 ; a&d engineering inc . , the samples were suspended by nylon thread and placed two - by - two inside a plastic vessel with a wide opening containing 7.5 ml of distilled and deionized water , taking care to avoid any contact between them and the inner surface of the container . after this period , the samples were removed from the containers , rinsed with distilled and deionized water , blotted dry with absorbent paper , and placed in a dehumidifier for 24 h. afterwards , they were weighed again . the weight loss of each sample , expressed as percentage of the original mass ( m%=mi - mf ) , was taken as the solubility of the sealer . the solution in which the specimens were immersed was analyzed by atomic absorption spectrometry ( perkin elmer instruments gmbh , berlingen , germany ) to quantify the levels of na , k , ni and zn ions released in the solution . table 1 shows the mean values and standard deviations for the physicochemical properties ( setting time , flow , radiopacity , dimensional change following setting and solubility ) of the tested sealers . mean values and standard deviation of the physiochemical properties for each sealer same letters indicate statistical similarity ( p>0.05 ) nh : not set ; np : not performed the ansi / ada specification requires that the setting time of a sealer shall be within 10% of that stated by the manufacturers . in this study , ah plus and apexit plus the manufacturers of endomthasone and endofill do not mention the setting time of these sealers and polifil is still in experimental phase . statistical analysis demonstrated that all sealers displayed mean values statistically different among themselves ( p<0.05 ) and endomthasone and endofill presented , respectively , the longest and the shortest setting time . the ansi / ada specification requires that a sealer shall have a diameter of no less than 20 mm and all groups of this study conformed to the standards . statistical analysis showed that endofill and apexit plus presented the highest mean values ( p<0.05 ) and were statistically similar between themselves . the dimensional change of all sealers was greater than the values considered acceptable by ansi / ada . ah plus , polifil and endomthasone were statistically similar among themselves ( p>0.05 ) and were different from the other materials ( p<0.05 ) . all sealers are in agreement with ansi / ada standards . the distilled and deionized water used for the solubility test was analyzed by atomic absorption spectrometry . the concentration of na ions was higher for apexit plus , followed by ah plus , endomthasone and endofill . for k ions , the release was higher in endomthasone , followed by apexit plus , ah plus , polifil and endofill . metallic ions concentration ( mg / l ) found in the immersion liquid of the solubility test the ansi / ada specification requires that the setting time of a sealer shall be within 10% of that stated by the manufacturers . in this study , ah plus and apexit plus the manufacturers of endomthasone and endofill do not mention the setting time of these sealers and polifil is still in experimental phase . statistical analysis demonstrated that all sealers displayed mean values statistically different among themselves ( p<0.05 ) and endomthasone and endofill presented , respectively , the longest and the shortest setting time . the ansi / ada specification requires that a sealer shall have a diameter of no less than 20 mm and all groups of this study conformed to the standards . statistical analysis showed that endofill and apexit plus presented the highest mean values ( p<0.05 ) and were statistically similar between themselves . the dimensional change of all sealers was greater than the values considered acceptable by ansi / ada . ah plus , polifil and endomthasone were statistically similar among themselves ( p>0.05 ) and were different from the other materials ( p<0.05 ) . all sealers are in agreement with ansi / ada standards . the distilled and deionized water used for the solubility test was analyzed by atomic absorption spectrometry . the concentration of na ions was higher for apexit plus , followed by ah plus , endomthasone and endofill . for k ions , the release was higher in endomthasone , followed by apexit plus , ah plus , polifil and endofill . the physiochemical tests conducted in this study were based on the specification 57 of ansi / ada , following the modifications proposed by carvalho - jnior , et al . the images were obtained using the digora digital system ( digora software for windows ) , which requires a shorter exposure time and the operating system captures , processes , stores and measures the images . according to the ansi / ada , the setting time of a sealer should vary only 10% in relation to the established by the manufacturer . in the present study , ah plus and apexit plus are in agreement with ansi / ada standards . the manufacturers of endomthasone and endofill do not mention the setting time of these sealers and polifil is still in experimental phase , thus , no setting time has been defined yet . the setting time is primarily a control test on the stable behavior of a product and is dependent on the sealer components , particle size , room temperature , and relative humidity . the chemical agents used to promote the radiopacity of endomthasone ( lead oxide and bismuth subnitrate ) and ah plus ( zirconium oxide and calcium tungstate ) can be responsible for the longer setting time , since these radiopacifying agents have low solubility in water . sealapex did not set in the experimental phase of this study ( 1 week - period ) , which is in agreement with previous reports . ansi / ada establishes as 20 mm the acceptable minimum value for the diameter of the disc formed by the sealer . all materials of the present study were in accordance with the ansi / ada specification . dimensional change demonstrates , in percentage , the shrinkage or expansion of the material following setting . the dimensional change of all tested sealers was greater than the values considered acceptable by ansi / ada . ah plus , endomthasone and polifil presented expansion , while apexit plus and endofill presented contraction . however , this is an experimental sealer and , consequently , the mechanism related to water sorption and diffusion in the matrix is still not completely elucidated . ah plus , apexit plus and endofill had solubilization with mass loss . based on the results of this laboratory research , it may be concluded that : ah plus , apexit plus and endofill sealers are in accordance with ansi / ada standards . endomthasone 's manufacturer did not mention the setting time , polifil is an experimental sealer and sealapex did not set ; all tested sealers were in accordance with ansi / ada for flow , radiopacity and solubility ; the dimensional change of all sealers did not fulfill the ansi / ada requirements .

it is proving difficult to promote a high - level and fair distribution of health in the world s rapidly growing urban settings.1 urban health promotion is not simply a matter of the right interventions , or even the necessary resources . urban ( and indeed global ) health depends , to an important extent , on governance , the institutions and processes through which societies manage the course of events . there is a growing literature on governance and its impact on health.25 more importantly , people throughout the world are experimenting with governance strategies to improve urban life . this paper draws on a thematic review of urban governance innovation prepared for the knowledge network on urban settings of the who commission on the social determinants of health ( csdh).6 here we describe the concept of governance , highlight exemplary innovations and describe strategies urban governors can use to maximize their influence on the national and international decisions that structure urban life . we conclude with some observations on the limitations of local governance strategies and topics for further study . governance is the management of the course of events in a social system.7 in the urban setting , it is the sum of the many ways individuals and institutions , public and private , plan and manage the common affairs of the city.8 responsible , capable and fair public institutions continue to be vital to good governance , but governance can not be understood as the work only of government.9 governance is polycentric , distributed among multiple organizations exercising diverse forms of power . states do not enjoy a monopoly on governance , and themselves are often governed by non - state actors.10,11 governance thus comprises both institutions and procedures formalized in constitutions and laws , and the many institutions corporations , ngo coalitions and methods of power bribery , media campaigns that are only obscured by equating governance with government . students of governance increasingly emphasize complexity , both of contemporary social and economic systems and of the governance networks that have emerged to manage them . governance is understood not as an orderly , stable political science but as an art , a dynamic process of collective social improvisation in which a plethora of actors are striving to organize matters for their own advantage . any given contemporary governance system may be inefficient , corrupt or unresponsive to the needs of the governed . aside from efficiency in delivering good results , good governance is commonly defined in terms of practical virtues rooted in human rights and the principle that governors derive their authority from the people . these include participation , fairness , decency ( the degree to which rules are formed and implemented without humiliating or harming particular groups of people ) , accountability , sustainability and transparency.8 governance has always developed as an adaptation to social conditions . this process of adaptation has produced some marvelous and durable institutions , practices and norms , but it can not be expected that governance solutions devised in 1648 or 1787 or even 1948 will in every case be adequate to the conditions of the present.12 even the best forms of governance can be worn down by the unrelenting efforts of competing factions to game or capture the system.7 leading thinkers on governance have criticized the tendency to treat past ways of delivering good government as the only possible forms , emphasizing the need for democratic experimentalism and institutional innovation.1113 innovation is crucial because the prevailing design principles of democratic governance like separation of powers , or even a written constitutionare losing their vitality.12 real governance may so differ from the formal rules that the formal local government decision - making process may be largely irrelevant to what actually happens.14 for some , taking complexity seriously means embracing the polycentric character of governance and designing governance systems that emphasize social learning and coordination rather than centralized , top - down management.11,15 one of the most important consequences of recognizing governance beyond government is the possibility of applying the norms that have traditionally constrained only states to powerful private governors . in a world in which multinational corporations and wealthy foundations may wield as much power over certain decisions as governments , people are beginning to ask whether and how private governors can be required to be fair , transparent and accountable.16 governance is a necessary consideration in any program to understand and influence the social determinants of health.2,4 in the csdh s view , the level and distribution of health in a population depend on social and environmental factors operating at various levels of social organization and unfolding over time.17 governance reflects social structure and acts as one of the social mechanisms that sort people to unequal health outcomes by upholding existing distributions of resources like power , money and knowledge.18 those with the power to shape events in the community are able to organize matters in ways that benefit them and externalize undesirable effects on those less able to exert influence.19 participation in governance is at once a function and a catalyst of people s empowerment , and can therefore be seen as a pathway linking autonomy and social engagement to health.20 although data on the link between politics , policy and health are limited , there is some evidence that countries controlled by political parties with more egalitarian ideologies tend to have more economically redistributive policies and more equitable health outcomes.5 there is also evidence suggesting that participation in governance may be healthy for individuals and communities.21 our paper for the csdh examined cases of governance innovation in areas like policing , sanitation and housing . we divided what we saw into two categories , reinventing government and reinventing governance . reinventing government involves efforts to recalibrate traditional state institutions and practices to improve their capacity , often paradoxically by ceding the implementation of policy to non - state actors through devices like governance partnerships , self - regulation , and the use of markets as regulatory tools ( for example , environmental emissions trading schemes).22,23 many reinventing government schemes have been linked to a problematic neoliberal , smaller government ideology , and experience with measures like the privatization of water and sewer systems has not been entirely happy.24 we also found many instances of government reinvention that seemed to promote good government and healthy public policies . participatory public expenditure management ( ppem ) is an excellent example.25 over the last two decades , participatory budgeting in which citizens take part in budget planning , overseeing public expenditures , and monitoring delivery of goods and services has grown from a brazilian experiment to an international model for socially accountable urban governance.26 the reform of land - tenure registration systems is another example . uncertainty about ownership and tenure deprives poor people of the opportunity to convert property into capital for investment or home improvement , and undermines the incentive to improve housing stock and neighborhood amenities.27 reforms in property governance have simplified title registration systems and zoning rules.28 similar efforts have been made to eliminate bureaucratic barriers to small business entrepreneurship.29 reinventing governance aims more broadly to mobilize governors with little or no connection to the state.10,12,13 innovation has entailed developing new institutions and new tools of governance that can be placed at the disposal of stakeholders acting independently of or even in competition with traditional government institutions . these typically involve ngos deploying soft strategies like information sharing and shaming ( see , for example , the people s health movement s alternative global health report30 ) but also harder market strategies like voluntary product certification schemes.31 our review found many successful examples in the urban setting . community organizations have performed well in developing water and sewer systems , garbage collection , and local health promotion programs.3234 indeed , the world development report in 2006 called community participation probably the biggest influence on the success or failure of public sanitation facilities.35 unsatisfactory results from a thai government housing improvement program in the 1990s led to the creation of an independent public agency , the community organizations development institute ( codi ) . codi has a partnership structure , with a board of government and civil society representatives , but works primarily through organizations and networks in the target communities that plan and implement housing upgrades in their own neighborhoods . as of the end of 2004 , upgrading programs were proceeding on this model in 175 communities involving more than 14,000 households.36 the landscape of governance is littered with governance deficits , gaps between people s stake in governance and their access to governance institutions.37 many governance innovators have focused on developing models of governance that ensure that people have substantial and equal opportunities to participate directly in decisions that affect them.7,14,38 what has been called microgovernance involves seeding communities that have been excluded from governance with small institutions around which people can mobilize their knowledge and capacity.3 in south african townships , residents provide dispute resolution and community development services that traditional state bodies were failing to deliver through a new institution called the peace committee . in india , health promotion for sex workers has been built around collectives like the durbar mahila samanwaya committee in sonagachi.39 new governance practices like these not only change how specific activities are managed , but also potentially the dynamics of the larger urban governance system . urban governors do not control many of the important determinants of urban well - being , from the amount of the national budget available to meet urban needs to the business decisions of leaders in the global economy . reinventing urban governance for health is in practical terms a project of the weak . in the urban setting , it turns on poorer residents gaining a greater share of control and resources . at the national and global levels , it turns on local governors ability to influence the upstream determinants of their situation . john braithwaite has described a set of strategies that the winners in the global game of governance have used to advantage themselves , and asked how these methods of power used by the strong might be adapted by the weak.37,40 in this section of the paper , we apply these strategies to healthy urban governance . build and rebuild institutions of governance to increase participation and effectiveness.those who have little must manage what they have well . defining the territorial and subject matter jurisdiction of metropolitan government structures is also fundamental , although decades of effort have not produced a generally applicable solution.41 new governance strategies of privatization and self - regulation can be tools of more efficient public management . ppem , community policing and other practices that increase the meaningful involvement of stakeholders have shown good results , but do not replace and indeed may depend on the vitality of governance institutions outside the government . funding ( and minimizing legal barriers on ) ngos , microgovernance initiatives and institutional innovations that devolve power to stakeholders can all increase urban governance capacity by mobilizing the resources and capacities of communities that currently have no real access to governance.network governance.once a wide range of governance institutions are active , building connections among them and with compatriots in other urban settings allows the weak to increase their resources for advocacy and upstream governance . networks like the asian coalition for housing rights and slum / shack dwellers international ( sdi ) not only take local action but also work together to support each other from community to community within cities , from city to city within nations , and internationally.42(p 2),4345 the healthy cities movement and regional health promotion networks , through which areas in close geographic proximity pool resources and expertise , exemplify the type of cooperation necessary to global health.46concentrate technical competence at network nodes.effective networks of governance typically create nodes on the network that concentrate resources and technologies for the purpose of achieving a common goal.7,47 technical expertise and the capacity to rapidly gather , interpret and respond to information helps weaker actors compete successfully with stronger ones.37 the campaign of developing countries and hiv / aids advocates to mitigate the impact of the trips agreement on access to essential medicines demonstrated the importance of organizing networks around institutions of technical competence such as medecines sans frontieres and the consumer project on technology.48 another example is the health promotion foundation , like vichealth in australia , designed as a reliably funded source of expertise and advocacy in the cause of better health governance.49focus on forums where urban governors can be creative and assertive.many powerful governing institutions and systems are not concerned about the welfare of urban settings . if weak actors engage in governance in and through organizational nodes that others , particularly rich and powerful others , have established and work through , they will typically find themselves at a disadvantage because they will find that the agendas of others have been built into nodal processes.37 forum shifting may be defined as relocating interactions ( like negotiation or regulation ) from an institution of governance in which an actor encounters resistance to one where it is likely to achieve its objective . forum shifting has been an extremely useful tool of the powerful ; it can also work for weaker players like cities and ngos . local governments are forum shifting when , for example , they file law - suits against gun makers in places where provincial and national legislatures have rejected gun - control regulation.50 ppem creates a new forum for spending decisions in which non - state actors have more authority in relation to local government officials.51 ngos like peace committees and water cooperatives also represent a forum shift : unsatisfied with the services they are getting within a scheme of government provision , they create a new service - providing institution in which they have greater control over processes and outcomes.have a responsive regulatory strategy.this is a generic best practice of power in any forum . responsive regulation entails using the least expensive and intrusive form of action needed to secure compliance.52 dialogue is more efficient than threats ; threats are more efficient than actually imposing sanctions . but as braithwaite points out , a responsive strategy is more than good technique : it has two even deeper virtues . first , it represents a way of understanding governance in a democracy based on responding to peoples problems , environments , demands . the obligation to respond to others in the society is a vital example of the relational checks and balances that constrain power in a polycentric governance system . second , responsiveness embodies the capacity for learning and the openness to new information that are characteristic of effective governance institutions.53have a big stick and threaten to use it.even responsive regulators sometimes crack down hard . urban communities are weak in relation to national governments , multinational corporations and international donors , but they are not powerless . urban governors have a variety of sticks , from networked advocacy and shaming strategies , through regulations and taxes , to debt default . weak actors have proven reasonably effective at influencing global policy not just by speaking up for the interests of poorer regions in international fora , but by taking the battle into the media and domestic politics.40 build and rebuild institutions of governance to increase participation and effectiveness . defining the territorial and subject matter jurisdiction of metropolitan government structures is also fundamental , although decades of effort have not produced a generally applicable solution.41 new governance strategies of privatization and self - regulation can be tools of more efficient public management . ppem , community policing and other practices that increase the meaningful involvement of stakeholders have shown good results , but do not replace and indeed may depend on the vitality of governance institutions outside the government . funding ( and minimizing legal barriers on ) ngos , microgovernance initiatives and institutional innovations that devolve power to stakeholders can all increase urban governance capacity by mobilizing the resources and capacities of communities that currently have no real access to governance . once a wide range of governance institutions are active , building connections among them and with compatriots in other urban settings allows the weak to increase their resources for advocacy and upstream governance . networks like the asian coalition for housing rights and slum / shack dwellers international ( sdi ) not only take local action but also work together to support each other from community to community within cities , from city to city within nations , and internationally.42(p 2),4345 the healthy cities movement and regional health promotion networks , through which areas in close geographic proximity pool resources and expertise , exemplify the type of cooperation necessary to global health.46 concentrate technical competence at network nodes . effective networks of governance typically create nodes on the network that concentrate resources and technologies for the purpose of achieving a common goal.7,47 technical expertise and the capacity to rapidly gather , interpret and respond to information helps weaker actors compete successfully with stronger ones.37 the campaign of developing countries and hiv / aids advocates to mitigate the impact of the trips agreement on access to essential medicines demonstrated the importance of organizing networks around institutions of technical competence such as medecines sans frontieres and the consumer project on technology.48 another example is the health promotion foundation , like vichealth in australia , designed as a reliably funded source of expertise and advocacy in the cause of better health governance.49 focus on forums where urban governors can be creative and assertive . many powerful governing institutions and systems are not concerned about the welfare of urban settings . if weak actors engage in governance in and through organizational nodes that others , particularly rich and powerful others , have established and work through , they will typically find themselves at a disadvantage because they will find that the agendas of others have been built into nodal processes.37 forum shifting may be defined as relocating interactions ( like negotiation or regulation ) from an institution of governance in which an actor encounters resistance to one where it is likely to achieve its objective . forum shifting has been an extremely useful tool of the powerful ; it can also work for weaker players like cities and ngos . local governments are forum shifting when , for example , they file law - suits against gun makers in places where provincial and national legislatures have rejected gun - control regulation.50 ppem creates a new forum for spending decisions in which non - state actors have more authority in relation to local government officials.51 ngos like peace committees and water cooperatives also represent a forum shift : unsatisfied with the services they are getting within a scheme of government provision , they create a new service - providing institution in which they have greater control over processes and outcomes . responsive regulation entails using the least expensive and intrusive form of action needed to secure compliance.52 dialogue is more efficient than threats ; threats are more efficient than actually imposing sanctions . but as braithwaite points out , a responsive strategy is more than good technique : it has two even deeper virtues . first , it represents a way of understanding governance in a democracy based on responding to peoples problems , environments , demands . the obligation to respond to others in the society is a vital example of the relational checks and balances that constrain power in a polycentric governance system . second , responsiveness embodies the capacity for learning and the openness to new information that are characteristic of effective governance institutions.53 have a big stick and threaten to use it . even responsive urban communities are weak in relation to national governments , multinational corporations and international donors , but they are not powerless . urban governors have a variety of sticks , from networked advocacy and shaming strategies , through regulations and taxes , to debt default . weak actors have proven reasonably effective at influencing global policy not just by speaking up for the interests of poorer regions in international fora , but by taking the battle into the media and domestic politics.40 strong local governance has its pitfalls.54,55 those with greater resources of experience , money or skill can game the local system as they can a national government.56 the voices of the poorer , weaker , more socially marginal can be ignored . urban settings often have large populations of illegal internal or international migrants whose right to participate is contested.57 urbanites do not necessarily , or even most of the time , organize themselves and vote as urban dwellers , but rather act as members of ideological or ethnic blocks organized around national political issues that may reflect and worsen divisions at the local level.14 urban governors are part of a system that works best as it approaches the ideal of a virtuous circuit ( see figure 1 ) . at any level of social organization , governing institutions require the capacity to mobilize and coordinate local resources , but from a global governance perspective , vertical coordination and participation are essential . the best of interventions organized at the global level will suffer if the national , provincial and especially the local institutions of governance are bypassed or lack the capacity for effective implementation . even the imposition of good solutions in a top down manner , without real decision - making participation by those most affected , is paternalistic and illegitimate from a democratic perspective . good governance requires the interplay of power and constraint to forestall dysfunctional phenomena such as capture . good national and international governance can be a source of norms , and a recourse for those excluded from local decision making.55 national governments provide the policy environment in which local government and governance can innovate , or not.58,59 it is not even clear that empowering urban areas leads to greater attention to urban inequities : although central governments are unlikely to be generally more pro - poor than local governments , it may be easier for central governments to insist on pro - poor use of grant resources than for local governments to use their own resources in that way.14(p 14 ) local governments typically are short not just on cash but on properly trained bureaucrats with the skills and incentives to use their power productively . the strategies we have discussed here are hardly new to local health advocates ; the problem is that lack of resources can limit efficacy at every step . improving skills in governance , widening the repertoire of strategies , will make poor urbanites more effective , but local governors also need access to the resources controlled by higher levels of governance . the promotion of innovation in healthy urban governance will benefit from a significant investment in research and practice . on the research side , more support is required for the study of the design principles or grammars of successful governance , particularly outside of and in partnership with government.60 it is even more important to fund governance entrepreneurs reinventing governance in communities around the world , and to support ongoing community processes of governance reinvention . stories for researchers , governments and ngos unless they can be replicated at a sufficient scale to influence the condition of the mass of urbanites.58,61 international funders and governments speak about the importance of good governance and strong civil society , but investment in general governance capacity , unlinked to a particular categorical program or specific objective , is still too rare . people can learn to better improvise , but no amount of research or technology transfer will turn governance from an art into a science . it will be difficult to prove that any particular form or process of governance causes urban health to improve , yet we have more than enough reasons to prefer good governance over the alternative . good governance is not just valuable for the ends it promotes , but for the process of collective cultural imagination it represents . urban settings are places where the world can be re - imagined , where efforts at reform create the context for new ideas and further action , and where new norms are formed.62,63 the world s urban settings present an enormous opportunity to define and implement healthy public policy through governance innovation .

urban health promotion is not simply a matter of the right interventions , or even the necessary resources . urban ( and indeed global ) health depends to an important extent on governance , the institutions and processes through which societies manage the course of events . this paper describes the concept of governance , distinguishing between reforms aimed at improving how government works and innovations that more fundamentally reinvent governance by developing new institutions and processes of local stakeholder control . the paper highlights strategies urban governors can use to maximize their influence on the national and international decisions that structure urban life . it concludes with some observations on the limitations of local governance strategies and the importance of establishing a virtuous circuit of governance through which urban dwellers play a greater role in the formation and implementation of policy at the national and global levels .

INTRODUCTION GOVERNANCE AND THE SOCIAL DETERMINANTS OF HEALTH THE LANDSCAPE OF GOVERNANCE INNOVATION: FROM REINVENTING GOVERNMENT TO REINVENTING GOVERNANCE INNOVATIVE STRATEGIES FOR URBAN GOVERNANCE: HEALTHY AND POWERFUL CITIES CONCLUSION: CHALLENGES OF HEALTHY URBAN GOVERNANCE

it is proving difficult to promote a high - level and fair distribution of health in the world s rapidly growing urban settings.1 urban health promotion is not simply a matter of the right interventions , or even the necessary resources . urban ( and indeed global ) health depends , to an important extent , on governance , the institutions and processes through which societies manage the course of events . there is a growing literature on governance and its impact on health.25 more importantly , people throughout the world are experimenting with governance strategies to improve urban life . this paper draws on a thematic review of urban governance innovation prepared for the knowledge network on urban settings of the who commission on the social determinants of health ( csdh).6 here we describe the concept of governance , highlight exemplary innovations and describe strategies urban governors can use to maximize their influence on the national and international decisions that structure urban life . we conclude with some observations on the limitations of local governance strategies and topics for further study . governance is the management of the course of events in a social system.7 in the urban setting , it is the sum of the many ways individuals and institutions , public and private , plan and manage the common affairs of the city.8 responsible , capable and fair public institutions continue to be vital to good governance , but governance can not be understood as the work only of government.9 governance is polycentric , distributed among multiple organizations exercising diverse forms of power . states do not enjoy a monopoly on governance , and themselves are often governed by non - state actors.10,11 governance thus comprises both institutions and procedures formalized in constitutions and laws , and the many institutions corporations , ngo coalitions and methods of power bribery , media campaigns that are only obscured by equating governance with government . students of governance increasingly emphasize complexity , both of contemporary social and economic systems and of the governance networks that have emerged to manage them . any given contemporary governance system may be inefficient , corrupt or unresponsive to the needs of the governed . aside from efficiency in delivering good results , good governance is commonly defined in terms of practical virtues rooted in human rights and the principle that governors derive their authority from the people . this process of adaptation has produced some marvelous and durable institutions , practices and norms , but it can not be expected that governance solutions devised in 1648 or 1787 or even 1948 will in every case be adequate to the conditions of the present.12 even the best forms of governance can be worn down by the unrelenting efforts of competing factions to game or capture the system.7 leading thinkers on governance have criticized the tendency to treat past ways of delivering good government as the only possible forms , emphasizing the need for democratic experimentalism and institutional innovation.1113 innovation is crucial because the prevailing design principles of democratic governance like separation of powers , or even a written constitutionare losing their vitality.12 real governance may so differ from the formal rules that the formal local government decision - making process may be largely irrelevant to what actually happens.14 for some , taking complexity seriously means embracing the polycentric character of governance and designing governance systems that emphasize social learning and coordination rather than centralized , top - down management.11,15 one of the most important consequences of recognizing governance beyond government is the possibility of applying the norms that have traditionally constrained only states to powerful private governors . in a world in which multinational corporations and wealthy foundations may wield as much power over certain decisions as governments , people are beginning to ask whether and how private governors can be required to be fair , transparent and accountable.16 governance is a necessary consideration in any program to understand and influence the social determinants of health.2,4 in the csdh s view , the level and distribution of health in a population depend on social and environmental factors operating at various levels of social organization and unfolding over time.17 governance reflects social structure and acts as one of the social mechanisms that sort people to unequal health outcomes by upholding existing distributions of resources like power , money and knowledge.18 those with the power to shape events in the community are able to organize matters in ways that benefit them and externalize undesirable effects on those less able to exert influence.19 participation in governance is at once a function and a catalyst of people s empowerment , and can therefore be seen as a pathway linking autonomy and social engagement to health.20 although data on the link between politics , policy and health are limited , there is some evidence that countries controlled by political parties with more egalitarian ideologies tend to have more economically redistributive policies and more equitable health outcomes.5 there is also evidence suggesting that participation in governance may be healthy for individuals and communities.21 our paper for the csdh examined cases of governance innovation in areas like policing , sanitation and housing . reinventing government involves efforts to recalibrate traditional state institutions and practices to improve their capacity , often paradoxically by ceding the implementation of policy to non - state actors through devices like governance partnerships , self - regulation , and the use of markets as regulatory tools ( for example , environmental emissions trading schemes).22,23 many reinventing government schemes have been linked to a problematic neoliberal , smaller government ideology , and experience with measures like the privatization of water and sewer systems has not been entirely happy.24 we also found many instances of government reinvention that seemed to promote good government and healthy public policies . participatory public expenditure management ( ppem ) is an excellent example.25 over the last two decades , participatory budgeting in which citizens take part in budget planning , overseeing public expenditures , and monitoring delivery of goods and services has grown from a brazilian experiment to an international model for socially accountable urban governance.26 the reform of land - tenure registration systems is another example . uncertainty about ownership and tenure deprives poor people of the opportunity to convert property into capital for investment or home improvement , and undermines the incentive to improve housing stock and neighborhood amenities.27 reforms in property governance have simplified title registration systems and zoning rules.28 similar efforts have been made to eliminate bureaucratic barriers to small business entrepreneurship.29 reinventing governance aims more broadly to mobilize governors with little or no connection to the state.10,12,13 innovation has entailed developing new institutions and new tools of governance that can be placed at the disposal of stakeholders acting independently of or even in competition with traditional government institutions . these typically involve ngos deploying soft strategies like information sharing and shaming ( see , for example , the people s health movement s alternative global health report30 ) but also harder market strategies like voluntary product certification schemes.31 our review found many successful examples in the urban setting . community organizations have performed well in developing water and sewer systems , garbage collection , and local health promotion programs.3234 indeed , the world development report in 2006 called community participation probably the biggest influence on the success or failure of public sanitation facilities.35 unsatisfactory results from a thai government housing improvement program in the 1990s led to the creation of an independent public agency , the community organizations development institute ( codi ) . codi has a partnership structure , with a board of government and civil society representatives , but works primarily through organizations and networks in the target communities that plan and implement housing upgrades in their own neighborhoods . as of the end of 2004 , upgrading programs were proceeding on this model in 175 communities involving more than 14,000 households.36 the landscape of governance is littered with governance deficits , gaps between people s stake in governance and their access to governance institutions.37 many governance innovators have focused on developing models of governance that ensure that people have substantial and equal opportunities to participate directly in decisions that affect them.7,14,38 what has been called microgovernance involves seeding communities that have been excluded from governance with small institutions around which people can mobilize their knowledge and capacity.3 in south african townships , residents provide dispute resolution and community development services that traditional state bodies were failing to deliver through a new institution called the peace committee . in india , health promotion for sex workers has been built around collectives like the durbar mahila samanwaya committee in sonagachi.39 new governance practices like these not only change how specific activities are managed , but also potentially the dynamics of the larger urban governance system . urban governors do not control many of the important determinants of urban well - being , from the amount of the national budget available to meet urban needs to the business decisions of leaders in the global economy . reinventing urban governance for health is in practical terms a project of the weak . in the urban setting , it turns on poorer residents gaining a greater share of control and resources . at the national and global levels , it turns on local governors ability to influence the upstream determinants of their situation . john braithwaite has described a set of strategies that the winners in the global game of governance have used to advantage themselves , and asked how these methods of power used by the strong might be adapted by the weak.37,40 in this section of the paper , we apply these strategies to healthy urban governance . build and rebuild institutions of governance to increase participation and effectiveness.those who have little must manage what they have well . defining the territorial and subject matter jurisdiction of metropolitan government structures is also fundamental , although decades of effort have not produced a generally applicable solution.41 new governance strategies of privatization and self - regulation can be tools of more efficient public management . ppem , community policing and other practices that increase the meaningful involvement of stakeholders have shown good results , but do not replace and indeed may depend on the vitality of governance institutions outside the government . funding ( and minimizing legal barriers on ) ngos , microgovernance initiatives and institutional innovations that devolve power to stakeholders can all increase urban governance capacity by mobilizing the resources and capacities of communities that currently have no real access to governance.network governance.once a wide range of governance institutions are active , building connections among them and with compatriots in other urban settings allows the weak to increase their resources for advocacy and upstream governance . networks like the asian coalition for housing rights and slum / shack dwellers international ( sdi ) not only take local action but also work together to support each other from community to community within cities , from city to city within nations , and internationally.42(p 2),4345 the healthy cities movement and regional health promotion networks , through which areas in close geographic proximity pool resources and expertise , exemplify the type of cooperation necessary to global health.46concentrate technical competence at network nodes.effective networks of governance typically create nodes on the network that concentrate resources and technologies for the purpose of achieving a common goal.7,47 technical expertise and the capacity to rapidly gather , interpret and respond to information helps weaker actors compete successfully with stronger ones.37 the campaign of developing countries and hiv / aids advocates to mitigate the impact of the trips agreement on access to essential medicines demonstrated the importance of organizing networks around institutions of technical competence such as medecines sans frontieres and the consumer project on technology.48 another example is the health promotion foundation , like vichealth in australia , designed as a reliably funded source of expertise and advocacy in the cause of better health governance.49focus on forums where urban governors can be creative and assertive.many powerful governing institutions and systems are not concerned about the welfare of urban settings . if weak actors engage in governance in and through organizational nodes that others , particularly rich and powerful others , have established and work through , they will typically find themselves at a disadvantage because they will find that the agendas of others have been built into nodal processes.37 forum shifting may be defined as relocating interactions ( like negotiation or regulation ) from an institution of governance in which an actor encounters resistance to one where it is likely to achieve its objective . forum shifting has been an extremely useful tool of the powerful ; it can also work for weaker players like cities and ngos . local governments are forum shifting when , for example , they file law - suits against gun makers in places where provincial and national legislatures have rejected gun - control regulation.50 ppem creates a new forum for spending decisions in which non - state actors have more authority in relation to local government officials.51 ngos like peace committees and water cooperatives also represent a forum shift : unsatisfied with the services they are getting within a scheme of government provision , they create a new service - providing institution in which they have greater control over processes and outcomes.have a responsive regulatory strategy.this is a generic best practice of power in any forum . the obligation to respond to others in the society is a vital example of the relational checks and balances that constrain power in a polycentric governance system . second , responsiveness embodies the capacity for learning and the openness to new information that are characteristic of effective governance institutions.53have a big stick and threaten to use it.even responsive regulators sometimes crack down hard . urban communities are weak in relation to national governments , multinational corporations and international donors , but they are not powerless . urban governors have a variety of sticks , from networked advocacy and shaming strategies , through regulations and taxes , to debt default . weak actors have proven reasonably effective at influencing global policy not just by speaking up for the interests of poorer regions in international fora , but by taking the battle into the media and domestic politics.40 build and rebuild institutions of governance to increase participation and effectiveness . defining the territorial and subject matter jurisdiction of metropolitan government structures is also fundamental , although decades of effort have not produced a generally applicable solution.41 new governance strategies of privatization and self - regulation can be tools of more efficient public management . ppem , community policing and other practices that increase the meaningful involvement of stakeholders have shown good results , but do not replace and indeed may depend on the vitality of governance institutions outside the government . funding ( and minimizing legal barriers on ) ngos , microgovernance initiatives and institutional innovations that devolve power to stakeholders can all increase urban governance capacity by mobilizing the resources and capacities of communities that currently have no real access to governance . once a wide range of governance institutions are active , building connections among them and with compatriots in other urban settings allows the weak to increase their resources for advocacy and upstream governance . networks like the asian coalition for housing rights and slum / shack dwellers international ( sdi ) not only take local action but also work together to support each other from community to community within cities , from city to city within nations , and internationally.42(p 2),4345 the healthy cities movement and regional health promotion networks , through which areas in close geographic proximity pool resources and expertise , exemplify the type of cooperation necessary to global health.46 concentrate technical competence at network nodes . effective networks of governance typically create nodes on the network that concentrate resources and technologies for the purpose of achieving a common goal.7,47 technical expertise and the capacity to rapidly gather , interpret and respond to information helps weaker actors compete successfully with stronger ones.37 the campaign of developing countries and hiv / aids advocates to mitigate the impact of the trips agreement on access to essential medicines demonstrated the importance of organizing networks around institutions of technical competence such as medecines sans frontieres and the consumer project on technology.48 another example is the health promotion foundation , like vichealth in australia , designed as a reliably funded source of expertise and advocacy in the cause of better health governance.49 focus on forums where urban governors can be creative and assertive . many powerful governing institutions and systems are not concerned about the welfare of urban settings . if weak actors engage in governance in and through organizational nodes that others , particularly rich and powerful others , have established and work through , they will typically find themselves at a disadvantage because they will find that the agendas of others have been built into nodal processes.37 forum shifting may be defined as relocating interactions ( like negotiation or regulation ) from an institution of governance in which an actor encounters resistance to one where it is likely to achieve its objective . forum shifting has been an extremely useful tool of the powerful ; it can also work for weaker players like cities and ngos . but as braithwaite points out , a responsive strategy is more than good technique : it has two even deeper virtues . first , it represents a way of understanding governance in a democracy based on responding to peoples problems , environments , demands . the obligation to respond to others in the society is a vital example of the relational checks and balances that constrain power in a polycentric governance system . second , responsiveness embodies the capacity for learning and the openness to new information that are characteristic of effective governance institutions.53 have a big stick and threaten to use it . even responsive urban communities are weak in relation to national governments , multinational corporations and international donors , but they are not powerless . urban governors have a variety of sticks , from networked advocacy and shaming strategies , through regulations and taxes , to debt default . weak actors have proven reasonably effective at influencing global policy not just by speaking up for the interests of poorer regions in international fora , but by taking the battle into the media and domestic politics.40 strong local governance has its pitfalls.54,55 those with greater resources of experience , money or skill can game the local system as they can a national government.56 the voices of the poorer , weaker , more socially marginal can be ignored . urban settings often have large populations of illegal internal or international migrants whose right to participate is contested.57 urbanites do not necessarily , or even most of the time , organize themselves and vote as urban dwellers , but rather act as members of ideological or ethnic blocks organized around national political issues that may reflect and worsen divisions at the local level.14 urban governors are part of a system that works best as it approaches the ideal of a virtuous circuit ( see figure 1 ) . the best of interventions organized at the global level will suffer if the national , provincial and especially the local institutions of governance are bypassed or lack the capacity for effective implementation . even the imposition of good solutions in a top down manner , without real decision - making participation by those most affected , is paternalistic and illegitimate from a democratic perspective . good national and international governance can be a source of norms , and a recourse for those excluded from local decision making.55 national governments provide the policy environment in which local government and governance can innovate , or not.58,59 it is not even clear that empowering urban areas leads to greater attention to urban inequities : although central governments are unlikely to be generally more pro - poor than local governments , it may be easier for central governments to insist on pro - poor use of grant resources than for local governments to use their own resources in that way.14(p 14 ) local governments typically are short not just on cash but on properly trained bureaucrats with the skills and incentives to use their power productively . improving skills in governance , widening the repertoire of strategies , will make poor urbanites more effective , but local governors also need access to the resources controlled by higher levels of governance . on the research side , more support is required for the study of the design principles or grammars of successful governance , particularly outside of and in partnership with government.60 it is even more important to fund governance entrepreneurs reinventing governance in communities around the world , and to support ongoing community processes of governance reinvention . stories for researchers , governments and ngos unless they can be replicated at a sufficient scale to influence the condition of the mass of urbanites.58,61 international funders and governments speak about the importance of good governance and strong civil society , but investment in general governance capacity , unlinked to a particular categorical program or specific objective , is still too rare . people can learn to better improvise , but no amount of research or technology transfer will turn governance from an art into a science . it will be difficult to prove that any particular form or process of governance causes urban health to improve , yet we have more than enough reasons to prefer good governance over the alternative . good governance is not just valuable for the ends it promotes , but for the process of collective cultural imagination it represents .

alzheimer s disease ( ad ) is a progressive neurodegenerative disease that is clinically characterized by impairment of cognitive and functional abilities together with behavioral symptoms . the diagnosis of probable ad has classically been based on clinical criteria , such as those published in 1984 by the national institute of neurological and communicative disorders and stroke - alzheimer s disease and related disorders association ( nincds - adrda ) . according to those criteria , a definitive diagnosis of ad requires postmortem confirmation of specific neuropathological changes ( accumulation of neuritic plaques and neurofibrillary tangles containing hyperphosphorylated tau proteins ) . greater understanding of the pathology and course of ad has led to it being re - conceptualized as a disease continuum ( fig . 1 ) . it is believed that patients with ad experience a long asymptomatic ( preclinical ) phase in which neuropathological changes occur but cognitive ability is normal [ 35 ] , followed by a symptomatic ( prodromal or pre - dementia ) phase of progressive cognitive decline before the onset of functional impairment and overt dementia [ 69 ] . longitudinal follow - up studies of population - based community - dwelling individuals have shown that cognitive impairment can be detected well before the onset of dementia symptoms . as more people live into older age , the number of individuals with dementia is increasing [ 2 , 11 ] , and the economic , health , and social care costs of dementia are escalating [ 1113 ] . in response to this , dementia has become a priority area for coordinated action at the european union ( eu ) and global level [ 1416 ] . many countries now have national dementia strategies and government policies that emphasize early diagnosis and intervention [ 2 , 1720 ] . in recent years , there have been calls for a cultural shift in diagnosing ad at an earlier stage , before patients have crossed the threshold into dementia . the alzheimer cooperative valuation in europe ( alcove ) project has proposed that diagnosis should generally occur earlier than is currently common practice , at a time when patients and their family first notice changes in cognitive function and can use the information to make sense of what is happening , make lifestyle changes , and plan for the future ; the term timely diagnosis this is not the only definition proposed for timely diagnosis ; it can also be described as the diagnosis made at the right time for the individual patient , irrespective of the disease stage . for the current review , we defined timely diagnosis of ad as the diagnosis made at a time when individuals first become worried enough to seek help and come to the attention of clinicians because of concerns about changes in cognition , behavior , or functioning not necessarily resulting in dementia , and we extended the literature search to diagnosis at the prodromal or pre - dementia stage of ad ( t2 and t3 in fig . 1 ) [ 2 , 3 ] . timely diagnosis of ad differs from early diagnosis ( t1 in fig . 1 ) , which would require population or targeted screening to identify people in the asymptomatic phase of ad . the international working group ( iwg ) has proposed a new concept of ad with new diagnostic criteria based on the presence of biomarkers , allowing the identification of a prodromal stage ( also called the pre - dementia stage of ad ) and of preclinical states for ad [ 23 , 24 ] . based on these criteria , ad is now considered a clinico - biological entity that can be identified in vivo and no more reference to the dementia threshold is needed for the diagnosis of ad . in line with this new approach , the revised criteria for the clinical diagnosis of ad by the national institute on aging and alzheimer association ( nia - aa ) also considered a symptomatic pre - dementia phase of ad , referred to as mild cognitive impairment due to ad [ 25 , 26 ] . the iwg research criteria permit diagnosis of ad at the prodromal phase before patients have developed dementia ; these require evidence of both specific clinical features and in vivo biological evidence of an underlying abnormal pathology that is well - defined and detected using biomarkers ( table 1 ) . although these new researchcriteria require further validation , recent findings suggest that they have good specificity and are feasible for use in the clinical setting for the diagnosis of ad at the prodromal stage . there are many possible benefits of a timely and accurate diagnosis of ad for patients , caregivers , and society [ 11 , 22 ] . one of the main theoretical advantages of a timely diagnosis at the prodromal stage is the opportunity to achieve added value from earlier treatment or intervention with disease - modifying therapy in a clinical trial before the onset of dementia . this is entirely speculative at present as no effective disease - modifying therapies are available . early intervention has the potential to improve the quality of life of patients and their informal family caregivers , both of whom are often relieved once the patient is diagnosed [ 30 , 31 ] . a timely diagnosis at the prodromal stage may also improve patient access to support services or pathways of care and enable planning for the future . on the other hand , there are a number of conceivable risks or challenges associated with a timely diagnosis of ad , including ethical issues , competency questions , discrimination , and stigmatization [ 3235 ] . another concern is misdiagnosis , which can lead to inappropriate treatment of patients who could take unnecessary medications for ad or not receive correct therapy for potentially treatable disorders . the monetary costs to society of establishing systems for timely diagnosis and intervention may also be burdensome . our aim was to review the research literature to identify whether there are studies that demonstrate the benefits and potential risks of a timely diagnosis of ad for individuals who exhibit changes in cognition , behavior , or function but are not yet clearly demented . as more people live into older age , the number of individuals with dementia is increasing [ 2 , 11 ] , and the economic , health , and social care costs of dementia are escalating [ 1113 ] . in response to this , dementia has become a priority area for coordinated action at the european union ( eu ) and global level [ 1416 ] . many countries now have national dementia strategies and government policies that emphasize early diagnosis and intervention [ 2 , 1720 ] . in recent years , there have been calls for a cultural shift in diagnosing ad at an earlier stage , before patients have crossed the threshold into dementia . the alzheimer cooperative valuation in europe ( alcove ) project has proposed that diagnosis should generally occur earlier than is currently common practice , at a time when patients and their family first notice changes in cognitive function and can use the information to make sense of what is happening , make lifestyle changes , and plan for the future ; the term timely diagnosis this is not the only definition proposed for timely diagnosis ; it can also be described as the diagnosis made at the right time for the individual patient , irrespective of the disease stage . for the current review , we defined timely diagnosis of ad as the diagnosis made at a time when individuals first become worried enough to seek help and come to the attention of clinicians because of concerns about changes in cognition , behavior , or functioning not necessarily resulting in dementia , and we extended the literature search to diagnosis at the prodromal or pre - dementia stage of ad ( t2 and t3 in fig timely diagnosis of ad differs from early diagnosis ( t1 in fig . 1 ) , which would require population or targeted screening to identify people in the asymptomatic phase of ad . the international working group ( iwg ) has proposed a new concept of ad with new diagnostic criteria based on the presence of biomarkers , allowing the identification of a prodromal stage ( also called the pre - dementia stage of ad ) and of preclinical states for ad [ 23 , 24 ] . based on these criteria , ad is now considered a clinico - biological entity that can be identified in vivo and no more reference to the dementia threshold is needed for the diagnosis of ad . in line with this new approach , the revised criteria for the clinical diagnosis of ad by the national institute on aging and alzheimer association ( nia - aa ) also considered a symptomatic pre - dementia phase of ad , referred to as mild cognitive impairment due to ad [ 25 , 26 ] . the iwg research criteria permit diagnosis of ad at the prodromal phase before patients have developed dementia ; these require evidence of both specific clinical features and in vivo biological evidence of an underlying abnormal pathology that is well - defined and detected using biomarkers ( table 1 ) . although these new researchcriteria require further validation , recent findings suggest that they have good specificity and are feasible for use in the clinical setting for the diagnosis of ad at the prodromal stage . there are many possible benefits of a timely and accurate diagnosis of ad for patients , caregivers , and society [ 11 , 22 ] . one of the main theoretical advantages of a timely diagnosis at the prodromal stage is the opportunity to achieve added value from earlier treatment or intervention with disease - modifying therapy in a clinical trial before the onset of dementia . this is entirely speculative at present as no effective disease - modifying therapies are available . early intervention has the potential to improve the quality of life of patients and their informal family caregivers , both of whom are often relieved once the patient is diagnosed [ 30 , 31 ] . a timely diagnosis at the prodromal stage may also improve patient access to support services or pathways of care and enable planning for the future . on the other hand , there are a number of conceivable risks or challenges associated with a timely diagnosis of ad , including ethical issues , competency questions , discrimination , and stigmatization [ 3235 ] . another concern is misdiagnosis , which can lead to inappropriate treatment of patients who could take unnecessary medications for ad or not receive correct therapy for potentially treatable disorders . the monetary costs to society of establishing systems for timely diagnosis and intervention may also be burdensome . our aim was to review the research literature to identify whether there are studies that demonstrate the benefits and potential risks of a timely diagnosis of ad for individuals who exhibit changes in cognition , behavior , or function but are not yet clearly demented . a comprehensive literature search was performed to identify publications that investigated the benefits and risks of a timely diagnosis of ad at the prodromal ( pre - dementia ) stage . first , a broad literature search on the electronic databases medline and embase was performed , from their start dates to november 20 , 2013 , using the following search terms : early diagnosis ( subject heading ) and alzheimer or dementia . the term early diagnosis was used for the search to ensure capture of all relevant articles in this area as the terms timely diagnosis , prodromal ad , and pre dementia have only recently been introduced and defined . items selected for further assessment were articles published in the english language since january 1 , 2000 ( except for the inclusion of a few relevant papers published in the late 1990s ) . this provided 451 records , including studies , reviews , editorials , letters , and commentaries . meeting abstracts were not systematically searched , but the authors included any relevant meeting abstracts that they were aware of . the titles and abstracts of all 451 records identified were independently examined by at least two of the authors and rated on a 4-point scale ( 1 = limited , 2 = acceptable , 3 = good , 4 = excellent ) for potential relevance for inclusion in this review . for any articles considered possibly eligible or where uncertainty existed , further studies identified from the reference lists of the full articles reviewed and from other sources were obtained and examined to see if they should also be included . eligible articles were those reporting the results of studies investigating the benefits or challenges of a timely diagnosis of ad . these studies could be quantitative ( e.g. , cost studies ) or qualitative ( e.g. , surveys , focus groups ) , and there were no geographical exclusions . however , studies examining the development of or cost / benefit of the tools used to make an early / timely diagnosis ( e.g. , biomarkers ) were not included . of the 45 references selected by the authors , and assessed further for eligibility , nine were studies or surveys pertaining to the consequences of a timely diagnosis of ad and were included in the results . to verify that no relevant studies on the benefits or challenges of a timely diagnosis of ad were missed , a second comprehensive search of medline ( through pubmed ) and embase ( both accessed via proquest dialog ) from january 1 , 2000 to may 21 , 2014 using the search terms ( timely diagnosis ) and ( mild cognitive impairment or amnestic mild cognitive impairment ) was subsequently performed . after removing duplicates , the titles of the 31 records identified were assessed and nine abstracts were selected for further assessment of eligibility . none of these articles yielded findings on the consequences of a timely diagnosis of prodromal ad . a further search of medline and embase ( both accessed via proquest dialog ) to identify original clinical studies on the benefits and challenges of a timely diagnosis of ad was performed on june 2 , 2014 using the following search terms : dementia or alzheimer or alzheimer s and prodromal or pre - dementia or early symptoms and diagnosis . when the search was limited to clinical trials in embase and clinical trials was added as a search string in medline , 169 records remained after duplicates were removed . after assessment of the titles and abstracts , no additional studies were identified for inclusion in the results . note that the heterogeneity of the studies identified prevented us from performing a full systematic review , including meta - analysis , according to prisma ( preferred reporting items for systematic reviews and meta - analyses ) guidelines . figure 2 illustrates the article identification and selection process for the literature searches . because the identified studies have different aims , combination or comparison of the data in a systematic manner was not possible . included studies did not undergo any quality assessment ( e.g. , risk of bias ) . a comprehensive literature search was performed to identify publications that investigated the benefits and risks of a timely diagnosis of ad at the prodromal ( pre - dementia ) stage . first , a broad literature search on the electronic databases medline and embase was performed , from their start dates to november 20 , 2013 , using the following search terms : early diagnosis ( subject heading ) and alzheimer or dementia . the term early diagnosis was used for the search to ensure capture of all relevant articles in this area as the terms timely diagnosis , prodromal ad , and pre dementia have only recently been introduced and defined . items selected for further assessment were articles published in the english language since january 1 , 2000 ( except for the inclusion of a few relevant papers published in the late 1990s ) . this provided 451 records , including studies , reviews , editorials , letters , and commentaries . meeting abstracts were not systematically searched , but the authors included any relevant meeting abstracts that they were aware of . the titles and abstracts of all 451 records identified were independently examined by at least two of the authors and rated on a 4-point scale ( 1 = limited , 2 = acceptable , 3 = good , 4 = excellent ) for potential relevance for inclusion in this review . for any articles considered possibly eligible or where uncertainty existed , further studies identified from the reference lists of the full articles reviewed and from other sources were obtained and examined to see if they should also be included . eligible articles were those reporting the results of studies investigating the benefits or challenges of a timely diagnosis of ad . these studies could be quantitative ( e.g. , cost studies ) or qualitative ( e.g. , surveys , focus groups ) , and there were no geographical exclusions . however , studies examining the development of or cost / benefit of the tools used to make an early / timely diagnosis ( e.g. , biomarkers ) were not included . of the 45 references selected by the authors , and assessed further for eligibility , nine were studies or surveys pertaining to the consequences of a timely diagnosis of ad and were included in the results . to verify that no relevant studies on the benefits or challenges of a timely diagnosis of ad were missed , a second comprehensive search of medline ( through pubmed ) and embase ( both accessed via proquest dialog ) from january 1 , 2000 to may 21 , 2014 using the search terms ( timely diagnosis ) and ( mild cognitive impairment or amnestic mild cognitive impairment ) was subsequently performed . after removing duplicates , the titles of the 31 records identified were assessed and nine abstracts were selected for further assessment of eligibility . none of these articles yielded findings on the consequences of a timely diagnosis of prodromal ad . a further search of medline and embase ( both accessed via proquest dialog ) to identify original clinical studies on the benefits and challenges of a timely diagnosis of ad was performed on june 2 , 2014 using the following search terms : dementia or alzheimer or alzheimer s and prodromal or pre - dementia or early symptoms and diagnosis . when the search was limited to clinical trials in embase and clinical trials was added as a search string in medline , 169 records remained after duplicates were removed . after assessment of the titles and abstracts , no additional studies were identified for inclusion in the results . note that the heterogeneity of the studies identified prevented us from performing a full systematic review , including meta - analysis , according to prisma ( preferred reporting items for systematic reviews and meta - analyses ) guidelines . figure 2 illustrates the article identification and selection process for the literature searches . because the identified studies have different aims , combination or comparison of the data in a systematic manner was not possible . included studies did not undergo any quality assessment ( e.g. , risk of bias ) . nine studies related to the benefits or challenges of a timely diagnosis of ad were identified from the literature search . some of these studies included subjects in the pre - dementia stage of ad but none were specifically focused on diagnosing ad at the prodromal stage . one study was a survey of the psychological reactions of patients and their companions to receiving a diagnosis of dementia , while three studies focused on physicians or caregivers , not patients [ 3941 ] . these included a qualitative survey of caregivers or primary care physicians ( pcps ) on the perceived benefits and risks of an early recognition of dementia or alzheimer s dementia ; a survey of general practitioner ( gp ) attitudes toward diagnosing dementia that aimed to understand the low dementia diagnosis rate in certain regions in england ; and a randomized controlled study that examined the effects of a tailored educational intervention on dementia diagnosis and management in primary care . two studies were of memory clinic patients , looking at the effects of diagnosis made early in the course of the illness on institutionalization and mortality [ 42 , 43 ] . finally , three studies looked at the possible cost - benefits of an early diagnosis of dementia or alzheimer s dementia using simulation models and estimates of outcomes [ 4446 ] . using a pre / post survey design , carpenter et al . studied the reactions of 90 people and their companions upon receiving a diagnosis of dementia . the participants were people with and without memory or cognitive complaints seeking evaluation , and the companions were mostly the spouse ( 61% ) or child ( 22% ) of the person seeking dementia evaluation . symptoms of anxiety and depression were assessed using the state - trait anxiety inventory and geriatric depression scale , respectively , with patients and their companions being assessed separately . of the 90 patients who participated in the study , 31% were given a diagnosis of no dementia ( clinical dementiarating [ cdr ] = 0 ) , 46% were given a diagnosis of very mild dementia ( cdr = 0.5 ) , and 23% were given a diagnosis of mild dementia ( cdr = 1 ) . the results showed a substantial reduction in anxiety and no significant changes in depression or psychological distressin either patients or companions after receiving the diagnostic feedback , regardless of diagnostic outcome or severity . in the uk , iliffe et al . surveyed 990 pcps ( including gps , community nurses , practice nurses , community mental health nurses , and others ) on the perceived benefits and risks of an early recognition of dementia in general practice this study generated data from workshops using a nominal group approach , identifying benefits for patients , families and local services . reduced uncertainty and the opportunity to come to terms with the diagnosis , seek support , and avoid crises were the most relevant advantages identified for patients . the benefits for families included awareness of prognosis and disease course as well as the need to organize support , plan for the future , make appropriate legal arrangements , and optimize quality of life . the hazards identified for patients and families included fear , anxiety / depression , stigma , and altered relationships , while pcps expressed concerns about diagnostic errors and resource limitations , including shortfalls in local services , as more people received diagnoses early in the disease continuum . in an online survey where gps in primary care in norfolk and suffolk , england , were asked to rate the extent to which they agreed or disagreed with seven statements on dementia diagnosis , 85% of the 113 respondents agreed that it is beneficial to patients and their families to have a timely diagnosis of dementia . although this survey showed positive attitudes toward diagnosing dementia , it also found that participating gps were less confident in their knowledge about the availability of post - diagnostic support services for people with dementia and their carers , and were dissatisfied with locally available services . the authors commented that gps attitudes toward diagnosing and managing dementia were more positive since the 2009 national audit of gps in the uk . some studies have indicated that educational interventions for healthcare providers may improve the early recognition of dementia in primary care [ 48 , 49 ] and , therefore , may have subsequent benefits for patients and their caregivers . however , a recent study of an educational intervention tailored to the needs of an individual primary care practice that combined practice - based workshops and computer decision support systems ( evidem - ed study ) did not show positive results . this open - label cluster randomized controlled trial , which included more than 1000 patients with dementia seen in 23 general practices in england , had a pre / post - intervention design of educational intervention versus usual care . the educational program combined timely diagnosis of dementia and psychosocial support around the time of diagnosis with components appropriate to the later stages of the disease course , while the usual care control practices were provided with a summary of the uk national institute for health and care excellence dementia clinical guideline 42 . the results showed no significant difference in dementia management reviews before and after the educational intervention aimed to help physicians make a timely diagnosis ; there was also no change in case detection rates . a multidisciplinary memory clinic set up in sheffield , england , was designed to facilitate diagnosis and treatment of alzheimer s dementia early in the course of the disease through careful management of the diagnostic process with a focus on pre- and post - diagnostic counseling followed by psychosocial interventions . the effectiveness of this memory clinic model was investigated in a small retrospective cohort study of 30 patients with alzheimer s dementia in the memory clinic treatment group versus 30 ad patients in the control group who received standard care from other facilities . the median time to institutionalization was 26.5 months for the memory clinic group versus 17.5 months for the control group , demonstrating that early diagnosis of alzheimer s dementia can delay time to institutionalization , in this case by a median of 9 months . one study provides indirect evidence that diagnosis of ad at the earlier stage may prolong survival . in a cohort study of 970 patients with dementia ( including 663 with ad ) attending a memory clinic in france , survival analysis showed that a shorter time between first symptoms and first visit was associated with longer survival regardless of diagnosis ( risk ratio = 0.7 for each year earlier the first visit occurred , p < 0.0001 ) . three studies investigated the cost - benefits of early diagnosis and treatment of alzheimer s dementia [ 4446 ] . these studies questioned whether the costs associated with early identification and diagnostic evaluation can be offset by cost savings achieved by the use of early interventions that hypothetically slow disease progression and/or delay the time to institutionalization . banerjee and wittenberg performed a cost - benefit analysis of the croydon memory service model for early diagnosis and intervention in dementia . this british service model takes a multi - disciplinary and multi - agency approach and is designed to provide an early diagnosis , information , and help for people with dementia and their families . the costs of this service , if extrapolated nationally , were estimated at 220 million per year ( in 2007/2008 prices ) . also , with a theoretical reduction of 6% , 10% , and 20% in residential care home admission by year 10 after introducing the service model , potential annual savings to society were estimated at approximately 150 million , 245 million , and 490 million , respectively . in addition , it was estimated that the service model need only achieve a modest increase in average quality of life ( of between 0.01 and 0.02 quality adjusted life years [ qaly ] per person year ) together with a 10% reduction in institutionalization to be considered cost - effective . weimer and sager performed a cost - benefit analysis ( monte carlo model ) of early identification and treatment of alzheimer s dementia . the model estimated the net social benefits and net fiscal savings of early intervention with drug treatment , a caregiver intervention program , and a combination of both interventions . the results showed that the net benefits could be highest when patients received a diagnosis at the initial symptomatic stage of the disease and when drug treatment was combined with caregiver intervention : the mean net social , state fiscal , and federal fiscal benefits of drug treatment plus caregiver intervention for a 70-year - old married woman with a mini - mental state examination ( mmse ) score of 28 were estimated as $ us125,000 , $ 16,000 , and $ 34,000 , respectively . performed an economic evaluation of early assessment and treatment for ad in the uk using discrete event simulation based on data from the consortium to establish a registry for alzheimer s disease ( cerad ) , and patient - level data from seven donepezil clinical trials to simulate ad progression and the effects of treatment intervention . the model calculates direct costs of care and the indirect costs of caregiver time over a period of 10 years . qalys for patients and caregivers were also reported , as were incremental cost - effectiveness ratios ( cost / qaly ) . findings show that early assessment and treatment result in up - front costs of 4083 and 2402 per patient ( 2007 cost year ) , respectively , but this was offset by savings in patient care . the total expenditures ( for drugs , early assessment , direct care , and indirect costs ) were lower for the early assessment and treatment group ( 204,561 ) versus treatment without early assessment ( 209,837 ) , or no early assessment and no treatment ( 212,302 ) . reduced institutional care was the largest contributor to savings , and patients assessed and treated early remained in the community longer . compared with the no assessment / treatment group , early assessment reduced the time patients spent with low cognitive ability ( mmse scores < 10 ) by over 5 months . the analyses suggested that early assessment and treatment could have health benefits for the patient and might also be cost - effective . nine studies related to the benefits or challenges of a timely diagnosis of ad were identified from the literature search . some of these studies included subjects in the pre - dementia stage of ad but none were specifically focused on diagnosing ad at the prodromal stage . one study was a survey of the psychological reactions of patients and their companions to receiving a diagnosis of dementia , while three studies focused on physicians or caregivers , not patients [ 3941 ] . these included a qualitative survey of caregivers or primary care physicians ( pcps ) on the perceived benefits and risks of an early recognition of dementia or alzheimer s dementia ; a survey of general practitioner ( gp ) attitudes toward diagnosing dementia that aimed to understand the low dementia diagnosis rate in certain regions in england ; and a randomized controlled study that examined the effects of a tailored educational intervention on dementia diagnosis and management in primary care . two studies were of memory clinic patients , looking at the effects of diagnosis made early in the course of the illness on institutionalization and mortality [ 42 , 43 ] . finally , three studies looked at the possible cost - benefits of an early diagnosis of dementia or alzheimer s dementia using simulation models and estimates of outcomes [ 4446 ] . using a pre / post survey design , carpenter et al . studied the reactions of 90 people and their companions upon receiving a diagnosis of dementia . the participants were people with and without memory or cognitive complaints seeking evaluation , and the companions were mostly the spouse ( 61% ) or child ( 22% ) of the person seeking dementia evaluation . symptoms of anxiety and depression were assessed using the state - trait anxiety inventory and geriatric depression scale , respectively , with patients and their companions being assessed separately . of the 90 patients who participated in the study , 31% were given a diagnosis of no dementia ( clinical dementiarating [ cdr ] = 0 ) , 46% were given a diagnosis of very mild dementia ( cdr = 0.5 ) , and 23% were given a diagnosis of mild dementia ( cdr = 1 ) . the results showed a substantial reduction in anxiety and no significant changes in depression or psychological distressin either patients or companions after receiving the diagnostic feedback , regardless of diagnostic outcome or severity . in the uk , iliffe et al . surveyed 990 pcps ( including gps , community nurses , practice nurses , community mental health nurses , and others ) on the perceived benefits and risks of an early recognition of dementia in general practice . this study generated data from workshops using a nominal group approach , identifying benefits for patients , families and local services . reduced uncertainty and the opportunity to come to terms with the diagnosis , seek support , and avoid crises were the most relevant advantages identified for patients . the benefits for families included awareness of prognosis and disease course as well as the need to organize support , plan for the future , make appropriate legal arrangements , and optimize quality of life . the hazards identified for patients and families included fear , anxiety / depression , stigma , and altered relationships , while pcps expressed concerns about diagnostic errors and resource limitations , including shortfalls in local services , as more people received diagnoses early in the disease continuum . in an online survey where gps in primary care in norfolk and suffolk , england , were asked to rate the extent to which they agreed or disagreed with seven statements on dementia diagnosis , 85% of the 113 respondents agreed that it is beneficial to patients and their families to have a timely diagnosis of dementia . although this survey showed positive attitudes toward diagnosing dementia , it also found that participating gps were less confident in their knowledge about the availability of post - diagnostic support services for people with dementia and their carers , and were dissatisfied with locally available services . the authors commented that gps attitudes toward diagnosing and managing dementia were more positive since the 2009 national audit of gps in the uk . some studies have indicated that educational interventions for healthcare providers may improve the early recognition of dementia in primary care [ 48 , 49 ] and , therefore , may have subsequent benefits for patients and their caregivers . however , a recent study of an educational intervention tailored to the needs of an individual primary care practice that combined practice - based workshops and computer decision support systems ( evidem - ed study ) did not show positive results . this open - label cluster randomized controlled trial , which included more than 1000 patients with dementia seen in 23 general practices in england , had a pre / post - intervention design of educational intervention versus usual care . the educational program combined timely diagnosis of dementia and psychosocial support around the time of diagnosis with components appropriate to the later stages of the disease course , while the usual care control practices were provided with a summary of the uk national institute for health and care excellence dementia clinical guideline 42 . the results showed no significant difference in dementia management reviews before and after the educational intervention aimed to help physicians make a timely diagnosis ; there was also no change in case detection rates . a multidisciplinary memory clinic set up in sheffield , england , was designed to facilitate diagnosis and treatment of alzheimer s dementia early in the course of the disease through careful management of the diagnostic process with a focus on pre- and post - diagnostic counseling followed by psychosocial interventions . the effectiveness of this memory clinic model was investigated in a small retrospective cohort study of 30 patients with alzheimer s dementia in the memory clinic treatment group versus 30 ad patients in the control group who received standard care from other facilities . the median time to institutionalization was 26.5 months for the memory clinic group versus 17.5 months for the control group , demonstrating that early diagnosis of alzheimer s dementia can delay time to institutionalization , in this case by a median of 9 months . one study provides indirect evidence that diagnosis of ad at the earlier stage may prolong survival . in a cohort study of 970 patients with dementia ( including 663 with ad ) attending a memory clinic in france , survival analysis showed that a shorter time between first symptoms and first visit was associated with longer survival regardless of diagnosis ( risk ratio = 0.7 for each year earlier the first visit occurred , p < 0.0001 ) . three studies investigated the cost - benefits of early diagnosis and treatment of alzheimer s dementia [ 4446 ] . these studies questioned whether the costs associated with early identification and diagnostic evaluation can be offset by cost savings achieved by the use of early interventions that hypothetically slow disease progression and/or delay the time to institutionalization . banerjee and wittenberg performed a cost - benefit analysis of the croydon memory service model for early diagnosis and intervention in dementia . this british service model takes a multi - disciplinary and multi - agency approach and is designed to provide an early diagnosis , information , and help for people with dementia and their families . the costs of this service , if extrapolated nationally , were estimated at 220 million per year ( in 2007/2008 prices ) . also , with a theoretical reduction of 6% , 10% , and 20% in residential care home admission by year 10 after introducing the service model , potential annual savings to society were estimated at approximately 150 million , 245 million , and 490 million , respectively . in addition , it was estimated that the service model need only achieve a modest increase in average quality of life ( of between 0.01 and 0.02 quality adjusted life years [ qaly ] per person year ) together with a 10% reduction in institutionalization to be considered cost - effective . weimer and sager performed a cost - benefit analysis ( monte carlo model ) of early identification and treatment of alzheimer s dementia . the model estimated the net social benefits and net fiscal savings of early intervention with drug treatment , a caregiver intervention program , and a combination of both interventions . the results showed that the net benefits could be highest when patients received a diagnosis at the initial symptomatic stage of the disease and when drug treatment was combined with caregiver intervention : the mean net social , state fiscal , and federal fiscal benefits of drug treatment plus caregiver intervention for a 70-year - old married woman with a mini - mental state examination ( mmse ) score of 28 were estimated as $ us125,000 , $ 16,000 , and $ 34,000 , respectively . performed an economic evaluation of early assessment and treatment for ad in the uk using discrete event simulation based on data from the consortium to establish a registry for alzheimer s disease ( cerad ) , and patient - level data from seven donepezil clinical trials to simulate ad progression and the effects of treatment intervention . the model calculates direct costs of care and the indirect costs of caregiver time over a period of 10 years . qalys for patients and caregivers were also reported , as were incremental cost - effectiveness ratios ( cost / qaly ) . findings show that early assessment and treatment result in up - front costs of 4083 and 2402 per patient ( 2007 cost year ) , respectively , but this was offset by savings in patient care . the total expenditures ( for drugs , early assessment , direct care , and indirect costs ) were lower for the early assessment and treatment group ( 204,561 ) versus treatment without early assessment ( 209,837 ) , or no early assessment and no treatment ( 212,302 ) . reduced institutional care was the largest contributor to savings , and patients assessed and treated early remained in the community longer . compared with the no assessment / treatment group , early assessment reduced the time patients spent with low cognitive ability ( mmse scores < 10 ) by over 5 months . the analyses suggested that early assessment and treatment could have health benefits for the patient and might also be cost - effective . members of the scientific community , stakeholders ( e.g. , ad associations ) , regulators , and policy makers are , to varying degrees , encouraging a cultural shift toward making a timely diagnosis of ad at the initial symptomatic stages of the illness . despite this change in paradigm , our extensive and comprehensive review of the literature identified some studies highlighting the potential advantages of diagnosis and intervention early in the time course of dementia , but failed to find studies clearly focused on the benefits for patients , carers , or society of a timely diagnosis at the prodromal stage , before dementia sets in . in agreement with the 2011 world alzheimer report , we found that much of the literature outlining the benefits of timely diagnosis of ad is based on expert opinion rather than research evidence . the scarcity of published studies assessing the benefits and challenges of timely diagnosis could be due to the fact that the definition of ad as an entity that encompasses both pre - dementia and dementia phases is relatively recent and still not widely accepted . alternatively , some methodological issues could explain why few studies on this topic were identified . although our search of the literature was comprehensive and the search terms used should have been broad enough to capture most publications relevant to a timely diagnosis of ad , it is possible that some studies were missed because of the terminology used . in addition , some of the identified studies assessed the potential advantages of early recognition of cognitive decline without specifying the etiology of the dementia syndromes and could have included individuals with other types of dementia or mixed pathology . another limitation of our review is that the methodological quality of the studies was not rated , some of which may have had a high risk of bias . also , the heterogeneity of the studies identified prevented the performance of a fully systematic review including meta - analysis , according to prisma guidelines . several of the studies identified assessed the possible economic benefits of early diagnosis and treatment of alzheimer s dementia [ 4446 ] . these studies , however , were not evidence - based but based on models that estimated the cost - effectiveness of different theoretical interventions and/or outcomes . the significant economic impact of ad is expected to increase in the future with the trend toward diagnosis at the prodromal stage . a timely diagnosis offers the opportunity of introducing psychosocial interventions that may prolong the time people spend in initial stages of the disease and delay admission to residential long - term care homes , as indicated by the effectiveness of a multidisciplinary memory clinic model . data from a markov simulation model of intervention with a hypothetical disease - modifying therapy at the pre - dementia stage also support this hypothesis . as the costs associated with institutionalization and long - term residential care account for a large proportion of the total care costs of ad [ 52 , 53 ] , savings in overall healthcare costs may be achieved by extending the time a patient can remain living in the community . however , as societal costs of caring for community - dwelling patients with ad are primarily determined by the costs of caregiver informal care [ 13 , 54 , 55 ] , informal care costs must be taken into account together with the caregiver burden . although timely diagnosis and intervention are likely to incur greater up - front costs , economic modeling suggests that these may be offset by subsequent savings achieved primarily from a reduction in institutionalization ; additional benefits , also demonstrated in a cohort study in france , include prolonged patient survival and improved patient / carer quality of life [ 43 , 44 , 46 ] . simulation models of early intervention with hypothetical disease - modifying therapies indicate that substantial cost savings might be possible [ 56 , 57 ] . for example , the introduction in the uk of a hypothetical disease modifier that would delay the onset of dementia by 5 years ( from 2020 to 2025 ) is estimated to potentially reduce the number of people with dementia in 2030 by 36% , with a related highly significant decrease in the costs of care . however , the actual cost impact of delaying disease progression from pre - dementia through mild to severe ad , and of prolonging the time spent living in the community , still remains to be determined in longitudinal studies . thus , further investigation of the health and social care costs associated with timely diagnosis of ad at the prodromal stage is needed . people who seek health care generally prefer active strategies and certainty about their diagnosis and prognosis , even when effective drugs are not available . many of the potential benefits of timely diagnosis and treatment of ad from the studies identified in this review are dependent on the existence of a disease - modifying medication . unfortunately , such a medication does not yet exist and evidence - based studies will have to be performed if a proven disease - modifying treatment becomes available . the scant evidence base regarding the benefits of timely diagnosis of ad on outcomes in patients ( e.g. , subsequent disease progression , time to institutionalization , death ) or caregivers ( e.g. , burden , quality of life , depression / anxiety ) from published studies allows us to only speculate on the potential clinical benefits and risks of diagnosing patients at an earlier phase in the disease continuum , when they have symptoms but have not yet developed overt dementia . moreover , much of the following discussion is based on the assumption that a timely diagnosis of ad is feasible , even if the well - recognized problem of under - diagnosis of dementia at more severe stages in the current medical setting [ 2 , 40 ] demonstrates that this is not always the case . among the many conceivable benefits to patients , caregivers , healthcare providers , and society of a timely diagnosis of ad ( table 2 ) , one of the most important would appear to be that an earlier and improved diagnostic pathway may help patients avoid the medical nomadism that some experience during the diagnostic process , which begins with seeking help and ends with receiving a definitive diagnosis and treatment . prompt evaluation means that it might be possible to detect and treat other causes of memory problems ( e.g. , depression , anxiety , sleep disorders ) . as many older people have comorbidities , we can speculate that timely diagnosis of ad may help avoid prescription of medications that could actually worsen cognitive function . other important possible benefits of a timely diagnosis are that it may reduce feelings of uncertainty and anxiety in people with memory complaints and their families [ 38 , 60 ] , and may improve their quality of life and relationships . recent studies indicate that many people are in favor of early diagnostic testing for ad and would want a potential diagnosis of ad to be disclosed to them and their relatives [ 61 , 63 ] . for example , the value of knowing survey of public attitudes to early diagnosis of ad found that over 94% of the people surveyed ( in each of the four european countries where the question was asked ) wanted to be told if they had ad , and a recent qualitative study on people with cognitive decline , ranging from subjective memory problems to early dementia , showed that the overwhelming majority of participants were keen to know their diagnosis and its long - term consequences . moreover , as shown by carpenter et al . , patients and their caregivers report a reduction in anxiety and no increase in psychological distress after receiving a diagnosis of dementia in its initial stages . in addition , a recent focus group report found that a primary driver for people with memory problems to take part in a clinical trial for prodromal ad was to obtain an unambiguous diagnosis . early detection of dementia was considered the highest priority topic for dementia research in a recent survey conducted in scotland on ad patients , their carers , and the general public with an interest in dementia . gps and primary care health workers consider that patients and their families are the main beneficiaries of an earlier diagnosis [ 39 , 40 ] . theoretically , a timely diagnosis of ad could allow families to plan and prepare for the future . before the onset of dementia and while insight is preserved , patients may have the capacity to understand what is happening and make decisions about future living / care options , treatment , and financial and legal arrangements [ 33 , 68 ] . caregivers would be able to plan and organize future support , explore outside resources , and address safety concerns ( e.g. , driving ) , financial planning , legal issues ( e.g. , advance directives ) , and caretaking arrangements [ 31 , 39 , 69 ] . putative advantages of diagnosis at the earlier stages of the disease for local healthcare and social services are that they can better anticipate future demands . we can postulate that recognition of ad during the prodromal stage would allow physicians to offer therapies that address specific symptoms , such as anxiety or impaired sleep , and manage medications prescribed for comorbidities that may be inadvertently exacerbating dementia or other emerging symptoms.it can be speculated that a timely diagnosis of ad would also open up the opportunity of early intervention with disease - modifying therapies if they become available . a variety of possibly disease - modifying compounds in clinical development may theoretically increase the time spent in the pre - dementia to mild ad stages . once healthcare systems and processes that enable timely diagnosis of ad are established , data on the actual benefits to patients and their families can be collected . diagnosis of ad at the prodromal stage is very challenging in the current medical setting and made more difficult by numerous barriers . some of these barriers are relevant to only patients , caregivers , healthcare providers , or society , while others could be shared by all ( table 2 ) . some of the existing barriers to diagnosis of dementia may also apply to the timely diagnosis of ad at the prodromal stage . in particular , stigma associated with dementia is a major issue , whether it be public stigma , self - stigma ( which may deter individuals from seeking professional help ) , or family / caregiver stigma , all of which can negatively affect the quality of life of people with dementia and their caregivers [ 31 , 7072 ] . although there are reports of patients and caregivers expressing a positive attitude toward receiving a timely diagnosis of dementia , some physicians , especially in the primary care setting , cite various reasons explaining their reluctance to give a diagnosis of ad at the initial symptomatic stages of illness . for example , they say they do not consider it is beneficial for patients overall health and well - being , perceive there are no effective treatments , or consider that a diagnosis early in the disease continuum may actually be harmful to patients [ 39 , 7375 ] . while some patients and families may experience negative reactions to disclosure of a diagnosis of ad , including feelings of loss , anger , uncertainty , frustration , anxiety / depression , and catastrophic thinking ( although this has not been specifically investigated for patients given a diagnosis of prodromal ad ) [ 61 , 77 ] , other reports find no such long - term after - effects from a dementia diagnosis , and conclude that individual preferences for diagnosis disclosure should be taken into account [ 38 , 60 , 78 ] . concerns have been expressed that an early diagnosis of dementia may conceivably result in an increased suicide risk and request for physician - assisted suicide , although there is limited evidence to support this hypothesis . in a retrospective cohort study of patients aged 60 years and over with a diagnosis of dementia and who died by suicide during the study period ( 2001 to 2005 ) , subgroup analysis of 136 patients found that 75% of the suicides occurred in those with a new dementia diagnosis . a history of psychiatric hospitalization , diagnosis of depression , and prescription fills for antidepressant or anxiolytic medication were also associated with an increased risk of suicide . although more research is clearly needed on the risk for suicidal behavior in ad , a recent review by draper recommended that clinicians should be sensitive to the potential for suicide in vulnerable individuals , especially in the first few years after diagnosis . for healthcare providers , other possible drawbacks of diagnosis at the pre - dementia stage are an increased risk of misdiagnosis and uncertainty about the rate of progression to dementia , which can vary considerably among individuals . finally , memory problems and alterations of cognitive function can cause changes in personal identity , capacity , and autonomy ; these issues would presumably add to the ethical challenges associated with a timely diagnosis of ad at the prodromal stage ( table 3 ) [ 33 , 35 ] . to achieve a timely diagnosis at the initial stage of the disease before patients exhibit dementia symptoms , existing barriers to diagnosis will have to be removed . this would require pcps to be attuned to the early symptoms of ad , and healthcare professionals , stakeholders , and policy makers would need to take action to fill gaps in knowledge , skills , attitudes , and resources . clinical practice guidelines will need to be modified to align with the cultural shift to a timelydiagnosis of ad at the pre - dementia stage . for example , the most recent european federation of the neurological societies ( efns ) guidelines for the diagnosis and management of ad do not include diagnosis at the prodromal stage , and many of the screening tests recommended for assessing global cognition and activities of daily living may not be specific or sensitive enough for identifying prodromal ad . thus , the methods used for assessing people with memory problems to detect prodromal ad must be appropriate and accurate . establishing a clinical diagnosis of ad a timely diagnosis at the pre - dementia stage requires a coordinated diagnostic process involving a multidisciplinary team approach ; all of those involved will need to be aware of the subtle cognitive and non - cognitive changes that can precede the onset of dementia . therefore , the cultural shift toward timely diagnosis depends upon increasing public and professional awareness of the initial symptoms of ad , improving knowledge among healthcare professionals about the benefits of a timely diagnosis and early intervention , reducing the fear and stigma about dementia , normalizing the experience of dementia , and developing and implementing dementia strategies and integrated diagnostic services that enable timely diagnosis and provide post - diagnostic support , care , and interventions for patients and their families . some of this can be achieved through public awareness campaigns as well as targeted communication and education of healthcare professionals , while others will require system - wide and structural changes at the national level . we believe that timely diagnosis of ad at a time when people first seek for help being worried about changes in cognition , behavior , or functioning not necessarily resulting in dementia , has the potential to reduce the impact of no or delayed diagnosis or misdiagnosis . timely diagnosis at the prodromal stage of the disease could offer many potential benefits to patients and caregivers , especially the opportunity to obtain treatment to control symptoms , avoid medications that may worsen symptoms , and , possibly in the future , access to interventions that slow or lessen the disease process . patients could put into place advance care planning and make end - of - life decisions , consider changing unhealthy lifestyles , and seek better medical care . the findings of this literature review show that , at the current time , these ideas are mainly based on expert opinion and perhaps belief ; evidence is lacking , and further studies are needed to demonstrate not only that a timely diagnosis is feasible , but also that it has benefits . such evidence would support the cultural shift towards diagnosis at the pre - dementia stage of ad .

background : timely diagnosis of alzheimer s disease ( ad ) refers to a diagnosis at the stage when patients come to the attention of clinicians because of concerns about changes in cognition , behavior , or functioning and can be still free of dementia and functionally independent.objectives:to comprehensively review existing scientific evidence on the benefits and potential challenges of making a timely diagnosis of ad.methods:relevant studies were identified by searching electronic databases ( medline , embase ) and bibliographies for studies published in english between 1 january 2000 and 2 june 2014 on the consequences of a timely diagnosis of ad.results:nine studies were identified that investigated the consequences of diagnosing ad at the initial stages ; none were specifically focused on prodromal ad . a timely diagnosis potentially offers the opportunities of early intervention , implementation of coordinated care plans , better management of symptoms , patient safety , cost savings , and postponement of institutionalization . barriers to making a timely diagnosis include stigma , suicide risk , lack of training , diagnostic uncertainty , shortage of specialized diagnostic services , and the reluctance of healthcare providers to make a diagnosis when no effective disease - modifying options are available.conclusions:despite its potential benefits , few published studies have explored the advantages or risks of a timely diagnosis of ad . in light of the cultural shift toward diagnosis at the initial stage of the disease continuum , when the patient does not yet have dementia , more investigations are needed to evaluate the benefits and address the barriers that may impede making a timely ad diagnosis .

Introduction Moving to a timely diagnosis of AD at the prodromal stage Potential benefits or risks of timely diagnosis of AD Objectives Methods Search strategy and study selection Results Summary of literature search Perceived consequences of a timely diagnosis of AD Effects of interventions or models to facilitate a timely diagnosis of AD Cost benefits of early identification of AD Discussion Conclusions

alzheimer s disease ( ad ) is a progressive neurodegenerative disease that is clinically characterized by impairment of cognitive and functional abilities together with behavioral symptoms . the alzheimer cooperative valuation in europe ( alcove ) project has proposed that diagnosis should generally occur earlier than is currently common practice , at a time when patients and their family first notice changes in cognitive function and can use the information to make sense of what is happening , make lifestyle changes , and plan for the future ; the term timely diagnosis this is not the only definition proposed for timely diagnosis ; it can also be described as the diagnosis made at the right time for the individual patient , irrespective of the disease stage . for the current review , we defined timely diagnosis of ad as the diagnosis made at a time when individuals first become worried enough to seek help and come to the attention of clinicians because of concerns about changes in cognition , behavior , or functioning not necessarily resulting in dementia , and we extended the literature search to diagnosis at the prodromal or pre - dementia stage of ad ( t2 and t3 in fig . the international working group ( iwg ) has proposed a new concept of ad with new diagnostic criteria based on the presence of biomarkers , allowing the identification of a prodromal stage ( also called the pre - dementia stage of ad ) and of preclinical states for ad [ 23 , 24 ] . one of the main theoretical advantages of a timely diagnosis at the prodromal stage is the opportunity to achieve added value from earlier treatment or intervention with disease - modifying therapy in a clinical trial before the onset of dementia . this is entirely speculative at present as no effective disease - modifying therapies are available . on the other hand , there are a number of conceivable risks or challenges associated with a timely diagnosis of ad , including ethical issues , competency questions , discrimination , and stigmatization [ 3235 ] . our aim was to review the research literature to identify whether there are studies that demonstrate the benefits and potential risks of a timely diagnosis of ad for individuals who exhibit changes in cognition , behavior , or function but are not yet clearly demented . the alzheimer cooperative valuation in europe ( alcove ) project has proposed that diagnosis should generally occur earlier than is currently common practice , at a time when patients and their family first notice changes in cognitive function and can use the information to make sense of what is happening , make lifestyle changes , and plan for the future ; the term timely diagnosis this is not the only definition proposed for timely diagnosis ; it can also be described as the diagnosis made at the right time for the individual patient , irrespective of the disease stage . for the current review , we defined timely diagnosis of ad as the diagnosis made at a time when individuals first become worried enough to seek help and come to the attention of clinicians because of concerns about changes in cognition , behavior , or functioning not necessarily resulting in dementia , and we extended the literature search to diagnosis at the prodromal or pre - dementia stage of ad ( t2 and t3 in fig timely diagnosis of ad differs from early diagnosis ( t1 in fig . the international working group ( iwg ) has proposed a new concept of ad with new diagnostic criteria based on the presence of biomarkers , allowing the identification of a prodromal stage ( also called the pre - dementia stage of ad ) and of preclinical states for ad [ 23 , 24 ] . there are many possible benefits of a timely and accurate diagnosis of ad for patients , caregivers , and society [ 11 , 22 ] . one of the main theoretical advantages of a timely diagnosis at the prodromal stage is the opportunity to achieve added value from earlier treatment or intervention with disease - modifying therapy in a clinical trial before the onset of dementia . this is entirely speculative at present as no effective disease - modifying therapies are available . on the other hand , there are a number of conceivable risks or challenges associated with a timely diagnosis of ad , including ethical issues , competency questions , discrimination , and stigmatization [ 3235 ] . our aim was to review the research literature to identify whether there are studies that demonstrate the benefits and potential risks of a timely diagnosis of ad for individuals who exhibit changes in cognition , behavior , or function but are not yet clearly demented . a comprehensive literature search was performed to identify publications that investigated the benefits and risks of a timely diagnosis of ad at the prodromal ( pre - dementia ) stage . eligible articles were those reporting the results of studies investigating the benefits or challenges of a timely diagnosis of ad . of the 45 references selected by the authors , and assessed further for eligibility , nine were studies or surveys pertaining to the consequences of a timely diagnosis of ad and were included in the results . to verify that no relevant studies on the benefits or challenges of a timely diagnosis of ad were missed , a second comprehensive search of medline ( through pubmed ) and embase ( both accessed via proquest dialog ) from january 1 , 2000 to may 21 , 2014 using the search terms ( timely diagnosis ) and ( mild cognitive impairment or amnestic mild cognitive impairment ) was subsequently performed . none of these articles yielded findings on the consequences of a timely diagnosis of prodromal ad . a further search of medline and embase ( both accessed via proquest dialog ) to identify original clinical studies on the benefits and challenges of a timely diagnosis of ad was performed on june 2 , 2014 using the following search terms : dementia or alzheimer or alzheimer s and prodromal or pre - dementia or early symptoms and diagnosis . a comprehensive literature search was performed to identify publications that investigated the benefits and risks of a timely diagnosis of ad at the prodromal ( pre - dementia ) stage . eligible articles were those reporting the results of studies investigating the benefits or challenges of a timely diagnosis of ad . of the 45 references selected by the authors , and assessed further for eligibility , nine were studies or surveys pertaining to the consequences of a timely diagnosis of ad and were included in the results . to verify that no relevant studies on the benefits or challenges of a timely diagnosis of ad were missed , a second comprehensive search of medline ( through pubmed ) and embase ( both accessed via proquest dialog ) from january 1 , 2000 to may 21 , 2014 using the search terms ( timely diagnosis ) and ( mild cognitive impairment or amnestic mild cognitive impairment ) was subsequently performed . none of these articles yielded findings on the consequences of a timely diagnosis of prodromal ad . a further search of medline and embase ( both accessed via proquest dialog ) to identify original clinical studies on the benefits and challenges of a timely diagnosis of ad was performed on june 2 , 2014 using the following search terms : dementia or alzheimer or alzheimer s and prodromal or pre - dementia or early symptoms and diagnosis . nine studies related to the benefits or challenges of a timely diagnosis of ad were identified from the literature search . some of these studies included subjects in the pre - dementia stage of ad but none were specifically focused on diagnosing ad at the prodromal stage . one study was a survey of the psychological reactions of patients and their companions to receiving a diagnosis of dementia , while three studies focused on physicians or caregivers , not patients [ 3941 ] . these included a qualitative survey of caregivers or primary care physicians ( pcps ) on the perceived benefits and risks of an early recognition of dementia or alzheimer s dementia ; a survey of general practitioner ( gp ) attitudes toward diagnosing dementia that aimed to understand the low dementia diagnosis rate in certain regions in england ; and a randomized controlled study that examined the effects of a tailored educational intervention on dementia diagnosis and management in primary care . surveyed 990 pcps ( including gps , community nurses , practice nurses , community mental health nurses , and others ) on the perceived benefits and risks of an early recognition of dementia in general practice this study generated data from workshops using a nominal group approach , identifying benefits for patients , families and local services . the hazards identified for patients and families included fear , anxiety / depression , stigma , and altered relationships , while pcps expressed concerns about diagnostic errors and resource limitations , including shortfalls in local services , as more people received diagnoses early in the disease continuum . in an online survey where gps in primary care in norfolk and suffolk , england , were asked to rate the extent to which they agreed or disagreed with seven statements on dementia diagnosis , 85% of the 113 respondents agreed that it is beneficial to patients and their families to have a timely diagnosis of dementia . the educational program combined timely diagnosis of dementia and psychosocial support around the time of diagnosis with components appropriate to the later stages of the disease course , while the usual care control practices were provided with a summary of the uk national institute for health and care excellence dementia clinical guideline 42 . a multidisciplinary memory clinic set up in sheffield , england , was designed to facilitate diagnosis and treatment of alzheimer s dementia early in the course of the disease through careful management of the diagnostic process with a focus on pre- and post - diagnostic counseling followed by psychosocial interventions . the results showed that the net benefits could be highest when patients received a diagnosis at the initial symptomatic stage of the disease and when drug treatment was combined with caregiver intervention : the mean net social , state fiscal , and federal fiscal benefits of drug treatment plus caregiver intervention for a 70-year - old married woman with a mini - mental state examination ( mmse ) score of 28 were estimated as $ us125,000 , $ 16,000 , and $ 34,000 , respectively . performed an economic evaluation of early assessment and treatment for ad in the uk using discrete event simulation based on data from the consortium to establish a registry for alzheimer s disease ( cerad ) , and patient - level data from seven donepezil clinical trials to simulate ad progression and the effects of treatment intervention . nine studies related to the benefits or challenges of a timely diagnosis of ad were identified from the literature search . some of these studies included subjects in the pre - dementia stage of ad but none were specifically focused on diagnosing ad at the prodromal stage . one study was a survey of the psychological reactions of patients and their companions to receiving a diagnosis of dementia , while three studies focused on physicians or caregivers , not patients [ 3941 ] . these included a qualitative survey of caregivers or primary care physicians ( pcps ) on the perceived benefits and risks of an early recognition of dementia or alzheimer s dementia ; a survey of general practitioner ( gp ) attitudes toward diagnosing dementia that aimed to understand the low dementia diagnosis rate in certain regions in england ; and a randomized controlled study that examined the effects of a tailored educational intervention on dementia diagnosis and management in primary care . surveyed 990 pcps ( including gps , community nurses , practice nurses , community mental health nurses , and others ) on the perceived benefits and risks of an early recognition of dementia in general practice . the hazards identified for patients and families included fear , anxiety / depression , stigma , and altered relationships , while pcps expressed concerns about diagnostic errors and resource limitations , including shortfalls in local services , as more people received diagnoses early in the disease continuum . in an online survey where gps in primary care in norfolk and suffolk , england , were asked to rate the extent to which they agreed or disagreed with seven statements on dementia diagnosis , 85% of the 113 respondents agreed that it is beneficial to patients and their families to have a timely diagnosis of dementia . the educational program combined timely diagnosis of dementia and psychosocial support around the time of diagnosis with components appropriate to the later stages of the disease course , while the usual care control practices were provided with a summary of the uk national institute for health and care excellence dementia clinical guideline 42 . a multidisciplinary memory clinic set up in sheffield , england , was designed to facilitate diagnosis and treatment of alzheimer s dementia early in the course of the disease through careful management of the diagnostic process with a focus on pre- and post - diagnostic counseling followed by psychosocial interventions . the results showed that the net benefits could be highest when patients received a diagnosis at the initial symptomatic stage of the disease and when drug treatment was combined with caregiver intervention : the mean net social , state fiscal , and federal fiscal benefits of drug treatment plus caregiver intervention for a 70-year - old married woman with a mini - mental state examination ( mmse ) score of 28 were estimated as $ us125,000 , $ 16,000 , and $ 34,000 , respectively . performed an economic evaluation of early assessment and treatment for ad in the uk using discrete event simulation based on data from the consortium to establish a registry for alzheimer s disease ( cerad ) , and patient - level data from seven donepezil clinical trials to simulate ad progression and the effects of treatment intervention . , ad associations ) , regulators , and policy makers are , to varying degrees , encouraging a cultural shift toward making a timely diagnosis of ad at the initial symptomatic stages of the illness . despite this change in paradigm , our extensive and comprehensive review of the literature identified some studies highlighting the potential advantages of diagnosis and intervention early in the time course of dementia , but failed to find studies clearly focused on the benefits for patients , carers , or society of a timely diagnosis at the prodromal stage , before dementia sets in . in agreement with the 2011 world alzheimer report , we found that much of the literature outlining the benefits of timely diagnosis of ad is based on expert opinion rather than research evidence . the scarcity of published studies assessing the benefits and challenges of timely diagnosis could be due to the fact that the definition of ad as an entity that encompasses both pre - dementia and dementia phases is relatively recent and still not widely accepted . although our search of the literature was comprehensive and the search terms used should have been broad enough to capture most publications relevant to a timely diagnosis of ad , it is possible that some studies were missed because of the terminology used . a timely diagnosis offers the opportunity of introducing psychosocial interventions that may prolong the time people spend in initial stages of the disease and delay admission to residential long - term care homes , as indicated by the effectiveness of a multidisciplinary memory clinic model . simulation models of early intervention with hypothetical disease - modifying therapies indicate that substantial cost savings might be possible [ 56 , 57 ] . thus , further investigation of the health and social care costs associated with timely diagnosis of ad at the prodromal stage is needed . many of the potential benefits of timely diagnosis and treatment of ad from the studies identified in this review are dependent on the existence of a disease - modifying medication . , burden , quality of life , depression / anxiety ) from published studies allows us to only speculate on the potential clinical benefits and risks of diagnosing patients at an earlier phase in the disease continuum , when they have symptoms but have not yet developed overt dementia . moreover , much of the following discussion is based on the assumption that a timely diagnosis of ad is feasible , even if the well - recognized problem of under - diagnosis of dementia at more severe stages in the current medical setting [ 2 , 40 ] demonstrates that this is not always the case . among the many conceivable benefits to patients , caregivers , healthcare providers , and society of a timely diagnosis of ad ( table 2 ) , one of the most important would appear to be that an earlier and improved diagnostic pathway may help patients avoid the medical nomadism that some experience during the diagnostic process , which begins with seeking help and ends with receiving a definitive diagnosis and treatment . for example , the value of knowing survey of public attitudes to early diagnosis of ad found that over 94% of the people surveyed ( in each of the four european countries where the question was asked ) wanted to be told if they had ad , and a recent qualitative study on people with cognitive decline , ranging from subjective memory problems to early dementia , showed that the overwhelming majority of participants were keen to know their diagnosis and its long - term consequences . we can postulate that recognition of ad during the prodromal stage would allow physicians to offer therapies that address specific symptoms , such as anxiety or impaired sleep , and manage medications prescribed for comorbidities that may be inadvertently exacerbating dementia or other emerging symptoms.it can be speculated that a timely diagnosis of ad would also open up the opportunity of early intervention with disease - modifying therapies if they become available . some of the existing barriers to diagnosis of dementia may also apply to the timely diagnosis of ad at the prodromal stage . although there are reports of patients and caregivers expressing a positive attitude toward receiving a timely diagnosis of dementia , some physicians , especially in the primary care setting , cite various reasons explaining their reluctance to give a diagnosis of ad at the initial symptomatic stages of illness . for example , they say they do not consider it is beneficial for patients overall health and well - being , perceive there are no effective treatments , or consider that a diagnosis early in the disease continuum may actually be harmful to patients [ 39 , 7375 ] . while some patients and families may experience negative reactions to disclosure of a diagnosis of ad , including feelings of loss , anger , uncertainty , frustration , anxiety / depression , and catastrophic thinking ( although this has not been specifically investigated for patients given a diagnosis of prodromal ad ) [ 61 , 77 ] , other reports find no such long - term after - effects from a dementia diagnosis , and conclude that individual preferences for diagnosis disclosure should be taken into account [ 38 , 60 , 78 ] . finally , memory problems and alterations of cognitive function can cause changes in personal identity , capacity , and autonomy ; these issues would presumably add to the ethical challenges associated with a timely diagnosis of ad at the prodromal stage ( table 3 ) [ 33 , 35 ] . to achieve a timely diagnosis at the initial stage of the disease before patients exhibit dementia symptoms , existing barriers to diagnosis will have to be removed . clinical practice guidelines will need to be modified to align with the cultural shift to a timelydiagnosis of ad at the pre - dementia stage . for example , the most recent european federation of the neurological societies ( efns ) guidelines for the diagnosis and management of ad do not include diagnosis at the prodromal stage , and many of the screening tests recommended for assessing global cognition and activities of daily living may not be specific or sensitive enough for identifying prodromal ad . establishing a clinical diagnosis of ad a timely diagnosis at the pre - dementia stage requires a coordinated diagnostic process involving a multidisciplinary team approach ; all of those involved will need to be aware of the subtle cognitive and non - cognitive changes that can precede the onset of dementia . therefore , the cultural shift toward timely diagnosis depends upon increasing public and professional awareness of the initial symptoms of ad , improving knowledge among healthcare professionals about the benefits of a timely diagnosis and early intervention , reducing the fear and stigma about dementia , normalizing the experience of dementia , and developing and implementing dementia strategies and integrated diagnostic services that enable timely diagnosis and provide post - diagnostic support , care , and interventions for patients and their families . we believe that timely diagnosis of ad at a time when people first seek for help being worried about changes in cognition , behavior , or functioning not necessarily resulting in dementia , has the potential to reduce the impact of no or delayed diagnosis or misdiagnosis . timely diagnosis at the prodromal stage of the disease could offer many potential benefits to patients and caregivers , especially the opportunity to obtain treatment to control symptoms , avoid medications that may worsen symptoms , and , possibly in the future , access to interventions that slow or lessen the disease process . the findings of this literature review show that , at the current time , these ideas are mainly based on expert opinion and perhaps belief ; evidence is lacking , and further studies are needed to demonstrate not only that a timely diagnosis is feasible , but also that it has benefits . such evidence would support the cultural shift towards diagnosis at the pre - dementia stage of ad .

male c57bl/6j mice ( 810 weeks of age ) were purchased from jackson laboratories ( bar harbor , me ) . the mice were in the light 14 hours per day and in darkness 10 hours per day everyday of their life ( per routine animal housing protocol ) and were given free access to standard laboratory chow and water . to induce acute er stress , mice were treated with a single intraperitoneal ( ip ) injection of tunicamycin ( 0.5 mg / kg ) and were killed 6 hours later . to induce prolonged er stress , mice were treated with daily injections of tunicamycin ( 0.1 mg / kg ip ) for 5 days ( cumulative dose , 0.5 mg / kg ip ) . control mice were treated with vehicle ( 10% dimethyl sulfoxide [ dmso ] ip ) . the livers were excised rapidly , flushed with ice - cold saline , sectioned , and snap - frozen in liquid nitrogen . the small intestine was removed , flushed with ice - cold saline , and the terminal 5-cm segment was snap - frozen in liquid nitrogen . all animal protocols were approved by the northwestern university institutional animal care and use committee . hepatic bile acid concentration was measured using a bioquant total bile acid colorimetric assay ( bq kits , san diego , ca ) . plasma alanine aminotransferase level ( teco diagnostics , anaheim , ca ) was measured using a spectrophotometric assay per the manufacturer s protocol . total cholesterol content in liver homogenate was measured using an infinity spectrophotometric assay ( fisher scientific , middletown , va ) . plasma 7-hydroxy-4-cholesten-3 - 1 ( c4 ) measurement was performed at the mayo clinic immunochemical core lab ( rochester , mn ) . hepatic bile acid composition was measured by high - performance liquid chromatography as previously described . samples were spiked with glycocholic acid as an internal standard to control for extraction efficiency . the content of tauromuricholic acid , taurocholic acid , and taurochenodeoxycholic acid was calculated from a standard curve and reported as the percentage of total hepatic bile acids . human hepatoma ( hepg2 ) cells ( atcc , mannasas , va ) were cultured in dulbecco 's modified eagle medium ( dmem ) with 10% fetal bovine serum and maintained at 37c in 5% co2 . cells were grown to 80% confluence in 6-well plates and treated with 12 mol / l tunicamycin , 100 nmol / l thapsigargin ( sigma - aldrich , st . louis , mo ) , 5 mmol / l dl - homocysteine ( sigma - aldrich ) , or vehicle ( dmso / saline ) in serum - free dmem for 6 hours . to determine whether the effects of tunicamycin are dependent on c - jun - n - terminal kinase ( jnk ) or extracellular signaling - regulated kinase ( erk ) , hepg2 cells were treated with the jnk inhibitor sp600125 ( sigma - aldrich ) at a concentration of 25 mol / l or the mapk / erk kinase inhibitor pd184352 ( santa cruz biotechnology , dallas , tx ) at 1 mol / l or vehicle ( dmso / saline ) as previously described . one hour after exposure to sp600125 or pd184352 , cells were treated with tunicamycin ( 12 mol / l ; sigma - aldrich ) in serum - free dmem and incubated for an additional 6 hours . successful inhibition of jnk and erk activation was confirmed by western blot analysis as previously described . total rna from frozen liver , ileum , or cultured hepg2 cells was isolated using trizol reagent ( ambion life technologies , carlsbad , ca ) , and real - time quantitative polymerase chain reaction was performed as previously described.27 , 28 total protein was isolated from frozen liver samples and western blotting was performed as previously described.27 , 28 protein detection was performed using polyclonal rabbit antibodies to cyp7a1 ( proteintech , rosemont , il ) and glyceraldehyde-3-phosphate dehydrogenase ( cell signaling technology , danvers , ma ) . bound antibody was detected using goat anti - rabbit polyclonal horseradish - peroxidase antibody ( cell signaling technology ) and developed using enhanced chemiluminescence western blotting substrate ( cell signaling technology ) . densitometry was performed on individual samples using imagej software ( available : imagej.nih.gov/ij/ ; national institutes of health , bethesda , md ) . all authors had access to the study data and reviewed and approved the final manuscript . male c57bl/6j mice ( 810 weeks of age ) were purchased from jackson laboratories ( bar harbor , me ) . the mice were in the light 14 hours per day and in darkness 10 hours per day everyday of their life ( per routine animal housing protocol ) and were given free access to standard laboratory chow and water . to induce acute er stress , mice were treated with a single intraperitoneal ( ip ) injection of tunicamycin ( 0.5 mg / kg ) and were killed 6 hours later . to induce prolonged er stress , mice were treated with daily injections of tunicamycin ( 0.1 mg / kg ip ) for 5 days ( cumulative dose , 0.5 mg / kg ip ) . control mice were treated with vehicle ( 10% dimethyl sulfoxide [ dmso ] ip ) . the livers were excised rapidly , flushed with ice - cold saline , sectioned , and snap - frozen in liquid nitrogen . the small intestine was removed , flushed with ice - cold saline , and the terminal 5-cm segment was snap - frozen in liquid nitrogen . all animal protocols were approved by the northwestern university institutional animal care and use committee . hepatic bile acid concentration was measured using a bioquant total bile acid colorimetric assay ( bq kits , san diego , ca ) . plasma alanine aminotransferase level ( teco diagnostics , anaheim , ca ) was measured using a spectrophotometric assay per the manufacturer s protocol . total cholesterol content in liver homogenate was measured using an infinity spectrophotometric assay ( fisher scientific , middletown , va ) . plasma 7-hydroxy-4-cholesten-3 - 1 ( c4 ) measurement was performed at the mayo clinic immunochemical core lab ( rochester , mn ) . hepatic bile acid composition was measured by high - performance liquid chromatography as previously described . samples were spiked with glycocholic acid as an internal standard to control for extraction efficiency . the content of tauromuricholic acid , taurocholic acid , and taurochenodeoxycholic acid was calculated from a standard curve and reported as the percentage of total hepatic bile acids . human hepatoma ( hepg2 ) cells ( atcc , mannasas , va ) were cultured in dulbecco 's modified eagle medium ( dmem ) with 10% fetal bovine serum and maintained at 37c in 5% co2 . cells were grown to 80% confluence in 6-well plates and treated with 12 mol / l tunicamycin , 100 nmol / l thapsigargin ( sigma - aldrich , st . louis , mo ) , 5 mmol / l dl - homocysteine ( sigma - aldrich ) , or vehicle ( dmso / saline ) in serum - free dmem for 6 hours . to determine whether the effects of tunicamycin are dependent on c - jun - n - terminal kinase ( jnk ) or extracellular signaling - regulated kinase ( erk ) , hepg2 cells were treated with the jnk inhibitor sp600125 ( sigma - aldrich ) at a concentration of 25 mol / l or the mapk / erk kinase inhibitor pd184352 ( santa cruz biotechnology , dallas , tx ) at 1 mol / l or vehicle ( dmso / saline ) as previously described . one hour after exposure to sp600125 or pd184352 , cells were treated with tunicamycin ( 12 mol / l ; sigma - aldrich ) in serum - free dmem and incubated for an additional 6 hours . successful inhibition of jnk and erk activation was confirmed by western blot analysis as previously described . total rna from frozen liver , ileum , or cultured hepg2 cells was isolated using trizol reagent ( ambion life technologies , carlsbad , ca ) , and real - time quantitative polymerase chain reaction was performed as previously described.27 , 28 total protein was isolated from frozen liver samples and western blotting was performed as previously described.27 , 28 protein detection was performed using polyclonal rabbit antibodies to cyp7a1 ( proteintech , rosemont , il ) and glyceraldehyde-3-phosphate dehydrogenase ( cell signaling technology , danvers , ma ) . bound antibody was detected using goat anti - rabbit polyclonal horseradish - peroxidase antibody ( cell signaling technology ) and developed using enhanced chemiluminescence western blotting substrate ( cell signaling technology ) . densitometry was performed on individual samples using imagej software ( available : imagej.nih.gov/ij/ ; national institutes of health , bethesda , md ) . all authors had access to the study data and reviewed and approved the final manuscript . cyp7a1 is the primary bile acid synthetic enzyme controlling the rate - limiting step in the conversion of hepatic cholesterol to bile acids.29 , 30 to determine the effect of er stress on hepatic cyp7a1 expression , mice were treated with tunicamycin ( 0.5 mg / kg ip ) , a well - established er stress inducing agent in mice.31 , 32 , 33 mice treated with tunicamycin showed robust hepatic upr activation at 6 hours as evidenced by induction of glucose - regulated protein 78 kilodaltons , an er chaperone and master regulator of the upr , spliced x - box binding protein 1 , a major mediator of the inositol requiring enzyme 1 ( ire1 ) branch of the upr , and ccaat / enhancer binding protein homologous protein , a regulator of er stress - induced apoptosis34 , 35 , 36 , 37 ( table 1 ) . induction of hepatic er stress resulted in significant suppression of hepatic cyp7a1 messenger rna ( mrna ) and cyp7a1 protein expression ( figure 1a and b ) . the plasma concentration of c4 , a stable intermediate generated in the synthesis of bile acids from cholesterol , is an indicator of the activity of the cyp7a1-dependent bile acid synthetic pathway . consistent with the observed suppression of cyp7a1 , induction of er stress in mice reduced the plasma concentration of c4 ( figure 1c ) . although cyp7a1 regulates the major pathway of bile acid synthesis , bile acid synthesis also occurs via the alternative ( acidic ) pathway of bile acid synthesis involving sterol 27-hydroxylase and oxysterol 7--hydroxylase.38 , 39 , 40 we found that neither sterol 27-hydroxylase nor oxysterol 7 hydroxylase mrna expression was altered by induction of er stress ( figure 1d ) . there are significant differences in the expression levels and regulation of the cyp7a1 gene in mice and human beings . most notably , human beings lack an liver x receptor - response element in the cyp7a1 promoter , rendering human cyp7a1 unresponsive to dietary cholesterol.41 , 42 , 43 to exclude the possibility that the effects of er stress on cyp7a1 expression are specific to mice , we determined the effect of pharmacologic er stress on cyp7a1 expression in a human hepatoma cell line ( hepg2 ) . paralleling our findings in vivo , induction of er stress in hepg2 cells suppressed cyp7a1 expression ( figure 1e ) . to ensure that these effects are not specific to tunicamycin , we also treated hepg2 cells with 2 alternative pharmacologic er stress inducing agents , thapsigargin and homocysteine , for 6 hours . cyp7a1 expression was suppressed by 87% and 67% in hepg2 cells treated with thapsigargin and homocysteine , respectively ( figure 1e ) . a major mechanism of cyp7a1 regulation is via feedback inhibition from bile acids.44 , 45 , 46 specifically , bile acids bind to the farnesoid x receptor ( fxr ) in the ileum , stimulating release of fibroblast growth factor ( fgf ) 15/19 from the ileocyte , which subsequently acts in the liver to suppress cyp7a1 transcription.47 , 48 intestinal er stress , achieved through oral administration of tunicamycin to mice , has been shown to induce ileal fgf15 expression . we found that administration of intraperitoneal tunicamycin did not induce intestinal er stress at 6 hours ( table 1 ) . consistent with an absence of intestinal upr activation , we found no induction of ileal fgf15 expression in mice treated with intraperitoneal tunicamycin for 6 hours ( figure 2a ) . although activation of intestinal fxr - fgf15/19 signaling now is considered the major mechanism of bile acid feedback inhibition of cyp7a1 transcription , bile acids also inhibit cyp7a1 via activation of fxr within the liver , leading to induction of the small heterodimer partner ( shp ) . we found that induction of er stress in mice did not increase hepatic shp expression , indicating that tunicamycin does not suppress cyp7a1 via a fxr - shp dependent mechanism ( figure 2b ) . suppression of cyp7a1 expression is a feature of the hepatic inflammatory response.11 , 12 activation of cytokines such as tumor necrosis factor , interleukin ( il)1 , and il6 is associated with liver injury and has been shown to suppress hepatic cyp7a1 expression in vitro and in vivo.11 , 50 , 51 , 52 , 53 therefore , we considered the possibility that suppression of cyp7a1 expression by er stress is a manifestation of a broader stress response associated with inflammatory cytokine activation . we found no significant inflammatory cytokine activation at 6 hours after induction of er stress ( figure 2b ) . on the contrary , acute exposure to er stress suppressed hepatic tnf , il1 , and il6 expression . these data indicate that suppression of bile acid synthesis by er stress is independent of inflammatory cytokine activation . activation of jnk has been implicated in the inhibition of cyp7a1 by inflammatory cytokines.52 , 54 , 55 furthermore , it is well established that jnk activation occurs in response to er stress.26 , 56 , 57 to confirm whether er stress induced cyp7a1 suppression is dependent on jnk activation , we determined the effects of tunicamycin on hepg2 cells treated with the jnk inhibitor sp600125 . inhibition of jnk signaling in hepg2 cells did not prevent suppression of cyp7a1 by tunicamycin ( figure 2c ) . fgf15/19-mediated suppression of cyp7a1 expression is dependent on erk activation.58 , 59 we assessed whether er stress inhibition of erk in hepg2 cells using pd184352 did not prevent er stress induced suppression of cyp7a1 , thus providing additional evidence that the effect of er on hepatic bile acid synthesis is not dependent on fgf15/19 activation ( figure 2d ) . suppression of cyp7a1 expression is a major compensatory mechanism to prevent bile acid accumulation in the setting of cholestatic liver injury . several other compensatory mechanisms are activated in the liver in response to cholestasis , including suppression of bile acid uptake , induction of bile acid efflux , and enhanced biliary bile acid secretion . acute er stress increased hepatic expression of adenosine triphosphate binding cassette ( abc)b11 , the canalicular bile salt export pump , yet had no significant effect on the hepatic expression of ntcp , the basolateral transporter controlling sinusoidal uptake of bile acids into hepatocytes ( figure 3a ) . the multidrug resistance - associated protein 3 ( abcc3 ) is a basolateral bile acid efflux pump that is thought to have a protective role in the setting of cholestasis by removing bile acids from the cholestatic liver.60 , 61 the expression of abcc3 was increased in response to acute er stress ( figure 3a ) . having shown that hepatic abcb11 and abcc3 are activated in mice subjected to er stress , we next measured abcb11 and abcc3 expression in human hepatoma cells ( hepg2 ) treated with tunicamycin . paralleling our findings in vivo , induction of er stress with tunicamycin , thapsigargin , or homocysteine in hepg2 cells increased expression of abcb11 and abcc3 ( figure 3b and c ) . we have shown that induction of er stress suppresses the primary bile acid synthetic pathway and enhances expression of transporters responsible for the removal of bile acids from the liver . these pathways seemingly would converge to reduce the hepatic bile acid content over time . to test this hypothesis , we examined the effect of prolonged er stress on hepatic bile acid content . mice were treated with daily injections of low - dose tunicamycin ( 0.1 mg / kg ip ) for 5 days . similar to the pattern observed in mice acutely after induction of er stress , mice subjected to prolonged er stress showed induction of the hepatic upr associated with marked suppression of cyp7a1 and induction of abcc3 ( table 1 and figure 4a ) . consistent with suppressed cyp7a1-dependent bile acid synthesis , hepatic cholesterol content was increased and hepatic bile acid content was decreased in response to prolonged er stress ( figure 4b and c ) . the relative composition of the hepatic bile acids was unaffected by er stress ( figure 4d ) . unlike acute er stress , prolonged er stress suppressed hepatic expression of abcb11 and ntcp , bile acid transporters known to be suppressed by hepatic inflammation ( figure 4a ) . we found that prolonged er stress resulted in induction of the inflammatory cytokines il1 and il6 , suggesting activation of a more generalized hepatic inflammatory response in the setting of sustained er stress ( figure 4e ) . consistent with the development of hepatic inflammation , mice subjected to prolonged er stress showed a mild increase in plasma alanine aminotransferase level ( figure 4f ) and early ballooning degeneration on h&e - stained liver sections ( figure 4 g ) . prolonged er stress also resulted in induction of the intestinal upr , suggesting activation of a broader systemic stress response over time ( table 1 ) . cyp7a1 is the primary bile acid synthetic enzyme controlling the rate - limiting step in the conversion of hepatic cholesterol to bile acids.29 , 30 to determine the effect of er stress on hepatic cyp7a1 expression , mice were treated with tunicamycin ( 0.5 mg / kg ip ) , a well - established er stress inducing agent in mice.31 , 32 , 33 mice treated with tunicamycin showed robust hepatic upr activation at 6 hours as evidenced by induction of glucose - regulated protein 78 kilodaltons , an er chaperone and master regulator of the upr , spliced x - box binding protein 1 , a major mediator of the inositol requiring enzyme 1 ( ire1 ) branch of the upr , and ccaat / enhancer binding protein homologous protein , a regulator of er stress - induced apoptosis34 , 35 , 36 , 37 ( table 1 ) . induction of hepatic er stress resulted in significant suppression of hepatic cyp7a1 messenger rna ( mrna ) and cyp7a1 protein expression ( figure 1a and b ) . the plasma concentration of c4 , a stable intermediate generated in the synthesis of bile acids from cholesterol , is an indicator of the activity of the cyp7a1-dependent bile acid synthetic pathway . consistent with the observed suppression of cyp7a1 , induction of er stress in mice reduced the plasma concentration of c4 ( figure 1c ) . although cyp7a1 regulates the major pathway of bile acid synthesis , bile acid synthesis also occurs via the alternative ( acidic ) pathway of bile acid synthesis involving sterol 27-hydroxylase and oxysterol 7--hydroxylase.38 , 39 , 40 we found that neither sterol 27-hydroxylase nor oxysterol 7 hydroxylase mrna expression was altered by induction of er stress ( figure 1d ) . there are significant differences in the expression levels and regulation of the cyp7a1 gene in mice and human beings . most notably , human beings lack an liver x receptor - response element in the cyp7a1 promoter , rendering human cyp7a1 unresponsive to dietary cholesterol.41 , 42 , 43 to exclude the possibility that the effects of er stress on cyp7a1 expression are specific to mice , we determined the effect of pharmacologic er stress on cyp7a1 expression in a human hepatoma cell line ( hepg2 ) . paralleling our findings in vivo , induction of er stress in hepg2 cells suppressed cyp7a1 expression ( figure 1e ) . to ensure that these effects are not specific to tunicamycin , we also treated hepg2 cells with 2 alternative pharmacologic er stress inducing agents , thapsigargin and homocysteine , for 6 hours . cyp7a1 expression was suppressed by 87% and 67% in hepg2 cells treated with thapsigargin and homocysteine , respectively ( figure 1e ) . a major mechanism of cyp7a1 regulation is via feedback inhibition from bile acids.44 , 45 , 46 specifically , bile acids bind to the farnesoid x receptor ( fxr ) in the ileum , stimulating release of fibroblast growth factor ( fgf ) 15/19 from the ileocyte , which subsequently acts in the liver to suppress cyp7a1 transcription.47 , 48 intestinal er stress , achieved through oral administration of tunicamycin to mice , has been shown to induce ileal fgf15 expression . we found that administration of intraperitoneal tunicamycin did not induce intestinal er stress at 6 hours ( table 1 ) . consistent with an absence of intestinal upr activation , we found no induction of ileal fgf15 expression in mice treated with intraperitoneal tunicamycin for 6 hours ( figure 2a ) . although activation of intestinal fxr - fgf15/19 signaling now is considered the major mechanism of bile acid feedback inhibition of cyp7a1 transcription , bile acids also inhibit cyp7a1 via activation of fxr within the liver , leading to induction of the small heterodimer partner ( shp ) . we found that induction of er stress in mice did not increase hepatic shp expression , indicating that tunicamycin does not suppress cyp7a1 via a fxr - shp dependent mechanism ( figure 2b ) . suppression of cyp7a1 expression is a feature of the hepatic inflammatory response.11 , 12 activation of cytokines such as tumor necrosis factor , interleukin ( il)1 , and il6 is associated with liver injury and has been shown to suppress hepatic cyp7a1 expression in vitro and in vivo.11 , 50 , 51 , 52 , 53 therefore , we considered the possibility that suppression of cyp7a1 expression by er stress is a manifestation of a broader stress response associated with inflammatory cytokine activation . we found no significant inflammatory cytokine activation at 6 hours after induction of er stress ( figure 2b ) . on the contrary , acute exposure to er stress suppressed hepatic tnf , il1 , and il6 expression . these data indicate that suppression of bile acid synthesis by er stress is independent of inflammatory cytokine activation . activation of jnk has been implicated in the inhibition of cyp7a1 by inflammatory cytokines.52 , 54 , 55 furthermore , it is well established that jnk activation occurs in response to er stress.26 , 56 , 57 to confirm whether er stress induced cyp7a1 suppression is dependent on jnk activation , we determined the effects of tunicamycin on hepg2 cells treated with the jnk inhibitor sp600125 . inhibition of jnk signaling in hepg2 cells did not prevent suppression of cyp7a1 by tunicamycin ( figure 2c ) . fgf15/19-mediated suppression of cyp7a1 expression is dependent on erk activation.58 , 59 we assessed whether er stress inhibition of erk in hepg2 cells using pd184352 did not prevent er stress induced suppression of cyp7a1 , thus providing additional evidence that the effect of er on hepatic bile acid synthesis is not dependent on fgf15/19 activation ( figure 2d ) . suppression of cyp7a1 expression is a major compensatory mechanism to prevent bile acid accumulation in the setting of cholestatic liver injury . several other compensatory mechanisms are activated in the liver in response to cholestasis , including suppression of bile acid uptake , induction of bile acid efflux , and enhanced biliary bile acid secretion . acute er stress increased hepatic expression of adenosine triphosphate binding cassette ( abc)b11 , the canalicular bile salt export pump , yet had no significant effect on the hepatic expression of ntcp , the basolateral transporter controlling sinusoidal uptake of bile acids into hepatocytes ( figure 3a ) . the multidrug resistance - associated protein 3 ( abcc3 ) is a basolateral bile acid efflux pump that is thought to have a protective role in the setting of cholestasis by removing bile acids from the cholestatic liver.60 , 61 the expression of abcc3 was increased in response to acute er stress ( figure 3a ) . having shown that hepatic abcb11 and abcc3 are activated in mice subjected to er stress , we next measured abcb11 and abcc3 expression in human hepatoma cells ( hepg2 ) treated with tunicamycin . paralleling our findings in vivo , induction of er stress with tunicamycin , thapsigargin , or homocysteine in hepg2 cells increased expression of abcb11 and abcc3 ( figure 3b and c ) . we have shown that induction of er stress suppresses the primary bile acid synthetic pathway and enhances expression of transporters responsible for the removal of bile acids from the liver . these pathways seemingly would converge to reduce the hepatic bile acid content over time . to test this hypothesis , we examined the effect of prolonged er stress on hepatic bile acid content . mice were treated with daily injections of low - dose tunicamycin ( 0.1 mg / kg ip ) for 5 days . similar to the pattern observed in mice acutely after induction of er stress , mice subjected to prolonged er stress showed induction of the hepatic upr associated with marked suppression of cyp7a1 and induction of abcc3 ( table 1 and figure 4a ) . consistent with suppressed cyp7a1-dependent bile acid synthesis , hepatic cholesterol content was increased and hepatic bile acid content was decreased in response to prolonged er stress ( figure 4b and c ) . the relative composition of the hepatic bile acids was unaffected by er stress ( figure 4d ) . unlike acute er stress , prolonged er stress suppressed hepatic expression of abcb11 and ntcp , bile acid transporters known to be suppressed by hepatic inflammation ( figure 4a ) . we found that prolonged er stress resulted in induction of the inflammatory cytokines il1 and il6 , suggesting activation of a more generalized hepatic inflammatory response in the setting of sustained er stress ( figure 4e ) . consistent with the development of hepatic inflammation , mice subjected to prolonged er stress showed a mild increase in plasma alanine aminotransferase level ( figure 4f ) and early ballooning degeneration on h&e - stained liver sections ( figure 4 g ) . prolonged er stress also resulted in induction of the intestinal upr , suggesting activation of a broader systemic stress response over time ( table 1 ) . bile acids perform critical physiologic functions , however , when present in excess these molecules promote hepatotoxicity . in response to hepatocellular stress , induced anticholestatic mechanism we have identified is suppression of the cyp7a1-dependent bile acid synthetic pathway . we found that suppression of cyp7a1 occurs early after induction of er stress , in the absence of inflammatory cytokine activation . in addition , we showed that cyp7a1 suppression by er stress occurred in the absence of hepatic shp or ileal fgf15 activation . these data show that cyp7a1 suppression by er stress was independent of established fxr - dependent or cytokine - mediated pathways . moreover , these data strongly suggest that the observed effects of acute er stress on hepatic bile acid metabolism were a direct result of er stress and not simply a manifestation of a broader hepatic inflammatory response . we found that er stress also regulated the expression of hepatic basolateral and canalicular transporters . er stress acutely increased hepatic expression of abcc3 , a basolateral bile salt efflux pump , and abcb11 , the canalicular bile salt export pump . these findings suggest that , as part of a coordinated protective response , the hepatocyte removed toxic bile acids from the liver in response to er stress . consistent with this assertion was the finding that prolonged er stress lead to a reduction in hepatic bile acid content . contrary to the acute response to er stress , we found that sustained er stress led to suppression of abcb11 and ntcp . furthermore , we found that sustained er stress promoted a hepatic inflammatory response associated with cytokine activation . suppression of abcb11 and ntcp has been a well - established feature of the hepatic inflammatory response,12 , 13 , 62 , 63 , 64 , 65 and we speculate that suppression of these transporters with prolonged er stress is secondary to inflammatory cytokine activation . we have speculated that suppression of cyp7a1 is a direct result of upr activation yet it remains unclear which element(s ) of this highly intricate signaling cascade may be responsible for the observed phenotype . the upr is initiated through 3 er transmembrane receptors , pkr - like er kinase , activating transcription factor 6 , and ire1 and 1 master chaperone , glucose - regulated protein 78 kilodaltons . the ire1 pathway is the most evolutionarily conserved branch of the upr and is considered the major mediator of the adaptive response to cellular stress.66 , 67 , 68 it therefore is reasonable to hypothesize that the ire1 arm of the upr may mediate an adaptive response aimed at maintaining bile acid homeostasis . furthermore , although all 3 branches of the upr have been shown to modulate metabolic pathways , the ire1 pathway has emerged as the branch most strongly implicated in regulating hepatic lipid metabolism , which is linked intimately to bile acid metabolism.69 , 70 , 71 activated ire1 , functioning as an endonuclease , induces nonconventional splicing of the transcription factor x - box binding protein 1 , which has been implicated in the transcription of a myriad of genes involved in proteostasis and metabolic processes . it has become increasingly apparent , however , that the major regulatory effect of ire1 activation is attributable to regulated ire1-dependent mrna decay ( ridd ) . a rapidly increasing number of mrnas and micrornas involved in a diverse array of cellular processes have been identified as ridd targets.72 , 73 , 74 , 75 at least 2 such mrnas encode er - localized cytochrome p450 enzymes , cyp1a2 and cyp2e1 . these data raise the possibility that in the setting of er stress , cyp7a1 mrna may be targeted for destruction via ridd . the present work implicates er stress as a novel regulator of the major bile acid synthetic pathway . although the classic pathophysiologic consequence of disrupted bile acid homeostasis is cholestatic liver injury , altered bile acid metabolism now is known to impact numerous physiologic processes and may have broader implications for human metabolic disease . impaired bile acid metabolism has been implicated in the pathogenesis of diabetes and obesity , both of which are associated with er stress.77 , 78 , 79 the possibility that altered bile acid homeostasis is a mechanistic link between er stress and the development of metabolic disease warrants further investigation .

background & aimscholestasis promotes endoplasmic reticulum ( er ) stress in the liver , however , the effect of er stress on hepatic bile acid metabolism is unknown . we aim to determine the effect of er stress on hepatic bile acid synthesis and transport in mice.methodser stress was induced pharmacologically in c57bl/6j mice and human hepatoma ( hepg2 ) cells . the hepatic expression of genes controlling bile acid synthesis and transport was determined . to measure the activity of the primary bile acid synthetic pathway , the concentration of 7-hydroxy-4-cholesten-3 - 1 was measured in plasma.resultsinduction of er stress in mice and hepg2 cells rapidly suppressed the hepatic expression of the primary bile acid synthetic enzyme , cholesterol 7-hydroxylase . plasma levels of 7-hydroxy-4-cholesten-3 - 1 were reduced in mice subjected to er stress , indicating impaired bile acid synthesis . induction of er stress in mice and hepg2 cells increased expression of the bile salt export pump ( adenosine triphosphate binding cassette [ abc]b11 ) and a bile salt efflux pump ( abcc3 ) . the observed regulation of cyp7a1 , abcb11 , and abcc3 occurred in the absence of hepatic inflammatory cytokine activation and was not dependent on activation of hepatic small heterodimer partner or intestinal fibroblast growth factor 15 . consistent with suppressed bile acid synthesis and enhanced bile acid export from hepatocytes , prolonged er stress decreased the hepatic bile acid content in mice.conclusionsinduction of er stress in mice suppresses bile acid synthesis and enhances bile acid removal from hepatocytes independently of established bile acid regulatory pathways . these data show a novel function of the er stress response in regulating bile acid metabolism .

Materials and Methods Animals and Treatments Plasma and Hepatic Biochemical Analysis High-Performance Liquid Chromatography Assay Cell Culture Analysis of Gene and Protein Expression Statistical Analysis Results ER Stress Suppresses the Primary Bile Acid Synthetic Pathway Suppression of Suppression of Suppression of ER Stress Regulates Hepatic Bile Acid Transporters in Mice Prolonged ER Stress Reduces the Hepatic BileAcid Content in Mice Discussion

human hepatoma ( hepg2 ) cells ( atcc , mannasas , va ) were cultured in dulbecco 's modified eagle medium ( dmem ) with 10% fetal bovine serum and maintained at 37c in 5% co2 . human hepatoma ( hepg2 ) cells ( atcc , mannasas , va ) were cultured in dulbecco 's modified eagle medium ( dmem ) with 10% fetal bovine serum and maintained at 37c in 5% co2 . cyp7a1 is the primary bile acid synthetic enzyme controlling the rate - limiting step in the conversion of hepatic cholesterol to bile acids.29 , 30 to determine the effect of er stress on hepatic cyp7a1 expression , mice were treated with tunicamycin ( 0.5 mg / kg ip ) , a well - established er stress inducing agent in mice.31 , 32 , 33 mice treated with tunicamycin showed robust hepatic upr activation at 6 hours as evidenced by induction of glucose - regulated protein 78 kilodaltons , an er chaperone and master regulator of the upr , spliced x - box binding protein 1 , a major mediator of the inositol requiring enzyme 1 ( ire1 ) branch of the upr , and ccaat / enhancer binding protein homologous protein , a regulator of er stress - induced apoptosis34 , 35 , 36 , 37 ( table 1 ) . the plasma concentration of c4 , a stable intermediate generated in the synthesis of bile acids from cholesterol , is an indicator of the activity of the cyp7a1-dependent bile acid synthetic pathway . consistent with the observed suppression of cyp7a1 , induction of er stress in mice reduced the plasma concentration of c4 ( figure 1c ) . although cyp7a1 regulates the major pathway of bile acid synthesis , bile acid synthesis also occurs via the alternative ( acidic ) pathway of bile acid synthesis involving sterol 27-hydroxylase and oxysterol 7--hydroxylase.38 , 39 , 40 we found that neither sterol 27-hydroxylase nor oxysterol 7 hydroxylase mrna expression was altered by induction of er stress ( figure 1d ) . there are significant differences in the expression levels and regulation of the cyp7a1 gene in mice and human beings . most notably , human beings lack an liver x receptor - response element in the cyp7a1 promoter , rendering human cyp7a1 unresponsive to dietary cholesterol.41 , 42 , 43 to exclude the possibility that the effects of er stress on cyp7a1 expression are specific to mice , we determined the effect of pharmacologic er stress on cyp7a1 expression in a human hepatoma cell line ( hepg2 ) . paralleling our findings in vivo , induction of er stress in hepg2 cells suppressed cyp7a1 expression ( figure 1e ) . a major mechanism of cyp7a1 regulation is via feedback inhibition from bile acids.44 , 45 , 46 specifically , bile acids bind to the farnesoid x receptor ( fxr ) in the ileum , stimulating release of fibroblast growth factor ( fgf ) 15/19 from the ileocyte , which subsequently acts in the liver to suppress cyp7a1 transcription.47 , 48 intestinal er stress , achieved through oral administration of tunicamycin to mice , has been shown to induce ileal fgf15 expression . although activation of intestinal fxr - fgf15/19 signaling now is considered the major mechanism of bile acid feedback inhibition of cyp7a1 transcription , bile acids also inhibit cyp7a1 via activation of fxr within the liver , leading to induction of the small heterodimer partner ( shp ) . we found that induction of er stress in mice did not increase hepatic shp expression , indicating that tunicamycin does not suppress cyp7a1 via a fxr - shp dependent mechanism ( figure 2b ) . suppression of cyp7a1 expression is a feature of the hepatic inflammatory response.11 , 12 activation of cytokines such as tumor necrosis factor , interleukin ( il)1 , and il6 is associated with liver injury and has been shown to suppress hepatic cyp7a1 expression in vitro and in vivo.11 , 50 , 51 , 52 , 53 therefore , we considered the possibility that suppression of cyp7a1 expression by er stress is a manifestation of a broader stress response associated with inflammatory cytokine activation . we found no significant inflammatory cytokine activation at 6 hours after induction of er stress ( figure 2b ) . these data indicate that suppression of bile acid synthesis by er stress is independent of inflammatory cytokine activation . activation of jnk has been implicated in the inhibition of cyp7a1 by inflammatory cytokines.52 , 54 , 55 furthermore , it is well established that jnk activation occurs in response to er stress.26 , 56 , 57 to confirm whether er stress induced cyp7a1 suppression is dependent on jnk activation , we determined the effects of tunicamycin on hepg2 cells treated with the jnk inhibitor sp600125 . fgf15/19-mediated suppression of cyp7a1 expression is dependent on erk activation.58 , 59 we assessed whether er stress inhibition of erk in hepg2 cells using pd184352 did not prevent er stress induced suppression of cyp7a1 , thus providing additional evidence that the effect of er on hepatic bile acid synthesis is not dependent on fgf15/19 activation ( figure 2d ) . several other compensatory mechanisms are activated in the liver in response to cholestasis , including suppression of bile acid uptake , induction of bile acid efflux , and enhanced biliary bile acid secretion . acute er stress increased hepatic expression of adenosine triphosphate binding cassette ( abc)b11 , the canalicular bile salt export pump , yet had no significant effect on the hepatic expression of ntcp , the basolateral transporter controlling sinusoidal uptake of bile acids into hepatocytes ( figure 3a ) . the multidrug resistance - associated protein 3 ( abcc3 ) is a basolateral bile acid efflux pump that is thought to have a protective role in the setting of cholestasis by removing bile acids from the cholestatic liver.60 , 61 the expression of abcc3 was increased in response to acute er stress ( figure 3a ) . having shown that hepatic abcb11 and abcc3 are activated in mice subjected to er stress , we next measured abcb11 and abcc3 expression in human hepatoma cells ( hepg2 ) treated with tunicamycin . paralleling our findings in vivo , induction of er stress with tunicamycin , thapsigargin , or homocysteine in hepg2 cells increased expression of abcb11 and abcc3 ( figure 3b and c ) . we have shown that induction of er stress suppresses the primary bile acid synthetic pathway and enhances expression of transporters responsible for the removal of bile acids from the liver . to test this hypothesis , we examined the effect of prolonged er stress on hepatic bile acid content . similar to the pattern observed in mice acutely after induction of er stress , mice subjected to prolonged er stress showed induction of the hepatic upr associated with marked suppression of cyp7a1 and induction of abcc3 ( table 1 and figure 4a ) . consistent with suppressed cyp7a1-dependent bile acid synthesis , hepatic cholesterol content was increased and hepatic bile acid content was decreased in response to prolonged er stress ( figure 4b and c ) . unlike acute er stress , prolonged er stress suppressed hepatic expression of abcb11 and ntcp , bile acid transporters known to be suppressed by hepatic inflammation ( figure 4a ) . we found that prolonged er stress resulted in induction of the inflammatory cytokines il1 and il6 , suggesting activation of a more generalized hepatic inflammatory response in the setting of sustained er stress ( figure 4e ) . consistent with the development of hepatic inflammation , mice subjected to prolonged er stress showed a mild increase in plasma alanine aminotransferase level ( figure 4f ) and early ballooning degeneration on h&e - stained liver sections ( figure 4 g ) . prolonged er stress also resulted in induction of the intestinal upr , suggesting activation of a broader systemic stress response over time ( table 1 ) . cyp7a1 is the primary bile acid synthetic enzyme controlling the rate - limiting step in the conversion of hepatic cholesterol to bile acids.29 , 30 to determine the effect of er stress on hepatic cyp7a1 expression , mice were treated with tunicamycin ( 0.5 mg / kg ip ) , a well - established er stress inducing agent in mice.31 , 32 , 33 mice treated with tunicamycin showed robust hepatic upr activation at 6 hours as evidenced by induction of glucose - regulated protein 78 kilodaltons , an er chaperone and master regulator of the upr , spliced x - box binding protein 1 , a major mediator of the inositol requiring enzyme 1 ( ire1 ) branch of the upr , and ccaat / enhancer binding protein homologous protein , a regulator of er stress - induced apoptosis34 , 35 , 36 , 37 ( table 1 ) . induction of hepatic er stress resulted in significant suppression of hepatic cyp7a1 messenger rna ( mrna ) and cyp7a1 protein expression ( figure 1a and b ) . the plasma concentration of c4 , a stable intermediate generated in the synthesis of bile acids from cholesterol , is an indicator of the activity of the cyp7a1-dependent bile acid synthetic pathway . consistent with the observed suppression of cyp7a1 , induction of er stress in mice reduced the plasma concentration of c4 ( figure 1c ) . although cyp7a1 regulates the major pathway of bile acid synthesis , bile acid synthesis also occurs via the alternative ( acidic ) pathway of bile acid synthesis involving sterol 27-hydroxylase and oxysterol 7--hydroxylase.38 , 39 , 40 we found that neither sterol 27-hydroxylase nor oxysterol 7 hydroxylase mrna expression was altered by induction of er stress ( figure 1d ) . there are significant differences in the expression levels and regulation of the cyp7a1 gene in mice and human beings . most notably , human beings lack an liver x receptor - response element in the cyp7a1 promoter , rendering human cyp7a1 unresponsive to dietary cholesterol.41 , 42 , 43 to exclude the possibility that the effects of er stress on cyp7a1 expression are specific to mice , we determined the effect of pharmacologic er stress on cyp7a1 expression in a human hepatoma cell line ( hepg2 ) . paralleling our findings in vivo , induction of er stress in hepg2 cells suppressed cyp7a1 expression ( figure 1e ) . a major mechanism of cyp7a1 regulation is via feedback inhibition from bile acids.44 , 45 , 46 specifically , bile acids bind to the farnesoid x receptor ( fxr ) in the ileum , stimulating release of fibroblast growth factor ( fgf ) 15/19 from the ileocyte , which subsequently acts in the liver to suppress cyp7a1 transcription.47 , 48 intestinal er stress , achieved through oral administration of tunicamycin to mice , has been shown to induce ileal fgf15 expression . although activation of intestinal fxr - fgf15/19 signaling now is considered the major mechanism of bile acid feedback inhibition of cyp7a1 transcription , bile acids also inhibit cyp7a1 via activation of fxr within the liver , leading to induction of the small heterodimer partner ( shp ) . we found that induction of er stress in mice did not increase hepatic shp expression , indicating that tunicamycin does not suppress cyp7a1 via a fxr - shp dependent mechanism ( figure 2b ) . suppression of cyp7a1 expression is a feature of the hepatic inflammatory response.11 , 12 activation of cytokines such as tumor necrosis factor , interleukin ( il)1 , and il6 is associated with liver injury and has been shown to suppress hepatic cyp7a1 expression in vitro and in vivo.11 , 50 , 51 , 52 , 53 therefore , we considered the possibility that suppression of cyp7a1 expression by er stress is a manifestation of a broader stress response associated with inflammatory cytokine activation . we found no significant inflammatory cytokine activation at 6 hours after induction of er stress ( figure 2b ) . these data indicate that suppression of bile acid synthesis by er stress is independent of inflammatory cytokine activation . activation of jnk has been implicated in the inhibition of cyp7a1 by inflammatory cytokines.52 , 54 , 55 furthermore , it is well established that jnk activation occurs in response to er stress.26 , 56 , 57 to confirm whether er stress induced cyp7a1 suppression is dependent on jnk activation , we determined the effects of tunicamycin on hepg2 cells treated with the jnk inhibitor sp600125 . fgf15/19-mediated suppression of cyp7a1 expression is dependent on erk activation.58 , 59 we assessed whether er stress inhibition of erk in hepg2 cells using pd184352 did not prevent er stress induced suppression of cyp7a1 , thus providing additional evidence that the effect of er on hepatic bile acid synthesis is not dependent on fgf15/19 activation ( figure 2d ) . several other compensatory mechanisms are activated in the liver in response to cholestasis , including suppression of bile acid uptake , induction of bile acid efflux , and enhanced biliary bile acid secretion . acute er stress increased hepatic expression of adenosine triphosphate binding cassette ( abc)b11 , the canalicular bile salt export pump , yet had no significant effect on the hepatic expression of ntcp , the basolateral transporter controlling sinusoidal uptake of bile acids into hepatocytes ( figure 3a ) . the multidrug resistance - associated protein 3 ( abcc3 ) is a basolateral bile acid efflux pump that is thought to have a protective role in the setting of cholestasis by removing bile acids from the cholestatic liver.60 , 61 the expression of abcc3 was increased in response to acute er stress ( figure 3a ) . having shown that hepatic abcb11 and abcc3 are activated in mice subjected to er stress , we next measured abcb11 and abcc3 expression in human hepatoma cells ( hepg2 ) treated with tunicamycin . paralleling our findings in vivo , induction of er stress with tunicamycin , thapsigargin , or homocysteine in hepg2 cells increased expression of abcb11 and abcc3 ( figure 3b and c ) . we have shown that induction of er stress suppresses the primary bile acid synthetic pathway and enhances expression of transporters responsible for the removal of bile acids from the liver . to test this hypothesis , we examined the effect of prolonged er stress on hepatic bile acid content . similar to the pattern observed in mice acutely after induction of er stress , mice subjected to prolonged er stress showed induction of the hepatic upr associated with marked suppression of cyp7a1 and induction of abcc3 ( table 1 and figure 4a ) . consistent with suppressed cyp7a1-dependent bile acid synthesis , hepatic cholesterol content was increased and hepatic bile acid content was decreased in response to prolonged er stress ( figure 4b and c ) . unlike acute er stress , prolonged er stress suppressed hepatic expression of abcb11 and ntcp , bile acid transporters known to be suppressed by hepatic inflammation ( figure 4a ) . we found that prolonged er stress resulted in induction of the inflammatory cytokines il1 and il6 , suggesting activation of a more generalized hepatic inflammatory response in the setting of sustained er stress ( figure 4e ) . consistent with the development of hepatic inflammation , mice subjected to prolonged er stress showed a mild increase in plasma alanine aminotransferase level ( figure 4f ) and early ballooning degeneration on h&e - stained liver sections ( figure 4 g ) . prolonged er stress also resulted in induction of the intestinal upr , suggesting activation of a broader systemic stress response over time ( table 1 ) . in response to hepatocellular stress , induced anticholestatic mechanism we have identified is suppression of the cyp7a1-dependent bile acid synthetic pathway . we found that suppression of cyp7a1 occurs early after induction of er stress , in the absence of inflammatory cytokine activation . in addition , we showed that cyp7a1 suppression by er stress occurred in the absence of hepatic shp or ileal fgf15 activation . these data show that cyp7a1 suppression by er stress was independent of established fxr - dependent or cytokine - mediated pathways . moreover , these data strongly suggest that the observed effects of acute er stress on hepatic bile acid metabolism were a direct result of er stress and not simply a manifestation of a broader hepatic inflammatory response . er stress acutely increased hepatic expression of abcc3 , a basolateral bile salt efflux pump , and abcb11 , the canalicular bile salt export pump . consistent with this assertion was the finding that prolonged er stress lead to a reduction in hepatic bile acid content . suppression of abcb11 and ntcp has been a well - established feature of the hepatic inflammatory response,12 , 13 , 62 , 63 , 64 , 65 and we speculate that suppression of these transporters with prolonged er stress is secondary to inflammatory cytokine activation . furthermore , although all 3 branches of the upr have been shown to modulate metabolic pathways , the ire1 pathway has emerged as the branch most strongly implicated in regulating hepatic lipid metabolism , which is linked intimately to bile acid metabolism.69 , 70 , 71 activated ire1 , functioning as an endonuclease , induces nonconventional splicing of the transcription factor x - box binding protein 1 , which has been implicated in the transcription of a myriad of genes involved in proteostasis and metabolic processes . these data raise the possibility that in the setting of er stress , cyp7a1 mrna may be targeted for destruction via ridd . the present work implicates er stress as a novel regulator of the major bile acid synthetic pathway . impaired bile acid metabolism has been implicated in the pathogenesis of diabetes and obesity , both of which are associated with er stress.77 , 78 , 79 the possibility that altered bile acid homeostasis is a mechanistic link between er stress and the development of metabolic disease warrants further investigation .

the endocrine cells of the pancreas , located in the islets of langerhans , are responsible for blood glucose homeostasis , secreting hormones with differing and even opposing effects on blood glucose disposal . beta - cells , the most numerous islet cells , secrete the hormone insulin which reduces blood glucose levels by increasing peripheral uptake of glucose and by suppressing release of glucose from the liver . conversely , islet alpha - cells secrete the hormone glucagon which can increase blood glucose levels . glucagon mainly acts on the liver where it promotes glycogenolysis , releasing glucose from breakdown of glycogen stores and gluconeogenesis . optimal control of blood glucose levels depends on delicate changes in insulin production and secretion by the pancreatic beta - cells and on their capacity for large increases in secretion after meals , requiring large stores of insulin . it is of critical importance that islets maintain an adequate beta - cell mass in response to various changes . recent evidence has revealed that beta - cell replication plays a central role in maintaining adult beta - cell mass . in addition , rates of beta - cell proliferation change dynamically according to metabolic demand throughout life . however , replication of pre - existing beta - cells is not the only available mechanism for generating new beta - cells . in fact , a reasonable body of evidence supports the existence of four other potentially important contributors to adult beta - cell mass regulation : ( i ) differentiation from stem cells precursors , ( ii ) transdifferentiation from a non - beta - cell differentiated precursor , ( iii ) whole islet neogenesis on the plus side and apoptosis on the negative , and ( iv ) changes in beta - cell size [ 3 , 4 ] . however , the relative contribution of these processes in maintaining and expanding beta - cell mass is at present not well defined and varies between species [ 57 ] . during adult life , the beta - cell mass may have to adapt in the face of increased demands due to increases in body mass , pregnancy , or even loss of insulin sensitivity of peripheral tissues . if such compensatory adaptation is inadequate , then glucose homeostasis will be compromised and result in chronically elevated blood glucose , or diabetes [ 8 , 9 ] . it is well known that beta - cells proliferate extensively during late embryonic development , but the rate of replication slows during postnatal life . during adult life , beta - cell proliferation remarkably , this low rate of baseline proliferation can be increased significantly in response to pregnancy or obesity and is regarded as an adaptive mechanism in response to increasing systemic insulin demand . although important roles of insulin and glucose in beta - cell compensation have been suggested , the mechanism underlying this process is not well understood . in recent years , various groups have identified micrornas ( mirnas ) small molecules of noncoding rna that are able to regulate protein expression that contribute to beta - cell dysfunction and diabetes onset [ 1418 ] . type 2 diabetes ( t2d ) is characterized by hyperglycaemia resulting from impaired insulin secretion and/or impaired insulin action in peripheral tissues . t2d constitutes one of the greatest pandemics of our time , with 220 million people currently diagnosed , and 439 million people expected to be affected by 2030 . importantly , there is substantial evidence that beta - cell dysfunction plays a major role in the pathology of t2d . for this reason , great efforts are being made in order to develop new therapeutic strategies , such as beta - cell replacement or regenerative medicine . however , despite progress , most diabetic patients will still die prematurely as a direct result of their disease , its complications , or sometimes even its treatments . in fact , although one may hope that glp-1 analogues and improved lifestyle may eventually translate into a slowing of t2d progress , clinical trials data have been generally disappointing and confirm that the disease continues to progress [ 2225 ] . to date , no effective treatments for t1d based on slowing or reversing the natural history of the disease exist . thus we must rely on treatments that can maintain or restore blood glucose levels , and evidences that disease progression can be significantly arrested are scanty . thus , all t1d and over time most t2d patients will require exogenous insulin ( or in rare cases an islet transplant ) . it is not all negative , and short - term benefits ( usually progression from prediabetes to overt diabetes ) have been reported in small studies for metformin and acarbose with the common factor likely to be weight loss . trials with the pparg ligand rosiglitazone have also suggested that the progression of t2d may be retarded , but as the drug has been withdrawn , due to an increased risk of cardiovascular disease , this remains interesting rather than clinically useful . in the largest study of t2d to date ( ukpds - uk prospective diabetes study ( ukpds ) group ) , treatments did not slow progression of patients to additional drugs in order to maintain blood glucose levels at target levels . furthermore , beta - cells from donor pancreases are in such short supply that transplants can be provided only to a limited number of patients . ultimately , to cure diabetes , missing beta - cells that must be replaced , and in practical terms this would need to be done from within or a limitless source of beta - like cells must be developed for transplants . one way forward is to create beta - cells from alternative cell sources ( neogenesis , transdifferentiation , stem cells , etc . ) . such an approach requires further knowledge of the mechanisms that regulate pancreatic beta - cell mass . this review briefly outlines current knowledge of significant factors / nutrients regulating beta - cells mass , and their signal transduction pathways , with greater focus on postnatal regulation and the role of a new class of beta - cell mass regulators : the micrornas . how do beta - cells maintain their normal mass?beta - cell mass is increased by neogenesis ( differentiation from beta - cell precursor cells ) , proliferation , and cell hypertrophy ( increased cell size ) and is decreased by beta - cell death , primarily through apoptosis , and beta - cell atrophy ( decreased cell size , figure 1 ) . several studies have revealed that the primary mechanism by which new beta - cells arise during adulthood is through proliferation of existing beta - cells rather than neogenesis [ 7 , 27 ] . thus , organisms born with reduced beta - cell mass have fewer beta - cells available to enter the cell cycle later in life . under normal circumstances during adulthood , beta - cells are a slowly renewing population , with steady low levels of proliferation and apoptosis . however , beta - cell mass continuously expands over the lifespan of an organism , likely due to age - related increases in body weight and insulin resistance . a large number of hormones and nutrients have been shown to affect beta - cell mass , as extensively reviewed by nielsen et al . . as a result , the number of molecular pathways that have been implicated in beta mass regulation is also large . many studies have employed animal models , and it is important to note that significant differences between species have been observed . beta - cell mass is increased by neogenesis ( differentiation from beta - cell precursor cells ) , proliferation , and cell hypertrophy ( increased cell size ) and is decreased by beta - cell death , primarily through apoptosis , and beta - cell atrophy ( decreased cell size , figure 1 ) . several studies have revealed that the primary mechanism by which new beta - cells arise during adulthood is through proliferation of existing beta - cells rather than neogenesis [ 7 , 27 ] . thus , organisms born with reduced beta - cell mass have fewer beta - cells available to enter the cell cycle later in life . under normal circumstances during adulthood , beta - cells are a slowly renewing population , with steady low levels of proliferation and apoptosis . however , beta - cell mass continuously expands over the lifespan of an organism , likely due to age - related increases in body weight and insulin resistance . a large number of hormones and nutrients have been shown to affect beta - cell mass , as extensively reviewed by nielsen et al . . as a result , the number of molecular pathways that have been implicated in beta mass regulation is also large . many studies have employed animal models , and it is important to note that significant differences between species have been observed . beta - cell mass regulation is modulated by various environmental factors and nutrients including glucose , insulin , amino acids , fatty acids , and various other growth factors / hormones , such as igf - i , igf - ii , glucagon - like peptide-1 ( glp-1 ) 1 , glucagon , gastroinhibitory peptide ( gip ) , somatostatin ( sst ) , hgf and betacellulin , gastrin , cholecystokinin ( cck ) , growth hormone ( gh ) , prolactin ( prl ) , placental lactogen ( pl ) , and leptin , amongst others ( table 1 ) . these growth factors and nutrients can affect a variety of beta - cell functions suppress / stimulate beta - cell replication , survival , mass expansion , and differentiation through different intracellular pathways discussed later . glucose glucose is thought to be the most important determinant of beta - cell mass equilibrium [ 3136 ] . glucose is not only the pathological hallmark of diabetes , but it is also a potential contributor to further decline in beta - cell mass through what has been termed glucotoxicity . thus , in the face of evolving demand for insulin , if adaptation ( functional or numeric ) fails , then blood glucose levels rise , sequentially resulting in igt and then t2d . once glucose levels exceed the safe threshold , then further beta - cell death will likely arise .it has been reported that hyperglycaemia for a short time in rats induces beta - cells to enter the cell cycle accompanied by ~50% increase in beta - cell number by neogenesis [ 33 , 38 ] and suppression of beta - cell apoptosis [ 33 , 36 ] . in another study using prolonged periods of glucose infusion , an increase of beta - cell mass was also reported , although the origin of these cells has been disputed . while some authors describe proliferation of new beta - cells formed by neogenesis of precursor cells , other groups report an increase in beta - cell replication and hypertrophy as well as neogenesis , leading to sustained effects on beta - cell mass even after glucose infusion is stopped [ 35 , 39].to support the mitogen effect of glucose on beta - cells , porat and colleagues demonstrated in an animal model an increased replication rate of beta - cells correlated with an increased blood glucose level . moreover , this effect was mediated by a specific enzyme involved in glucose metabolism ( glycolysis ) in beta - cells , glucokinase ( gck ) . in fact , it was demonstrated that gck has a key role in beta - cell proliferation through membrane depolarization .in addition , glucose has been shown to promote beta - cell survival by suppressing a constitutive apoptotic program in vitro . moreover , it was reported that mild hyperglycaemia can affect beta - cell phenotype with a progressive loss of beta - cell differentiation markers ( pdx-1 , beta2/neurod , nkx6.1 , and hnf-1 ) and genes involved in glucose sensing ( glut-2 and glucokinase ) [ 37 , 42 ] . on the other hand , some genes that are normally suppressed in beta - cells ( e.g. , c - myc and its gene target , lactate dehydrogenase - a ) are increased . this proapoptotic effect of glucose may also involve altered calcium homeostasis , activation of caspases by cytokines , such as il-1 which is secreted by human islets in the presence of high glucose and leads to beta - cell apoptosis or fas - fas - ligand interactions , reactive oxygen species , and c - myc [ 46 , 47 ] . intriguingly , c - myc has been shown to trigger apoptosis through all of these pathways in other systems , including in particular the activation of some caspases ( caspase 8 and 9 ) , fas - fas - ligand signalling , and reactive oxygen species ( reviewed in ) .therefore , paradoxically , it appears that glucose may have both an inhibitory and stimulating effect on beta - cell apoptosis . however , it appears that both the level and duration of hyperglycaemia are crucial in determining the fate of the beta - cell . for example , prolonged hyperglycaemia appears to have a proapoptotic effect , a process referred to as glucose toxicity . chronic hyperglycemia may thus be injurious to beta - cells and contribute to the development of t2d . increased beta - cell apoptosis and reduced beta - cell mass have been observed in humans with t2d also , in keeping with the preclinical data in [ 6 , 5053 ] . this difference in beta - cell mass between t2d patients and matched controls is particularly evident when those are matched for obesity ( obese patients without t2d have increased beta - cell mass compared to lean controls ) . glucose is thought to be the most important determinant of beta - cell mass equilibrium [ 3136 ] . glucose is not only the pathological hallmark of diabetes , but it is also a potential contributor to further decline in beta - cell mass through what has been termed glucotoxicity . thus , in the face of evolving demand for insulin , if adaptation ( functional or numeric ) fails , then blood glucose levels rise , sequentially resulting in igt and then t2d . once glucose levels exceed the safe threshold , then further beta - cell death will likely arise . it has been reported that hyperglycaemia for a short time in rats induces beta - cells to enter the cell cycle accompanied by ~50% increase in beta - cell number by neogenesis [ 33 , 38 ] and suppression of beta - cell apoptosis [ 33 , 36 ] . in another study using prolonged periods of glucose infusion , an increase of beta - cell mass was also reported , although the origin of these cells has been disputed . while some authors describe proliferation of new beta - cells formed by neogenesis of precursor cells , other groups report an increase in beta - cell replication and hypertrophy as well as neogenesis , leading to sustained effects on beta - cell mass even after glucose infusion is stopped [ 35 , 39 ] . to support the mitogen effect of glucose on beta - cells , porat and colleagues demonstrated in an animal model an increased replication rate of beta - cells correlated with an increased blood glucose level . moreover , this effect was mediated by a specific enzyme involved in glucose metabolism ( glycolysis ) in beta - cells , glucokinase ( gck ) . in fact , it was demonstrated that gck has a key role in beta - cell proliferation through membrane depolarization . in addition , glucose has been shown to promote beta - cell survival by suppressing a constitutive apoptotic program in vitro . moreover , it was reported that mild hyperglycaemia can affect beta - cell phenotype with a progressive loss of beta - cell differentiation markers ( pdx-1 , beta2/neurod , nkx6.1 , and hnf-1 ) and genes involved in glucose sensing ( glut-2 and glucokinase ) [ 37 , 42 ] . on the other hand , some genes that are normally suppressed in beta - cells ( e.g. , c - myc and its gene target , lactate dehydrogenase - a ) are increased . this proapoptotic effect of glucose may also involve altered calcium homeostasis , activation of caspases by cytokines , such as il-1 which is secreted by human islets in the presence of high glucose and leads to beta - cell apoptosis or fas - fas - ligand interactions , reactive oxygen species , and c - myc [ 46 , 47 ] . intriguingly , c - myc has been shown to trigger apoptosis through all of these pathways in other systems , including in particular the activation of some caspases ( caspase 8 and 9 ) , fas - fas - ligand signalling , and reactive oxygen species ( reviewed in ) . therefore , paradoxically , it appears that glucose may have both an inhibitory and stimulating effect on beta - cell apoptosis . however , it appears that both the level and duration of hyperglycaemia are crucial in determining the fate of the beta - cell . for example , prolonged hyperglycaemia appears to have a proapoptotic effect , a process referred to as glucose toxicity . chronic hyperglycemia may thus be injurious to beta - cells and contribute to the development of t2d . increased beta - cell apoptosis and reduced beta - cell mass have been observed in humans with t2d also , in keeping with the preclinical data in [ 6 , 5053 ] . this difference in beta - cell mass between t2d patients and matched controls is particularly evident when those are matched for obesity ( obese patients without t2d have increased beta - cell mass compared to lean controls ) . various authors have shown in various in vivo studies that insulin alone can stimulate beta - cell mass , whereas other authors have found that it promotes growth only in the presence of hyperglycaemia [ 34 , 36 , 5456 ] . in a physiological condition , it seems reasonable that during a period of increased insulin demand , insulin itself could stimulate beta - cell growth , creating a positive feedback loop . further studies have suggested a positive role of insulin in cell regeneration [ 57 , 58].genetically altered mice in which the insulin receptor was knocked out exhibited a decreased beta - cell mass in adults and diabetes onset . insulin resistance , leading to hyperinsulinemia , stimulates an increase in beta - cell mass.in contrast , as previously described [ 60 , 61 ] , insulin can also impose negative effect on beta - cell mass and/or proliferation . for example , rats with insulinomas resulting in hyperinsulinemia and hypoglycaemia were found to have smaller beta - cells , as well as reduced beta - cell numbers ( in the islets without tumour ) , suggesting that a sensor mechanisms exist that can co - ordinate beta - cell mass across islets and possibly even distal grafts . increased rates of beta - cell apoptosis were evident [ 60 , 61 ] , but interestingly , beta - cell mass regenerated within days of tumour excision . this demonstrates that excess insulin with a negative feedback is able to inhibit beta - cell growth.these observations indicate that insulin can control beta - cell population dynamics , although the mechanism is still unclear . various authors have shown in various in vivo studies that insulin alone can stimulate beta - cell mass , whereas other authors have found that it promotes growth only in the presence of hyperglycaemia [ 34 , 36 , 5456 ] . in a physiological condition , it seems reasonable that during a period of increased insulin demand , insulin itself could stimulate beta - cell growth , creating a positive feedback loop . further studies have suggested a positive role of insulin in cell regeneration [ 57 , 58 ] . genetically altered mice in which the insulin receptor was knocked out exhibited a decreased beta - cell mass in adults and diabetes onset . insulin resistance , leading to hyperinsulinemia , stimulates an increase in beta - cell mass . in contrast , as previously described [ 60 , 61 ] , insulin can also impose negative effect on beta - cell mass and/or proliferation . for example , rats with insulinomas resulting in hyperinsulinemia and hypoglycaemia were found to have smaller beta - cells , as well as reduced beta - cell numbers ( in the islets without tumour ) , suggesting that a sensor mechanisms exist that can co - ordinate beta - cell mass across islets and possibly even distal grafts . increased rates of beta - cell apoptosis were evident [ 60 , 61 ] , but interestingly , beta - cell mass regenerated within days of tumour excision . this demonstrates that excess insulin with a negative feedback is able to inhibit beta - cell growth . these observations indicate that insulin can control beta - cell population dynamics , although the mechanism is still unclear . free fatty acidsanother proposed stimulus for beta - cell mass regulation has been free fatty acids ( ffas ) . it is well documented that chronic high levels of ffas , which are often accompanied by obesity , contributes to the pathophysiology of insulin resistance and t2d [ 64 , 65 ] . it is reported that acute ffa exposure stimulates insulin secretion , while prolonged ffa exposure decreases glucose - stimulated insulin secretion [ 66 , 67 ] and induces insulin resistance and beta - cell dysfunction in both animal models and humans [ 6972].animal studies have been conflicting with some arguing that ffas have a positive trophic effect on beta - cells , whilst others suggest the contrary . however , precise molecular mechanisms linking ffa to beta - cell dysfunction have yet to be fully elucidated.it was reported that a prolonged in vitro exposure of isolated islets or insulin - secreting cell lines to elevated levels of fatty acids is associated with inhibition of glucose - induced insulin secretion [ 67 , 73 , 74 ] , impairment of insulin gene expression [ 7578 ] , and induction of cell death by apoptosis both in vitro [ 7988 ] and in vivo studies .importantly , in vitro [ 76 , 88 , 89 ] and in vivo [ 90 , 91 ] studies have provided evidence that lipotoxicity only occurs in the presence of concomitantly elevated glucose levels . a number of studies have shown that fatty acids can induce beta - cell death by apoptosis in the presence of high glucose . in vitro , saturated fatty acids induce beta - cell apoptosis [ 82 , 86 , 88 ] , whereas unsaturated fatty acids are usually protective [ 81 , 82 , 88 ] . recently , it was observed by various authors that oxidative stress is implicated in the pathogenesis of beta - cell dysfunction induced by ffas . it has been suggested that increased reactive oxygen species ( ros ) levels are the important trigger for beta - cell dysfunction . under diabetic conditions , ros is increased in many tissues and organs and cause various forms of tissue damage in patients with diabetes . it is considered that enhanced ros generation may act as a link between ffa and beta - cell dysfunction . another proposed stimulus for beta - cell mass regulation has been free fatty acids ( ffas ) . it is well documented that chronic high levels of ffas , which are often accompanied by obesity , contributes to the pathophysiology of insulin resistance and t2d [ 64 , 65 ] . it is reported that acute ffa exposure stimulates insulin secretion , while prolonged ffa exposure decreases glucose - stimulated insulin secretion [ 66 , 67 ] and induces insulin resistance and beta - cell dysfunction in both animal models and humans [ 6972 ] . animal studies have been conflicting with some arguing that ffas have a positive trophic effect on beta - cells , whilst others suggest the contrary . however , precise molecular mechanisms linking ffa to beta - cell dysfunction have yet to be fully elucidated . it was reported that a prolonged in vitro exposure of isolated islets or insulin - secreting cell lines to elevated levels of fatty acids is associated with inhibition of glucose - induced insulin secretion [ 67 , 73 , 74 ] , impairment of insulin gene expression [ 7578 ] , and induction of cell death by apoptosis both in vitro [ 7988 ] and in vivo studies . importantly , in vitro [ 76 , 88 , 89 ] and in vivo [ 90 , 91 ] studies have provided evidence that lipotoxicity only occurs in the presence of concomitantly elevated glucose levels . a number of studies have shown that fatty acids can induce beta - cell death by apoptosis in the presence of high glucose . in vitro , saturated fatty acids induce beta - cell apoptosis [ 82 , 86 , 88 ] , whereas unsaturated fatty acids are usually protective [ 81 , 82 , 88 ] . recently , it was observed by various authors that oxidative stress is implicated in the pathogenesis of beta - cell dysfunction induced by ffas . it has been suggested that increased reactive oxygen species ( ros ) levels are the important trigger for beta - cell dysfunction . under diabetic conditions , ros is increased in many tissues and organs and cause various forms of tissue damage in patients with diabetes . it is considered that enhanced ros generation may act as a link between ffa and beta - cell dysfunction . glucagon - like peptide-1 glucagon - like peptide-1 ( glp-1 ) is produced and secreted in response to fat and glucose from intestinal l - cells , located in the distal ileum and colon . preclinical studies reveal potential benefits of glp-1 receptor agonist treatment in individuals with t2d , which include the promotion of beta - cell proliferation and reduced beta - cell apoptosis . these preclinical results indicate that glp-1 could be beneficial in treating patients with t2d.however , the role of endogenous glp-1 in stimulating beta - cell proliferation is less clear . data regarding the regenerative role of glp-1 and related agonists are controversial.however , there is strong evidence to suggest that glp-1 receptor activation can protect beta - cells from apoptosis [ 9497 ] and equally convincing evidence of a beta - cell mitogenic effect of glp-1 [ 93 , 94 , 98 , 99].it was reported that exogenous glp-1 treatment enhances beta - cell replication in normoglycaemic rats and mice [ 101105].however , while there are reports of glp-1-mediated islet neogenesis [ 98 , 106109 ] , recent statements by researchers examining the in vivo effects of glp-1 call into question a neogenic response [ 110112].glp-1 treatment stimulates beta - cell replication in multiple rodent models of obesity - induced diabetes , including both intact [ 113 , 114 ] and defective leptin signalling [ 94 , 115117 ] models . in addition , some authors have demonstrated that exogenous glp-1 treatment promotes beta - cell replication in models of beta - cell regeneration and obesity - induced diabetes [ 118 , 119].moreover , glp-1 prevents beta - cell death in mouse models of beta - cell loss , in particular , in both unstressed mice and in streptozotocin - induced beta - cell toxicity [ 95 , 120 ] . in summary , exogenous glp-1 treatment stimulates beta - cell proliferation in multiple rodent species and models of beta - cell mitogenesis . data to support a role for endogenous glp-1 signalling in beta - cell proliferation are less clear . glucagon - like peptide-1 ( glp-1 ) is produced and secreted in response to fat and glucose from intestinal l - cells , located in the distal ileum and colon . preclinical studies reveal potential benefits of glp-1 receptor agonist treatment in individuals with t2d , which include the promotion of beta - cell proliferation and reduced beta - cell apoptosis . however , the role of endogenous glp-1 in stimulating beta - cell proliferation is less clear . data regarding the regenerative role of glp-1 and related agonists are controversial . however , there is strong evidence to suggest that glp-1 receptor activation can protect beta - cells from apoptosis [ 9497 ] and equally convincing evidence of a beta - cell mitogenic effect of glp-1 [ 93 , 94 , 98 , 99 ] . it was reported that exogenous glp-1 treatment enhances beta - cell replication in normoglycaemic rats and mice [ 101105 ] . however , while there are reports of glp-1-mediated islet neogenesis [ 98 , 106109 ] , recent statements by researchers examining the in vivo effects of glp-1 call into question a neogenic response [ 110112 ] . glp-1 treatment stimulates beta - cell replication in multiple rodent models of obesity - induced diabetes , including both intact [ 113 , 114 ] and defective leptin signalling [ 94 , 115117 ] models . in addition , some authors have demonstrated that exogenous glp-1 treatment promotes beta - cell replication in models of beta - cell regeneration and obesity - induced diabetes [ 118 , 119 ] . moreover , glp-1 prevents beta - cell death in mouse models of beta - cell loss , in particular , in both unstressed mice and in streptozotocin - induced beta - cell toxicity [ 95 , 120 ] . in summary , exogenous glp-1 treatment stimulates beta - cell proliferation in multiple rodent species and models of beta - cell mitogenesis . data to support a role for endogenous glp-1 signalling in beta - cell proliferation are less clear . glucose - dependent insulinotropic polypeptide glucose - dependent insulinotropic polypeptide ( gip ) is a gut hormone synthesized and secreted from intestinal k - cells , which reside in the duodenum and jejunum . it was previously reported that if gip promotes beta - cell proliferation during obesity , then gip inhibition should reduce beta - cell mass .however , it is unclear whether the primary effect of gip antagonism is on the adipocyte or the beta - cell . few studies implicate gip in in vivo beta - cell proliferation [ 119 , 122 ] . however , one report demonstrated that gip treatment increases islet size and number in the leptin ob / ob mouse . the latter study did not discriminate between beta - cell proliferation , apoptosis , or islet neogenesis . in a human patient , elevated fasting plasma gip correlated with islet cell hyperplasia . despite the limited in vivo evidence , in vitro experiments demonstrate that gip enhances beta - cell mitogenesis [ 125128].furthermore , the role of gip in the reduction of beta - cell apoptosis in vivo is more clearly defined than its role in beta - cell proliferation.various studies have also demonstrated that exogenous gip treatment prevents beta - cell apoptosis during severe obesity [ 123 , 129 ] and streptozotocin - induced diabetes [ 120 , 129 ] . glucose - dependent insulinotropic polypeptide ( gip ) is a gut hormone synthesized and secreted from intestinal k - cells , which reside in the duodenum and jejunum . it was previously reported that if gip promotes beta - cell proliferation during obesity , then gip inhibition should reduce beta - cell mass . however , it is unclear whether the primary effect of gip antagonism is on the adipocyte or the beta - cell . few studies implicate gip in in vivo beta - cell proliferation [ 119 , 122 ] . however , one report demonstrated that gip treatment increases islet size and number in the leptin ob / ob mouse . the latter study did not discriminate between beta - cell proliferation , apoptosis , or islet neogenesis . in a human patient , elevated fasting plasma gip correlated with islet cell hyperplasia . despite the limited in vivo evidence , in vitro experiments furthermore , the role of gip in the reduction of beta - cell apoptosis in vivo is more clearly defined than its role in beta - cell proliferation . various studies have also demonstrated that exogenous gip treatment prevents beta - cell apoptosis during severe obesity [ 123 , 129 ] and streptozotocin - induced diabetes [ 120 , 129 ] . insulin - like growth factor i and iithe role of insulin - like growth factor i ( igf - i ) and insulin - like growth factor ii ( igf - ii ) in beta - cell mass regulation has long been accepted [ 130132 ] . igf - i has been shown to increase the number of replicating beta - cells in rodent islets by up to 6% of the islet cell population [ 133135 ] and to induce differentiated pancreatic beta - cell growth . however , igf - i appears only to be effective at inducing beta - cell proliferation under physiologically relevant glucose concentration range : 6 mm to 18 mm . thus , igf - i potentiates the mitogenic effect of glucose on beta - cell proliferation.interestingly , overexpression of igf - i in beta - cells is associated with increased beta - cell proliferation , but not mass . in contrast , transgenic mice overexpressing igf - ii exhibit an increase in beta - cell mass due to augmented proliferation . taken together , there is clear evidence that the igf molecules are important factors in beta - cell proliferation and mass.seemingly paradoxically , beta - cell - specific igf - i knockout mice showed a delayed onset of t2d induced by a high - fat diet , accompanied by enlarged pancreatic islets , increased insulin mrna levels , and preserved sensitivity to insulin . moreover , given the dominant role of circulating igfs on tissue mass homeostasis in other systems , the significance of this is uncertain . concerning igf - ii , it has been reported that mice overexpressing this hormone display an increase in beta - cell mass , hyperinsulinemia , and increased glucose - stimulated insulin secretion . these results suggest that igf - ii may have some potential as an islet growth factor . the role of insulin - like growth factor i ( igf - i ) and insulin - like growth factor ii ( igf - ii ) in beta - cell mass regulation has long been accepted [ 130132 ] . igf - i has been shown to increase the number of replicating beta - cells in rodent islets by up to 6% of the islet cell population [ 133135 ] and to induce differentiated pancreatic beta - cell growth . however , igf - i appears only to be effective at inducing beta - cell proliferation under physiologically relevant glucose concentration range : 6 mm to 18 mm . thus , igf - i potentiates the mitogenic effect of glucose on beta - cell proliferation . interestingly , overexpression of igf - i in beta - cells is associated with increased beta - cell proliferation , but not mass . in contrast , transgenic mice overexpressing igf - ii exhibit an increase in beta - cell mass due to augmented proliferation . taken together , there is clear evidence that the igf molecules are important factors in beta - cell proliferation and mass . seemingly paradoxically , beta - cell - specific igf - i knockout mice showed a delayed onset of t2d induced by a high - fat diet , accompanied by enlarged pancreatic islets , increased insulin mrna levels , and preserved sensitivity to insulin . moreover , given the dominant role of circulating igfs on tissue mass homeostasis in other systems , the significance of this is uncertain . concerning igf - ii , it has been reported that mice overexpressing this hormone display an increase in beta - cell mass , hyperinsulinemia , and increased glucose - stimulated insulin secretion . these results suggest that igf - ii may have some potential as an islet growth factor . cholecystokinin cholecystokinin ( cck ) is able to stimulate beta - cell proliferation in vivo , as reported by different authors . in fact , various studies have demonstrated that the administration of cck-8 after streptozotocin treatment in rats expands beta - cell mass via proliferation , in association with fasting plasma insulin increasing , and fasting plasma glucose reduction . at the same time similarly , in a rat model , loss of cck resulted in decreased islet size and reduced beta - cell mass through increased beta - cell death . however , overexpression of cck in mouse and human islets has no effect on replication . cholecystokinin ( cck ) is able to stimulate beta - cell proliferation in vivo , as reported by different authors . in fact , various studies have demonstrated that the administration of cck-8 after streptozotocin treatment in rats expands beta - cell mass via proliferation , in association with fasting plasma insulin increasing , and fasting plasma glucose reduction . at the same time similarly , in a rat model , loss of cck resulted in decreased islet size and reduced beta - cell mass through increased beta - cell death . however , overexpression of cck in mouse and human islets has no effect on replication . parathyroid hormone - related proteina recent study has demonstrated that acute and systemic administration of the first 36 amino acids of parathyroid hormone - related protein ( pthrp ( 136 ) ) in rodents can stimulate beta - cell proliferation and enhance beta - cell mass in vivo , without negatively affecting beta - cell function or survival . previous studies have demonstrated that rip - pthrp transgenic mice producing the full - length pthrp ( 1139 ) in beta - cells show an increase in beta - cell mass , proliferation and islet number , and improvement of glucose homeostasis [ 145147 ] . in addition , in vitro studies ( in rodent insulinoma cell lines and primary human beta - cells ) have shown that pthrp ( 136 ) is sufficient to enhance not only proliferation , but also function , increasing insulin mrna and protein , and augmenting glucose - stimulated insulin secretion [ 148150 ] . however , it is notable that pthrp knockout mice have normal beta - cell mass as compared to controls , suggesting that pthrp may not be crucial for normal beta - cell mass in vivo . however , the explanation of this result should be found on the possibility that the pthrp was absent in islets but it could be produced in other islet cell types , resulting in a paracrine compensatory mechanism .these findings suggest that pthrp may provide a possible target for gene therapeutic strategies designed to increase beta - cell mass and function . a recent study has demonstrated that acute and systemic administration of the first 36 amino acids of parathyroid hormone - related protein ( pthrp ( 136 ) ) in rodents can stimulate beta - cell proliferation and enhance beta - cell mass in vivo , without negatively affecting beta - cell function or survival . previous studies have demonstrated that rip - pthrp transgenic mice producing the full - length pthrp ( 1139 ) in beta - cells show an increase in beta - cell mass , proliferation and islet number , and improvement of glucose homeostasis [ 145147 ] . in addition , in vitro studies ( in rodent insulinoma cell lines and primary human beta - cells ) have shown that pthrp ( 136 ) is sufficient to enhance not only proliferation , but also function , increasing insulin mrna and protein , and augmenting glucose - stimulated insulin secretion [ 148150 ] . however , it is notable that pthrp knockout mice have normal beta - cell mass as compared to controls , suggesting that pthrp may not be crucial for normal beta - cell mass in vivo . however , the explanation of this result should be found on the possibility that the pthrp was absent in islets but it could be produced in other islet cell types , resulting in a paracrine compensatory mechanism . these findings suggest that pthrp may provide a possible target for gene therapeutic strategies designed to increase beta - cell mass and function . ghrelin ghrelin is a stomach - derived hormone involved in glucose metabolism and has been shown to regulate cellular differentiation and proliferation in various systems . in beta - cells , ghrelin treatment has been shown to increase expression of insulin and pdx1 , as well as beta - cell replication in a diabetes model . moreover , it has been recently reported that both acylated- and unacylated - ghrelin can on the one hand promote cell proliferation while on the other inhibit apoptosis of beta - cells , including human pancreatic islets induced by serum starvation and/or cytokine 's stimulation [ 153 , 154 ] . ghrelin is a stomach - derived hormone involved in glucose metabolism and has been shown to regulate cellular differentiation and proliferation in various systems . in beta - cells , ghrelin treatment has been shown to increase expression of insulin and pdx1 , as well as beta - cell replication in a diabetes model . moreover , it has been recently reported that both acylated- and unacylated - ghrelin can on the one hand promote cell proliferation while on the other inhibit apoptosis of beta - cells , including human pancreatic islets induced by serum starvation and/or cytokine 's stimulation [ 153 , 154 ] . mice with heterozygous deletion for men1 develop pancreatic hyperplasia that coincides with hyperinsulinemia and hypoglycaemia , with an increased islet cell growth through a progressive reduction in expression of some cell growth suppressors , such as p18 and p27 . it was reported that the regulation of p18 and p27 by menin is through histone methylation [ 158 , 159 ] . moreover , mutation of men1 can induce acceleration in s - phase entry and consequently an enhancement of cell proliferation in pancreatic islets . recently , it has been reported that menin is also able to increase caspase-8 expression and that caspase-8 is critical for the maintenance of beta - cell mass under physiological conditions . more recently , menin has been demonstrated to play an important role during pregnancy , but this will be discussed below . mice with heterozygous deletion for men1 develop pancreatic hyperplasia that coincides with hyperinsulinemia and hypoglycaemia , with an increased islet cell growth through a progressive reduction in expression of some cell growth suppressors , such as p18 and p27 . it was reported that the regulation of p18 and p27 by menin is through histone methylation [ 158 , 159 ] . moreover , mutation of men1 can induce acceleration in s - phase entry and consequently an enhancement of cell proliferation in pancreatic islets . recently , it has been reported that menin is also able to increase caspase-8 expression and that caspase-8 is critical for the maintenance of beta - cell mass under physiological conditions . more recently , menin has been demonstrated to play an important role during pregnancy , but this will be discussed below . leptin leptin is one of the most important cytokines produced by the ob gene and secreted by adipose tissue , and it plays an important role in controlling food intake / satiety and body energy balance ( 51 ) through the leptin - receptor ( ob - r ) . ob - r is also present in pancreatic tissue , and it has been proposed that leptin may increase beta - cell proliferation of insulin - secreting cell line .leptin has also been reported to prevent pancreatic beta - cells from inducible apoptosis , and this may partially account for islet hypertrophy in obese rodents and patients . this was associated with a reduction of triglyceride accumulation , an inhibition of nitric oxide production , and a restoration of bcl-2 expression in the face of fatty acid insult . this may be a possible mechanism for its antiapoptotic effect [ 164166].in the past , it was reported that leptin acted via the jak / stat and mapk pathways and has been shown to increase beta - cell proliferation in cell line studies and foetal pancreatic tissue [ 167 , 168].however , recent findings indicate that leptin mainly acts through the pi3k - akt signalling pathway in beta - cell proliferation [ 169171 ] . all these results suggest that leptin has a protective role on beta - cell function . however , the effect of leptin on apoptosis has been conflicting . in vitro , prolonged exposure to leptin has been associated with impaired beta - cell function , and increased rates of apoptosis .furthermore , in contrast to the observations in rodent animal , leptin is likely to have a deleterious effect on human islet function . for instance , it was reported that in isolated islets from mice , leptin was able to increase cell number through the suppression of apoptosis meanwhile in human islets in vitro leptin impairs insulin secretion and induces apoptosis of beta - cells in the presence of 20 mm glucose via activation of c - jnk . overall , leptin is likely to exert diverse effects in the regulation of pancreatic beta - cell function and proliferation . therefore , further research is still required to clarify its distinct role in various conditions . leptin is one of the most important cytokines produced by the ob gene and secreted by adipose tissue , and it plays an important role in controlling food intake / satiety and body energy balance ( 51 ) through the leptin - receptor ( ob - r ) . ob - r is also present in pancreatic tissue , and it has been proposed that leptin may increase beta - cell proliferation of insulin - secreting cell line . leptin has also been reported to prevent pancreatic beta - cells from inducible apoptosis , and this may partially account for islet hypertrophy in obese rodents and patients . this was associated with a reduction of triglyceride accumulation , an inhibition of nitric oxide production , and a restoration of bcl-2 expression in the face of fatty acid insult . this may be a possible mechanism for its antiapoptotic effect [ 164166 ] . in the past , it was reported that leptin acted via the jak / stat and mapk pathways and has been shown to increase beta - cell proliferation in cell line studies and foetal pancreatic tissue [ 167 , 168 ] . however , recent findings indicate that leptin mainly acts through the pi3k - akt signalling pathway in beta - cell proliferation [ 169171 ] . all these results suggest that leptin has a protective role on beta - cell function . however , the effect of leptin on apoptosis has been conflicting . in vitro , prolonged exposure to leptin has been associated with impaired beta - cell function , and increased rates of apoptosis . furthermore , in contrast to the observations in rodent animal , leptin is likely to have a deleterious effect on human islet function . for instance , it was reported that in isolated islets from mice , leptin was able to increase cell number through the suppression of apoptosis meanwhile in human islets in vitro leptin impairs insulin secretion and induces apoptosis of beta - cells in the presence of 20 mm glucose via activation of c - jnk . overall , leptin is likely to exert diverse effects in the regulation of pancreatic beta - cell function and proliferation . therefore , further research is still required to clarify its distinct role in various conditions . this novel compound can mimic the effects of insulin , by binding insulin - receptor , and exerts protective effects on pancreatic beta - cells acting via the pi3k / akt and mapk pathways . through these pathways , visfatin would appear to have the potential to regulate plasma glucose levels , as well as beneficial effects on beta - cell mass . overall , visfatin may have a protective effect on pancreatic beta - cell mass , but further research is necessary to clarify its distinct roles . this novel compound can mimic the effects of insulin , by binding insulin - receptor , and exerts protective effects on pancreatic beta - cells acting via the pi3k / akt and mapk pathways . through these pathways , visfatin would appear to have the potential to regulate plasma glucose levels , as well as beneficial effects on beta - cell mass . overall , visfatin may have a protective effect on pancreatic beta - cell mass , but further research is necessary to clarify its distinct roles . placental hormonesthese hormones ( especially placental lactogen and prolactin ) are principally responsible for the changes in beta - cell mass during pregnancy ( reviewed in [ 177 , 178 ] ) . these hormones stimulate beta - cell proliferation in isolated islets , and beta - cell mass is reduced 2642% in receptor deficient mice as described in a later . these hormones ( especially placental lactogen and prolactin ) are principally responsible for the changes in beta - cell mass during pregnancy ( reviewed in [ 177 , 178 ] ) . these hormones stimulate beta - cell proliferation in isolated islets , and beta - cell mass is reduced 2642% in receptor deficient mice as described in a later . cxs have been shown to affect cell proliferation and survival in different cell types [ 180182 ] . in particular , it was reported that the cx36 isoform is the only one expressed in rodent beta - cells . a recent study performed by klee et al . , using connexins - inducible / knockout transgenic mouse models , reported a key role in beta - cell mass regulation by demonstrating that the native cx36 does not alter islet size or insulin content , whereas the cx43 isoform increases both parameters , and cx32 has a similar effect only when combined with the gh . cxs have been shown to affect cell proliferation and survival in different cell types [ 180182 ] . in particular , it was reported that the cx36 isoform is the only one expressed in rodent beta - cells . a recent study performed by klee et al . , using connexins - inducible / knockout transgenic mouse models , reported a key role in beta - cell mass regulation by demonstrating that the native cx36 does not alter islet size or insulin content , whereas the cx43 isoform increases both parameters , and cx32 has a similar effect only when combined with the gh . bone morphogenetic proteinsas recently reported , there are some new mediators that are responsible of proliferation / apoptosis of beta - cells . bone morphogenetic proteins ( bmps ) are growth factors involved in the pancreas mass homeostasis both during embryonic development and adult life [ 185 , 186 ] . the binding of these molecules , in particular bmp1 and bmp4 , to their specific receptors induce , the phosphorylation and nuclear translocation of smads . in addition , there were identified different target genes able to regulate different transcription factors ( id1 , id2 , id3 , id4 ) and proteins involved both in the proliferation and apoptosis processes ( including gk , irs , e2f , smad7/9 , hes-1 ) . this is the reason why bmps have a double effect , both proliferative and proapoptotic . as recently reported , there are some new mediators that are responsible of proliferation / apoptosis of beta - cells . bone morphogenetic proteins ( bmps ) are growth factors involved in the pancreas mass homeostasis both during embryonic development and adult life [ 185 , 186 ] . the binding of these molecules , in particular bmp1 and bmp4 , to their specific receptors induce , the phosphorylation and nuclear translocation of smads . in addition , there were identified different target genes able to regulate different transcription factors ( id1 , id2 , id3 , id4 ) and proteins involved both in the proliferation and apoptosis processes ( including gk , irs , e2f , smad7/9 , hes-1 ) . this is the reason why bmps have a double effect , both proliferative and proapoptotic . an ever expanding literature suggests that the above - described hormonal and nonhormonal factors are able to regulate beta - cell mass by cell cycle regulatory proteins and a range of key signalling pathways . of particular interest in the light of recent data are mitogenic and apoptotic signalling pathways regulated by the transcription factor c - myc , downstream targets involved in g1/s transition [ 187195 ] , and various relevant pathways , including pi3k - akt and mapk . in particular , the pi3k - akt pathway is an important mediator of beta - cell mass increase [ 5 , 136 , 196 ] . in fact , pi3k - akt is known to play a major role in preventing c - myc apoptosis in many cell types and to promote hypertrophy , hyperplasia , and neogenesis . phosphatidylinositol 3-kinase / akt pathwaythere are many signal transduction pathways involved in the regulation of beta - cell mass , with phosphatidylinositol 3-kinase ( pi3k ) and akt signalling being one of the best defined . this pathway is activated by factors , such as glucose , insulin , igfs , and glp-1 [ 136 , 199 ] , and is able to regulate beta - cell size , proliferation , and apoptosis . following ligand - receptor binding , insulin receptor substrate ( irs ) is phosphorylated and is able to interact with various downstream targets . the most important target of irs is pi3k , which in turn can activate pdk1 ( via a secondary messenger ) , resulting in the activation of akt [ 200 , 201 ] . akt plays diverse roles in the beta - cell , including activation of glycogen synthase kinase-3 ( gsk3 , described later ) , and phosphorylation and nuclear exclusion of forkhead transcription factor o1 ( foxo1 ) , ultimately leading to cell growth / replication and suppression of apoptosis ( described in figure 2).this is further evidenced in irs2 mice , whereby beta - cell proliferation was achieved by ablation of one allele of foxo1 .it was reported that pdx1 transcription is regulated positively by forkhead transcription factor a2 ( foxa2 ) but negatively by foxo1 [ 204 , 205 ] . in fact , these 2 forkhead transcription factors share common dna - binding sites in the pdx1 promoter , foxo1 competes with foxa2 for the same binding site resulting in the inhibition of pdx1 transcription . moreover , nuclear translocation of foxo1 excludes pdx1 from the nucleus , or vice versa [ 204 , 206 ] . in addition , constitutively active nuclear foxo1 has been shown to inhibit beta - cell proliferation in the face of insulin resistance . this serves as a second mechanism by which foxo1 inhibits beta - cell proliferation.recently , studies of beta - cells exposed to low nutrition revealed an additional beneficial role of foxo1 activation beta - cell proliferation without any changes in apoptosis compared to the control group . moreover , this study revealed that pi3k and mapk signalling pathways are dampened and that the induction of cyclind1 expression by activated foxo1 in low nutrition is responsible for the improved proliferation of beta - cells .various studies using animal models to evaluate the role of different molecules on the pi3k / akt pathway and beta - cell mass regulation have been performed . both irs1 and 2 are critical for beta - cell growth and survival ( previously mentioned ) , and they occupy a key position between the insulin receptor and downstream signaling elements . irs1 knockout mice develop severe insulin resistance [ 209 , 210 ] and compensate for insulin resistance by increasing beta - cell mass . although irs2 mice are born with only a slightly reduced beta - cell mass , they later develop diabetes due to a marked increase in spontaneous apoptosis and reduced survival of beta - cells . it , therefore , appears that irs2 plays an important role in maintaining beta - cell homeostasis.furthermore , the role of akt has been studied in beta - cells using genetically altered mice in which akt is knocked out or overexpressed . akt knockout mice displayed an increase of blood glycaemia and insulin resistance , although there have been conflicting reports on the effect on compensatory beta - cell growth . overexpressing akt in transgenic mice led to a significant increase in beta - cell mass [ 5 , 213 ] . in addition , akt has been shown to promote beta - cell survival in vivo , suggesting an important role for akt in beta - cell mass regulation.the role of foxo1 on signal transduction was further evidenced in different mouse models . it was demonstrated that constitutively active form of foxo1 protects cells from oxidative - stress - induced damage , and using another mouse model , it was demonstrated that deletion of one allele of foxo1 resulted in a marked increase in the number , but not the size .the role of pdx1 in beta - cell biology has been predominately examined in knockout studies . however , inactivating mutations in one pdx-1 allele have yielded conflicting results , although a critical level of pdx-1 is thought to be required in order to maintain beta - cell mass that [ 215 , 216 ] . there are many signal transduction pathways involved in the regulation of beta - cell mass , with phosphatidylinositol 3-kinase ( pi3k ) and akt signalling being one of the best defined . this pathway is activated by factors , such as glucose , insulin , igfs , and glp-1 [ 136 , 199 ] , and is able to regulate beta - cell size , proliferation , and apoptosis . following ligand - receptor binding , insulin receptor substrate ( irs ) is phosphorylated and is able to interact with various downstream targets . the most important target of irs is pi3k , which in turn can activate pdk1 ( via a secondary messenger ) , resulting in the activation of akt [ 200 , 201 ] . akt plays diverse roles in the beta - cell , including activation of glycogen synthase kinase-3 ( gsk3 , described later ) , and phosphorylation and nuclear exclusion of forkhead transcription factor o1 ( foxo1 ) , ultimately leading to cell growth / replication and suppression of apoptosis ( described in figure 2 ) . this is further evidenced in irs2 mice , whereby beta - cell proliferation was achieved by ablation of one allele of foxo1 . it was reported that pdx1 transcription is regulated positively by forkhead transcription factor a2 ( foxa2 ) but negatively by foxo1 [ 204 , 205 ] . in fact , these 2 forkhead transcription factors share common dna - binding sites in the pdx1 promoter , foxo1 competes with foxa2 for the same binding site resulting in the inhibition of pdx1 transcription . moreover , nuclear translocation of foxo1 excludes pdx1 from the nucleus , or vice versa [ 204 , 206 ] . in addition , constitutively active nuclear foxo1 has been shown to inhibit beta - cell proliferation in the face of insulin resistance . recently , studies of beta - cells exposed to low nutrition revealed an additional beneficial role of foxo1 activation beta - cell proliferation without any changes in apoptosis compared to the control group . moreover , this study revealed that pi3k and mapk signalling pathways are dampened and that the induction of cyclind1 expression by activated foxo1 in low nutrition is responsible for the improved proliferation of beta - cells . various studies using animal models to evaluate the role of different molecules on the pi3k / akt pathway and beta - cell mass regulation have been performed . both irs1 and 2 are critical for beta - cell growth and survival ( previously mentioned ) , and they occupy a key position between the insulin receptor and downstream signaling elements . irs1 knockout mice develop severe insulin resistance [ 209 , 210 ] and compensate for insulin resistance by increasing beta - cell mass . although irs2 mice are born with only a slightly reduced beta - cell mass , they later develop diabetes due to a marked increase in spontaneous apoptosis and reduced survival of beta - cells . it , therefore , appears that irs2 plays an important role in maintaining beta - cell homeostasis . furthermore , the role of akt has been studied in beta - cells using genetically altered mice in which akt is knocked out or overexpressed . akt knockout mice displayed an increase of blood glycaemia and insulin resistance , although there have been conflicting reports on the effect on compensatory beta - cell growth . overexpressing akt in transgenic mice led to a significant increase in beta - cell mass [ 5 , 213 ] . in addition , akt has been shown to promote beta - cell survival in vivo , suggesting an important role for akt in beta - cell mass regulation . it was demonstrated that constitutively active form of foxo1 protects cells from oxidative - stress - induced damage , and using another mouse model , it was demonstrated that deletion of one allele of foxo1 resulted in a marked increase in the number , but not the size . the role of pdx1 in beta - cell biology has been predominately examined in knockout studies . however , inactivating mutations in one pdx-1 allele have yielded conflicting results , although a critical level of pdx-1 is thought to be required in order to maintain beta - cell mass that [ 215 , 216 ] . gsk3glycogen synthase kinase-3 ( gsk3 ) , a constitutively active serine / threonine kinase , was the first substrate shown to be phosphorylated by akt ( 8) . mice overexpressing a constitutively active form of gsk3 in beta - cells display reduced levels of pdx-1 protein . in vitro studies confirmed the role of gsk3 in regulating pdx-1 protein stability , for example , in min6 cells , gsk3 has been shown to phosphorylate pdx-1 leading to its proteasomal degradation . these results highlighted a new mechanism , whereby signalling through the pi3k / akt / gsk3 pathway regulates beta - cell growth . in addition to the modulation of pdx-1 stability , gsk3 can also regulate beta cell proliferation through phosphorylation and degradation of cell cycle proteins , such as cyclin d1 and/or p27 [ 218 , 219 ] . other targets of gsk3 include numerous transcription factors involved in the cell cycle including c - myc and c - jun . therefore , in response to different growth factors , akt prevents gsk3 phosphorylation of cyclin d , thereby promoting cell cycle progression and subsequent mitogenesis ( figure 2 ) . glycogen synthase kinase-3 ( gsk3 ) , a constitutively active serine / threonine kinase , was the first substrate shown to be phosphorylated by akt ( 8) . mice overexpressing a constitutively active form of gsk3 in beta - cells display reduced levels of pdx-1 protein . in vitro studies confirmed the role of gsk3 in regulating pdx-1 protein stability , for example , in min6 cells , gsk3 has been shown to phosphorylate pdx-1 leading to its proteasomal degradation . these results highlighted a new mechanism , whereby signalling through the pi3k / akt / gsk3 pathway regulates beta - cell growth . in addition to the modulation of pdx-1 stability , gsk3 can also regulate beta cell proliferation through phosphorylation and degradation of cell cycle proteins , such as cyclin d1 and/or p27 [ 218 , 219 ] . other targets of gsk3 include numerous transcription factors involved in the cell cycle including c - myc and c - jun . therefore , in response to different growth factors , akt prevents gsk3 phosphorylation of cyclin d , thereby promoting cell cycle progression and subsequent mitogenesis ( figure 2 ) . mitogen - activated protein - kinase ( mapk ) pathwayirs can activate the raf - mek - erk pathway ( aka the mitogen - activated protein - kinase ( mapk ) pathway , figure 2 ) which plays a crucial role in development , cellular differentiation , and mitogenesis . firstly , irs engages the adaptor molecule growth factor receptor - bound protein-2 ( grb2 ) via its sh2 domain [ 222 , 223 ] . in turn , grb2 binds to the murine son - of - sevenless-1 protein ( msos ) , which is able to activate ras protein [ 222 , 223 ] . it then , associates with raf serine kinase , which subsequently phosphorylates mitogen activated protein kinase - kinase ( mek ) resulting in the activation of erk / mapk . activated erk / mapk can then activate other protein kinases or migrate to the nucleus to activate transcription factors , such as c - jun , c - myc and c - fos , important in the cell cycle . however , the ras / mapk pathway can also be activated independently of irs via direct binding of grb2 to the receptor 's sh2 domain [ 222 , 223 ] . irs can activate the raf - mek - erk pathway ( aka the mitogen - activated protein - kinase ( mapk ) pathway , figure 2 ) which plays a crucial role in development , cellular differentiation , and mitogenesis . firstly , irs engages the adaptor molecule growth factor receptor - bound protein-2 ( grb2 ) via its sh2 domain [ 222 , 223 ] . in turn , grb2 binds to the murine son - of - sevenless-1 protein ( msos ) , which is able to activate ras protein [ 222 , 223 ] . it then , associates with raf serine kinase , which subsequently phosphorylates mitogen activated protein kinase - kinase ( mek ) resulting in the activation of erk / mapk . activated erk / mapk can then activate other protein kinases or migrate to the nucleus to activate transcription factors , such as c - jun , c - myc and c - fos , important in the cell cycle . however , the ras / mapk pathway can also be activated independently of irs via direct binding of grb2 to the receptor 's sh2 domain [ 222 , 223 ] . janus kinase / signal transducers and activators of transcription pathwayanother important pathway in beta - cell biology is the jak / stat pathway . both growth hormone ( gh ) and prolactin have been shown to mediate their effects via this pathway [ 226 , 227 ] . once activated , janus kinases ( jaks ) are able to phosphorylate signal transducers and activators of transcriptions ( stats ) which enter the nucleus to regulate transcription of target genes . in particular , jak2 and stat-5 have been shown to regulate beta - cell growth and survival [ 226228 ] , as well as to reduce apoptosis . more recently , it was recognized that the jak / stat pathway is regulated by suppressors of cytokine signaling ( socs ) . these suppressor proteins inhibit stat phosphorylation by binding to phosphorylated jaks , or compete with stats for phosphotyrosine binding sites on cytokine receptors . its effects have resulted in reduced beta - cell proliferation in a sex - dependent manner . both growth hormone ( gh ) and prolactin have been shown to mediate their effects via this pathway [ 226 , 227 ] . once activated , janus kinases ( jaks ) are able to phosphorylate signal transducers and activators of transcriptions ( stats ) which enter the nucleus to regulate transcription of target genes . in particular , jak2 and stat-5 have been shown to regulate beta - cell growth and survival [ 226228 ] , as well as to reduce apoptosis . more recently , it was recognized that the jak / stat pathway is regulated by suppressors of cytokine signaling ( socs ) . these suppressor proteins inhibit stat phosphorylation by binding to phosphorylated jaks , or compete with stats for phosphotyrosine binding sites on cytokine receptors . its effects have resulted in reduced beta - cell proliferation in a sex - dependent manner . c - myc the cellular proto - oncogene , c - myc , encodes the protein c - myc whose key biological function is to promote cell cycle progression . however , this enigmatic protein appears to be a key player in various other biological processes , such as differentiation , cell death , and angiogenesis . recent studies by several laboratories , including our own , support the notion that c - myc may have a central role in the regulation of beta - cell mass required for replication ( during the g1/s transition ) , cell growth , and apoptosis .it is likely that blood glucose levels influence the regulation and expression of c - myc in beta - cells . in fact , it was observed that expression of c - myc was stimulated by high glucose in a ca - dependent manner and also by camp . therefore , taken together , these studies support the opinion that high blood glucose levels may activate beta - cell replication through c - myc expression.besides , the actual mechanism by which hyperglycaemia activates c - myc is fully clear . it is known that cell signalling of c - myc pass through cyclin e - cdk2 activity early in the g1 phase of the cell cycle , an essential event in c - myc - induced g1-s progression [ 234 , 235].in particular , it is well known that ccnd2 ( which encodes cyclin d2 ) and cdk4 are direct target genes of c - myc [ 236 , 237 ] . expression of ccnd2 and cdk4 leads to sequestration of p27 , the cdk inhibitor , in cyclin d2cdk4 complexes . the subsequent degradation of p27 has been shown to involve two other c - myc target genes , cul-1 and cks . by preventing the binding of p27 to cyclin e - cdk2 complexes , c - myc allows inhibitor - free cyclin e - cdk2 complexes to become accessible to phosphorylation by cyclin activating kinase ( cak ) . increased cdk2 and cdk4 activities would result in rb hyperphosphorylation and subsequent release of e2f from rb.however , the final result of c - myc activation on beta - cell is peculiar and differs in various tissues . for example , in contrast of skin , the predominant effect of activating c - myc in pancreatic beta - cells of adult transgenic mice ( cmycer ) is apoptosis and not proliferation , resulting in development of diabetes [ 46 , 238].however , concerning cell signalling mechanism involved in cmyc - induced apoptosis , d cyclins do not appear to be required for c - myc - induced apoptosis in vitro , indicating that these two major functions of c - myc ( proliferation and apoptosis ) may involve , at least in part , distinct sets of downstream mediators . the cellular proto - oncogene , c - myc , encodes the protein c - myc whose key biological function is to promote cell cycle progression . however , this enigmatic protein appears to be a key player in various other biological processes , such as differentiation , cell death , and angiogenesis . recent studies by several laboratories , including our own , support the notion that c - myc may have a central role in the regulation of beta - cell mass required for replication ( during the g1/s transition ) , cell growth , and apoptosis . it is likely that blood glucose levels influence the regulation and expression of c - myc in beta - cells . in fact , it was observed that expression of c - myc was stimulated by high glucose in a ca - dependent manner and also by camp . therefore , taken together , these studies support the opinion that high blood glucose levels may activate beta - cell replication through c - myc expression . besides , the actual mechanism by which hyperglycaemia activates c - myc is fully clear . it is known that cell signalling of c - myc pass through cyclin e - cdk2 activity early in the g1 phase of the cell cycle , an essential event in c - myc - induced g1-s progression [ 234 , 235 ] . in particular , it is well known that ccnd2 ( which encodes cyclin d2 ) and cdk4 are direct target genes of c - myc [ 236 , 237 ] . expression of ccnd2 and cdk4 leads to sequestration of p27 , the cdk inhibitor , in cyclin d2cdk4 complexes . the subsequent degradation of p27 has been shown to involve two other c - myc target genes , cul-1 and cks . by preventing the binding of p27 to cyclin e - cdk2 complexes , c - myc allows inhibitor - free cyclin e - cdk2 complexes to become accessible to phosphorylation by cyclin activating kinase ( cak ) . increased cdk2 and cdk4 activities would result in rb hyperphosphorylation and subsequent release of e2f from rb . however , the final result of c - myc activation on beta - cell is peculiar and differs in various tissues . for example , in contrast of skin , the predominant effect of activating c - myc in pancreatic beta - cells of adult transgenic mice ( cmycer ) is apoptosis and not proliferation , resulting in development of diabetes [ 46 , 238 ] . however , concerning cell signalling mechanism involved in cmyc - induced apoptosis , d cyclins do not appear to be required for c - myc - induced apoptosis in vitro , indicating that these two major functions of c - myc ( proliferation and apoptosis ) may involve , at least in part , distinct sets of downstream mediators . calcineurin / nuclear factor of activated t - cellsmore recently a new important pathway able to regulate beta - cell mass and function has been identified . the key components of this pathway are ca / calmodulin - dependent protein phosphatase 2-b ( or calcineurin - b ) and nuclear factor of activated t cells ( nfat).in beta - cells calcineurin is activated by an increase of cytosolic ca , once activated , it can dephosphorylate nfat transcription factors , which translocate into the nucleus where bind to consensus nfat binding elements on specific gene promoters resulting in the activation of gene transcription . on the contrary , some specific nfat kinase can phosphorylate nfat proteins and then inactivate and exporting them from nucleus.in particular , it was reported in transgenic nfat activated mice that nfat in beta - cells can induce proliferation activating transcription of genes coding for cyclin d1 , cyclin d2 , and cdk4 .more recently , another possible way to regulate the beta - cell mass has been reported . in fact , it was observed that calcineurin / nfat pathway is implicated in the glucose regulation of irs-2 gene expression . in particular , nfat activated can bind to irs-2 promoter enhancing its transcription and it was prevented by specific inhibition of nfat . unfortunately , until now , the mechanisms involved in the regulation of beta - cell cycle by calcineurin are partially understood.the important role of calcineurin / nfat on beta - cell proliferation was highlighted using different transgenic models . using mice with beta - cell - specific deletion of the calcineurin phosphatase regulatory subunit , calcineurin b1 ( cnb1 ) , has shown a reduction of beta - cell function , proliferation and mass , and it coincided with reduced expression of normal regulators of beta - cell proliferation . similarly , long - term activation of calcineurin induces impaired glucose tolerance by alterations in beta - cell mass , and the activation of calcineurin signalling negatively affects proliferation and survival of beta - cells .on the contrary , in normal adult beta - cells , conditional nfat activation has shown to promote expression of cell - cycle regulators resulting in an increase of beta - cell proliferation and mass , resulting in hyperinsulinaemia . more recently a new important pathway able to regulate beta - cell mass and function has been identified . the key components of this pathway are ca / calmodulin - dependent protein phosphatase 2-b ( or calcineurin - b ) and nuclear factor of activated t cells ( nfat ) . in beta - cells calcineurin is activated by an increase of cytosolic ca , once activated , it can dephosphorylate nfat transcription factors , which translocate into the nucleus where bind to consensus nfat binding elements on specific gene promoters resulting in the activation of gene transcription . on the contrary , some specific nfat kinase can phosphorylate nfat proteins and then inactivate and exporting them from nucleus . in particular , it was reported in transgenic nfat activated mice that nfat in beta - cells can induce proliferation activating transcription of genes coding for cyclin d1 , cyclin d2 , and cdk4 . more recently , another possible way to regulate the beta - cell mass has been reported . in fact , it was observed that calcineurin / nfat pathway is implicated in the glucose regulation of irs-2 gene expression . in particular , nfat activated can bind to irs-2 promoter enhancing its transcription and it was prevented by specific inhibition of nfat . unfortunately , until now , the mechanisms involved in the regulation of beta - cell cycle by calcineurin are partially understood . the important role of calcineurin / nfat on beta - cell proliferation was highlighted using different transgenic models . using mice with beta - cell - specific deletion of the calcineurin phosphatase regulatory subunit , calcineurin b1 ( cnb1 ) , has shown a reduction of beta - cell function , proliferation and mass , and it coincided with reduced expression of normal regulators of beta - cell proliferation . similarly , long - term activation of calcineurin induces impaired glucose tolerance by alterations in beta - cell mass , and the activation of calcineurin signalling negatively affects proliferation and survival of beta - cells . on the contrary , in normal adult beta - cells , conditional nfat activation has shown to promote expression of cell - cycle regulators resulting in an increase of beta - cell proliferation and mass , resulting in hyperinsulinaemia . the critical point in the beta - cell cycle seems to be the g1s checkpoint . at this point , the cell is committed to dna replication , and it is regulated by the retinoblastoma protein ( rb ) . rb itself is inactivated by a group of serine threonine protein kinases , the cyclin - dependant kinases ( cdks ) including cdk4/6 and cdk2 , which in turn are negatively regulated by cyclin - dependent kinase inhibitors ( cdkis ) . rb is a member of a family of pocket proteins which is able to block g1s transition by binding to e2f transcriptional activator proteins . when liberated from rb , e2f transcription factor elicits its activity in controlling the transition from g1 to s phase . mice deleted for e2f-1 display defective pancreatic growth and islet dysfunction . in e2f-1 and e2f-2 double knockout mice , both exocrine and endocrine glands had atrophied by 3 months of age , a feature not seen in single knockouts at the same time point . the cyclin - dependent kinase cdk4 and 6 are activated by cyclin d ( in particular , d1 and d2 ) whereas cdk2 is activated by cyclin e. both cyclins d1 and d2 are essential for normal postnatal islet growth . in adult islets , global deletion of cyclin d2 fails to stimulate adequate compensatory upregulation of cyclin d1 within islets and drastically damages postnatal beta - cell proliferation , islet mass , and decreases glucose tolerance [ 7 , 189 ] . the importance of cyclin d1 has been highlighted using transgenic mice that overexpress cyclin d1 . in this animal model , cdk4 has also been shown to be a key regulator of beta - cell cycle . knockout studies have shown a very specific phenotype , with beta - cell hypoplasia and severe diabetes [ 191 , 194 , 245 ] , whilst studies using an activating mutation in cdk4 , rendering it resistant to p16 , have resulted in pancreatic hyperplasia , which demonstrated normal physiology [ 191 , 192 ] . other studies have shown up to a three - fold increase in beta - cell proliferation with cdk4 overexpression [ 191 , 193 ] . the cdks are inhibited by two groups of cdk inhibitors ( cdkis ) which are also expressed in islets , ink4 proteins ( p16 , p15 , p18 , p19 inhibit cdk 4 and cdk6 ) , and cip / kip proteins ( p21 , p27 and p57 ) . whilst ink4 family proteins promote cell cycle arrest , p21 and p27 are integrated into the cyclin d / cdk4 complex that is able to arrest cell cycle progression through dephosphorylation of rb protein , on the other hand , they are able to inhibit cyclin e / a cdk2 complex resulting in a block of phosphorylation of rb protein . various studies have looked at the effects of cdkis on islets . in p21 mice , islets were phenotypically and metabolically comparable to their wildtype counterparts , possibly due to upregulation of p57 , whereas loss of function of p57 has been associated with hyperinsulinaemia at infancy . actively inhibiting the effect of p18 , using knockout mice resulted in beta - cell hyperplasia of up to 40% that appears to be cdk4 dependent . in contrast , p27 has been shown to partially reverse the islet phenotype independent of cdk4 . overexpression of p27 in beta - cells of transgenic mice impairs beta - cell proliferation , resulting in decreased beta - cell mass and diabetes . furthermore , p27 mice increases beta - cell proliferation doubling their beta - cell mass at birth , and this expansion was accompanied by increased insulin secretion . micrornas ( mirnas ) are small 1923 nucleotide noncoding rna molecules that act as posttranscriptional regulators of different genes involved in various cellular processes , such as apoptosis , differentiation , and proliferation . this regulation pass through generally , mirnas can regulate protein synthesis either by repressing translation and/or by degradation through deadenylation of mrna targets [ 250 , 251 ] . the role of mirnas in beta - cell mass regulation is not yet fully understood . it was reported that its overexpression attenuates proliferation of beta - cells and glucose - induced insulin secretion [ 1618 ] . indeed , ectopic expression of mir-375 in diabetic pancreatic beta - cells results in increased susceptibility to fatty acid induced apoptosis . moreover , it was reported that in ob / ob mice in which mir-375 was deleted , they develop a marked decrease in beta - cell mass , which results in a severe insulin - deficient diabetes not found in ob / ob mice . therefore , it is becoming clear that mir-375 targets a suit of genes that negatively regulate cell growth and proliferation and that aberrant loss of this mirna leads to dramatic reduction of beta - cell mass . furthermore , it was reported that overexpressions of both mir-34a and mir-146a are involved in programmed cell death , in particular , in apoptotic processes . in fact , prolonged beta - cell exposure to palmitate ( ffa ) and proinflammatory cytokines changes / increases the expression of mir-34a and mir-146a , and treatment with anti - mir34a or anti - mir148 diminished the number of death cells in the presence of apoptotic stimulus . the effect on the regulation of apoptosis may be due to the capacity of mir-34 to control the expression of the antiapoptotic protein bcl2 , meanwhile mir-146a may control the apoptotic process through the regulation of the nf - kb pathway . in addition to maintaining beta - cell mass under normal circumstances , as just discussed , an organism must also be able to alter its beta - cell mass in accordance with insulin 's requirements . in particular conditions , such as pregnancy and obesity , beta - cell mass enlargement is observed . however , when compensatory beta - cell mass expansion is inadequate , gestational diabetes in the case of pregnancy and t2d in the case of obesity are the results . although the majority of humans do not become diabetic in these circumstances , a significant part of the population is predisposed to beta - cell failure , for currently unknown reasons . the precise mechanism of beta - cell mass maintenance and expansion , that is , proliferation , neogenesis , or increase in size , has been elucidated only in part [ 178 , 254 , 255 ] . different studies in rodents found a two- to five - fold increase in beta - cell mass during gestation [ 255 , 256 ] and demonstrated an involvement of both beta - cell hypertrophy [ 178 , 256 , 257 ] and proliferation [ 256 , 257 ] . van assche et al . have reported that in humans during pregnancy the beta - cell mass increased 2.4-fold compared with nonpregnant women . unfortunately , human data are limited to few studies using a small number of subjects . butler et al . have reported an approximately 1.4-fold increase in beta - cell mass using 18 women . in contrast to rodents , beta - cell mass expansion in humans was achieved by the formation of new islets or islet neogenesis . the main stimuli for beta - cell proliferation during pregnancy are placental lactogen ( pl ) , prolactin ( prl ) , growth hormone ( gh ) [ 178 , 259 ] , and serotonin . postpartum , in rodents , beta - cell mass returns to normal levels within 10 days through increased beta - cell apoptosis , decreased beta - cell proliferation , and reduced beta - cell size . placental lactogenplacental lactogen ( pl ) hormone has been implicated as the primary factor responsible for the enhanced islet mass and function that occur during pregnancy [ 178 , 259 ] . pl interacts with receptors in the prl / gh receptor family , stimulating the jak-2/stat-5 intracellular signalling pathway [ 261 , 262 ] . prior studies performed in vitro and over the short term suggested that pl is a more powerful islet mitogen than gh or prl [ 178 , 263 ] . these data were confirmed in in vivo studies using transgenic mice expressing mouse pl-1 under the control of the rat insulin promoter ( rip - mpl1 ) [ 259 , 264 ] . the expansion of beta - cell mass in rip - mpl1 was attributed principally to a two - fold increase in beta - cell proliferation and a 20% increase in beta - cell size ( hypertrophy ) . interestingly , islet number was not significantly increased when compared to wild - type mice . these findings were supported by a pl receptor knockout study , which showed a significant reduction in beta - cell mass . placental lactogen ( pl ) hormone has been implicated as the primary factor responsible for the enhanced islet mass and function that occur during pregnancy [ 178 , 259 ] . pl interacts with receptors in the prl / gh receptor family , stimulating the jak-2/stat-5 intracellular signalling pathway [ 261 , 262 ] . prior studies performed in vitro and over the short term suggested that pl is a more powerful islet mitogen than gh or prl [ 178 , 263 ] . these data were confirmed in in vivo studies using transgenic mice expressing mouse pl-1 under the control of the rat insulin promoter ( rip - mpl1 ) [ 259 , 264 ] . the expansion of beta - cell mass in rip - mpl1 was attributed principally to a two - fold increase in beta - cell proliferation and a 20% increase in beta - cell size ( hypertrophy ) . interestingly , islet number was not significantly increased when compared to wild - type mice . these findings were supported by a pl receptor knockout study , which showed a significant reduction in beta - cell mass . prolactinprolactin ( prl ) , a hormone , acts through its specific receptor , prolactin - receptor ( prlr ) to induce beta - cell proliferation in vitro . targeted deletion of the prlr reduces beta - cell mass and mildly impairs insulin secretion . the pivotal role of the prlr for beta - cell adaptation during pregnancy was demonstrated using pregnant mice heterozygous for the prlr null mutant . it has been extensively demonstrated in different in vitro studies that prl binding to prlr is able to activate different signaling pathways such as stat5 , mapk , and irs / pi3k resulting in enhancing beta - cell mass during pregnancy [ 267269 ] . prolactin ( prl ) , a hormone , acts through its specific receptor , prolactin - receptor ( prlr ) to induce beta - cell proliferation in vitro . targeted deletion of the prlr reduces beta - cell mass and mildly impairs insulin secretion . the pivotal role of the prlr for beta - cell adaptation during pregnancy was demonstrated using pregnant mice heterozygous for the prlr null mutant . it has been extensively demonstrated in different in vitro studies that prl binding to prlr is able to activate different signaling pathways such as stat5 , mapk , and irs / pi3k resulting in enhancing beta - cell mass during pregnancy [ 267269 ] . meninmore recently , menin ( a tumour suppressor encoded by the men1 gene ) has been linked to the regulation of beta - cell proliferation during pregnancy . expression of menin , p18 , and p27 are reduced during pregnancy in maternal islets , thus leading to islet mass expansion to meet the increased metabolic demand . after birth , interestingly , menin is regulated by prolactin , supporting menin as an important mediator of beta - cell proliferation during pregnancy . it has also been proposed that , during pregnancy , activation of jak2 and akt , in response to prolactin , leads to increased p21 expression , whereas menin and p18 expression are suppressed . it is well known that akt and p21 induce beta - cell proliferation , meanwhile the downregulation of menin and p18 allows for enhanced beta - cell proliferation . more recently , menin ( a tumour suppressor encoded by the men1 gene ) has been linked to the regulation of beta - cell proliferation during pregnancy . expression of menin , p18 , and p27 are reduced during pregnancy in maternal islets , thus leading to islet mass expansion to meet the increased metabolic demand . after birth , interestingly , menin is regulated by prolactin , supporting menin as an important mediator of beta - cell proliferation during pregnancy . it has also been proposed that , during pregnancy , activation of jak2 and akt , in response to prolactin , leads to increased p21 expression , whereas menin and p18 expression are suppressed . it is well known that akt and p21 induce beta - cell proliferation , meanwhile the downregulation of menin and p18 allows for enhanced beta - cell proliferation . serotonina recent study by kim et al . shows that beta - cells , like serotoninergic neurons , are able to synthesize , store , and secrete serotonin ( 5-ht ) as well as express the specific serotonin receptors ( 5-htr2b and 5-htr1d ) . these authors reported that , in pregnant mice , 5-htr2bexpression increased significantly during midgestation ( day 6 to 16 ) and normalized at the end of gestation , whereas 5-htr1d expression increased at the end of gestation ( day 17 ) and postpartum . notably , increased 5-htr2bexpression closely correlated with the period of increased beta - cell proliferation , and increased 5-htr1dexpression correlated with the cessation of beta - cell proliferation and regression of beta - cell mass . moreover , it was reported that in pregnant mice stimulation with high concentrations of prolactin induces a strong increase in the serotonin expression compared with nonpregnant control mice . these results suggest that during pregnancy lactogenic signalling induces serotonin expression and synthesis in islets able to stimulate beta - cell proliferation through the 5-htr2b pathway . shortly before parturition , expression of 5-htr2b decreases and 5-htr1d expression increases , resulting in the reduction of beta - cell proliferation and beta - cell mass . in this way , a recent study by kim et al . shows that beta - cells , like serotoninergic neurons , are able to synthesize , store , and secrete serotonin ( 5-ht ) as well as express the specific serotonin receptors ( 5-htr2b and 5-htr1d ) . these authors reported that , in pregnant mice , 5-htr2bexpression increased significantly during midgestation ( day 6 to 16 ) and normalized at the end of gestation , whereas 5-htr1d expression increased at the end of gestation ( day 17 ) and postpartum . notably , increased 5-htr2bexpression closely correlated with the period of increased beta - cell proliferation , and increased 5-htr1dexpression correlated with the cessation of beta - cell proliferation and regression of beta - cell mass . moreover , it was reported that in pregnant mice stimulation with high concentrations of prolactin induces a strong increase in the serotonin expression compared with nonpregnant control mice . these results suggest that during pregnancy lactogenic signalling induces serotonin expression and synthesis in islets able to stimulate beta - cell proliferation through the 5-htr2b pathway . shortly before parturition , expression of 5-htr2b decreases and 5-htr1d expression increases , resulting in the reduction of beta - cell proliferation and beta - cell mass . in this way , beta - cell mass returns rapidly to prepregnancy levels . obesity is linked / correlated with insulin resistance and associated with compensatory physiological response at the level of beta - cell mass increase . as previously described , when compensatory beta - cell mass expansion is inadequate , t2d in the case of obesity is consequential . diet - induced obesity results in insulin resistance and beta - cell mass expansion in humans and mice . in nondiabetic animal models of obesity , for example , the c57bl/6 mouse strain which is notoriously susceptible to these effects , there is a 2.2-fold increase in beta - cell mass and proliferation after 4 months of a high - fat diet versus a control diet . meanwhile , in the zucker diabetic fatty ( zdf ) fa / fa rat , beta - cell mass increased four folds compared with lean controls . however , these mice eventually become diabetic and lose their beta - cell mass due to increased beta - cell apoptosis and reduced beta - cell proliferation . in genetic models of obesity and insulin resistance , there is a compensatory expansion of beta - cell mass . for example , in mice lacking a functional leptin receptor ( db / db mice ) , beta - cell mass exhibits two - fold beta - cell mass increase . the zucker rat ( fa / fa ) also has a homozygous mutation in the gene encoding the leptin receptor . zdf rats exhibit increased beta - cell mass and increased beta - cell proliferation prior to the onset of diabetes , but increased beta - cell apoptosis prevents them from adequately expanding their beta - cell mass after the onset of diabetes , despite continued high rates of beta - cell proliferation . this phenotype contrasts with that observed in nondiabetic zucker fatty ( zf ) rats , which possess the same mutation as zdf rats and also become obese and insulin resistant but do not develop diabetes due to sufficient beta - cell mass expansion through increased beta - cell proliferation , neogenesis , and hypertrophy . on the basis of available research findings that we have discussed above , we can propose that ( i ) postnatal beta - cell mass sizes in response to changing metabolic demands , ( ii ) is carried out by an interaction of beta - cell replication ( proliferation and/or neogenesis ) and apoptosis , and ( iii ) this process is regulated by different growth factors / nutrients that interact between them . actually , our present knowledge to the understanding of proliferation , neogenesis , and apoptosis is still incomplete . the balance between apoptosis and replication seems to be pivotal in a right beta - cell mass maintenance . failure in this balance results in beta - cell dysfunction and consequently diabetes onset . because of the worldwide diffusion of this pathology , new therapies able to restore the original beta - cell mass and its functionality are under continuous development . islet transplantation is already under clinical investigation but for the foreseeable future it will be very restricted in application due to the limited supplies of human cadaveric islets for transplantation . as a result , there is intense interest in developing other sources of new beta - cells and identification of new molecules able to restore beta - cell mass in diabetics .

pancreatic beta - cells , which secrete the hormone insulin , are the key arbiters of glucose homeostasis . defective beta - cell numbers and/or function underlie essentially all major forms of diabetes and must be restored if diabetes is to be cured . thus , the identification of the molecular regulators of beta - cell mass and a better understanding of the processes of beta - cell differentiation and proliferation may provide further insight for the development of new therapeutic targets for diabetes . this review will focus on the principal hormones and nutrients , as well as downstream signalling pathways regulating beta - cell mass in the adult . furthermore , we will also address more recently appreciated regulators of beta - cell mass , such as micrornas .

1. Introduction 2. Postnatal Beta-Cell Regulation 3. Role of MicroRNAs on the Beta-Cell Mass Regulation 4. Beta-Cell Mass Plasticity 5. Conclusions

beta - cells , the most numerous islet cells , secrete the hormone insulin which reduces blood glucose levels by increasing peripheral uptake of glucose and by suppressing release of glucose from the liver . during adult life , the beta - cell mass may have to adapt in the face of increased demands due to increases in body mass , pregnancy , or even loss of insulin sensitivity of peripheral tissues . for this reason , great efforts are being made in order to develop new therapeutic strategies , such as beta - cell replacement or regenerative medicine . such an approach requires further knowledge of the mechanisms that regulate pancreatic beta - cell mass . this review briefly outlines current knowledge of significant factors / nutrients regulating beta - cells mass , and their signal transduction pathways , with greater focus on postnatal regulation and the role of a new class of beta - cell mass regulators : the micrornas . thus , organisms born with reduced beta - cell mass have fewer beta - cells available to enter the cell cycle later in life . a large number of hormones and nutrients have been shown to affect beta - cell mass , as extensively reviewed by nielsen et al . thus , organisms born with reduced beta - cell mass have fewer beta - cells available to enter the cell cycle later in life . a large number of hormones and nutrients have been shown to affect beta - cell mass , as extensively reviewed by nielsen et al . beta - cell mass regulation is modulated by various environmental factors and nutrients including glucose , insulin , amino acids , fatty acids , and various other growth factors / hormones , such as igf - i , igf - ii , glucagon - like peptide-1 ( glp-1 ) 1 , glucagon , gastroinhibitory peptide ( gip ) , somatostatin ( sst ) , hgf and betacellulin , gastrin , cholecystokinin ( cck ) , growth hormone ( gh ) , prolactin ( prl ) , placental lactogen ( pl ) , and leptin , amongst others ( table 1 ) . glucose glucose is thought to be the most important determinant of beta - cell mass equilibrium [ 3136 ] . while some authors describe proliferation of new beta - cells formed by neogenesis of precursor cells , other groups report an increase in beta - cell replication and hypertrophy as well as neogenesis , leading to sustained effects on beta - cell mass even after glucose infusion is stopped [ 35 , 39].to support the mitogen effect of glucose on beta - cells , porat and colleagues demonstrated in an animal model an increased replication rate of beta - cells correlated with an increased blood glucose level . this proapoptotic effect of glucose may also involve altered calcium homeostasis , activation of caspases by cytokines , such as il-1 which is secreted by human islets in the presence of high glucose and leads to beta - cell apoptosis or fas - fas - ligand interactions , reactive oxygen species , and c - myc [ 46 , 47 ] . glucose is thought to be the most important determinant of beta - cell mass equilibrium [ 3136 ] . in another study using prolonged periods of glucose infusion , an increase of beta - cell mass was also reported , although the origin of these cells has been disputed . while some authors describe proliferation of new beta - cells formed by neogenesis of precursor cells , other groups report an increase in beta - cell replication and hypertrophy as well as neogenesis , leading to sustained effects on beta - cell mass even after glucose infusion is stopped [ 35 , 39 ] . to support the mitogen effect of glucose on beta - cells , porat and colleagues demonstrated in an animal model an increased replication rate of beta - cells correlated with an increased blood glucose level . this proapoptotic effect of glucose may also involve altered calcium homeostasis , activation of caspases by cytokines , such as il-1 which is secreted by human islets in the presence of high glucose and leads to beta - cell apoptosis or fas - fas - ligand interactions , reactive oxygen species , and c - myc [ 46 , 47 ] . various authors have shown in various in vivo studies that insulin alone can stimulate beta - cell mass , whereas other authors have found that it promotes growth only in the presence of hyperglycaemia [ 34 , 36 , 5456 ] . insulin resistance , leading to hyperinsulinemia , stimulates an increase in beta - cell mass.in contrast , as previously described [ 60 , 61 ] , insulin can also impose negative effect on beta - cell mass and/or proliferation . for example , rats with insulinomas resulting in hyperinsulinemia and hypoglycaemia were found to have smaller beta - cells , as well as reduced beta - cell numbers ( in the islets without tumour ) , suggesting that a sensor mechanisms exist that can co - ordinate beta - cell mass across islets and possibly even distal grafts . for example , rats with insulinomas resulting in hyperinsulinemia and hypoglycaemia were found to have smaller beta - cells , as well as reduced beta - cell numbers ( in the islets without tumour ) , suggesting that a sensor mechanisms exist that can co - ordinate beta - cell mass across islets and possibly even distal grafts . however , precise molecular mechanisms linking ffa to beta - cell dysfunction have yet to be fully elucidated.it was reported that a prolonged in vitro exposure of isolated islets or insulin - secreting cell lines to elevated levels of fatty acids is associated with inhibition of glucose - induced insulin secretion [ 67 , 73 , 74 ] , impairment of insulin gene expression [ 7578 ] , and induction of cell death by apoptosis both in vitro [ 7988 ] and in vivo studies .importantly , in vitro [ 76 , 88 , 89 ] and in vivo [ 90 , 91 ] studies have provided evidence that lipotoxicity only occurs in the presence of concomitantly elevated glucose levels . despite the limited in vivo evidence , in vitro experiments demonstrate that gip enhances beta - cell mitogenesis [ 125128].furthermore , the role of gip in the reduction of beta - cell apoptosis in vivo is more clearly defined than its role in beta - cell proliferation.various studies have also demonstrated that exogenous gip treatment prevents beta - cell apoptosis during severe obesity [ 123 , 129 ] and streptozotocin - induced diabetes [ 120 , 129 ] . despite the limited in vivo evidence , in vitro experiments furthermore , the role of gip in the reduction of beta - cell apoptosis in vivo is more clearly defined than its role in beta - cell proliferation . igf - i has been shown to increase the number of replicating beta - cells in rodent islets by up to 6% of the islet cell population [ 133135 ] and to induce differentiated pancreatic beta - cell growth . thus , igf - i potentiates the mitogenic effect of glucose on beta - cell proliferation.interestingly , overexpression of igf - i in beta - cells is associated with increased beta - cell proliferation , but not mass . parathyroid hormone - related proteina recent study has demonstrated that acute and systemic administration of the first 36 amino acids of parathyroid hormone - related protein ( pthrp ( 136 ) ) in rodents can stimulate beta - cell proliferation and enhance beta - cell mass in vivo , without negatively affecting beta - cell function or survival . previous studies have demonstrated that rip - pthrp transgenic mice producing the full - length pthrp ( 1139 ) in beta - cells show an increase in beta - cell mass , proliferation and islet number , and improvement of glucose homeostasis [ 145147 ] . however , the explanation of this result should be found on the possibility that the pthrp was absent in islets but it could be produced in other islet cell types , resulting in a paracrine compensatory mechanism .these findings suggest that pthrp may provide a possible target for gene therapeutic strategies designed to increase beta - cell mass and function . previous studies have demonstrated that rip - pthrp transgenic mice producing the full - length pthrp ( 1139 ) in beta - cells show an increase in beta - cell mass , proliferation and islet number , and improvement of glucose homeostasis [ 145147 ] . these findings suggest that pthrp may provide a possible target for gene therapeutic strategies designed to increase beta - cell mass and function . in beta - cells , ghrelin treatment has been shown to increase expression of insulin and pdx1 , as well as beta - cell replication in a diabetes model . moreover , it has been recently reported that both acylated- and unacylated - ghrelin can on the one hand promote cell proliferation while on the other inhibit apoptosis of beta - cells , including human pancreatic islets induced by serum starvation and/or cytokine 's stimulation [ 153 , 154 ] . in beta - cells , ghrelin treatment has been shown to increase expression of insulin and pdx1 , as well as beta - cell replication in a diabetes model . recently , it has been reported that menin is also able to increase caspase-8 expression and that caspase-8 is critical for the maintenance of beta - cell mass under physiological conditions . recently , it has been reported that menin is also able to increase caspase-8 expression and that caspase-8 is critical for the maintenance of beta - cell mass under physiological conditions . overall , leptin is likely to exert diverse effects in the regulation of pancreatic beta - cell function and proliferation . overall , leptin is likely to exert diverse effects in the regulation of pancreatic beta - cell function and proliferation . through these pathways , visfatin would appear to have the potential to regulate plasma glucose levels , as well as beneficial effects on beta - cell mass . overall , visfatin may have a protective effect on pancreatic beta - cell mass , but further research is necessary to clarify its distinct roles . through these pathways , visfatin would appear to have the potential to regulate plasma glucose levels , as well as beneficial effects on beta - cell mass . overall , visfatin may have a protective effect on pancreatic beta - cell mass , but further research is necessary to clarify its distinct roles . an ever expanding literature suggests that the above - described hormonal and nonhormonal factors are able to regulate beta - cell mass by cell cycle regulatory proteins and a range of key signalling pathways . phosphatidylinositol 3-kinase / akt pathwaythere are many signal transduction pathways involved in the regulation of beta - cell mass , with phosphatidylinositol 3-kinase ( pi3k ) and akt signalling being one of the best defined . this pathway is activated by factors , such as glucose , insulin , igfs , and glp-1 [ 136 , 199 ] , and is able to regulate beta - cell size , proliferation , and apoptosis . moreover , this study revealed that pi3k and mapk signalling pathways are dampened and that the induction of cyclind1 expression by activated foxo1 in low nutrition is responsible for the improved proliferation of beta - cells .various studies using animal models to evaluate the role of different molecules on the pi3k / akt pathway and beta - cell mass regulation have been performed . although irs2 mice are born with only a slightly reduced beta - cell mass , they later develop diabetes due to a marked increase in spontaneous apoptosis and reduced survival of beta - cells . there are many signal transduction pathways involved in the regulation of beta - cell mass , with phosphatidylinositol 3-kinase ( pi3k ) and akt signalling being one of the best defined . this pathway is activated by factors , such as glucose , insulin , igfs , and glp-1 [ 136 , 199 ] , and is able to regulate beta - cell size , proliferation , and apoptosis . moreover , this study revealed that pi3k and mapk signalling pathways are dampened and that the induction of cyclind1 expression by activated foxo1 in low nutrition is responsible for the improved proliferation of beta - cells . although irs2 mice are born with only a slightly reduced beta - cell mass , they later develop diabetes due to a marked increase in spontaneous apoptosis and reduced survival of beta - cells . in particular , jak2 and stat-5 have been shown to regulate beta - cell growth and survival [ 226228 ] , as well as to reduce apoptosis . recent studies by several laboratories , including our own , support the notion that c - myc may have a central role in the regulation of beta - cell mass required for replication ( during the g1/s transition ) , cell growth , and apoptosis .it is likely that blood glucose levels influence the regulation and expression of c - myc in beta - cells . for example , in contrast of skin , the predominant effect of activating c - myc in pancreatic beta - cells of adult transgenic mice ( cmycer ) is apoptosis and not proliferation , resulting in development of diabetes [ 46 , 238].however , concerning cell signalling mechanism involved in cmyc - induced apoptosis , d cyclins do not appear to be required for c - myc - induced apoptosis in vitro , indicating that these two major functions of c - myc ( proliferation and apoptosis ) may involve , at least in part , distinct sets of downstream mediators . recent studies by several laboratories , including our own , support the notion that c - myc may have a central role in the regulation of beta - cell mass required for replication ( during the g1/s transition ) , cell growth , and apoptosis . for example , in contrast of skin , the predominant effect of activating c - myc in pancreatic beta - cells of adult transgenic mice ( cmycer ) is apoptosis and not proliferation , resulting in development of diabetes [ 46 , 238 ] . on the contrary , some specific nfat kinase can phosphorylate nfat proteins and then inactivate and exporting them from nucleus.in particular , it was reported in transgenic nfat activated mice that nfat in beta - cells can induce proliferation activating transcription of genes coding for cyclin d1 , cyclin d2 , and cdk4 .more recently , another possible way to regulate the beta - cell mass has been reported . unfortunately , until now , the mechanisms involved in the regulation of beta - cell cycle by calcineurin are partially understood.the important role of calcineurin / nfat on beta - cell proliferation was highlighted using different transgenic models . using mice with beta - cell - specific deletion of the calcineurin phosphatase regulatory subunit , calcineurin b1 ( cnb1 ) , has shown a reduction of beta - cell function , proliferation and mass , and it coincided with reduced expression of normal regulators of beta - cell proliferation . similarly , long - term activation of calcineurin induces impaired glucose tolerance by alterations in beta - cell mass , and the activation of calcineurin signalling negatively affects proliferation and survival of beta - cells .on the contrary , in normal adult beta - cells , conditional nfat activation has shown to promote expression of cell - cycle regulators resulting in an increase of beta - cell proliferation and mass , resulting in hyperinsulinaemia . more recently a new important pathway able to regulate beta - cell mass and function has been identified . unfortunately , until now , the mechanisms involved in the regulation of beta - cell cycle by calcineurin are partially understood . using mice with beta - cell - specific deletion of the calcineurin phosphatase regulatory subunit , calcineurin b1 ( cnb1 ) , has shown a reduction of beta - cell function , proliferation and mass , and it coincided with reduced expression of normal regulators of beta - cell proliferation . similarly , long - term activation of calcineurin induces impaired glucose tolerance by alterations in beta - cell mass , and the activation of calcineurin signalling negatively affects proliferation and survival of beta - cells . on the contrary , in normal adult beta - cells , conditional nfat activation has shown to promote expression of cell - cycle regulators resulting in an increase of beta - cell proliferation and mass , resulting in hyperinsulinaemia . overexpression of p27 in beta - cells of transgenic mice impairs beta - cell proliferation , resulting in decreased beta - cell mass and diabetes . therefore , it is becoming clear that mir-375 targets a suit of genes that negatively regulate cell growth and proliferation and that aberrant loss of this mirna leads to dramatic reduction of beta - cell mass . in addition to maintaining beta - cell mass under normal circumstances , as just discussed , an organism must also be able to alter its beta - cell mass in accordance with insulin 's requirements . in particular conditions , such as pregnancy and obesity , beta - cell mass enlargement is observed . however , when compensatory beta - cell mass expansion is inadequate , gestational diabetes in the case of pregnancy and t2d in the case of obesity are the results . the expansion of beta - cell mass in rip - mpl1 was attributed principally to a two - fold increase in beta - cell proliferation and a 20% increase in beta - cell size ( hypertrophy ) . the expansion of beta - cell mass in rip - mpl1 was attributed principally to a two - fold increase in beta - cell proliferation and a 20% increase in beta - cell size ( hypertrophy ) . shows that beta - cells , like serotoninergic neurons , are able to synthesize , store , and secrete serotonin ( 5-ht ) as well as express the specific serotonin receptors ( 5-htr2b and 5-htr1d ) . shortly before parturition , expression of 5-htr2b decreases and 5-htr1d expression increases , resulting in the reduction of beta - cell proliferation and beta - cell mass . shows that beta - cells , like serotoninergic neurons , are able to synthesize , store , and secrete serotonin ( 5-ht ) as well as express the specific serotonin receptors ( 5-htr2b and 5-htr1d ) . shortly before parturition , expression of 5-htr2b decreases and 5-htr1d expression increases , resulting in the reduction of beta - cell proliferation and beta - cell mass . in nondiabetic animal models of obesity , for example , the c57bl/6 mouse strain which is notoriously susceptible to these effects , there is a 2.2-fold increase in beta - cell mass and proliferation after 4 months of a high - fat diet versus a control diet . zdf rats exhibit increased beta - cell mass and increased beta - cell proliferation prior to the onset of diabetes , but increased beta - cell apoptosis prevents them from adequately expanding their beta - cell mass after the onset of diabetes , despite continued high rates of beta - cell proliferation . on the basis of available research findings that we have discussed above , we can propose that ( i ) postnatal beta - cell mass sizes in response to changing metabolic demands , ( ii ) is carried out by an interaction of beta - cell replication ( proliferation and/or neogenesis ) and apoptosis , and ( iii ) this process is regulated by different growth factors / nutrients that interact between them . because of the worldwide diffusion of this pathology , new therapies able to restore the original beta - cell mass and its functionality are under continuous development . as a result , there is intense interest in developing other sources of new beta - cells and identification of new molecules able to restore beta - cell mass in diabetics .

autism spectrum disorders ( asds ) are characterized by impairments in social interaction , communication , and restricted or repetitive behaviors and interests ( dsm - iv : american psychiatric association , 1994 ) . neuroimaging research has revealed a broad network of regional brain abnormalities in asd , including frontal , parietal , and limbic regions , the basal ganglia , and the cerebellum ( amaral et al . , 2008 ) . cerebellar structural and functional differences are consistently reported in asd , suggesting that cerebellar dysfunction may be important in the etiology of the disorder ( allen et al . , 2004 ; courchesne , 1997 ; fatemi et al . , 2012 ) . supporting this , almost all post - mortem analyses of asd individuals have reported reduced purkinje cell size and number regardless of age , sex , or cognitive ability ( bailey et al . , 1998 ; although neuroimaging meta - analyses suggest that several different regions of the cerebellum are affected in asd ( duerden et al . , 2012 ; stoodley , 2014 ; yu et al . , 2011 ) , no study has used both voxel - based and lobular region of interest analyses to examine structural differences within the cerebella of autistic individuals , while also assessing the relationship between these cerebellar subregions and core asd symptoms . anatomically , the cerebellum is divided into ten lobules ( lobules i x ) and three lobes : the anterior lobe ( lobules i v ) , the posterior lobe ( lobules vi ix ) , and the flocculonodular lobe ( lobule x ; fig . anatomical , clinical , and neuroimaging studies support the idea that regions within the cerebellum have functionally distinct roles in movement , cognition and affective processing ( stoodley and schmahmann , 2010 ) . somatomotor representations of the body are found in the anterior lobe and lobule viii , which interconnect with sensorimotor areas of the cerebral cortex and are engaged during sensorimotor tasks ( stoodley and schmahmann , 2010 ) . the large posterior lobe , including lobules vi and vii ( which is subdivided into crus i , crus ii and viib ) , receives input from prefrontal and parietal association areas and is engaged during cognitive tasks ( strick et al . , 2009 recent functional connectivity data show that the majority of the cerebellum is functionally connected to association networks involved in cognitive and affective processes , rather than somatomotor networks ( buckner et al . , 2011 ) . clinical outcomes also reflect the topography seen in healthy controls , as lesions involving posterior regions of cerebellum can lead to difficulties in executive functioning , language , memory and affect , while damage to the anterior cerebellum can result in motor impairments with minimal cognitive effects ( schmahmann and sherman , 1998 ) . based on these data , the putative role of the human cerebellum has been expanded to include higher order cognitive and affective processes ( ito , 2008 ; stoodley and schmahmann , 2009 ; strick et al . , 2009 ) . the unique patterns of connectivity of different cerebellar subregions result in a functional topography , whereby different regions process different types of information ( stoodley , 2012 ; strick et al . , 2009 ) . this topography is of importance when considering the localization of cerebellar structural and functional differences in asd , and may be beneficial in the interpretation of cerebellar findings in asd . neuroimaging studies comparing asd with typically developing ( td ) structural mri studies have described hypoplasia of the posterior vermis in asd ( carper and courchesne , 2000 ; courchesne et al . , 1988 , 1994 , 2011 ; murakami et al . , 1989 ) , and meta - analyses of voxel - based morphometry ( vbm ) studies have reported consistent gray matter ( gm ) decreases in right crus i , lobule viii , and lobule ix ( duerden et al . , 2012 ; stoodley , 2014 ; yu et al . , 2011 ) . decreased gm is less commonly reported in regions such as left crus i , and sometimes overall increased cerebellar gm is noted ( duerden et al . , 2012 ; yu et al . , 2011 ) . functional mri studies have revealed reduced activation in the cerebellum in asd during social , language , and motor tasks . individuals with asd underactivate crus i while processing facial and vocal stimuli ( wang et al . , 2007 ) language tasks elicit abnormal activation in lobule vii in autistic individuals during core aspects of communication such as semantic processing ( harris et al . lastly , children with asd fail to engage the anterior cerebellum ( lobule iv / v ) during motor tasks when compared to their td peers ( mostofsky et al . while convergence across studies exists , there remains significant variation among cerebellar neuroimaging findings in asd . malformations of the cerebellar vermis are associated with social and affective disorders , while cerebellar hemisphere malformations are linked to expressive language , gross motor , and executive functioning deficits , symptoms relevant to asd ( bolduc et al . , 2012 ; tavano et al . , 2007 ) . premature infants sustaining cerebellar damage have a 40-fold increase in positive asd screens relative to controls ( limperopoulos et al . , 2007 ) . further , individuals with tuberous sclerosis ( tsc ) have high rates of asd symptoms ( gillberg et al . , 1994 ; hunt and shepherd , 1993 ; smalley et al . , 1992 ; wing and gould , 1979 ) which have been specifically related to tubers located within the cerebellum ( weber et al . , 2000 the links between cerebellar dysfunction and asd symptomology have led some to posit that autism might be a disease of the cerebellum ( rogers et al . , 2013 ) . however , few studies have examined the role of specific cerebellar subregions in autism , and even fewer have investigated correlations between regional gm and behavioral measures in asd ( kosaka et al . , 2010 ; riva et al . thus far , most studies have examined differences at the hemispheric level , and studies investigating regional differences often have not localized findings to particular cerebellar lobules . given the emerging functional topography of the cerebellum and the various cerebellar regions implicated in autism pathophysiology , it is important to investigate more discrete subdivisions within the cerebellum and to consider their functional relevance . the present study investigates cerebellar structure in asd and links the structural findings to the core symptoms of the disorder . to our knowledge , this is the first study to examine the cerebellum in autism at both a voxel - based and lobular level by using two complementary approaches voxel - based morphometry ( vbm ) ( ashburner and friston , 2000 ; good et al . , 2001 ) and the spatially unbiased infratentorial template ( suit ) ( diedrichsen et al . , 2009 ; diedrichsen , 2006 ) for lobular region of interest ( roi ) volumetric analysis . moreover , this is the first study to correlate cerebellar lobular volumes with behavioral measures . unlike roi approaches , vbm allows for a precise , voxel - level examination of the cerebellum in the context of the whole brain in an unbiased , operator - independent manner . complementing this approach , the suit template and atlas allow for a roi - based examination of cerebellar substructure by providing a high - resolution atlas and template of the human cerebellum and brainstem . the more commonly - used mni template provides little contrast for the cerebellum and cerebellar structures , while the suit template preserves the anatomical detail of the cerebellum and allows for better localization of cerebellar findings . as lobules are anatomically ( rather than functionally ) defined , vbm can elucidate with millimeter resolution how structure is related to asd symptomology without the confines of lobular boundaries , and might reveal gm differences that span or cross lobules . on the other hand , the suit roi method provides an excellent template for measuring specific cerebellar lobules and may be more statistically powerful than the vbm approach ; however , the more gross lobular measures might hide subtle gm differences between groups . because there can be differences in results when voxel - based vs. roi approaches are employed , using both of these techniques in the same dataset helps to establish structural differences that converge across analysis methods . therefore , combining vbm and suit methods in the same dataset allows for an examination of the cerebellum at the voxel - level and lobular - level , capitalizing on the strengths of each approach . for both approaches seventy children aged 813 years participated in this study : 35 children with asd ( 30 males ; mean age = 10.4 1.6 years ; 32 right - handed , 3 left - handed ) and 35 td children ( 21 males ; mean age = 10.4 1.5 years ; 32 right - handed , 2 left - handed , 1 mixed dominance ) . when there were multiple exact or closest - aged td participants , participants were matched by sex . participants were recruited as part of an on - going study conducted by the center for neurodevelopment and imaging research ( cnir ) at the kennedy krieger institute . sources of recruitment included advertisements posted in the community , local pediatricians ' and psychologists ' offices , local schools , social service organizations , chapters of the autism society of america , the interactive autism network ( ian ) database , outpatient clinics at kennedy krieger institute , and word of mouth . written consent was obtained from a parent / guardian and assent was obtained from each child . none of the children had intellectual disability , seizure or other neurological disorder , any severe chronic medical disorder , diagnosed genetic disorder , or psychotic disorder . in the td group , additional exclusions included any psychiatric disorder ( except specific or social phobia ) , speech and language disorder , broader autism phenotype effects ( piven and palmer , 1997 ) , and a family history of first - degree relatives with asd . intellectual ability was assessed using the wechsler intelligence scale for children 4th edition ( wisc - iv ; wechsler , 2003 ) . all td subjects and 33/35 asd subjects had full scale iqs ( fsiqs ) above 79 . two asd subjects with fsiqs below 79 were also included based on verbal comprehension index ( vci ) scores of 85 or greater . this is in line with recommendations to individualize measures best suited for estimation of cognitive abilities in children with asd ( mottron , 2004 ) . participants with a diagnosis of asd were assessed by a master 's level or higher psychologist who was reliable according to research criteria . the autism diagnostic interview revised ( adi - r ; lord et al . , 1994 ) and the autism diagnostic observation schedule generic ( ados - g ; lord et al . , 2000 ) all participants met dsm - iv criteria for asd based on the ados - g or adi - r and the clinical impression of the investigators . for each participant , a high - resolution t1-weighted mp - rage was acquired on a philips 3 t achieva mri scanner ( best , netherlands ) using an 8-channel head coil ( tr = 7.99 ms , te = 3.76 ms , flip angle = 8 , voxel size = 1 mm isotropic ) . scans containing significant motion artifacts or poor gray / white matter differentiation were excluded from a larger sample to produce the current dataset . voxel based morphometry ( vbm ) was used to identify differences in regional gm volume between the autism and td groups using spm8 implemented in matlab 2012b . t1 anatomical images were pre - processed using an optimized vbm procedure ( ashburner and friston , 2000 ; good et al . , 2001 ; 2005 ) including : examination of each image for gross anatomical abnormalities and scanner artifacts ; setting the origin to the anterior commissure ; segmentation into gray matter ( gm ) , white matter ( wm ) , and cerebrospinal fluid ( csf ) using new segment in spm8 ; creation of a study - specific template by importing parameter files produced during segmentation into dartel ; affine transformation of segmented tissues into mni space ; and standard smoothing with an 8 mm fwhm gaussian kernel . the smoothed , modulated , normalized data were used in the statistical analyses . the modulation step was added so that the final vbm statistics reflect volume differences rather than concentration differences in gm ( good et al . , 2001 ; lobular volumes were calculated using the suit toolbox ( diedrichsen et al . , 2009 ; diedrichsen , 2006 ) implemented in spm8 . the procedure involved : cropping and isolating the cerebellum from the t1 anatomical images ; normalizing each cropped image into suit space ; reslicing the probabilistic cerebellar atlas into individual subject space using the deformation parameters from normalization ; and calculating the number of voxels in each lobule in the resliced images . this process therefore resulted in 28 volumetric gm measurements , reflecting the ten bilateral lobules ( i x right and i x left ; lobules i iv are combined into one measure , and lobule vii is divided into viib , crus i and crus ii ; lobule viii is divided into viiia and viiib ) and vermis lobules vi x . total intracranial volume ( tiv ) was calculated by summing the total gm , wm , and csf volumes . two - tailed t - tests were performed to identify any group differences in total gm , total wm , total csf , and tiv . regional differences in gm between groups were assessed using the general linear model ( glm ) in spm8 . smoothed , modulated , normalized gm images were entered into a voxel - wise two - sample t - test analysis . results were thresholded at p < 0.001 ( uncorrected ) with an extent threshold of 50 voxels to control for type i error . an absolute threshold mask of 0.2 was used to avoid edge effects at the borders of gm and wm . total intracranial volume ( tiv = gm + wm + csf ) was entered as a covariate . gender was also entered as a covariate in the statistical model due to significantly different gender distributions in the asd and td groups ( = 4.62 , p = 0.032 ) . lobular volumes were corrected for total cerebellar volume ( individual lobular volumes / total cerebellar volume ) and two - tailed t - tests were performed to identify group differences . multiple regression analyses were conducted in spm8 to identify correlations between the adi and ados scores and regional gm volume . results were thresholded at p < 0.005 ( uncorrected ) with an extent threshold of 50 voxels . multiple regression analyses were conducted between gm and ados scores for 32 subjects and between gm and adi scores for 33 subjects due to missing data . for the lobular volumes , spearman rank - order correlations between lobular volumes and ados and adi behavioral scores were calculated in spss ( ibm spss statistics ) . no significant effects of diagnosis were observed for tiv ( p = 0.077 , asd > td ) , total gm ( p = 0.114 ) , or total wm ( p = 0.090 , asd > td ) . a significant effect of diagnosis was observed for total csf volume ( p = 0.037 , asd > td ) . because tiv and total wm approached significance , tiv was entered as a covariate for subsequent analyses . children with asd showed significantly reduced gm in right crus i / ii ( fig . right crus i / ii was the largest cluster in the brain in which asd < td ( mni 24 70 39 , t = 3.91 , k = 170 ) , with the second - highest t - value . there were no cerebellar regions where children with asd showed greater gm relative to td children . similar to the vbm data , the lobular analysis revealed that right crus i was significantly smaller in children with asd ( 11.16% of cerebellar volume ) compared to td children ( 11.70% of cerebellar volume , p = 0.033 ) . vermis viiia and vermis viiib were significantly larger in children with asd ( vermis viiia , 0.90% of cerebellar volume ; vermis viiib , 0.43% ) when compared to td children ( vermis viiia , 0.80% of cerebellar volume ; vermis viiib , 0.40% ; p = 0.005 and p = 0.014 , respectively ) . multiple regression analyses revealed significant negative correlations between regional cerebellar gm volume and ados and adi scores , such that smaller gm volumes were associated with higher ( more impaired ) scores ( table 1 ) . higher severity ados social + communication scores correlated with reduced gm in right lobule vi / crus i and right lobule viiia / viiib ( cyan , fig . poorer ados social interaction scores correlated with reduced gm in several clusters in vermis i iv ; vermis v vi ; right lobule i iv ; right lobule vi / crus i / crus ii ( largest and most significant cluster in the brain ) ; right lobule viiia / viiib ; left lobule viiia / viiib ; and left lobule viiib / ix ( purple , fig . 3 ) . higher severity ados stereotyped behaviors and restricted interests scores correlated with reduced gm in right crus i / ii ( green , fig . 3 ) , and more severe adi restricted , repetitive , and stereotyped behaviors scores correlated with reduced gm in right lobules i v ( yellow , fig . 3 ) . higher severity adi , social interaction scores correlated with reduced gm in right lobules i iv ( blue , fig . 4 shows these findings overlaid on the 7-network functional connectivity maps established by buckner et al . correlations with core asd symptoms converged on right crus i / ii , where there was significantly reduced gm in the asd group in both the vbm and lobular analyses ( fig . consistent with the vbm results , smaller vermis vi correlated with higher severity ados social scores ( table 2 ) . smaller left and right lobules viiib correlated with more severe ados stereotyped behaviors and restricted interest scores . lastly , larger vermis viib and viiia correlated with higher severity adi restricted , repetitive and stereotyped behavior scores . we investigated the localization of cerebellar structural differences in asd and the relationship between cerebellar gray matter , lobular volumes , and core autistic symptoms . to our knowledge , this is the first study to examine structural differences in the cerebellum in children with asd using two independent methods : whole - brain vbm and lobular volume analysis using the suit atlas . further , in each analysis we examined the correlations between behavioral scores and structural measures . across analysis methods , right crus i / ii was consistently reduced in asd participants when compared to td participants . in the vbm analysis , reductions in the right crus i / ii cluster predicted the severity of core asd symptoms . in several cerebellar regions , more abnormal gm findings were associated with more severe asd symptoms , suggesting that these structural differences are functionally significant . these findings support the involvement of the cerebellum in asd , and further identify specific cerebellar subregions where structural differences are found . the most consistent finding was in right crus i / ii , where both whole - brain vbm and lobular analyses revealed smaller gm volumes in asd relative to the td group . in particular , the vbm results showed that reduced gm in right crus i predicted higher severity on all ados subscales , suggesting that this region might be a valid potential cerebellar biomarker in asd . these findings are also consistent with previous studies : meta - analyses of gm volume differences in asd have reported decreased gm in right crus i ( duerden et al . , 2012 ; stoodley , 2014 ; yu et al . , 2011 ) , and reduced gm in right crus i / ii was found to correlate with increased repetitive behaviors , poorer communication scores , and poorer social interaction scores in both children and adults with asd ( riva et al . , 2013 ; rojas et al . , 2006 crus i and crus ii are anatomically and functionally connected to prefrontal and parietal regions of the cerebral cortex ( buckner et al . , 2011 ; hoover , and strick , 1999 ; kelly and strick , 2003 ; strick et al . , 2009 ) , and resting - state functional connectivity data suggest that crus i is functionally connected to ventral attention , frontoparietal , and default mode networks ( buckner et al . , 2011 ) . reduced connectivity within these networks , particularly the default mode and frontoparietal networks , has been related to impaired social and communicative abilities in asd ( assaf et al . , 2010 ; redcay et al . , task - based fmri studies reveal right crus i activation during executive function paradigms , language tasks and subtasks supporting language , and emotional processing ( mathiak et al . , 2002 , 2004 ; sen et al . , 2011 ; stoodley , 2012 ; stoodley and schmahmann , 2009 ) and a recent meta - analysis found that this region is involved in abstract social cognition ( van overwalle et al . , 2014 ) . in asd , abnormal activation in right crus i / ii is found during language and executive function measures , and during processing of facial and vocal stimuli ( harris et al . , 2006 ; solomon et al . , 2009 ; wang et al . , 2007 ) . ( 2014 ) found a prominent decrease of connectivity in asd between right crus i and supratentorial language regions , including the supplementary motor area ( sma ) , inferior frontal gyrus , and dorsolateral prefrontal cortex . these data , together with the results of our correlation analyses , suggest that structural differences in right crus i / ii could impact the core social and communication deficits in asd . healthy individuals engage lobule vi and crus i during motor imitation and when copying emotional facial expressions ( dapretto et al . the task - specific increase in right crus ii activation during imitation , together with the significant psychophysiological interactions between right crus i and the superior temporal sulcus ( sts ) , implies connections between right crus i and cortical regions involved in biological motion processing ( jack et al . , 2011 ) . significant psychophysiological interactions were also found between left crus ii and the superior parietal lobe ( spl ) , which is implicated in attentional processes underlying imitation ( jack et al . , 2011 ) . others report enhanced synchronization between right lobule vi / crus i and left superior temporal cortex prior to movement , suggesting that the cerebellum is involved in early motor planning during observation as well as imitation execution ( kessler et al . , behaviorally , individuals with autism are impaired in imitation and praxis ( edwards , 2014 ; mostofsky et al . , 2006 , 2009 ; mller et al . , 2003 ) , and gm differences in crus i and ii could result in underconnectivity with multiple cortical areas involved in imitation and praxis . the resulting deficits might underlie some of the social , communication , and motor impairments in autism ( mostofsky et al . poorer adi social interaction scores were related to decreased gm in anterior cerebellar lobules i v , and poorer ados social interaction scores correlated with decreased gm in vermis i v and vermis vi vii . these findings are consistent with previous work showing decreased volume in vermis i v in language - impaired individuals with autism ( hodge et al . , 2010 ) . this region of the cerebellum is typically associated with motor function , although recent functional connectivity mapping of the anterior cerebellum suggests connections with both motor and limbic regions of the cerebral cortex ( buckner et al . , 2011 ) . supporting this , in our study the adi repetitive behavior cluster was located within the proposed somatomotor region ( fig . 4e ) , and adi social scores correlated with a cluster falling within the limbic network ( fig . 4d ) . motor symptoms are common in individuals with asd ( gidley larson and mostofsky , 2008 ) , and are among some of the earliest identifiable signs distinguishing children with autism from their td peers ( landa and garrett - mayer , 2006 ) . consistent with our structural findings , functional imaging studies show that individuals with autism fail to recruit cerebellar lobules iv / v during a motor task ( mostofsky et al . , importantly , motor impairment in autism often co - occurs with verbal impairment ( noterdaeme et al . , 2010 ) and can be predictive of social , communication , and repetitive behavior impairments ( leonard et al . , 2014 ; linkenauger et al . , 2012 ; travers et al . , 2013 ) . altogether , these data support supports our finding that the greater the gm reduction in the anterior cerebellum , the more impaired the adi and ados social interaction scores . ados social interaction scores also correlated with a cluster in the posterior vermis . decreased volume of vermis vi vii is inversely related to the volume of the frontal cortex in asd ( carper and courchesne , 2000 ) , suggesting that abnormalities in this posterior midline cerebellar region might have knock - on effects on frontally - mediated cognitive skills involved in social interaction . lobular volumes of vermis viiia and vermis viiib were increased in children with asd , and increased lobular volumes in vermis viib and viiia were associated with increased repetitive behaviors . while hypoplasia of the posterior vermis has been reported in asd ( courchesne et al . , 1988 ; levitt et al . , 1999 ) , a small subset of studies have noted hyperplasia of the posterior vermis in autism . ( 2007 ) found an increase in gm in posterior vermis viii in individuals with asd , and hyperplasia of the posterior vermis has also been noted in fragile x individuals with autism ( kaufmann et al . , 2003 ) . it has been suggested that hyperplasia in vermis vi and vii may only be evident in a subset ( ~11% ) of individuals with asd ( akshoomoff et al . , 2004 ; courchesne et al . , poor registration , different normalization techniques , and the relatively small size of the vermal lobules may account for differences in localization to specific posterior vermal lobules between studies . in addition , factors such as iq have been shown to mediate vermis size in asd ( courchesne et al . , 1994 ) . although courchesne et al . ( 1994 ) observed that all instances of vermal hyperplasia occurred in individuals with iqs less than 70 , little is known about the conditions under which hyperplasia occurs . vii have been shown to correlate with increased repetitive behaviors and decreased exploratory actions ( pierce and courchesne , 2001 ) , consistent with our findings . in children with asd , we found that smaller lobular volumes in bilateral viiib were associated with higher ratings of repetitive behaviors and restricted interests , consistent with the proposed role of cerebellar lobule viii in sensorimotor function . this finding is not unprecedented , as a previous study reported that decreased gm in lobule viii was associated with impaired social interaction and communication scores ( rojas et al . , 2006 ) . in healthy individuals , lobule viii is activated during a variety of cognitive tasks , including verb generation and working memory paradigms ( stoodley and schmahmann , 2009 ) . specifically , it has been proposed that lobule viii is involved in cerebro - cerebellar loops important in sustaining the phonological store ( chen and desmond , 2005 ) , suggesting that reduced gm in lobule viii in asd might impair cerebro - cerebellar loops important in working memory . working memory impairments are thought to exacerbate social and communication deficits in children with asd ( d'urso et al . , 2014 ) , and our finding that reduced gm in lobule viiia was associated with impairments in social interaction and social + communication scores support this hypothesis . gm reductions in lobule ix also were associated with increased social / communicative impairments . while the function of lobule ix is not well defined , buckner et al . ( 2011 ) suggest it may form a tertiary representation of cerebral cortical networks based on its functional connectivity patterns ( kelly and strick , 2003 ) . lobule ix is functionally connected to areas such as the posterior cingulate , thalamus , and angular gyrus , and is part of the default mode network ( bernard et al . , 2012 ; lobule ix is highly functionally connected to crus i , viiia , and the anterior cerebellum ( bernard et al . , 2012 ) , , 2012 ; stoodley , 2014 ; yu et al . , 2011 ) . in healthy individuals , lobule ix is activated in response to conflict experienced when breaking with social norms ( klucharev et al . , 2009 ) . therefore , decreased gm in lobule ix might inhibit proper default mode network connectivity , leading to dysfunction in brain areas involved in social and communication processes . the combined evidence from the voxel - based and lobular analyses provides further support that autism is associated with structural differences in the cerebellum , and that these differences have functional relevance . it is thought that , due to its stereotyped neuronal architecture , the cerebellum performs the same operation on any input it receives ( see ito , 2006 ) . the computation performed by the cerebellum ( which schmahmann calls the universal cerebellar transform ; schmahmann , 1991 ) can be applied to many types of information involved in motor , cognitive and affective processing ( ito , 2006 ; schmahmann , 1991 ) . cerebellar output , therefore , is determined by the input received via its anatomical connections with different regions of the brain . damage or developmental abnormalities affecting the cerebellum could impede the basic processing of the cerebellum , with knock - on effects on cerebellar modulation of cerebro - cerebellar loops in a way that is relevant to asd symptomology . for example , preterm infants with injury to the cerebellum had impaired growth of the contralateral cerebral cortex ( limperopoulos et al . , 2010 ) , as well as significantly impaired expressive language , delayed receptive language , cognitive deficits , and motor disabilities ( limperopoulos et al . , 2007 ) . in the current study , the degree of gm reduction in discrete regions of the cerebellum was correlated with the severity of social , communication , and repetitive behavior impairments on autism diagnostic scales . this suggests that the processing provided by the cerebellum is relevant to a range of asd symptoms , and not only motor behaviors . further , the findings indicate that the degree of cerebellar abnormality predicts the severity of asd symptoms . reduced regional cerebellar gm might impair specific cerebro - cerebellar loops , linking the cerebellum to the multiple , distributed neural circuits underlying the disorder . our data support this concept , as cerebellar regions associated with poorer ados and adi scores included limbic , fronto - parietal , somatomotor and default mode networks . it has been suggested that the cerebellum may be important in setting up distant cortical networks in the brain during development ( see wang et al . , 2014 ) , indicating that structural deficits in the cerebellum might have long - term effects on cortical specialization . disruptions in specific cerebro - cerebellar loops might impair proper specialization of cortical regions involved in language , social interaction , and motor control in asd , leading to long - term impairments in these domains . therefore , the gm differences in the cerebellum could potentially impact the cerebral cortical networks that support both motor and non - motor language and social cognitive functions ( van overwalle et al . , 2014 ) . the results from the vbm and suit methods were not always consistent , which may be due to the differences in the resolution of these two approaches . for example , suit analyses revealed increased volumes in vermis viiia and viiib , while no increases were found in these regions in the vbm analysis . increased vermis viiia and viiib volumes have been reported in a previous vbm study ( salmond et al . , 2007 ) , but this finding is not consistent across studies ( e.g. , riva et al . , 2013 ) . it might be the case that in the current analysis , increased vermis volume was only apparent at the lobular level because the gm differences did not meet our cluster threshold in the vbm analysis . in fact , when using a more lenient voxel - level threshold without a cluster correction , a small cluster ( k = 36 ; p = 0.026 ) of increased gm was evident in vermal viiia / viiib . therefore , it is possible that inconsistencies between the two approaches reflect the differences in statistical power in voxel - based vs. roi analyses . of particular interest for future research is the specific contribution of the right crus i / ii region where we found that decreased gm correlated with impaired social interaction , impaired communication , and increased repetitive behaviors the hallmarks of an asd diagnosis . future research will aim to elucidate the specific contribution of this area to asd symptomology . the following is the supplementary data related to this article.table s1whole - brain results for vbm group comparison between asd and td .

neuroanatomical differences in the cerebellum are among the most consistent findings in autism spectrum disorder ( asd ) , but little is known about the relationship between cerebellar dysfunction and core asd symptoms . the newly - emerging existence of cerebellar sensorimotor and cognitive subregions provides a new framework for interpreting the functional significance of cerebellar findings in asd . here we use two complementary analyses whole - brain voxel - based morphometry ( vbm ) and the suit cerebellar atlas to investigate cerebellar regional gray matter ( gm ) and volumetric lobular measurements in 35 children with asd and 35 typically - developing ( td ) children ( mean age 10.4 1.6 years ; range 813 years ) . to examine the relationships between cerebellar structure and core asd symptoms , correlations were calculated between scores on the autism diagnostic observation schedule ( ados ) and autism diagnostic interview ( adi ) and the vbm and volumetric data . both vbm and the suit analyses revealed reduced gm in asd children in cerebellar lobule vii ( crus i / ii ) . the degree of regional and lobular gray matter reductions in different cerebellar subregions correlated with the severity of symptoms in social interaction , communication , and repetitive behaviors . structural differences and behavioral correlations converged on right cerebellar crus i / ii , a region which shows structural and functional connectivity with fronto - parietal and default mode networks . these results emphasize the importance of the location within the cerebellum to the potential functional impact of structural differences in asd , and suggest that gm differences in cerebellar right crus i / ii are associated with the core asd profile .

Introduction Materials and methods Results Discussion

autism spectrum disorders ( asds ) are characterized by impairments in social interaction , communication , and restricted or repetitive behaviors and interests ( dsm - iv : american psychiatric association , 1994 ) . neuroimaging research has revealed a broad network of regional brain abnormalities in asd , including frontal , parietal , and limbic regions , the basal ganglia , and the cerebellum ( amaral et al . cerebellar structural and functional differences are consistently reported in asd , suggesting that cerebellar dysfunction may be important in the etiology of the disorder ( allen et al . , 1998 ; although neuroimaging meta - analyses suggest that several different regions of the cerebellum are affected in asd ( duerden et al . , 2011 ) , no study has used both voxel - based and lobular region of interest analyses to examine structural differences within the cerebella of autistic individuals , while also assessing the relationship between these cerebellar subregions and core asd symptoms . anatomically , the cerebellum is divided into ten lobules ( lobules i x ) and three lobes : the anterior lobe ( lobules i v ) , the posterior lobe ( lobules vi ix ) , and the flocculonodular lobe ( lobule x ; fig . the large posterior lobe , including lobules vi and vii ( which is subdivided into crus i , crus ii and viib ) , receives input from prefrontal and parietal association areas and is engaged during cognitive tasks ( strick et al . , 2009 recent functional connectivity data show that the majority of the cerebellum is functionally connected to association networks involved in cognitive and affective processes , rather than somatomotor networks ( buckner et al . this topography is of importance when considering the localization of cerebellar structural and functional differences in asd , and may be beneficial in the interpretation of cerebellar findings in asd . neuroimaging studies comparing asd with typically developing ( td ) structural mri studies have described hypoplasia of the posterior vermis in asd ( carper and courchesne , 2000 ; courchesne et al . , 1989 ) , and meta - analyses of voxel - based morphometry ( vbm ) studies have reported consistent gray matter ( gm ) decreases in right crus i , lobule viii , and lobule ix ( duerden et al . functional mri studies have revealed reduced activation in the cerebellum in asd during social , language , and motor tasks . malformations of the cerebellar vermis are associated with social and affective disorders , while cerebellar hemisphere malformations are linked to expressive language , gross motor , and executive functioning deficits , symptoms relevant to asd ( bolduc et al . , 2000 the links between cerebellar dysfunction and asd symptomology have led some to posit that autism might be a disease of the cerebellum ( rogers et al . however , few studies have examined the role of specific cerebellar subregions in autism , and even fewer have investigated correlations between regional gm and behavioral measures in asd ( kosaka et al . given the emerging functional topography of the cerebellum and the various cerebellar regions implicated in autism pathophysiology , it is important to investigate more discrete subdivisions within the cerebellum and to consider their functional relevance . the present study investigates cerebellar structure in asd and links the structural findings to the core symptoms of the disorder . to our knowledge , this is the first study to examine the cerebellum in autism at both a voxel - based and lobular level by using two complementary approaches voxel - based morphometry ( vbm ) ( ashburner and friston , 2000 ; good et al . unlike roi approaches , vbm allows for a precise , voxel - level examination of the cerebellum in the context of the whole brain in an unbiased , operator - independent manner . complementing this approach , the suit template and atlas allow for a roi - based examination of cerebellar substructure by providing a high - resolution atlas and template of the human cerebellum and brainstem . the more commonly - used mni template provides little contrast for the cerebellum and cerebellar structures , while the suit template preserves the anatomical detail of the cerebellum and allows for better localization of cerebellar findings . on the other hand , the suit roi method provides an excellent template for measuring specific cerebellar lobules and may be more statistically powerful than the vbm approach ; however , the more gross lobular measures might hide subtle gm differences between groups . because there can be differences in results when voxel - based vs. roi approaches are employed , using both of these techniques in the same dataset helps to establish structural differences that converge across analysis methods . therefore , combining vbm and suit methods in the same dataset allows for an examination of the cerebellum at the voxel - level and lobular - level , capitalizing on the strengths of each approach . for both approaches seventy children aged 813 years participated in this study : 35 children with asd ( 30 males ; mean age = 10.4 1.6 years ; 32 right - handed , 3 left - handed ) and 35 td children ( 21 males ; mean age = 10.4 1.5 years ; 32 right - handed , 2 left - handed , 1 mixed dominance ) . sources of recruitment included advertisements posted in the community , local pediatricians ' and psychologists ' offices , local schools , social service organizations , chapters of the autism society of america , the interactive autism network ( ian ) database , outpatient clinics at kennedy krieger institute , and word of mouth . in the td group , additional exclusions included any psychiatric disorder ( except specific or social phobia ) , speech and language disorder , broader autism phenotype effects ( piven and palmer , 1997 ) , and a family history of first - degree relatives with asd . the autism diagnostic interview revised ( adi - r ; lord et al . , 1994 ) and the autism diagnostic observation schedule generic ( ados - g ; lord et al . , 2000 ) all participants met dsm - iv criteria for asd based on the ados - g or adi - r and the clinical impression of the investigators . voxel based morphometry ( vbm ) was used to identify differences in regional gm volume between the autism and td groups using spm8 implemented in matlab 2012b . , 2001 ; 2005 ) including : examination of each image for gross anatomical abnormalities and scanner artifacts ; setting the origin to the anterior commissure ; segmentation into gray matter ( gm ) , white matter ( wm ) , and cerebrospinal fluid ( csf ) using new segment in spm8 ; creation of a study - specific template by importing parameter files produced during segmentation into dartel ; affine transformation of segmented tissues into mni space ; and standard smoothing with an 8 mm fwhm gaussian kernel . the procedure involved : cropping and isolating the cerebellum from the t1 anatomical images ; normalizing each cropped image into suit space ; reslicing the probabilistic cerebellar atlas into individual subject space using the deformation parameters from normalization ; and calculating the number of voxels in each lobule in the resliced images . this process therefore resulted in 28 volumetric gm measurements , reflecting the ten bilateral lobules ( i x right and i x left ; lobules i iv are combined into one measure , and lobule vii is divided into viib , crus i and crus ii ; lobule viii is divided into viiia and viiib ) and vermis lobules vi x . children with asd showed significantly reduced gm in right crus i / ii ( fig . right crus i / ii was the largest cluster in the brain in which asd < td ( mni 24 70 39 , t = 3.91 , k = 170 ) , with the second - highest t - value . similar to the vbm data , the lobular analysis revealed that right crus i was significantly smaller in children with asd ( 11.16% of cerebellar volume ) compared to td children ( 11.70% of cerebellar volume , p = 0.033 ) . vermis viiia and vermis viiib were significantly larger in children with asd ( vermis viiia , 0.90% of cerebellar volume ; vermis viiib , 0.43% ) when compared to td children ( vermis viiia , 0.80% of cerebellar volume ; vermis viiib , 0.40% ; p = 0.005 and p = 0.014 , respectively ) . higher severity ados social + communication scores correlated with reduced gm in right lobule vi / crus i and right lobule viiia / viiib ( cyan , fig . poorer ados social interaction scores correlated with reduced gm in several clusters in vermis i iv ; vermis v vi ; right lobule i iv ; right lobule vi / crus i / crus ii ( largest and most significant cluster in the brain ) ; right lobule viiia / viiib ; left lobule viiia / viiib ; and left lobule viiib / ix ( purple , fig . higher severity ados stereotyped behaviors and restricted interests scores correlated with reduced gm in right crus i / ii ( green , fig . 3 ) , and more severe adi restricted , repetitive , and stereotyped behaviors scores correlated with reduced gm in right lobules i v ( yellow , fig . higher severity adi , social interaction scores correlated with reduced gm in right lobules i iv ( blue , fig . correlations with core asd symptoms converged on right crus i / ii , where there was significantly reduced gm in the asd group in both the vbm and lobular analyses ( fig . consistent with the vbm results , smaller vermis vi correlated with higher severity ados social scores ( table 2 ) . we investigated the localization of cerebellar structural differences in asd and the relationship between cerebellar gray matter , lobular volumes , and core autistic symptoms . to our knowledge , this is the first study to examine structural differences in the cerebellum in children with asd using two independent methods : whole - brain vbm and lobular volume analysis using the suit atlas . across analysis methods , right crus i / ii was consistently reduced in asd participants when compared to td participants . in the vbm analysis , reductions in the right crus i / ii cluster predicted the severity of core asd symptoms . in several cerebellar regions , more abnormal gm findings were associated with more severe asd symptoms , suggesting that these structural differences are functionally significant . these findings support the involvement of the cerebellum in asd , and further identify specific cerebellar subregions where structural differences are found . the most consistent finding was in right crus i / ii , where both whole - brain vbm and lobular analyses revealed smaller gm volumes in asd relative to the td group . in particular , the vbm results showed that reduced gm in right crus i predicted higher severity on all ados subscales , suggesting that this region might be a valid potential cerebellar biomarker in asd . these findings are also consistent with previous studies : meta - analyses of gm volume differences in asd have reported decreased gm in right crus i ( duerden et al . , 2011 ) , and reduced gm in right crus i / ii was found to correlate with increased repetitive behaviors , poorer communication scores , and poorer social interaction scores in both children and adults with asd ( riva et al . , 2006 crus i and crus ii are anatomically and functionally connected to prefrontal and parietal regions of the cerebral cortex ( buckner et al . , 2009 ) , and resting - state functional connectivity data suggest that crus i is functionally connected to ventral attention , frontoparietal , and default mode networks ( buckner et al . , task - based fmri studies reveal right crus i activation during executive function paradigms , language tasks and subtasks supporting language , and emotional processing ( mathiak et al . in asd , abnormal activation in right crus i / ii is found during language and executive function measures , and during processing of facial and vocal stimuli ( harris et al . ( 2014 ) found a prominent decrease of connectivity in asd between right crus i and supratentorial language regions , including the supplementary motor area ( sma ) , inferior frontal gyrus , and dorsolateral prefrontal cortex . these data , together with the results of our correlation analyses , suggest that structural differences in right crus i / ii could impact the core social and communication deficits in asd . the task - specific increase in right crus ii activation during imitation , together with the significant psychophysiological interactions between right crus i and the superior temporal sulcus ( sts ) , implies connections between right crus i and cortical regions involved in biological motion processing ( jack et al . , 2003 ) , and gm differences in crus i and ii could result in underconnectivity with multiple cortical areas involved in imitation and praxis . the resulting deficits might underlie some of the social , communication , and motor impairments in autism ( mostofsky et al . poorer adi social interaction scores were related to decreased gm in anterior cerebellar lobules i v , and poorer ados social interaction scores correlated with decreased gm in vermis i v and vermis vi vii . this region of the cerebellum is typically associated with motor function , although recent functional connectivity mapping of the anterior cerebellum suggests connections with both motor and limbic regions of the cerebral cortex ( buckner et al . 4e ) , and adi social scores correlated with a cluster falling within the limbic network ( fig . motor symptoms are common in individuals with asd ( gidley larson and mostofsky , 2008 ) , and are among some of the earliest identifiable signs distinguishing children with autism from their td peers ( landa and garrett - mayer , 2006 ) . , 2010 ) and can be predictive of social , communication , and repetitive behavior impairments ( leonard et al . decreased volume of vermis vi vii is inversely related to the volume of the frontal cortex in asd ( carper and courchesne , 2000 ) , suggesting that abnormalities in this posterior midline cerebellar region might have knock - on effects on frontally - mediated cognitive skills involved in social interaction . lobular volumes of vermis viiia and vermis viiib were increased in children with asd , and increased lobular volumes in vermis viib and viiia were associated with increased repetitive behaviors . , 1999 ) , a small subset of studies have noted hyperplasia of the posterior vermis in autism . ( 2007 ) found an increase in gm in posterior vermis viii in individuals with asd , and hyperplasia of the posterior vermis has also been noted in fragile x individuals with autism ( kaufmann et al . , poor registration , different normalization techniques , and the relatively small size of the vermal lobules may account for differences in localization to specific posterior vermal lobules between studies . ( 1994 ) observed that all instances of vermal hyperplasia occurred in individuals with iqs less than 70 , little is known about the conditions under which hyperplasia occurs . in children with asd , we found that smaller lobular volumes in bilateral viiib were associated with higher ratings of repetitive behaviors and restricted interests , consistent with the proposed role of cerebellar lobule viii in sensorimotor function . this finding is not unprecedented , as a previous study reported that decreased gm in lobule viii was associated with impaired social interaction and communication scores ( rojas et al . specifically , it has been proposed that lobule viii is involved in cerebro - cerebellar loops important in sustaining the phonological store ( chen and desmond , 2005 ) , suggesting that reduced gm in lobule viii in asd might impair cerebro - cerebellar loops important in working memory . , 2014 ) , and our finding that reduced gm in lobule viiia was associated with impairments in social interaction and social + communication scores support this hypothesis . lobule ix is functionally connected to areas such as the posterior cingulate , thalamus , and angular gyrus , and is part of the default mode network ( bernard et al . , 2012 ; lobule ix is highly functionally connected to crus i , viiia , and the anterior cerebellum ( bernard et al . therefore , decreased gm in lobule ix might inhibit proper default mode network connectivity , leading to dysfunction in brain areas involved in social and communication processes . the combined evidence from the voxel - based and lobular analyses provides further support that autism is associated with structural differences in the cerebellum , and that these differences have functional relevance . for example , preterm infants with injury to the cerebellum had impaired growth of the contralateral cerebral cortex ( limperopoulos et al . in the current study , the degree of gm reduction in discrete regions of the cerebellum was correlated with the severity of social , communication , and repetitive behavior impairments on autism diagnostic scales . this suggests that the processing provided by the cerebellum is relevant to a range of asd symptoms , and not only motor behaviors . further , the findings indicate that the degree of cerebellar abnormality predicts the severity of asd symptoms . reduced regional cerebellar gm might impair specific cerebro - cerebellar loops , linking the cerebellum to the multiple , distributed neural circuits underlying the disorder . our data support this concept , as cerebellar regions associated with poorer ados and adi scores included limbic , fronto - parietal , somatomotor and default mode networks . , 2014 ) , indicating that structural deficits in the cerebellum might have long - term effects on cortical specialization . disruptions in specific cerebro - cerebellar loops might impair proper specialization of cortical regions involved in language , social interaction , and motor control in asd , leading to long - term impairments in these domains . therefore , the gm differences in the cerebellum could potentially impact the cerebral cortical networks that support both motor and non - motor language and social cognitive functions ( van overwalle et al . the results from the vbm and suit methods were not always consistent , which may be due to the differences in the resolution of these two approaches . for example , suit analyses revealed increased volumes in vermis viiia and viiib , while no increases were found in these regions in the vbm analysis . it might be the case that in the current analysis , increased vermis volume was only apparent at the lobular level because the gm differences did not meet our cluster threshold in the vbm analysis . therefore , it is possible that inconsistencies between the two approaches reflect the differences in statistical power in voxel - based vs. roi analyses . of particular interest for future research is the specific contribution of the right crus i / ii region where we found that decreased gm correlated with impaired social interaction , impaired communication , and increased repetitive behaviors the hallmarks of an asd diagnosis .

in insect taxa which use acoustic signals for intraspecific communication , the recognition of the acoustic signals is crucial for their fitness , since it enables species identification and prevents hybridization ( gerhardt and huber 2002 ; greenfield 2002 ; bradbury and vehrencamp 2011 ) . however , ears also evolved in insects without acoustic signals , indicating that other functions of hearing may also include the detection of cues from predators , or the detection of hosts in the case of parasitoids . the anatomical and physiological diversity of insect ears we know so far is impressive , with perhaps 19 independent evolutionary origins , as is the diversity of body parts on which they evolved , including legs , wings , mouth parts or the abdomen ( hoy and robert 1996 ; yager 1999 ; yack 2004 ) . the identification of the acoustic signal as species - specific , or the discrimination between different signal variants , is only one part of the task : the signal should also be correctly localized . the negative fitness consequences for a prey escaping into the wrong direction in the face of a predator are obvious , similar to wrong or distorted directionality in the process of finding and approaching a mate . therefore , we should expect that the evolution of these ears and the neuronal network processing the directional cues enabled insects to localize a sound source reasonably well . in the case of particle velocity receivers such as the antennae of mosquitoes , or filiform hairs on the cerci directionality is no problem because such receivers are inherently directional . they respond to the particle velocity ( and thus a vectorial ) component of near - field sound . the problem with the task of directional hearing is obvious , however , with tympanate hearing ; it is basically a biophysical one associated with the small size of most insects . insects equipped with bilateral pairs of tympanate ears could principally make use of binaural cues for sound localization , like all other animals with two ears . however , their small size and hence interaural distance result in only minute interaural time differences ( itds ) . at the same time acoustic theory predicts that significant diffraction for the establishment of reasonable interaural intensity differences ( iids ) occurs only when the ratio of body size to the wavelength of sound exceeds a value of 0.1 ( morse and ingard 1968 ; robert 2005 ) . again , the small body size of insects in relation to the relatively large wavelength of the sound signals used for communication thus often limits or even prevents the establishment of reasonably large iids through diffraction . in my review , i will only shortly cover the biophysical aspects of directional hearing ; more complete descriptions of the biomechanics of sound propagation and the generation of cues for directional hearing in insects can be found in excellent earlier reviews ( michelsen 1992 , 1998 ; robert and hoy 1998 ; robert 2005 for all kind of receivers ; michelsen and larsen 2008 for pressure difference receivers ) . instead , after shortly reviewing the different kind of receivers in insects , i will focus on aspects of directional hearing which received relatively little attention in previous reviews , namely the evolution of a pressure difference receiver , 3d - hearing , directional hearing outdoors , and directional hearing for auditory scene analysis . if sound can act on only one side of the tympanal membrane , ears operate as pure pressure receivers . when insects are large compared to the wavelength of the sound frequencies of relevant signals , the insect s body can generate diffractive effects , resulting in a reduced pressure at the contralateral ear . iids as large as 40 db have been reported for a large noctuid moth with ultrasonic frequencies used by echolocating bats ( payne et al . 1966 ) . compared to the minimum values of about 1 db necessary for reliable directional responses ( see below ) such iids are rather large . in pressure difference receivers , sound can act on both sides of the tympanal membrane , requiring two ( or even more ) acoustic inputs which conduct pressure waves also to the internal side of the tympanum . thus , the force driving the tympanal membrane is the difference between the external and internal pressures . since the internal pressure component travels either through some body tissue , air sacs or in tracheal tubes , various degrees of attenuation or amplification and phase shift can occur relative to the external component . theoretically , evolutionary modifications of anatomical structures ( see below ) resulting in the proper amplitude and phase shifts between internal and external pressure components could create highly directional ears despite unfavorable ratios of body size to the wavelength of sound . since the internal sound conduction can depend on frequency , insect ears can act as pressure difference receivers at low frequencies and as a pressure receiver at higher frequencies . the locust ( grasshopper ) ear is such a case ( michelsen and rohrseitz 1995 ; schul et al . together with the other well - studied pressure difference receiver , the cricket ear ( see below ) , the grasshopper example highlights the importance of proper phase relationships between the external and internal sound component(s ) for producing significant directionality . the ears of the tachinid fly ormia ochracea are an example for mechanically coupled ears . they are so close together that both iids and itds appear to be much too small for a physical basis of directional hearing . for example , the interaural distance of 520 m would create no more than 1.45 s of it d , and significant diffractive effects can not play a role given the unfavorable ratio of body size to wavelength of sound ( robert et al . the flies show very accurate acoustic localization behavior in flight ( see below ) and while walking ( mason et al . 2001 ; mller and robert 2001 ) . the mystery of the directionality of these ears has been solved by the finding that due to the mechanical coupling of the tympana by a flexible cuticular lever the two tympanal membranes move out of phase at frequencies relevant for the fly ( miles et al . the mechanical it d of 5060 s thus created is much larger than the acoustical it d of 1.45 s . the larger interaural differences can then be processed by the nervous system ( for a detailed review on how these mechanical cues are used for the reliable neural coding of sound direction see robert and gpfert 2002 ; robert 2005 ) . a necessary word of caution should emphasize that the actual strategy of finding a host in nature may be more complex , as shown for another parasitoid fly emblemasoma auditrix ( lakes - harlan and khler 2003 ) . in an open area , more than half of the animals flew directly to the loudspeaker ( broadcasting a model song of their cicada host ) , but the presence of single landmarks reduced the percentage of direct flights to the target greatly . future studies will show whether this reflects species differences or the influence of a more or less complex structured environment . insects usually base their phonotactic approaches on a binaural comparison . whatever the biophysical mechanism creating binaural cues for sound localization , what are the minimum iids and itds that result in reliable directional responses in insects ? in dichotic experiments one can apply lateralized stimuli to either hearing organ without stimulating the opposite one in a wide range of intensities , so that the minimum interaural cues involved in localization can be tested . this is a difficult task in insects when body size and hence interaural disparities are minute and/or the two hearing organs are acoustically coupled . a tentative behavioral approach with a grasshopper indicated that the insects auditory system can make use of iids in the order of 12 db ( von helversen and rheinlaender 1988 ) . using a dichotic ear stimulation device for freely moving katydid females ( cross - talk barrier of about 50 db ) , females turned to the stronger stimulated side starting with a 1 db difference between both ears ( rheinlaender et al . the ability to resolve such small iids corroborates quantitative data on the accuracy of phonotactic approaches of the same insect , where females may experience only small stimulus angles of 610 , which create only small iids in the order of 12 db , but still make significant correct turns . the directionality of phonotaxis in female g. bimaculatus walking on an open - loop trackball system indicated even smaller interaural differences for reliable lateralization ( schneich and hedwig 2010 ; see below).fig . 1 a female gampsocleis gratiosa with a dichotic ear stimulation device . each speaker is connected via plastic tubes to the large spiracular opening of the acoustic trachea of the respective ipsilateral side , so that sound can be separately applied through the leg trachea to the inner side of each ear . b results of behavioural dichotic stimulation experiments with four females , in which the interaural intensity difference of a stimulus was varied . note that with an iid of 1 db there is a significant turn to the more strongly stimulated side , and with 23 db difference very few incorrect turns are made ( from rheinlaender et al . each speaker is connected via plastic tubes to the large spiracular opening of the acoustic trachea of the respective ipsilateral side , so that sound can be separately applied through the leg trachea to the inner side of each ear . b results of behavioural dichotic stimulation experiments with four females , in which the interaural intensity difference of a stimulus was varied . note that with an iid of 1 db there is a significant turn to the more strongly stimulated side , and with 23 db difference very few incorrect turns are made ( from rheinlaender et al . 2005 ) but what about the use of itds as a cue for sound localization ? the interaural disparity in insects can be extremely small , hence the available itds amount to only 35 s ( short - horned grasshoppers ) , or only 1.45 s ( parasitoid fly ) . these values of itds are so strongly constrained by body size and ear separation , that it had been accepted for a long time that such small itds can not be used for neuronal processing of sound direction . mrchen ( 1980 ) thus suggested that insects do not use the physical itds , but the physiological time differences in the binaural receptor fibre discharges instead . he examined in locust auditory receptor fibres the effect that the latency of sensory excitation is dependent on stimulus intensity ( imaizumi and pollack 2001 for a similar finding in crickets ) . the high negative correlation between response strength and latency results in a direction - dependent latency shift in the afferent activity . thus , the difference in the time of arrival of action potential activity on both sides of the nervous system may reach values of 56 ms for ipsi - versus contralateral sound , which exceeds the value of the physical it d between the ears by almost 1,000 times . the author therefore suggested that response strength and response latency can be regarded as equivalent directional cues for sound direction . similarly , in the study by schneich and hedwig ( 2010 ) the interaural intensity differences were closely reflected in the response latencies of the auditory afferent activity . the overall bilateral latency difference at an angular deviation of 30 was 1.28 ms whereas the actual interaural difference in it d is less than 15 s . the hypothesis of the dual mode of directional coding was examined using dichotic stimulation of the tympana in the locust ( rheinlaender and mrchen 1979 ) . they demonstrated a time - intensity trading phenomenon similar to the one reported for vertebrates ( erulkar 1972 ) , although the time cue was in the order of milliseconds , rather than microseconds as indicated by the physical itds . shifting the contralateral stimulus only 24 ms ahead of the ipsilateral one could drive the activity of an interneuron from maximal excitation into total inhibition . in the quasi - dichotic stimulus situation mentioned above , the behavioral resolution of chorthippus biguttulus males for itds was also in the order of 1 ms : when both stimuli were presented at equal loudness , but one speaker was leading the other by only 0.5 ms this resulted in significant turns towards the leading side ( von helversen and rheinlaender 1988 ) . all together , these experiments confirm that the dual mode of directional coding provided by auditory receptor fibres is indeed used at the first site of synaptic processing . although microseconds do not matter because the physical time delays between the ears are too small to be of relevance ( but see results for the ormia fly below ) , the onset of binaural arrival of receptor activity at these synapses ( the physiological time delay ) may be quite important for directional coding . whatever the magnitude of these binaural differences : how are behaviorally relevant itds and iids encoded in the discharge differences of afferents and interneurons of the auditory pathway ? the high behavioral precision discussed above for some acoustic insects is surprising when considering the low number of receptors in each ear and the high variability of receptor responses ( ronacher and krahe 2000 ) . moreover , even fewer elements are available at the level of interneurons . in their attempt to determine the neuronal correlates of such small iids for grasshoppers , ronacher and krahe ( 2000 ) noted that an iid of 1.5 db corresponds to a spike count difference of only 1 spike per response ( for a stimulus of 100 ms ) in a bilateral pair of receptors , but that the error probabilities for a decision based on these differences are larger than those observed in behavior . their conclusion was that the insect has to integrate information from up to 13 receptors to arrive at the observed behavioral precision . the prediction has been tested indirectly by stradner and rmer ( 2008 ) for a pair of first - order local interneurons ( omega - neurons ) in katydids , which integrate excitatory inputs from most of the auditory receptors in the ipsilateral ear ( rmer et al . 1988 ) , and inhibitory inputs from the contralateral side mediated by its mirror - image counterpart on the other side ( selverston et al . 1985 ; molina and stumpner 2005 ) . using an independent ear stimulation paradigm stradner and rmer ( 2008 ) demonstrated that starting with an iid of 1 db , the discharge differences in this pair of interneuron were large and significant , with the louder side being more strongly excited . thus small , behaviorally relevant iids are available for these insects from simple spike count differences in pairs of auditory interneurons ( for detailed reviews of neural processing of directional information see hennig et al . ( 2004 ) , and hedwig and pollack ( 2008 ) . the comparison of the two species with hyperacute directional hearing , the field cricket g. bimaculatus and the fly ormia ochracea may illustrate the limits of processing binaural cues for directional hearing known so far . both species have been tested under ideal acoustic conditions under open - loop conditions on a trackball ( mason et al . females of both species not only reliably discriminated the side of acoustic stimulation at angles of sound incidence starting 12 from the animal s longitudinal body axis ( indicative of correct lateralization ) , but also precisely walked towards the sound source . in the cricket study , the tympanic membrane oscillations of the ears revealed between 0 and 30 a linear increasing function of interaural amplitude differences with a slope of 0.4 db/. such small iids were closely reflected in the bilateral latency difference of responses of auditory afferents , which was 1.28 ms compared with the physical it d of less than 15 s ( schneich and hedwig 2010 ) . the linear latency gradient of 42 s/ in the frontal zone of the female cricket appears to be small , but the ormia fly even exploits a gradient of only 3.5 s/. as mentioned above , in the fly s ear the interaural distance of 520 m would create no more than 1.45 s physical it d . the coupling of the tympana has the effect that the two tympanal membranes move out of phase at the cf of the cricket song , which is the relevant frequency for the parasitoid fly ( miles et al . the mechanical it d of 50 s is much larger than the acoustical it d of 1.45 s . however , with the small stimulus angles they can discriminate in the frontal zone , the flies have to deal with acoustical itds as small as 50 ns , and 2 s for the mechanical it d . to process such small time differences adequately , the primary afferents exhibit properties different from those of other acoustic insects ( oshinsky and hoy 2002 ) . they are not spontaneously active , and respond with one action potential to acoustic stimuli with very high temporal acuity . the variation in the timing of the single spike was remarkably low with 95 s . and similar to the findings in locusts and crickets ( see above ) , there is an inverse relationship between the latency of the response and stimulus amplitude . in this way , physiological time differences in spike timing are generated between ipsi - and contralateral afferents with a magnitude of about 600 s , again much larger than the physical itds ( oshinsky and hoy 2002 ; review in robert 2005 ) . the anatomical basis for a functional pressure difference receiver as it is found in field crickets is probably the most complex one described so far , with a modified tracheal system where three important sound pressures interact at the anterior tympanum : the external sound pressure , the internal sound pressure from the ipsilateral spiracle via the leg trachea to the tympanum , and a second internal component originating from the contralateral spiracle . the tracheal connection between both sides includes a double membrane ( septum ) at the midline enhancing the time delay in the internal interaural transmission ( lhe and kleindienst 1994 ; michelsen and lhe 1995 ) . these three sound components need to interact with the proper amplitude and phase relationship to guarantee a frequency - dependent directionality at the low carrier frequencies employed by most crickets its frequency dependency poses another problem to the evolution of hearing , namely to match the best frequency of directionality with the frequency sensitivity of the ear : ideally both should be tuned to the cf of the male calling song . comparative data show that a mismatch between the sensitivity and directionality tuning is not uncommon in crickets , and that independent variation of both filters is possible ( kostarakos et al . by shifting their attention away from the commonly studied field crickets to the rich variety of other cricket species schmidt et al . ( 2011 ) and schmidt and rmer ( 2011 ) described cases where both the sharpness of frequency tuning and directional tuning was enhanced , its mismatch reduced , and increased iids were recorded . this appears to be the result of a high selection pressure of species competing for the acoustic communication channel in the nocturnal rainforest ( rmer 2013 ) . given that the anatomical arrangement of the acoustic tracheal system provides the basis for the pressure difference receiver a comparative analysis of 40 species from three different superfamilies sheds some light on its evolution ( schmidt and rmer 2013 ) . one of the most conspicuous features is related to modifications of the transverse trachea providing the contralateral input to the internal surface of the tympanum . the most basic form was found in a member of the superfamily rhaphidophoridae , a primary non - hearing species troglophilus neglectus , with no transverse trachea at all . three gryllacrididae species have an unspecialized connecting trachea with no acoustic vesicle and septum . within the gryllidae , an impressive anatomical transformation has taken place with the appearance of an acoustic vesicle . a striking structural modification is a double acoustic vesicle in rainforest species where large iids and almost perfect match between directional and sensitivity tuning had been found before ( schmidt et al . however , demonstrating the striking variability in the anatomical structures of the acoustic tracheal system is only the first step in reconstructing the evolution of the pressure difference receiver in crickets . relevant time and phase shifts leading to constructive and destructive interference at the tympanal membrane will depend on numerous parameters such as spiracle opening ( sound entrance ) , cross - section and length of tracheal branches ( i.e. , transverse and leg trachea ) , their relative position to one another , the size of the acoustic vesicle and biomechanical properties of the medial septum ( fletcher 2007 ) . currently we know very little about the contribution of each of these parameters for the directionality and its frequency tuning , but variation of some of these factors appears to be part of the anatomical diversity seen so far ( schmidt and rmer 2013 ) . to identify the complete apparatus for the propagation of both ipsilateral and contralateral sound components to the tympanal membrane a complete 3d-ct analysis of these tracheal components , including quantitative measurements of tube length , volume and diameter although the perception of source height , i.e. , elevation and depression , is certainly significant for many acoustic insects due to their habitat and lifestyle , surprisingly few studies have addressed spatial hearing in insects so far . in contrast to other taxa , where the detection of the elevation of a sound source depends either on pinnae ( in mammals ) or on ear asymmetries in owls ( review knudsen and konishi 1979 ; heffner and heffner 1992 ) , insects ( and frogs ) do not have ear structures specialized for the perception of elevated sound sources . yet , as shown for the polynesian field cricket teleogryllus oceanicus , they may not be essential for spatial hearing ( wyttenbach and hoy 1997 ) . the authors used the ultrasound avoidance response of flying crickets as part of their startle / avoidance behavior ( moiseff et al . 1978 ) , and determined the minimum audible angle as a measure of spatial auditory acuity . but in comparison to the acuity in azimuth , which was 11.25 from frontal and 45 from lateral , the acuity was much reduced in elevation ( 45 in the front and rear , and 60 to 90 from lateral ) . the task ( and success ) in the avoidance response of a flying cricket is just to fly away from the ultrasound stimulus , but most likely not exactly into the opposite direction , so that such acuity is sufficient for an adaptive behavior . however , we would expect a better three - dimensional acuity when it comes to positive phonotaxis . this is evident in a behavioral study by mller and robert ( 2001 ) with the remarkable phonotactic accuracy of the parasitoid fly o. ochracea when locating its host during flight . using sophisticated techniques to follow freely flying female flies towards the target broadcasting the hosts calling song , the authors identified three distinct phases : a brief takeoff phase ; a cruising phase in which course and altitude remain quite constant ; and a terminal landing phase . the terminal phase starts when the angle of source depression is about 7590 ( mller and robert 2001 ; their fig . even more surprising was the finding that the acoustic stimulus could be interrupted when the fly was on its way to the loudspeaker , but the fly still initiated the final descent phase correctly and landed close to the now silent loudspeaker . this suggests that the fly had acquired enough information to navigate accurately to the sound source at the time when the stimulus was switched off . further experiments with elevated sound sources demonstrated that the fly s auditory system is capable of finding a sound source in the three - dimensional space , and not just on the ground . so far we do not know the proximate mechanisms enabling the small fly to perform such accurate three - dimensional localization . spatial hearing is also essential for many tree crickets and katydids ( and probably some grasshoppers ) as well , since their microhabitats are trees and bushes , where biologically important sounds may arise from positions above and below the receiver , in addition to different locations in azimuth . ( 2007 ) used the duetting mode of communication between the sexes of the katydid leptophyes punctatissima , ( robinson et al . 1986 ; heller and von helversen 1986 ; bailey 2003 ) to attract phonotactically orienting males through an artificial , three - dimensional grid system towards acoustic models of the female replies . loudspeakers broadcasting these replies were positioned either at an elevated or depressed location ( by 45 each ) relative to the starting point , or in the azimuth . the grid system was designed so that each point in space could be reached by the male with almost equal probability , and did not favor any phonotactic path . all males tested reached the three speaker positions in space with only little deviation from the shortest possible path , and there was no statistical difference in quantitative measures of phonotaxis towards the elevated , depressed or horizontal speaker . a further attempt to investigate spatial hearing in the same katydid species was made using a walking compensator ( ofner et al . 2007 ) , which allowed correlating the degree of stimulus elevation from 0 ( azimuth ) to 90 ( exactly above the insect ) with quantitative measures of the orienting movement . with increasing loudspeaker elevation the males meandered more , and there were more turns to the wrong side with increasing loudspeaker elevation . however , most males performed phonotaxis with a high acuity up to an elevation of 60 ; some individuals were very accurate in their approach even at a source elevation of 75. when males were tested in response to sound sources elevated by 90 ( no binaural cues available ) , they circled strongly under the sound source and deviated much more from the axis of loudspeaker orientation compared to smaller elevation angles . a peculiar behavior was observed with males under poor directional cues : males tilted their head and thorax in a forward direction , often associated with a shift of the longitudinal body axis by up to 30 to each side and also bending the dorso - ventral axis to left and right ( fig . the authors interpreted this behavior as a kind of directional scanning , similar to that of vertebrates moving their heads to localize a sound source . by doing so they might actively induce changes in binaural directional cues ( see below and fig . this is based on the fact that the main acoustic input to the ear is via the acoustic spiracle and leg trachea , guiding in particular high - frequency sound to the inner side of the ear ( michelsen et al . 1994 ; michelsen 1998).fig . 2 a reconstruction of the body posture of males of l. punctatissima while they exhibit the tilting behavior . the numbered arrows indicate ( 1 ) the torque movement , ( 2 ) turning on the spot with a certain yaw angle and ( 3 ) a roll of the body axis to either side ( from ofner et al . b color - coded iids in the peripheral auditory system of l. punctatissima created by a male turning to right or left at different tilting ( head and thorax down ) angles , with the sound source at a frontal position in the horizontal plane ( from kostarakos et al . 2007 ) a reconstruction of the body posture of males of l. punctatissima while they exhibit the tilting behavior . the numbered arrows indicate ( 1 ) the torque movement , ( 2 ) turning on the spot with a certain yaw angle and ( 3 ) a roll of the body axis to either side ( from ofner et al . b color - coded iids in the peripheral auditory system of l. punctatissima created by a male turning to right or left at different tilting ( head and thorax down ) angles , with the sound source at a frontal position in the horizontal plane ( from kostarakos et al . ( 2007 ) determined the peripheral spatial directionality of the ear using physiological methods . whereas maximal iids of 18 db occurred between ipsi- and contralateral stimulation in the azimuth , these values decreased in a more or less systematic fashion with increasing elevation or depression of the sound source , so that the gradient of iids could be used by the male for his directional decisions during phonotaxis . imagine a male approaching an elevated female from some distance , the perceived elevation angle might be 30 , and by regularly meandering by 30 in the horizontal plane , this would create iids of about 8 db . approaching in the horizontal plane , the perceived elevation angle will increase with decreasing distance , and the same meandering will create smaller iids of 4 db at a source elevation angle of 75. only if the male follows the source elevation by climbing up , he will be able to operate with the largest possible iids for a given turn angle . how can the male solve the problem of ambiguity in peripheral directional cues , since depressed and elevated sound sources may result in almost the same iids ? two possible solutions have been suggested : one requires some kind of memory , by comparing the previous iid with the current one , so that a false decision , e.g. to climb down may result in a much more elevated angle , and corresponding smaller iids . such information could be used to correct his path and climb upwards to the source the next time . the core of this model for spatial orientation is the suggestion , that it requires a sequential analysis of binaural cues , thus involving memory . this is supported by a study on the lesser wax moth achroia grisella ( greenfield et al . 2002 ) , where all interaural acoustic differences for females orienting toward the male advertisement call have been experimentally removed . their findings also suggest that receivers may adjust their phonotactic movement in accordance with a sequential comparison of auditory input . in the case of the parasitoid fly orienting so precisely towards a sound source that has been switched off before ( see above ) some kind of memory must be involved as well , but of course different from the one involving sequential analysis ; both deserving more attention in the future . the second possibility for the approach of the katydid l. punctatissima is to avoid the wrong direction in the case of ambiguous information , by performing acoustic scanning beforehand . figure 2 illustrates this effect for the tilting ( head down ) component , with a sound source fixed in the horizontal plane of the male . largest changes in iids occur when the male turns right or left and does either not tilt its body or by only 30. however , there is a substantial loss in directionality with tilting angles between 60 and 90. the opposite would occur with a depressed sound source , because tilting would bring the source closer to the males horizontal plane , and thus would create large iids . iids have to be translated into binaural discharge differences of neurons to be processed by the auditory pathway . simultaneous recordings of the activity of a pair of directionally - sensitive interneurons allowed kostarakos et al . ( 2007 ) determining such binaural discharge differences in response to the female reply exactly under the same conditions as experienced by males performing phonotaxis in the artificial grid system and on the walking belt ( rheinlaender et al . 2007 ; ofner et al . such discharge differences are large and reliable in the azimuth with lateral stimulation , and decrease gradually with more frontal stimulation , and also with elevation and depression of sound sources . notably , at 60 or 75 elevation , these differences decreased to only one ap / stimulus . we should remember that at 75 elevation on the walking belt , the spatial acuity of most males strongly declined , but some males appeared to use such small bilateral differences for reliable phonotaxis . all together , these data demonstrate that the spatial acuity of the katydid is quite remarkable considering that the acoustic stimulus available for the orienting male is extremely short ( 1 ms ) and the number of receptor cells that are involved in the processing of directional information is rather small . moreover , receptors respond to such a short stimulus with only one action potential / stimulus , once it is above threshold ( hardt 1988 ) . thus , there is neither a dynamic intensity response function of a single receptor , nor is graded information available to changing stimulus angles . this is not a peculiar property of receptors in this species ; the intensity response curve of auditory receptors in other insects would be similar in response to a 1-ms stimulus . note also that the phasic response of receptors is due to the short stimulus duration , and not an intrinsic property , as in the case of the ormia fly ( see above ) . the only remaining information is the number of afferents being activated , as a result of their absolute sensitivity and/or tuning ( hardt 1988 ) . since the pioneering work by morton ( 1975 ) and wiley and richards ( 1978 ) there is an increasing awareness in the literature on acoustic communication about the properties of the transmission channel for sound signals , which may affect their amplitude , frequency spectrum and temporal pattern , in addition to masking noise which may decrease the signal - to - noise - ratio ( snr ) for detection ( brumm 2013 ) . as a result , the broadcast signal may differ substantially from the one available for a receiver at some distance . at the same time , however , the directional cues of sound under natural conditions have been largely neglected . in the previous section we have learned that some field crickets or parasitoid flies show hyperacute directional hearing when tested on a trackball under open - loop conditions in the laboratory , and that iids as small as 0.51 db , or physiological itds of only 0.51 ms may be sufficient for reliable directional responses . little is known about how well such cues are preserved after transmission of the signal in natural habitats . the technical difficulties in measuring such cues in the field let rheinlaender and rmer ( 1986 ) develop a so - called biological microphone ( adapted from roeders pioneering work on moth hearing the echolocation calls of bats ; roeder 1967 ) . by recording the activity of a pair of directionally - sensitive interneurons in the natural habitat of a katydid they could show that ( 1 ) directional information may be provided at remarkable communication distances , but ( 2 ) that directionality strongly depends on the spatial configuration between sender and receiver . notably , they found positions in the habitat where the animal could detect the signal , but was unable to localize it . a similar attempt was used to study the directionality in various types of habitat for short - horned grasshoppers ( gilbert and elsner 2000 ) . using long - term recordings of tympanal nerve activity in a portable electrophysiological preparation in chorthippus biguttulus , the directional dependence of the activity was monitored first in the laboratory under free field conditions , and then in various natural habitats of the species . on gravel and in sparse vegetation , the general patterns of directionality were quite similar to those in the free sound field of the laboratory , although the amount of iids calculated from the nerve activity was reduced . strongest differences were found in dense vegetation , both with respect to the maximum iids and the location of the minima in nerve activity , which not always occurred on the contralateral side , as is typical for the free sound field . field crickets live and communicate on the ground , which may create specific problems for communication . kostarakos and rmer ( 2010 ) examined directional hearing of crickets under these conditions again using a neurophysiological approach , by monitoring the activity of a pair of directionally sensitive , bilaterally homologous neurons . one major finding was again , that the directional information encoded in these neurons varied with distance , but there was no simple directional gradient on the transmission channel . moreover , when they analyzed the consistency of the neuronal directional cues over time , they found large variations in the amount of directionality within a time window of a minute , so that the binaural discharge difference at the same receiver position varied for example from 6 to 30 aps over time . similar strong variations occurred with respect to latency differences ( kostarakos and rmer 2010 ) . these approaches provided direct experimental evidence that directional sensitivity strongly depends on the spatial configuration between sender and receiver , and it is not only an inherent property of the insect s auditory system , as suggested from laboratory experiments . if directional cues under real world conditions are distorted as these data suggest , how do insects then perform in behavior ? since most data on localization behavior , and the underlying biophysics and neurophysiology are available for the field cricket g. bimaculatus , i will compare the localization performance of this species under outdoor conditions with the one in the laboratory . apparently , this performance is different in the various behavioral paradigms used for quantifying the acuity of localization . data from compensated walking on a closed - loop trackball system , as well as orientation in a y - maze indicated that field crickets are unable to discriminate the side of sound incidence within a frontal area of uncertainty covering 25 in azimuth ( weber et al . 1981 ; rheinlaender and bltgen 1982 ; weber and thorson 1989 ) , or 1014 ( bailey and thomson 1977 ; oldfield 1980 ) . however , the acuity of phonotaxis in female g. bimaculatus on an open - loop trackball system was much higher ( schneich and hedwig 2010 ) . their biophysical and neurophysiological measurements revealed a reliable gradient of iids with a slope of 0.4 db/ between 0 and 30 , also reflected in bilateral differences of afferent nerve responses . the authors concluded that under ideal conditions these crickets achieve directional hyperacuity that rivals best directional hearing in mammals and birds , or the one reported for the fly o. ochracea ( mason et al . reliably approach the target speaker broadcasting a standard model of the calling song in a no - choice trial , although for the shortest possible straight path females walked a considerable detour ( on average about 1 m for the shortest straight path of 2 m ; hirtenlehner and rmer 2014 ) . such detour is significantly higher outdoors , when compared with similar experiments in a laboratory arena . apart from the discontinuous gradients in intensity and directionality outlined above , one further reason for such differences between lab and outdoor experiments is the physical nature of the grassland in which females have to move and orient . even if females perceive reliable sensory information about the location of a stimulus , they might be forced by dense patches of grass or larger obstacles on the ground to move into another , even wrong direction . michelsen and rohrseitz ( 1997 ) provided a possible explanation for the fact that field crickets perform reasonably well under such conditions . they quantified the degradation of directional cues in a grassland habitat by measuring the sound amplitude and phase close to the ears of grasshopper carcasses for different directions of sound incidence with probe microphones . the degradation increased with frequency and distance from the sound source and with distance from the ground . thus , the cricket ear as a pressure difference receiver exploiting these phase cues may be particularly suited overcoming the degradation of directional cues . the distorted directional cues under outdoor conditions are also evident when we compare results of two - choice experiments conducted in the lab and field ( hirtenlehner and rmer 2014 ) . in general , for a significant choice larger differences in spl , cf or call rate are necessary in the field . as expected , the open - loop trackball system requires the smallest differences , with experiments in laboratory arenas in between ( for differences in spl : 5 , 1 and 3 db , respectively ) . clearly , female crickets and other acoustic insects benefit from an auditory system with high directional precision under unfavorable outdoor conditions . as evident from the previous sections , the issue of directional hearing is always strongly associated with the two important tasks for a receiver , namely to use directional information for approaching towards a mate , or in the case of a predation , to initiate appropriate escape responses away from the predator . we often forget , however , that directional hearing also contributes to auditory scene analysis , by separating sound sources according to their spatial location , as well as in reducing the effect of masking , by so - called spatial release from masking . for auditory scene analysis , a receiver groups and segregates all incoming sounds either according to their similarity or their differences in a number of sound parameters , signaling in acoustic insects often occurs in groups ( choruses ) of many individuals of the same and/or different species with the result that a complex sonic and ultrasonic background exists over which individuals must then communicate . field measurements in populations of the katydid t. viridissima revealed that the problem for a receiving insect may be quite complex , requiring them to discriminate the individual calls of up to four nearby males and more than ten others within hearing range , some of which are quite similar in amplitude ( rmer and krusch 2000 ) . how are these different acoustic objects represented in the cns of a receiver , and how does directional hearing facilitate the separation of sound sources ? one result was that the auditory world of the katydid is sharply divided into two hemispheres , with a pronounced separation along the longitudinal body axis . even with an angular separation of only 7.5 off the midline axis , two sound signals are better represented in the activity of the respective ipsilateral neuron . the strength of separation is given by the peripheral directionality of the ear , and by enhancement as a result of lateral inhibition . this mechanism , referred to as spatial release from masking , can also improve the detection and discrimination of signals in noise when the masker is spatially separated from the signal ( klump 1996 ) . again ; it is based on the directionality of the receivers hearing system and contributes to the segregation of sound sources . 2001 ; bregman 1990 ) and the detection and discrimination of conspecific signals from heterospecifics in anurans ; for a review in vertebrates see bee and micheyl ( 2008 ) . schmidt and rmer ( 2011 ) studied spatial release from masking for two species of rainforest crickets , which suffer from high nocturnal background noise . laboratory experiments yielded an average signal - to - noise - ratio ( snr ) of 8 db , when masker and signal were broadcasted from the same side ( owing to the sharp tuning to the cf of the conspecific signal ) . however , displacing the masker by 180 from the signal improved snrs by further 69 db . surprisingly , experiments carried out directly in the nocturnal rainforest yielded snrs of about 23 db compared with those in the laboratory with the same masker , where snrs reached only 14.5 and 16 db in both species . the authors conclude that conventional speaker playbacks in the lab do not properly reconstruct the masking noise situation in a spatially realistic manner , since they use playbacks of natural or artificial noise and broadcast it to either one or the other auditory side , whereas under real world conditions the same noise is spatially separated , with multiple sound sources affecting a receiver from different directions in the azimuth and elevation / depression . thus , without knowledge of the receiver properties and the spatial release mechanisms the detrimental effect of noise may be strongly overestimated . the contribution of the peripheral directionality of the ear and additional central nervous bilateral interactions ( through lateral inhibition ) for spatial release from masking is currently unknown .

when insects communicate by sound , or use acoustic cues to escape predators or detect prey or hosts they have to localize the sound in most cases , to perform adaptive behavioral responses . in the case of particle velocity receivers such as the antennae of mosquitoes , directionality is no problem because such receivers are inherently directional . insects equipped with bilateral pairs of tympanate ears could principally make use of binaural cues for sound localization , like all other animals with two ears . however , their small size is a major problem to create sufficiently large binaural cues , with respect to both interaural time differences ( itds , because interaural distances are so small ) , but also with respect to interaural intensity differences ( iids ) , since the ratio of body size to the wavelength of sound is rather unfavorable for diffractive effects . in my review , i will only shortly cover these biophysical aspects of directional hearing . instead , i will focus on aspects of directional hearing which received relatively little attention previously , the evolution of a pressure difference receiver , 3d - hearing , directional hearing outdoors , and directional hearing for auditory scene analysis .

Introduction Pressure and pressure difference receivers, and mechanically coupled receivers Minimum binaural cues for sound localization IIDs and ITDs as represented at the neuronal level The evolution of the pressure difference receiver in crickets Directionality in the third dimension Directional hearing under natural outdoor conditions Directional hearing beyond mate finding or escaping predators: auditory scene analysis

in insect taxa which use acoustic signals for intraspecific communication , the recognition of the acoustic signals is crucial for their fitness , since it enables species identification and prevents hybridization ( gerhardt and huber 2002 ; greenfield 2002 ; bradbury and vehrencamp 2011 ) . however , ears also evolved in insects without acoustic signals , indicating that other functions of hearing may also include the detection of cues from predators , or the detection of hosts in the case of parasitoids . the anatomical and physiological diversity of insect ears we know so far is impressive , with perhaps 19 independent evolutionary origins , as is the diversity of body parts on which they evolved , including legs , wings , mouth parts or the abdomen ( hoy and robert 1996 ; yager 1999 ; yack 2004 ) . therefore , we should expect that the evolution of these ears and the neuronal network processing the directional cues enabled insects to localize a sound source reasonably well . in the case of particle velocity receivers such as the antennae of mosquitoes , or filiform hairs on the cerci directionality is no problem because such receivers are inherently directional . they respond to the particle velocity ( and thus a vectorial ) component of near - field sound . the problem with the task of directional hearing is obvious , however , with tympanate hearing ; it is basically a biophysical one associated with the small size of most insects . insects equipped with bilateral pairs of tympanate ears could principally make use of binaural cues for sound localization , like all other animals with two ears . however , their small size and hence interaural distance result in only minute interaural time differences ( itds ) . at the same time acoustic theory predicts that significant diffraction for the establishment of reasonable interaural intensity differences ( iids ) occurs only when the ratio of body size to the wavelength of sound exceeds a value of 0.1 ( morse and ingard 1968 ; robert 2005 ) . again , the small body size of insects in relation to the relatively large wavelength of the sound signals used for communication thus often limits or even prevents the establishment of reasonably large iids through diffraction . in my review , i will only shortly cover the biophysical aspects of directional hearing ; more complete descriptions of the biomechanics of sound propagation and the generation of cues for directional hearing in insects can be found in excellent earlier reviews ( michelsen 1992 , 1998 ; robert and hoy 1998 ; robert 2005 for all kind of receivers ; michelsen and larsen 2008 for pressure difference receivers ) . instead , after shortly reviewing the different kind of receivers in insects , i will focus on aspects of directional hearing which received relatively little attention in previous reviews , namely the evolution of a pressure difference receiver , 3d - hearing , directional hearing outdoors , and directional hearing for auditory scene analysis . when insects are large compared to the wavelength of the sound frequencies of relevant signals , the insect s body can generate diffractive effects , resulting in a reduced pressure at the contralateral ear . theoretically , evolutionary modifications of anatomical structures ( see below ) resulting in the proper amplitude and phase shifts between internal and external pressure components could create highly directional ears despite unfavorable ratios of body size to the wavelength of sound . since the internal sound conduction can depend on frequency , insect ears can act as pressure difference receivers at low frequencies and as a pressure receiver at higher frequencies . together with the other well - studied pressure difference receiver , the cricket ear ( see below ) , the grasshopper example highlights the importance of proper phase relationships between the external and internal sound component(s ) for producing significant directionality . they are so close together that both iids and itds appear to be much too small for a physical basis of directional hearing . for example , the interaural distance of 520 m would create no more than 1.45 s of it d , and significant diffractive effects can not play a role given the unfavorable ratio of body size to wavelength of sound ( robert et al . in an open area , more than half of the animals flew directly to the loudspeaker ( broadcasting a model song of their cicada host ) , but the presence of single landmarks reduced the percentage of direct flights to the target greatly . whatever the biophysical mechanism creating binaural cues for sound localization , what are the minimum iids and itds that result in reliable directional responses in insects ? this is a difficult task in insects when body size and hence interaural disparities are minute and/or the two hearing organs are acoustically coupled . a tentative behavioral approach with a grasshopper indicated that the insects auditory system can make use of iids in the order of 12 db ( von helversen and rheinlaender 1988 ) . note that with an iid of 1 db there is a significant turn to the more strongly stimulated side , and with 23 db difference very few incorrect turns are made ( from rheinlaender et al . b results of behavioural dichotic stimulation experiments with four females , in which the interaural intensity difference of a stimulus was varied . note that with an iid of 1 db there is a significant turn to the more strongly stimulated side , and with 23 db difference very few incorrect turns are made ( from rheinlaender et al . 2005 ) but what about the use of itds as a cue for sound localization ? these values of itds are so strongly constrained by body size and ear separation , that it had been accepted for a long time that such small itds can not be used for neuronal processing of sound direction . mrchen ( 1980 ) thus suggested that insects do not use the physical itds , but the physiological time differences in the binaural receptor fibre discharges instead . thus , the difference in the time of arrival of action potential activity on both sides of the nervous system may reach values of 56 ms for ipsi - versus contralateral sound , which exceeds the value of the physical it d between the ears by almost 1,000 times . the author therefore suggested that response strength and response latency can be regarded as equivalent directional cues for sound direction . similarly , in the study by schneich and hedwig ( 2010 ) the interaural intensity differences were closely reflected in the response latencies of the auditory afferent activity . the hypothesis of the dual mode of directional coding was examined using dichotic stimulation of the tympana in the locust ( rheinlaender and mrchen 1979 ) . they demonstrated a time - intensity trading phenomenon similar to the one reported for vertebrates ( erulkar 1972 ) , although the time cue was in the order of milliseconds , rather than microseconds as indicated by the physical itds . in the quasi - dichotic stimulus situation mentioned above , the behavioral resolution of chorthippus biguttulus males for itds was also in the order of 1 ms : when both stimuli were presented at equal loudness , but one speaker was leading the other by only 0.5 ms this resulted in significant turns towards the leading side ( von helversen and rheinlaender 1988 ) . although microseconds do not matter because the physical time delays between the ears are too small to be of relevance ( but see results for the ormia fly below ) , the onset of binaural arrival of receptor activity at these synapses ( the physiological time delay ) may be quite important for directional coding . 1988 ) , and inhibitory inputs from the contralateral side mediated by its mirror - image counterpart on the other side ( selverston et al . ( 2004 ) , and hedwig and pollack ( 2008 ) . the comparison of the two species with hyperacute directional hearing , the field cricket g. bimaculatus and the fly ormia ochracea may illustrate the limits of processing binaural cues for directional hearing known so far . females of both species not only reliably discriminated the side of acoustic stimulation at angles of sound incidence starting 12 from the animal s longitudinal body axis ( indicative of correct lateralization ) , but also precisely walked towards the sound source . however , with the small stimulus angles they can discriminate in the frontal zone , the flies have to deal with acoustical itds as small as 50 ns , and 2 s for the mechanical it d . to process such small time differences adequately , the primary afferents exhibit properties different from those of other acoustic insects ( oshinsky and hoy 2002 ) . the anatomical basis for a functional pressure difference receiver as it is found in field crickets is probably the most complex one described so far , with a modified tracheal system where three important sound pressures interact at the anterior tympanum : the external sound pressure , the internal sound pressure from the ipsilateral spiracle via the leg trachea to the tympanum , and a second internal component originating from the contralateral spiracle . these three sound components need to interact with the proper amplitude and phase relationship to guarantee a frequency - dependent directionality at the low carrier frequencies employed by most crickets its frequency dependency poses another problem to the evolution of hearing , namely to match the best frequency of directionality with the frequency sensitivity of the ear : ideally both should be tuned to the cf of the male calling song . however , demonstrating the striking variability in the anatomical structures of the acoustic tracheal system is only the first step in reconstructing the evolution of the pressure difference receiver in crickets . , transverse and leg trachea ) , their relative position to one another , the size of the acoustic vesicle and biomechanical properties of the medial septum ( fletcher 2007 ) . to identify the complete apparatus for the propagation of both ipsilateral and contralateral sound components to the tympanal membrane a complete 3d-ct analysis of these tracheal components , including quantitative measurements of tube length , volume and diameter although the perception of source height , i.e. but in comparison to the acuity in azimuth , which was 11.25 from frontal and 45 from lateral , the acuity was much reduced in elevation ( 45 in the front and rear , and 60 to 90 from lateral ) . the task ( and success ) in the avoidance response of a flying cricket is just to fly away from the ultrasound stimulus , but most likely not exactly into the opposite direction , so that such acuity is sufficient for an adaptive behavior . even more surprising was the finding that the acoustic stimulus could be interrupted when the fly was on its way to the loudspeaker , but the fly still initiated the final descent phase correctly and landed close to the now silent loudspeaker . this suggests that the fly had acquired enough information to navigate accurately to the sound source at the time when the stimulus was switched off . further experiments with elevated sound sources demonstrated that the fly s auditory system is capable of finding a sound source in the three - dimensional space , and not just on the ground . spatial hearing is also essential for many tree crickets and katydids ( and probably some grasshoppers ) as well , since their microhabitats are trees and bushes , where biologically important sounds may arise from positions above and below the receiver , in addition to different locations in azimuth . loudspeakers broadcasting these replies were positioned either at an elevated or depressed location ( by 45 each ) relative to the starting point , or in the azimuth . with increasing loudspeaker elevation the males meandered more , and there were more turns to the wrong side with increasing loudspeaker elevation . however , most males performed phonotaxis with a high acuity up to an elevation of 60 ; some individuals were very accurate in their approach even at a source elevation of 75. when males were tested in response to sound sources elevated by 90 ( no binaural cues available ) , they circled strongly under the sound source and deviated much more from the axis of loudspeaker orientation compared to smaller elevation angles . b color - coded iids in the peripheral auditory system of l. punctatissima created by a male turning to right or left at different tilting ( head and thorax down ) angles , with the sound source at a frontal position in the horizontal plane ( from kostarakos et al . b color - coded iids in the peripheral auditory system of l. punctatissima created by a male turning to right or left at different tilting ( head and thorax down ) angles , with the sound source at a frontal position in the horizontal plane ( from kostarakos et al . whereas maximal iids of 18 db occurred between ipsi- and contralateral stimulation in the azimuth , these values decreased in a more or less systematic fashion with increasing elevation or depression of the sound source , so that the gradient of iids could be used by the male for his directional decisions during phonotaxis . imagine a male approaching an elevated female from some distance , the perceived elevation angle might be 30 , and by regularly meandering by 30 in the horizontal plane , this would create iids of about 8 db . approaching in the horizontal plane , the perceived elevation angle will increase with decreasing distance , and the same meandering will create smaller iids of 4 db at a source elevation angle of 75. only if the male follows the source elevation by climbing up , he will be able to operate with the largest possible iids for a given turn angle . how can the male solve the problem of ambiguity in peripheral directional cues , since depressed and elevated sound sources may result in almost the same iids ? the core of this model for spatial orientation is the suggestion , that it requires a sequential analysis of binaural cues , thus involving memory . in the case of the parasitoid fly orienting so precisely towards a sound source that has been switched off before ( see above ) some kind of memory must be involved as well , but of course different from the one involving sequential analysis ; both deserving more attention in the future . the second possibility for the approach of the katydid l. punctatissima is to avoid the wrong direction in the case of ambiguous information , by performing acoustic scanning beforehand . figure 2 illustrates this effect for the tilting ( head down ) component , with a sound source fixed in the horizontal plane of the male . largest changes in iids occur when the male turns right or left and does either not tilt its body or by only 30. however , there is a substantial loss in directionality with tilting angles between 60 and 90. the opposite would occur with a depressed sound source , because tilting would bring the source closer to the males horizontal plane , and thus would create large iids . such discharge differences are large and reliable in the azimuth with lateral stimulation , and decrease gradually with more frontal stimulation , and also with elevation and depression of sound sources . we should remember that at 75 elevation on the walking belt , the spatial acuity of most males strongly declined , but some males appeared to use such small bilateral differences for reliable phonotaxis . all together , these data demonstrate that the spatial acuity of the katydid is quite remarkable considering that the acoustic stimulus available for the orienting male is extremely short ( 1 ms ) and the number of receptor cells that are involved in the processing of directional information is rather small . note also that the phasic response of receptors is due to the short stimulus duration , and not an intrinsic property , as in the case of the ormia fly ( see above ) . since the pioneering work by morton ( 1975 ) and wiley and richards ( 1978 ) there is an increasing awareness in the literature on acoustic communication about the properties of the transmission channel for sound signals , which may affect their amplitude , frequency spectrum and temporal pattern , in addition to masking noise which may decrease the signal - to - noise - ratio ( snr ) for detection ( brumm 2013 ) . at the same time , however , the directional cues of sound under natural conditions have been largely neglected . in the previous section we have learned that some field crickets or parasitoid flies show hyperacute directional hearing when tested on a trackball under open - loop conditions in the laboratory , and that iids as small as 0.51 db , or physiological itds of only 0.51 ms may be sufficient for reliable directional responses . by recording the activity of a pair of directionally - sensitive interneurons in the natural habitat of a katydid they could show that ( 1 ) directional information may be provided at remarkable communication distances , but ( 2 ) that directionality strongly depends on the spatial configuration between sender and receiver . notably , they found positions in the habitat where the animal could detect the signal , but was unable to localize it . using long - term recordings of tympanal nerve activity in a portable electrophysiological preparation in chorthippus biguttulus , the directional dependence of the activity was monitored first in the laboratory under free field conditions , and then in various natural habitats of the species . strongest differences were found in dense vegetation , both with respect to the maximum iids and the location of the minima in nerve activity , which not always occurred on the contralateral side , as is typical for the free sound field . kostarakos and rmer ( 2010 ) examined directional hearing of crickets under these conditions again using a neurophysiological approach , by monitoring the activity of a pair of directionally sensitive , bilaterally homologous neurons . similar strong variations occurred with respect to latency differences ( kostarakos and rmer 2010 ) . since most data on localization behavior , and the underlying biophysics and neurophysiology are available for the field cricket g. bimaculatus , i will compare the localization performance of this species under outdoor conditions with the one in the laboratory . the authors concluded that under ideal conditions these crickets achieve directional hyperacuity that rivals best directional hearing in mammals and birds , or the one reported for the fly o. ochracea ( mason et al . they quantified the degradation of directional cues in a grassland habitat by measuring the sound amplitude and phase close to the ears of grasshopper carcasses for different directions of sound incidence with probe microphones . thus , the cricket ear as a pressure difference receiver exploiting these phase cues may be particularly suited overcoming the degradation of directional cues . as expected , the open - loop trackball system requires the smallest differences , with experiments in laboratory arenas in between ( for differences in spl : 5 , 1 and 3 db , respectively ) . as evident from the previous sections , the issue of directional hearing is always strongly associated with the two important tasks for a receiver , namely to use directional information for approaching towards a mate , or in the case of a predation , to initiate appropriate escape responses away from the predator . we often forget , however , that directional hearing also contributes to auditory scene analysis , by separating sound sources according to their spatial location , as well as in reducing the effect of masking , by so - called spatial release from masking . for auditory scene analysis , a receiver groups and segregates all incoming sounds either according to their similarity or their differences in a number of sound parameters , signaling in acoustic insects often occurs in groups ( choruses ) of many individuals of the same and/or different species with the result that a complex sonic and ultrasonic background exists over which individuals must then communicate . how are these different acoustic objects represented in the cns of a receiver , and how does directional hearing facilitate the separation of sound sources ? again ; it is based on the directionality of the receivers hearing system and contributes to the segregation of sound sources . the authors conclude that conventional speaker playbacks in the lab do not properly reconstruct the masking noise situation in a spatially realistic manner , since they use playbacks of natural or artificial noise and broadcast it to either one or the other auditory side , whereas under real world conditions the same noise is spatially separated , with multiple sound sources affecting a receiver from different directions in the azimuth and elevation / depression .

it focuses mainly on midwestern agriculture , but to the extent possible other regions will be discussed . the theoretical underpinnings and economic discussions can be found in other sources . before beginning any discussion on economies of size the concept of economies of size means that the average cost per unit of production decreases as the size of the farm increases . the economies can occur because the farmer is able to spread more production over the same level of fixed expenses . or , economies of size can occur when a farm is able to obtain volume discounts for inputs such as seed or fertilizer . an example would be the cost for pollution monitoring around a swine production facility . if the farm is required to monitor the groundwater around the facility for contamination , they must put in a well and monitoring equipment , which represent fixed costs and can serve a large number of pigs . as the number of pigs sold increases , the costs for this aspect of production would decrease . and , as a result , monitoring in this fashion would actually provide a cost advantage to a larger operation . one is economies of scale , which measure what happens if all inputs are increased by the same proportion . if costs per unit go down , there are increasing economies of scale , and if the costs per unit remain the same , there are constant returns to scale . the other concept is economies of scope , which refer to reducing costs for using resources by spreading the resources over more than one enterprise . hofstrand summarized the concepts of size and scope by noting that size spreads fixed resources over more units of output whereas scope spreads the cost of a given set of resources or skills over more than one product or enterprise . discussion of economies of size in production agriculture and the desirability of small farms has ebbed and flowed over time . one of this country 's earliest political debates centered on the conflicting views held by thomas jefferson and alexander hamilton regarding land ownership . jefferson argued for family ownership of farms as a means of ensuring interest in the democratic process . hamilton argued for selling the land to the highest bidder as a means of paying off the revolutionary war debt . jefferson won the debate and the united states followed the principal objective of promoting family farms . in the early part of the 20th century problems emerged for this ideal , problems related to economies of size . in 1909 president roosevelt formed the country life commission to address the issues of poverty in rural america . the commission found that in spite of the advances and money spent up until that time agriculture is not commercially as profitable as it is entitled to be for the labor and energy that the farmer expends and the risks that he assumes and the farmer is almost necessarily handicapped in the development of his business because his capital small and the volume of his transactions limited ; and he usually stands particularly alone against organized interests ( report of commission on country life , 1909 , in wunderlich , p. 146 ) . this commission report renewed the efforts to improve the lives of the farmers by increasing their productivity . the us economy moved through two world wars and the great depression after the parity price period . farm incomes fell after world war ii and again there was unrest in the countryside . in 1954 president eisenhower ordered the us dept of agriculture to prepare a report on the state of the agricultural economy . the report concluded that expanding agricultural productivity was the key and one of the recommendations to increase agricultural productivity was to allow farms to expand in order to take advantage of new labor - reducing technologies . it was argued that the expansion of small farms could be facilitated using programs designed to retrain and move agricultural workers to non - farm industries . others have noted that while some of the implemented government policies were successful , most of the reductions in farm labor and the corresponding increases in farm size were the result of changing technology . at the beginning of the 20th century there were concerns over the need to add more technology to production agriculture . these were followed in the middle of the century by a feeling that the new technology had already removed the need for much of the labor and what we needed to do was move agricultural workers to non - farm industries . towards the end of the 20th century , new fears were raised that family farms were losing ground and the move to increase productivity by decreasing labor may have yielded undesirable consequences . a small farms commission , created by the secretary of agriculture in 1998 , urged the government to recognize small farms and their contributions to society and actively support them . dedicated to optimizing the labor and ingenuity of small farm operators and the biological assets of their farms using less capital - intensive investments . ( p. 31 ) over time in the united states economies of size with respect to substituting capital for labor have been encouraged and discouraged . research and technology have been the primary drivers in changing the situation with respect to economies of size . in spite of the small farms commission report , we continue to move toward a dual agriculture with many small farms and relatively few large farms . the census of agriculture is the best source for a consistently gathered set of agricultural data for the entire country . the census provides an opportunity to examine several aspects of the issues related to economies of size . the first agricultural census was taken in 1840 and it was conducted every 10 years until 1920 . since the bureau of the census conducted the census of agriculture until 1996 when the responsibility was transferred to the usda primarily because the census bureau was going to increase the size needed for an operation to be considered a farm . one of the major difficulties with examining national farm - level data is how to determine what is considered a farm . for the purposes of the census , a farm is defined as any place from which $ 1000 or more of agricultural products were produced and sold , or normally would have been sold , during the census year . ( p. ix ) this definition has been used since 1974 and has created many problems and inconsistencies when people use or try to interpret the census data . inflation alone would suggest that the cutoff point should be raised to at least $ 5000 . another problem is the change in census coverage initiated by the usda in 2002 in an effort to survey more very small farms . they did not change the definition ; they simply chose to count more people . the change in coverage has led to a change in the composition of the farms considered in the census . figure 1 shows the distribution of farms and sales in the united states based on sales categories . notice in this figure the very smallest category , sales of less than $ 1000 , makes up over 30% of the farms . it is important to remember that the definition of a farm is any place that could have sold the farms are assigned points based on the agricultural enterprises and if the points total 1000 , they are included as farms in the count . one pig has a point value of 150 , so raising 7 pigs would classify a place as being a farm . a horse has a point value of 200 whether or not it is sold , which means that 5 horses classify a place as a farm . figure 1 shows that the majority of farms are small and they account for very little of the agricultural sales in the united states . the largest two categories , those with sales over $ 500,000 , represented 5% of us farms and had 74% of the sales in 2007 . at the other end of the spectrum , farms with sales less than $ 5000 accounted for 50% of the farms and generated less than 1% of the sales . it is interesting to note that government payments are greater than sales for the smallest group . for farms in the smallest size group over 90% of it is important to keep the size issue in mind when working with the census data . in many contexts , discussions about average farms are almost meaningless without making some distinction regarding the size of farm . with this caveat in mind there are two ways to use the census data to examine economies of size in us agriculture . the first item of interest is presented in figure 2 , which shows the dollar value of sales for every dollar of expenses reported . the more sales a farm can generate for each dollar of expense , the more profitable it will be . such efficiency gains can be thought of as economies of size at the aggregate level . notice in figure 2 that the amount of sales per dollar of expenditure increases rapidly until reaching approximately $ 100,000 in sales . once this level of sales is reached the ratio flattens out , indicating no further gains in efficiency with respect to expenses relative to sales . it is interesting to note in figure 2 that the ratio tends to grow smaller with the largest size group . figure 2 shows that there are very definite gains in efficiency as a farm increases in size but the rate of gain slows and actually decreases as size increases . figure 3 also illustrates the extent of economies of size present in the us farm sector . this figure shows the percentage of farms with a positive net cash farm income in 2007 . it looks very similar to figure 2 in that there are rapid increases in the percentage of farms showing positive net cash farm income , but this increase flattens out starting at around $ 100,000 in sales . economies of size for agricultural production in iowa can best be illustrated using data from the iowa farm business association ( ifba ) , a record - keeping service for iowa farmers . these farm operations would be more representative of the larger farms as shown in the census data . figure 4 shows the total economic costs of production per bushel of corn for farms in the ifba based on the number of row crop acres farmed . in figure 4 the cost per bushel decreases over the smaller acre categories but flattens out by 800 row crop acres . figure 5 shows the average cost per bushel for soybeans based on the number of row crop acres . figures 4 and 5 display what is known as an l - shaped average cost curve . that means there are initial economies of size but these size advantages dissipate , and then costs remain relatively flat over a range of sizes . most normative studies of crop farms , both early and recent , while identifying economies of size , have given little evidence that the cost curve deviates significantly from a sagging l shape . ( p. 206 ) hallam further discussed the economies of size in agricultural production : the general conclusion is that while some economies of size or scale may exist for livestock farms that significant economies , at least as conventionally defined , do not exist for most crop production activities . while differences in efficiency and growth paths differ among firms , few of these seem to be directly related to economies of size and scale . one observation by hallam was the discrepancy between farmers with respect to costs of production . this is illustrated in figure 6 , which shows the costs per bushel for corn when the cost groups are divided into thirds . figure 6 reports an almost $ 1.50 per bushel difference in costs of production between the high - cost producers and the low - cost producers . figure 7 presents the number of corn acres for the farms in each cost group . figure 7 shows that the high - cost producers had the fewest corn acres , whereas the middle - cost producers had the greatest number of corn acres . figures 6 and 7 reveal a couple of distinguishing characteristics that are important to remember when considering production agriculture . first , there is considerable variation among farms , even farms producing the same crop . the second observation is that the number of acres is not a guarantee of lower cost production . many other factors determine whether a farmer is a low - cost producer per unit of output . hallam noted that livestock production appears to be different with respect to economies of size . livestock production efficiencies and the changing structure of animal agriculture have been discussed in many places , most recently by macdonald and mcbride . figure 8 shows the costs of production per hundredweight for farms in the iowa farm business association based on the number of pigs marketed . figure 9 presents the cost per hundredweight of pork produced based on the number of sows . they show a more extreme drop in costs of production as size increases when compared to the crop costs shown in figures 4 and 5 . technological changes and changes in production have led to the dramatic decreases in costs of production . farrow - to - finish swine producers can achieve essentially the same profitability per unit of production across a wide range of volumes of production . ( p. 1 ) macdonald and mcbride note , there are substantial economies of scale up to certain threshold sizes , and farms can operate efficiently at sizes that are much larger than the thresholds . ( p. 36 ) there appears to be two major differences between animal and crop production with respect to economies of size . one of the major differences is the vertical integration of the industry , which has led to a major consolidation in the processing industry . the declining number of packing plants illustrates the consolidation in the processing industries . in 1976 there were 221 and 182 cattle and swine processing plants , respectively , a decrease of 74% in the cattle plants and a 63% decrease in swine processing plants . the decrease in the number of plants has been coupled with an increase in the number of animals slaughtered by size of plants . for example , in 1976 , 9% of the cattle were slaughtered in plants slaughtering more than 500,000 head per year . by 1998 in 1976 , 2% of the swine were slaughtered in plants slaughtering more than 1 million head per year . by 1998 one of the biggest components of these economies of size is procurement of the animals . as the packing plants have gotten larger , they have demanded a larger volume from the individual producer . this favors the larger - scale producers and requires either an increase in the size of production or some form of joint marketing . production contracts have become commonplace and today the majority of pork is raised under some form of contract . smaller producers may have similar costs of production but access to the market has become a significant problem . the second major factor that has led to the increased size of animal production operations is the ability to pass along costs to those not making the production decisions . these external costs are generally not calculated in the costs of production or measuring the efficiency of the large scale production . macdonald and mcbride discussed these external costs , which generally fall into two major categories . confinement of large numbers of animals generates manure and odor and has the potential to cause water and air quality deterioration . a second potential external cost concerns the threat to food safety from the use of prophylactic antibiotics . large numbers of animals housed together have greater potential for disease . routine antibiotic use can increase the chances for antibiotic resistance to develop and for contamination of the product . the severity of these problems depends on the individual operator , and the potential for problems increases as the concentration of the animals increase . the specialization of production often ignores the benefits and advantages of the synergy that exists in agriculture . crops can feed the animals , animal manure can provide nutrients for the crops , and the farmers can add value to their production with a more holistic approach . increased environmental costs , food safety concerns , undesirable odor , and concentration of the food supply are just a few of the concerns that have arisen with the change in philosophy of producing animals and animal products . animal agriculture is changing rapidly from midwestern enterprises of diversified farming to those which manufacture muscle protein . ( p. 1 ) this industrialized approach has led us to many of the problems and issues we face today . if farms display an l - shaped average cost curve and expansion does not lower the costs of production , why are we seeing such expansion in farm size ? the basic answer is that farms are getting larger because they do not incur diseconomies of size . as the farm increases in size the cost per unit of output remains relatively flat . as the number of units of output increases and there is no significant difference in the cost of production , income will increase . for example , using the average acreage shown in figure 4 , a farm with 800 acres of corn would have an average total cost of production of approximately $ 3.00 per bushel . if the yield was 200 bushels per acre and the price of corn was $ 3.50 per bushel , the farm would make $ 80,000 [ ( $ 3.50-$3.00 ) 200 800 ] . but , if the same farm had 1,500 acres of corn they would make $ 150,000 [ ( $ 3.50-$3.00 ) 200 1500 ] . such expansion is possible because of the labor - saving technologies that have been developed for production agriculture . figures 10 and 11 show the hours of labor required for corn and soybeans per acre and per 100 bushels , respectively . figure 10 illustrates the tremendous decline in labor requirements per acre , especially since the end of wwii . this decrease is primarily due to adoption of mechanical technologies , in particular the tractor . figure 11 shows the major decrease in labor requirements per unit of production that occurred between 1939 and 1949 . this decrease was due to the changes in technologies available , especially seed and fertilizers . number of hours to produce 100 bushels of corn and soybeans . as the amount of labor required per acre has declined , the number of acres that a farmer can farm has increased . although there will be considerable variation , a conservative estimation of the hours of fieldwork for corn and soybeans is approximately one hour per acre . using this estimate , farming 800 acres would take approximately 800 h of labor and farming 1500 acres would take 1500 h of labor . a 40-hour - a - week job for 50 weeks a year would be 2000 h. of course , the hours of labor for crop production are not evenly distributed over the year . crop production labor has two heavy periods : in the spring at planting time and in the fall during harvest . this makes direct comparisons difficult , but such a comparison does provide some order of magnitude regarding the hours of labor available . in addition to labor - saving technologies , other technologies have been developed that have lead to economies of size and thus an increase in the size of farms . a statewide comparison of the returns to herbicide - resistant soybeans and conventional soybeans in iowa found there was no significant difference in returns . given these findings , why was there such rapid adoption of herbicide - resistant soybeans ? there were essentially no herbicide - resistant acres planted in 1996 , and yet in just 4 or 5 years more than 90% of the soybean acres were using this technology . farmers had a variety of reasons , including ease of harvest and ability to cover more acres . perhaps most telling was the farmer who said that he planted them because they offered weed management that any idiot could do . new machines are capable of covering far more acres in less time and they are able to perform different jobs . strip - tillage is an example of a popular recent practice made possible by changes in equipment . strip - tillage saves soil , but its primary appeal for many is the decrease in labor needed . if costs increase and the resulting changes in revenue do not offset the increases , the profit margins narrow and farmers need to cover more acres to maintain their incomes . technology enables one person to farm more acres , but as more acres are farmed , the costs increase . so , they must adopt technology that allows them to farm even more acres and the cycle escalates further . bulk purchases allow the larger farm to acquire the same input but at a reduced cost . the cost reduction reflects the lower transaction costs for the input supplier : less paperwork , less handling , lower shipping costs , and so forth . in addition , the risk of default or non - payment increases as the number of purchasers increases . it could be argued that even though the probability of the risk of a default increases with more purchasers , the loss associated with any single default would go down and so the expected value would be the same . however , it would be easier and cheaper for the supplier to monitor fewer purchasers and so the probability of any single farm defaulting would be reduced . another advantage for larger farms is the ability to more fully utilize labor and employ labor - saving technology . some technologies such as machinery might be cheaper for a smaller farm if they used smaller equipment . but , other technologies such as yield monitors , soil sampling , and weather stations have relatively fixed costs , and the more bushels or units of production , the cheaper the cost of technology per unit . information technologies , including marketing , would be relatively fixed in price ; the more units of production , the lower the cost per unit . some smaller farms will find it uneconomical to employ these technologies relative to their larger counterparts . one of the major drawbacks is the changing nature of farming as the farm size grows . as a farm gets larger , the farmer changes from being someone actively involved with the agronomic / animal husbandry aspects of farming to being one who is a personnel and office manager . whether or not this is a good move depends on the goals of the individual farmer . the united states is no longer seeing an increase in farmland and instead is actually seeing a decrease as available land is converted to urban and other uses . this means that if farmers are going to expand the amount of land being farmed , they have to travel greater distances . in iowa traveling 50 to 100 miles to farm a parcel of land is becoming more common . the farther a farmer has to travel to reach land being farmed , the greater the cost . equipment has to be hauled adding transportation costs , and extra time is needed to move the equipment from farm to farm . a drawback to traveling greater distances and increasing the amount of acres farmed in general is the loss of time to pay attention to details . studies examining the characteristics of high - profit farms relative to low - profit farms show that management is a key factor . a farmer who is farming many acres will have to be quick about it and can not take the time to ensure that equipment is functioning at the optimum level . another serious drawback to farming more acres or handling more livestock is the inability to fully understand the unique characteristics of each unit of the operation . technology in global positioning and guidance systems can help overcome , this but there are still nuances that can be captured only by personal observation over time . too often with large acreages , farmers do not have the time to observe the land they are farming . almost every study has found that the average cost curve of most agricultural production is l - shaped . that is , costs decrease over a certain size range , but then they become flat . increases in size beyond where the curve becomes flat lead to increased income but not increased efficiency . studies also have shown that the curve may start to increase after certain sizes are reached . one is that agriculture has such tight margins and farmers must increase the volume of production if they are to produce an adequate income . in many cases they get bigger equipment so they can farm more acres . as they farm more acres they have to adopt techniques that increase their costs but also lower their profit margins . as the farmers profit margins tighten they need to have more acres to generate an adequate income . with more acres they need bigger equipment so they can farm more acres . the capital can buy technology that makes life and the job easier but the technology comes with a price . the substitution of capital for labor has led to many changes in agricultural production . unfortunately , today we are seeing situations where the capital is substituting for management . the farmer 's comment that herbicide - resistant soybeans offered him weed management that any idiot could do is a perfect example . they must continually evaluate and manage , but what they manage now are chemicals and methods to overcome natural systems . they do not use management that helps them work with the biological assets of their farms using less capital - intensive assets . ( p. 31 ) the technology being developed today is technology that favors larger farms . it either has a high fixed cost and/or technology that enables one person to cover more acres . the technology we have developed has led to the cheapest out - of - pocket food supply in the world . in the united states today we spend less of our disposable income on food ( 9.8% in 2007 ) than anywhere else in the world . the farmers share of the food dollar has actually been declining , and in 2006 of the farmer received just 19 cents of every $ 1 spent on food in the united states . technological advances have led to the lower portion of disposable income being spent on food and to the farmer receiving a lower share of the food dollar . but , it is important to recognize that increases in disposable income also have contributed to these trends . food has a low - income elasticity ; in other words , after basic food needs are met , increases in income will lead to lower amounts of income being spent on food . this concept is known as engle 's law , which states that with constant tastes and preferences as the level of income rises , the proportion of income spent on food decreases . this concept , named after the statistician ernst engle , does not imply that expenditure on food will not change but that the proportion of income spent on food will decrease . in spite of the increases in income , technology and outcomes of larger farms capturing current economies of size include questions about the stability of the food supply and potential monopolization of food production . based on the 2007 census , farms with sales over $ 500,000 accounted for 5% of the farms and 74% of all agricultural sales . this means that just 116,286 farms accounted for almost three fourths of all the value of sales of agricultural products in the country . the changing structure of agriculture can lead to a breakdown in competition and maybe even monopolization of food production . an industry with numerous producers may be more likely to supply food in both good and bad economic times . ( p. 41 ) he went on to note that this could be due to profits elsewhere , bad management , labor unrest , attempts to manipulate the market , weather problems in concentrated areas , etc . the recent cases of salmonella in tomatoes , sprouts , peppers , meat , and peanut butter are examples of what can occur . it can be argued that larger farms are better able to afford the technology necessary to prevent such food contamination . this harkens back to the ability to spread fixed costs over more output , thus lowering average costs of production . there are arguments that can be made about food contamination episodes , but one thing is clear : large - scale production can have large - scale impacts when problems do occur . illnesses and the costs of massive recalls are not likely to be factored into most discussions of the advantages of larger farms and agricultural operations . environmental impacts are another factor not clearly accounted for in the discussion regarding economies of scale . it is interesting that some of the production technologies producing lower soil erosion also promote more chemical usage and produce labor savings which leads to economies of size . no - till and strip - till are examples of such conservation - friendly production technologies . monitoring requirements , fencing regulations , and other regulations generally have a large component of their costs as fixed costs . this means that the larger - scale operations will have more units of output over which to spread the costs , thus lowering the average costs of production . rural communities / life , intrinsic rural values , and socioeconomic welfare are all being affected by the changing structure of agriculture . larger farm sizes spurred by economies of scale have had a significant impact on rural america as witnessed by the formation of the small farms commission . economies of size exist in production agriculture for a variety of reasons and have far - reaching consequences for food production and rural america 's future . whether the economies of size would exist to the same extent if we accounted for all costs is debatable . research that favors large - scale farming and reducing the amount of labor needed has led to many of the economies of size . what would happen if we made better use of the biological nature of production agriculture , researched technologies that favored small - scale production , and accounted for all costs is unknown . one thing is certain : the current path with its emphasis on economies of size has produced many undesirable consequences .

economies of size refer to the ability of a farm to lower costs of production by increasing production . agriculture production displays an l - shaped average cost curve where costs are lower initially but reach a point where no further gains are achieved . spreading fixed costs , bulk purchases , and marketing power are cited as reasons for economies of size . labor - reducing technologies may be the primary reason . most studies do not include the external costs from prophylactic antibiotic use , impact on rural communities , and environmental damage associated with large - scale production . these can contribute to the economies of size .

INTRODUCTION CENSUS DATA ECONOMIES OF SIZE IN IOWA DISCUSSION CONCLUSIONS

it focuses mainly on midwestern agriculture , but to the extent possible other regions will be discussed . the theoretical underpinnings and economic discussions can be found in other sources . before beginning any discussion on economies of size the concept of economies of size means that the average cost per unit of production decreases as the size of the farm increases . the economies can occur because the farmer is able to spread more production over the same level of fixed expenses . or , economies of size can occur when a farm is able to obtain volume discounts for inputs such as seed or fertilizer . an example would be the cost for pollution monitoring around a swine production facility . if the farm is required to monitor the groundwater around the facility for contamination , they must put in a well and monitoring equipment , which represent fixed costs and can serve a large number of pigs . as the number of pigs sold increases , the costs for this aspect of production would decrease . one is economies of scale , which measure what happens if all inputs are increased by the same proportion . if costs per unit go down , there are increasing economies of scale , and if the costs per unit remain the same , there are constant returns to scale . the other concept is economies of scope , which refer to reducing costs for using resources by spreading the resources over more than one enterprise . hofstrand summarized the concepts of size and scope by noting that size spreads fixed resources over more units of output whereas scope spreads the cost of a given set of resources or skills over more than one product or enterprise . discussion of economies of size in production agriculture and the desirability of small farms has ebbed and flowed over time . one of this country 's earliest political debates centered on the conflicting views held by thomas jefferson and alexander hamilton regarding land ownership . jefferson argued for family ownership of farms as a means of ensuring interest in the democratic process . hamilton argued for selling the land to the highest bidder as a means of paying off the revolutionary war debt . in the early part of the 20th century problems emerged for this ideal , problems related to economies of size . in 1909 president roosevelt formed the country life commission to address the issues of poverty in rural america . this commission report renewed the efforts to improve the lives of the farmers by increasing their productivity . the us economy moved through two world wars and the great depression after the parity price period . in 1954 president eisenhower ordered the us dept of agriculture to prepare a report on the state of the agricultural economy . the report concluded that expanding agricultural productivity was the key and one of the recommendations to increase agricultural productivity was to allow farms to expand in order to take advantage of new labor - reducing technologies . others have noted that while some of the implemented government policies were successful , most of the reductions in farm labor and the corresponding increases in farm size were the result of changing technology . at the beginning of the 20th century there were concerns over the need to add more technology to production agriculture . these were followed in the middle of the century by a feeling that the new technology had already removed the need for much of the labor and what we needed to do was move agricultural workers to non - farm industries . towards the end of the 20th century , new fears were raised that family farms were losing ground and the move to increase productivity by decreasing labor may have yielded undesirable consequences . dedicated to optimizing the labor and ingenuity of small farm operators and the biological assets of their farms using less capital - intensive investments . ( p. 31 ) over time in the united states economies of size with respect to substituting capital for labor have been encouraged and discouraged . research and technology have been the primary drivers in changing the situation with respect to economies of size . the census of agriculture is the best source for a consistently gathered set of agricultural data for the entire country . the census provides an opportunity to examine several aspects of the issues related to economies of size . the first agricultural census was taken in 1840 and it was conducted every 10 years until 1920 . since the bureau of the census conducted the census of agriculture until 1996 when the responsibility was transferred to the usda primarily because the census bureau was going to increase the size needed for an operation to be considered a farm . one of the major difficulties with examining national farm - level data is how to determine what is considered a farm . for the purposes of the census , a farm is defined as any place from which $ 1000 or more of agricultural products were produced and sold , or normally would have been sold , during the census year . ( p. ix ) this definition has been used since 1974 and has created many problems and inconsistencies when people use or try to interpret the census data . another problem is the change in census coverage initiated by the usda in 2002 in an effort to survey more very small farms . the change in coverage has led to a change in the composition of the farms considered in the census . figure 1 shows the distribution of farms and sales in the united states based on sales categories . notice in this figure the very smallest category , sales of less than $ 1000 , makes up over 30% of the farms . it is important to remember that the definition of a farm is any place that could have sold the farms are assigned points based on the agricultural enterprises and if the points total 1000 , they are included as farms in the count . one pig has a point value of 150 , so raising 7 pigs would classify a place as being a farm . a horse has a point value of 200 whether or not it is sold , which means that 5 horses classify a place as a farm . at the other end of the spectrum , farms with sales less than $ 5000 accounted for 50% of the farms and generated less than 1% of the sales . with this caveat in mind there are two ways to use the census data to examine economies of size in us agriculture . the more sales a farm can generate for each dollar of expense , the more profitable it will be . such efficiency gains can be thought of as economies of size at the aggregate level . once this level of sales is reached the ratio flattens out , indicating no further gains in efficiency with respect to expenses relative to sales . figure 2 shows that there are very definite gains in efficiency as a farm increases in size but the rate of gain slows and actually decreases as size increases . figure 3 also illustrates the extent of economies of size present in the us farm sector . economies of size for agricultural production in iowa can best be illustrated using data from the iowa farm business association ( ifba ) , a record - keeping service for iowa farmers . figure 4 shows the total economic costs of production per bushel of corn for farms in the ifba based on the number of row crop acres farmed . figure 5 shows the average cost per bushel for soybeans based on the number of row crop acres . figures 4 and 5 display what is known as an l - shaped average cost curve . that means there are initial economies of size but these size advantages dissipate , and then costs remain relatively flat over a range of sizes . most normative studies of crop farms , both early and recent , while identifying economies of size , have given little evidence that the cost curve deviates significantly from a sagging l shape . ( p. 206 ) hallam further discussed the economies of size in agricultural production : the general conclusion is that while some economies of size or scale may exist for livestock farms that significant economies , at least as conventionally defined , do not exist for most crop production activities . while differences in efficiency and growth paths differ among firms , few of these seem to be directly related to economies of size and scale . one observation by hallam was the discrepancy between farmers with respect to costs of production . this is illustrated in figure 6 , which shows the costs per bushel for corn when the cost groups are divided into thirds . figure 6 reports an almost $ 1.50 per bushel difference in costs of production between the high - cost producers and the low - cost producers . figure 7 shows that the high - cost producers had the fewest corn acres , whereas the middle - cost producers had the greatest number of corn acres . figures 6 and 7 reveal a couple of distinguishing characteristics that are important to remember when considering production agriculture . first , there is considerable variation among farms , even farms producing the same crop . the second observation is that the number of acres is not a guarantee of lower cost production . hallam noted that livestock production appears to be different with respect to economies of size . livestock production efficiencies and the changing structure of animal agriculture have been discussed in many places , most recently by macdonald and mcbride . figure 8 shows the costs of production per hundredweight for farms in the iowa farm business association based on the number of pigs marketed . figure 9 presents the cost per hundredweight of pork produced based on the number of sows . they show a more extreme drop in costs of production as size increases when compared to the crop costs shown in figures 4 and 5 . technological changes and changes in production have led to the dramatic decreases in costs of production . farrow - to - finish swine producers can achieve essentially the same profitability per unit of production across a wide range of volumes of production . ( p. 1 ) macdonald and mcbride note , there are substantial economies of scale up to certain threshold sizes , and farms can operate efficiently at sizes that are much larger than the thresholds . ( p. 36 ) there appears to be two major differences between animal and crop production with respect to economies of size . one of the major differences is the vertical integration of the industry , which has led to a major consolidation in the processing industry . the decrease in the number of plants has been coupled with an increase in the number of animals slaughtered by size of plants . by 1998 one of the biggest components of these economies of size is procurement of the animals . this favors the larger - scale producers and requires either an increase in the size of production or some form of joint marketing . production contracts have become commonplace and today the majority of pork is raised under some form of contract . smaller producers may have similar costs of production but access to the market has become a significant problem . the second major factor that has led to the increased size of animal production operations is the ability to pass along costs to those not making the production decisions . these external costs are generally not calculated in the costs of production or measuring the efficiency of the large scale production . macdonald and mcbride discussed these external costs , which generally fall into two major categories . large numbers of animals housed together have greater potential for disease . routine antibiotic use can increase the chances for antibiotic resistance to develop and for contamination of the product . the severity of these problems depends on the individual operator , and the potential for problems increases as the concentration of the animals increase . the specialization of production often ignores the benefits and advantages of the synergy that exists in agriculture . crops can feed the animals , animal manure can provide nutrients for the crops , and the farmers can add value to their production with a more holistic approach . increased environmental costs , food safety concerns , undesirable odor , and concentration of the food supply are just a few of the concerns that have arisen with the change in philosophy of producing animals and animal products . ( p. 1 ) this industrialized approach has led us to many of the problems and issues we face today . if farms display an l - shaped average cost curve and expansion does not lower the costs of production , why are we seeing such expansion in farm size ? the basic answer is that farms are getting larger because they do not incur diseconomies of size . as the number of units of output increases and there is no significant difference in the cost of production , income will increase . for example , using the average acreage shown in figure 4 , a farm with 800 acres of corn would have an average total cost of production of approximately $ 3.00 per bushel . if the yield was 200 bushels per acre and the price of corn was $ 3.50 per bushel , the farm would make $ 80,000 [ ( $ 3.50-$3.00 ) 200 800 ] . such expansion is possible because of the labor - saving technologies that have been developed for production agriculture . figures 10 and 11 show the hours of labor required for corn and soybeans per acre and per 100 bushels , respectively . this decrease is primarily due to adoption of mechanical technologies , in particular the tractor . figure 11 shows the major decrease in labor requirements per unit of production that occurred between 1939 and 1949 . this decrease was due to the changes in technologies available , especially seed and fertilizers . number of hours to produce 100 bushels of corn and soybeans . as the amount of labor required per acre has declined , the number of acres that a farmer can farm has increased . although there will be considerable variation , a conservative estimation of the hours of fieldwork for corn and soybeans is approximately one hour per acre . in addition to labor - saving technologies , other technologies have been developed that have lead to economies of size and thus an increase in the size of farms . there were essentially no herbicide - resistant acres planted in 1996 , and yet in just 4 or 5 years more than 90% of the soybean acres were using this technology . farmers had a variety of reasons , including ease of harvest and ability to cover more acres . new machines are capable of covering far more acres in less time and they are able to perform different jobs . strip - tillage is an example of a popular recent practice made possible by changes in equipment . if costs increase and the resulting changes in revenue do not offset the increases , the profit margins narrow and farmers need to cover more acres to maintain their incomes . technology enables one person to farm more acres , but as more acres are farmed , the costs increase . so , they must adopt technology that allows them to farm even more acres and the cycle escalates further . bulk purchases allow the larger farm to acquire the same input but at a reduced cost . the cost reduction reflects the lower transaction costs for the input supplier : less paperwork , less handling , lower shipping costs , and so forth . it could be argued that even though the probability of the risk of a default increases with more purchasers , the loss associated with any single default would go down and so the expected value would be the same . another advantage for larger farms is the ability to more fully utilize labor and employ labor - saving technology . some technologies such as machinery might be cheaper for a smaller farm if they used smaller equipment . but , other technologies such as yield monitors , soil sampling , and weather stations have relatively fixed costs , and the more bushels or units of production , the cheaper the cost of technology per unit . information technologies , including marketing , would be relatively fixed in price ; the more units of production , the lower the cost per unit . some smaller farms will find it uneconomical to employ these technologies relative to their larger counterparts . as a farm gets larger , the farmer changes from being someone actively involved with the agronomic / animal husbandry aspects of farming to being one who is a personnel and office manager . this means that if farmers are going to expand the amount of land being farmed , they have to travel greater distances . the farther a farmer has to travel to reach land being farmed , the greater the cost . equipment has to be hauled adding transportation costs , and extra time is needed to move the equipment from farm to farm . studies examining the characteristics of high - profit farms relative to low - profit farms show that management is a key factor . too often with large acreages , farmers do not have the time to observe the land they are farming . almost every study has found that the average cost curve of most agricultural production is l - shaped . one is that agriculture has such tight margins and farmers must increase the volume of production if they are to produce an adequate income . in many cases they get bigger equipment so they can farm more acres . as the farmers profit margins tighten they need to have more acres to generate an adequate income . with more acres they need bigger equipment so they can farm more acres . the substitution of capital for labor has led to many changes in agricultural production . they do not use management that helps them work with the biological assets of their farms using less capital - intensive assets . the technology we have developed has led to the cheapest out - of - pocket food supply in the world . in the united states today we spend less of our disposable income on food ( 9.8% in 2007 ) than anywhere else in the world . the farmers share of the food dollar has actually been declining , and in 2006 of the farmer received just 19 cents of every $ 1 spent on food in the united states . technological advances have led to the lower portion of disposable income being spent on food and to the farmer receiving a lower share of the food dollar . but , it is important to recognize that increases in disposable income also have contributed to these trends . food has a low - income elasticity ; in other words , after basic food needs are met , increases in income will lead to lower amounts of income being spent on food . this concept is known as engle 's law , which states that with constant tastes and preferences as the level of income rises , the proportion of income spent on food decreases . in spite of the increases in income , technology and outcomes of larger farms capturing current economies of size include questions about the stability of the food supply and potential monopolization of food production . based on the 2007 census , farms with sales over $ 500,000 accounted for 5% of the farms and 74% of all agricultural sales . the changing structure of agriculture can lead to a breakdown in competition and maybe even monopolization of food production . an industry with numerous producers may be more likely to supply food in both good and bad economic times . ( p. 41 ) he went on to note that this could be due to profits elsewhere , bad management , labor unrest , attempts to manipulate the market , weather problems in concentrated areas , etc . the recent cases of salmonella in tomatoes , sprouts , peppers , meat , and peanut butter are examples of what can occur . it can be argued that larger farms are better able to afford the technology necessary to prevent such food contamination . this harkens back to the ability to spread fixed costs over more output , thus lowering average costs of production . there are arguments that can be made about food contamination episodes , but one thing is clear : large - scale production can have large - scale impacts when problems do occur . illnesses and the costs of massive recalls are not likely to be factored into most discussions of the advantages of larger farms and agricultural operations . environmental impacts are another factor not clearly accounted for in the discussion regarding economies of scale . it is interesting that some of the production technologies producing lower soil erosion also promote more chemical usage and produce labor savings which leads to economies of size . no - till and strip - till are examples of such conservation - friendly production technologies . monitoring requirements , fencing regulations , and other regulations generally have a large component of their costs as fixed costs . this means that the larger - scale operations will have more units of output over which to spread the costs , thus lowering the average costs of production . rural communities / life , intrinsic rural values , and socioeconomic welfare are all being affected by the changing structure of agriculture . larger farm sizes spurred by economies of scale have had a significant impact on rural america as witnessed by the formation of the small farms commission . economies of size exist in production agriculture for a variety of reasons and have far - reaching consequences for food production and rural america 's future . whether the economies of size would exist to the same extent if we accounted for all costs is debatable . research that favors large - scale farming and reducing the amount of labor needed has led to many of the economies of size . what would happen if we made better use of the biological nature of production agriculture , researched technologies that favored small - scale production , and accounted for all costs is unknown . one thing is certain : the current path with its emphasis on economies of size has produced many undesirable consequences .

dopamine neurons play a pivotal role in several brain functions , including cognition , reward , and motor output ( jentsch et al . , 1997 , servan - schreiber et al . , 1998 , katz , 1979 , lippa et al . , 1973 , graeff , 1966 , andn and strmbom , 1974 , westermann and staib , 1976 ) . changes in dopamine activity have been implicated in , e.g. , schizophrenia , addiction , and parkinson disease ( stevens et al . , 1974 , seeman , 2013 , compton et al . , 1996 , ungless et al . , 2010 , a physiologically appropriate dopamine output depends , to a great extent , on homeostatic mechanisms . these mechanisms are only partly understood but includes immediate feedback through autoreceptors ( cragg and greenfield , 1997 ) . dopamine is also an important signaling molecule in the hypothalamus , where it inhibits prolactin release from the anterior pituitary ( freeman et al . , 2000 ) . the main source of neuroendocrine dopamine is the tuberoinfundibular dopamine ( tida ) neurons , located in the dorsomedial hypothalamic arcuate nucleus ( dmarc ) . tida neurons release dopamine into the portal capillaries at the median eminence ( me ) for transport to the anterior pituitary gland ( lyons and broberger , 2014 ) . patterned dopaminergic inhibition within the lactotrophic axis is essential for successful reproduction , as evidenced by the impaired fertility and other sexual side effects associated with the hyperprolactinaemia commonly seen in patients treated with antipsychotics with dopamine antagonist properties ( holt and peveler , 2011 ) . the pituitary consequences of tida neuron activation are well established ( fuxe , 1963 ) . however , the actions of dopamine on tida neurons have been the subject of only a few studies and remain poorly understood . biochemical studies have indicated that tida neurons may be under inhibitory influence by dopamine receptors of the type 2 family ( d2r ) ( berry and gudelsky , 1991 , lin et al . , 2000 , liang et al . , 2014 ) , but other investigators have found no effect ( demarest and moore , 1979a , timmerman et al . , 1995 ) or even disinhibition via d2r ( durham et al . , 1996 ) . how autoreceptors affect tida membrane properties and network activity has not been studied . in the ventral tegmental area ( vta ) and the substantia nigra ( sn ) of the midbrain , where this issue has been studied in more depth , autoinhibition appears to protect dopamine neurons from runaway excitation ( bjrklund and lindvall , 1975 , aghajanian and bunney , 1977 , paladini et al . , 2003 , beckstead et al . , 2004 , gentet and williams , 2007 ) . this has been shown to involve d2r - mediated activation of hyperpolarizing k conductances ( silva and bunney , 1988 ) . tida neurons share several features with their mesencephalic counterparts , including co - transmitters , such as gaba ( everitt et al . , 1984 ) and neurotensin ( ibata et al . , 1983 ) , and the ability to exhibit both tonic and phasic discharge configurations . it is not known , however , whether mechanisms of autoregulation are similar or different in tida cells . intriguingly , recent in vitro studies have revealed that tida neurons in the male rat discharge in an oscillating pattern , which can be switched to tonic discharge by hormones and neurotransmitters ( lyons et al . , 2010 , lyons et al . , 2012 , briffaud et al . , 2015 ) that , in in vivo studies , have correlated to prolactin release and lactation . this phenomenon raises the question of whether and how dopamine participates in the maintenance of this rhythmic behavior . the important question of how an oscillating circuit tunes its activity to internal feedback is poorly understood and ideally studied in a spontaneously active preparation . furthermore , understanding the homeostatic regulation of tida neurons is clinically important , as several of the most troubling side effects associated with antipsychotic , as well as antidepressant , drugs derive from their ability to raise serum prolactin ( madhusoodanan et al . , 2010 , the results we present here suggest the existence of a ultra - short autoinhibitory loop in tida neurons encompassing both pre - and postsynaptic mechanisms that is continuously adjusted to ambient somatodendritic dopamine levels . the tida neurons in the male rat exhibit stereotyped electrophysiological properties , including anomalous inward rectification , an a - like current conductance , and a slow ( 0.050.07 hz ) oscillation . this electrical profile correlates to the expression of tyrosine hydroxylase and can reliably be used to identify these cells for in vitro recordings ( lyons et al . , 2010 ) . using these criteria , we first sought to determine how tida network behavior is affected by agonists of the dopamine receptors , using whole - cell recordings . application of dopamine ( 20 m ) induced a reversible increase of the oscillation cycle duration ( + 7.66 1.47 s ; n = 5 ; p < 0.05 versus control ; anova ; figures 1ba1bd ; table s1 ) . to identify the different components of the cycle , a custom written analysis program was developed ( figure 1a ; supplemental experimental procedures ) . thus , the dopamine - induced slowing of the oscillation was found to result primarily from an increase in phase 1 ( i.e. , initial slow depolarization from nadir : + 5.03 1.13 s ; n = 5 ; p < 0.05 versus control ; anova ; figure 1bb ; table s1 ) but also from a smaller increase in phase 3 duration ( spiking phase : + 2.92 0.71 s ; n = 5 ; p < 0.05 versus control ; anova ; figure 1bb ; table s1 ) . the duration of phases 2 ( fast depolarization ) and 4 ( repolarization ) were not affected by the application of dopamine . application of the non - selective dopamine receptor agonist apomorphine ( 20 m ) resulted in similar changes in oscillation cycle properties , as seen with dopamine ( n = 5/5 ; figures 1ca1cd ; table s1 ) . application of the d2r agonist , 20 m quinpirole ( qp ) also caused changes to oscillation parameters indistinguishable from those observed following dopamine application ( n = 8/8 ; figure 1e ; table s1 ) . in the presence of the d1-type - receptor ( d1r)-specific agonist , skf-81,297 ( 10 m ) ( reavill et al . , 1993 ) , however , oscillation parameters were not significantly altered ( n = 5/5 ; figure 1d ; table s1 ) . as it could not , at this point , be excluded that ongoing dopamine release in the slice might be acting on d1 receptors diminishing an effect of exogenous agonists ( dewey et al . , 1992 ) , we also tested the d1r antagonist , sch-23,390 ( 10 m ) ( bourne , 2001 ) , but prolonged application of this compound did not affect oscillation parameters ( n = 7/7 ; table s1 ) . last , we tested whether d2r activation alters rhythmicity in the presence of fast - ionotropic blockade ( fib ) ( 100 m picrotoxin [ ptx ] , 10 m cnqx , and 25 m ap-5 ) . in these conditions , 20 m qp led to a prolongation of cycle duration ( 5.99 0.91 s ; n = 6 ; p < 0.01 versus fib ) by increasing phase 1 and phase 3 durations ( figure s2 ; table s1 ) . these data identify d2r as an autoreceptor that modulates tida membrane properties and slows down network rhythm . these findings raised the question of whether there is ongoing d2r activation due to endogenous dopamine release in the spontaneously active slice . application of the selective d2r antagonist eticlopride ( 1 m ) resulted in a depolarization of the tida oscillation nadir ( + 2.46 0.77 mv ; n = 5 ; p < 0.05 versus control ; figures 2aa and 2ab ; table s1 ) concomitant with a decrease in phase 1 duration ( 1.88 0.65 s ; n = 5 ; p < 0.05 versus control ; figure 2ab ; table s1 ) and an increase of phase 3 duration ( + 1.51 0.37 s ; n = 5 ; p < 0.05 versus control ; figure 2ab ; table s1 ) . the cycle duration remained unchanged ( 0.64 0.56 s ; n = 5 ; p > 0.05 versus control ; figure 2ab ; table s1 ) , as predicted by the opposite changes in the duration of phases 1 and 3 . application of a second d2r antagonist , the typical antipsychotic haloperidol ( 1 m ) , had similar effects ( n = 5/5 ; figures 2ba and 2bb ; table s1 ) . closer examination of the individual action potentials ( aps ) revealed that application of either eticlopride or haloperidol induced a statistically significant decrease in amplitude and broadening of the aps ( figures 2ad and 2bd ; table s2 ) . this effect on amplitude and half - width increased in size from the first to the fifth ap ( figures 2ad and 2bd ; table s2 ) . the effect on the waveform of the fifth ap of a cycle is demonstrated in figures 2ac and 2bc . similar changes in ap waveform have previously been described in midbrain dopamine neurons as a prodrome of the depolarization block ( db ) ( bikson et al . , 2003 , , 2015 , richards et al . , 1997 ) induced by antipsychotics ( grace and bunney , 1986 ) . this similarity , and the depolarized nadir induced by eticlopride , prompted us to examine the effects of higher concentrations of the d2r antagonists . application of eticlopride ( 10 m , n = 5/5 ) or haloperidol ( 10 m , n = 11/11 ) resulted in a gradual depolarization and ultimate collapse of the oscillation ( figures 2ca and 2cb ) . ap amplitude gradually decreased and was eventually followed by a complete loss of aps concurrent with the collapse of the oscillation ( figures 2ca and 2cb ) . injection of depolarizing square current pulses resulted only in a single , low - amplitude ap , verifying that the capacity for regenerative firing was compromised ( figure 2cc ) . collectively , these results suggest that tida neurons respond to d2r antagonists by excitation , leading to db in a manner highly similar to what has been described in midbrain dopamine neurons following chronic application of dopamine antagonist antipsychotics ( grace and bunney , 1986 ) . the partly opposite effects of d2r agonists and antagonists suggest that the baseline behavior of tida neurons may depend on continuous dopaminergic feedback . to further explore endogenous dopamine release in the dmarc we investigated the effect of blocking the dopamine transporter ( dat ) . evidence has been presented for both the absence ( demarest and moore , 1979b , annunziato et al . , 1980 ) and existence ( demaria et al . , 2000 , boss et al . , 1997 ) of functional dopamine reuptake in these cells . we addressed this issue directly by applying a dat blocker , either gbr-12783 or methylphenidate , and recorded the electrophysiological response . in the presence of gbr-12783 ( 1 m ) and methylphenidate ( 50 m ) , tida neurons exhibited changes similar to those elicited by d2r agonists , i.e. , dopamine , apomorphine , and qp , and slowed down oscillation frequency ( gbr-12783 , n = 8/8 ; methylphenidate , n = 5/5 ; figures 2da and 2db ; table s1 ) . while both phases 1 and 3 were significantly prolonged in the presence of methylphenidate , the application of gbr-12783 only resulted in a significantly increased duration of phase 1 . these data suggest the existence of endogenous release of dopamine from tida neurons and the presence of functional dopamine reuptake at the somatic level in these cells . next , we sought to identify the membrane actions that underlie the d2r - mediated modulation of tida neurons . application of tetrodotoxin ( ttx ; 0.5 m ) abolishes the tida oscillation ( lyons et al . , 2010 ) likely owing to the inhibition of a persistent na current . under these conditions , and with membrane potential set to 60 mv via negative current injection , application of qp ( 20 m ) resulted in a hyperpolarization of 4.19 0.78 mv ( n = 6 ; p < 0.01 versus control ; figures 3a and 3c ) . this hyperpolarization was attenuated when qp was applied to cells recorded with an intracellular solution containing cs ( p < 0.05 versus qp ) ( 1.69 0.45mv ; n = 9 ; p < 0.05 versus control ; figures 3a3c ) . while cs is known to block h current ( ih ) , the ability of intracellularly applied cs to attenuate this current is controversial ( harris and constanti , 1995 ) . thus , to more specifically test whether the phase 1 prolongation by d2r activation is partly an effect involving ih , qp was co - applied with the ih blocker zd-7288 ( 10 m ) . under these conditions , application of qp failed to significantly increase phase 1 duration , whereas a prominent prolongation of phase 3 remained ( figures 3d and 3e ) . these findings suggest a d2r - mediated decrease of ih . to characterize the ionic currents modulated by d2r agonism , tida neurons were recorded in voltage - clamp mode in the presence of ttx ( 0.5 m ) at a holding potential ( vhold ) of 60 mv , i.e. , at which the qp - induced hyperpolarization had been observed . application of qp ( 20 m ) did not significantly affect holding current under these conditions ( 0.49 1.5 pa ; n = 6 ; p > 0.05 versus control ; figure 3f ) . to determine current changes that might occur throughout the membrane potential spectrum , a voltage - clamp ramp protocol of 500-ms durations was used , allowing the identification of d2r - mediated currents throughout the voltage spectrum of 115 mv to 0 mv ( figure 3ga ) . digital subtraction of the ramp performed with control versus qp ( figure 3 gb ) revealed a qp - induced current ( iqp ) that was a non - reversing net outward current , active only at membrane potentials positive of approximately 45 mv ( n = 5 ; figure 3gc ) , increasing with depolarization within the range tested ( vhold = 0 mv ; + 124.83 34.97 pa ) . at a vhold negative of 50 mv no prominent iqp is evoked , suggesting that the cs - sensitive current underlying the phase 1 depolarization ( discussed earlier ) is not of sufficient amplitude to be isolated under these conditions . the iqp may reflect the activation of an outward current and/or the inhibition of an inward current . as the i - v relationship of iqp is similar to that of a reversed ica ( nowycky et al . , 1985 , fedulova et al . , 1985 ) , we tested whether d2r stimulation decreased ca flux in tida neurons , using a voltage ramp protocol . application of qp ( 20 m ) was followed by a substantial and significant decrease of the high - voltage - activated ( hva ) ca currents ( 52.71 9.09 pa ; n = 7 ; p < 0.01 versus control ; figure 4a ) . control experiments did not identify a prominent low - voltage - activated , t - type ca current in tida neurons ( figure 4a ) . application of the l - type - specific blocker nimodipine ( 10 m ) , however , attenuated the ca current ( 62.8 19.04 pa ; n = 5 ; p < 0.05 versus control , anova ; figure 4b ) . under l - type ca channel blockade , the qp - induced decrease in hva current was significantly diminished ( 34.6 9.42 pa ; n = 5 ; p < 0.05 versus nimodipine , anova ; figure 4b ) . application of the n - type - specific ca channel blocker -conotoxin gvia ( 1 m ) decreased the ca currents ( 71.4 22.72 pa ; n = 5 ; p < 0.05 versus control , anova ; figure 4c ) . under n - type ca channel blockade , application of qp ( 20 m ) resulted in a small , but significant , decrease of the remaining ca current ( 7.8 1.91 pa ; n = 5 ; p < 0.05 versus control , anova ; figure 4c ) . last , upon co - application of nimodipine and -conotoxin gvia , the ca current decreased dramatically ( 119.4 6.94 pa ; n = 5 ; p < 0.001 versus control , one - way anova ; figure 4d ) . in these conditions , application of qp ( 20 m ) failed to induce a significant decrease of the remaining ca current ( 10.6 5.32 pa ; n = 5 ; p > 0.05 versus control , anova ; figure 4d ) . to assess whether this d2r - mediated decrease in l - type ca currents could prolong phase 3 duration , short applications of low - ca / high - mg artificial cerebrospinal fluid ( acsf ) were performed . a reversible phase 3 prolongation was demonstrated under these conditions ( 59.98 24.45 s ; n = 7 ; p < 0.05 versus control ; figures 4ea and 4eb ) . similarly , application of nimodipine resulted in prolongation of phase 3 ( 18.33 7.61 s ; n = 8 ; p < 0.05 versus control ) in current - clamp ( n = 4 ) as well as voltage - clamp ( n = 4 ) recordings ( figures 4fa4fc ) . as ca influx through l - type channels has commonly been implicated in the activation of ca - dependent k channels ( e.g. , ( marrion and tavalin , 1998 ) , we last applied the big conductance ca - activated k channel ( bk ) antagonist , charybdotoxin ( 200 nm ) . in the presence of charybdotoxin , phase 3 was also significantly increased in duration ( 6.41 1.93 s ; n = 8 ; p < 0.05 versus control ) . these data suggest that tida d2r activation leads to inhibition of the l - type ( cav1.1 - 1.4 ) and n - type ( cav2.2 ) hva ca channels accounting for the prolongation of phase 3 , possibly via the interplay of ca and bk channels responsible for phase 3 termination . next , we investigated whether , in addition to postsynaptic modulation of tida membrane currents , d2r stimulation also affects synaptic input to these neurons . the frequency of inhibitory synaptic input in tida neurons is higher in phase 3 than in phase 1 ( lyons et al . , 2010 ) ( figures 5aa5ac ; phase 1 : spontaneous inhibitory post - synaptic current ( sipsc ) inter - event interval ( iei ) = 335.5 56.57 ms ; phase 3 : sipsc iei = 223 45.18 ms ; difference in means sem , 112.5 32.55 ms ; p < 0.05 ; n = 5 ) . this discrepancy may reflect different sources of inhibitory input onto tida neurons across the oscillatory cycle . the iei for sipscs in the presence of qp ( 20 m ) was significantly decreased during phase 1 ( 125 40.09 ms ; n = 5 ; figures 5ba and 5bb ; ks-2 , p < 0.05 versus control ; t test , p < 0.05 versus control ; figures 5ba and 5bb ) but significantly increased during phase 3 ( + 60.43 21.62 ms ; n = 5 ; figures 5ca and 5cb ; ks-2 , p < 0.05 versus control ; t test , p < 0.05 versus control ; figures 5ca and 5cb ) . in contrast , the amplitude of sipsc was not affected by qp application during either phase 1 ( + 1.39 3.19 pa ; n = 5 ; figure 5bc ; ks-2 , p > 0.05 versus control ; t test , p > 0.05 versus control ; figures 5ba and 5bc ) or phase 3 ( + 0.34 4.75 pa ; n = 5 ; figure 5cc ; ks-2 , p > 0.05 ; t test , p > 0.05 ; figures 5ca and 5cc ) . analysis of spontaneous excitatory postsynaptic currents ( sepscs ) revealed no significant changes ( figure s3 ) . likewise , miniature ipscs and epscs ( recorded in the presence of 0.5 m ttx ) were affected neither in iei nor in amplitude ( figure s4 ) . taken together , these data suggest that d2r stimulation increases inhibitory input during phase 1 , while it decreases it during phase 3 . the qp - induced increase in sipsc frequency during phase 1 suggests the possibility that the d2r - mediated decrease of oscillation frequency may be partly caused by augmented synaptic inhibition . if so , the effect of qp on phase 1 should be diminished or abolished in the absence of gabaa - mediated inhibition . thus , ptx ( 100 m ) was bath applied prior to qp ( 20 m ) application . under these conditions , the qp - mediated increase in cycle duration via prolongation of phase 1 was diminished by 48% ( figures 6a and 6c6e ; n = 6 ) . importantly , the qp - induced increase in phase 3 duration was not affected by ptx application ( figure 6e ; n = 6 ; p > 0.05 ) . the combined blockade of gabaa ( by ptx ) and gabab receptors ( by the antagonist , cgp-55845 ; 10 m ) did not produce any additional attenuation of the qp - mediated increase in cycle period or phase 1 or 3 duration , compared to ptx alone ( n = 5 ; figures 6b6e ; table s1 ) . these data demonstrate that increased gabaa transmission contributes to dopaminergic autoregulation of the tida oscillation frequency , acting within the same membrane potential spectrum as , and likely parallel to , a cs - sensitive hyperpolarizing postsynaptic current ( figures 3a3c ) . such regulation has generally been studied in the context of multicomponent feedback loops , involving actions of the corresponding pituitary hormone or a peripheral target hormone on the hypothalamic master , prolactin can stimulate both dopamine production ( clemens and meites , 1968 , hkfelt and fuxe , 1972 , demarest et al . , 1986 , gonzalez et al . , 1988 ) and ap discharge and waveform ( lyons et al . , 2012 , brown et al . , 2012 ) in tida cells to increase the suppression of its own release in the pituitary . however , the possibility that parvocellular neurons can autoregulate their own activity , as a means of immediate homeostatic tuning , has received little attention . in gonadotropin - releasing - hormone ( gnrh)-expressing neurons ( bedran de castro et al . , 1985 ) involving modification of the electrical activity ( xu et al . , 2008 ) . in tida neurons , the literature offers conflicting evidence for ( berry and gudelsky , 1991 , liang and pan , 2001 ) and against ( demarest and moore , 1979a , durham et al . , 1997 , timmerman et al . , 1995 ) importantly , the possibility that membrane properties and network activity are targeted by autoreceptors has not been explored on identified tida neurons . our experiments reveal that increasing d2r activation causes two coincident changes in the tida duty cycle : a prolonged duration of phase 1 , resulting in a slower oscillation , followed by a minor prolongation of phase 3 . the similar reconfiguration of the oscillation following the exogenous application of qp , as when ambient endogenous dopamine was increased by gbr-12783 and methylphenidate induced dat blockade ( although the former prolonged only phase 1 ) , strongly indicates that these effects are not simply pharmacological . it should be stressed that the present results do not unequivocally rule out dopaminergic sources other than tida neurons as the endogenous ligand source for autoreceptors . indeed , the term autoreceptor commonly refers to a receptor whose ligand is produced by the same neuron ; it does not , as such , posit that the ligand is exclusively supplied by that cell . however , given that the slice preparation used here likely includes little of dopamine populations outside of the a12 group , the results using receptor and dat blockers favor the interpretation that tida neurons are capable of autoregulation . our results further suggest that dats are located at release sites within the dmarc , i.e. , where the cell bodies of tidas are located . the functional expression of dat within the tida system has been controversial , with arguments presented both for its absence ( lookingland et al . , 1987 ) and for its presence ( boss et al . , 1997 , demaria et al . , 2000 however , this discussion has largely been based on the assumption that tida dats are located on axon terminal release sites ( i.e. , in the me ) . therefore , the point in question may not be whether tidas have dats but rather the site of the transporter s actions . our finding in which somatic whole - cell electrophysiology responds to dat blockade in the same manner as following d2r stimulation indicates that dat may be present and functional also on release sites that target the tida cell - body region . ultrastructural studies have shown that recurrent axon collaterals from tida neurons do not appear to form synaptic contacts , but extensive dendrodendritic and somatic contacts exist among these cells ( piotte et al . , 1985 ) . this anatomical organization is thus consistent with local release of dopamine from dendrites and/or non - synapse forming axon collaterals in the service of autoregulation , analogous to what has been observed in the midbrain dopamine neurons ( bjrklund and lindvall , 1975 , geffen et al . the modulation of phases 1 and 3 by d2r stimulation is mediated via distinct effects . at a membrane potential of 60 mv , i.e. , within the phase 1 range , d2r activation induces a hyperpolarization , suggesting the contribution of direct post - synaptic actions . the current underlying the relatively modest ( albeit with an impact at the network level ) hyperpolarization is difficult to extract in voltage - clamp gap - free or ramp recordings ; in tida neurons , this has yet only been reported with currents inducing several - fold larger changes in membrane potential ( lyons et al . pharmacological experiments , however , showed that the hyperpolarization is diminished by intracellular cs , implicating an increased k conductance and/or an inhibition of the hyperpolarization - activated cation current ( ih ) . evidence for each has been reported from midbrain dopamine neurons ( lacey et al . , 1987 , momiyama et al . , 1993 , jiang et al . , 1993 ) . here , the increase in phase 1 duration by d2r activation was abolished during ih blockade , suggesting ih as a mediator of the effect of d2r activation during phase 1 . an increased frequency of sipscs was also observed by d2r activation , raising the possibility that augmented phasic inhibition contributes to the slowing of this phase . notably , the increase in the duration of phase 1 was significantly attenuated when qp was applied during gabaa blockade ( by ptx ) . thus , simultaneous increases in presynaptic inhibition and postsynaptic k or ih - mediated outward current underlie the changes in phase 1 that primarily account for the decreased frequency resulting from an increased dopaminergic tone . as tida neurons do not discharge during phase 1 , the increased inhibitory input is likely generated by another local neuronal population . phase 3 occupies a relatively depolarized membrane potential space , overlapping with the changes in hva ca currents that followed d2r activation in tida neurons . previous work has suggested the existence of l - type ca currents in these cells ( lyons et al . , our results confirm this and reveal the presence of n - type ca channels in tida neurons ; the existence of p / q and r - type ca channels remains to be investigated . we show that reductions in both l- and n - type ca currents contribute to the hva iqp . attenuated inward currents may seem counterintuitive as an explanation for the extended depolarized plateau of phase 3 . however , tida neurons also exhibit ca - dependent k currents ( lyons et al . , 2012 ) , and pharmacological inhibition of ik(ca ) results in an increased duration of phase 3 , as shown in figure 4 . thus , the attenuation of hva ca currents accounts for the prolonged depolarization and discharge during d2r autoreceptor activation in tida neurons , possibly due to a decreased activation the ik(ca ) that ultimately causes repolarization to the nadir . the decreased synaptic inhibition during phase 3 ( figure 5ca ) , though it does not in itself significantly contribute to prolonging the depolarized state , may add a synergistic net excitatory influence . the mechanisms we identify here suggest an additional level of control in the neuroendocrine system where tida neurons homeostatically tune their activity to variations in the ambient extracellular levels of dopamine at the somatodendritic compartment . as local dopamine volume transmission increases ( mimicked by qp administration or dat inhibition ) , in addition , the gap junction connectivity between tida neurons ( lyons et al . , 2010 ) may provide efficient transduction of the d2r - induced changes throughout the network . ( 2003 ) have shown that when midbrain dopamine neurons are depleted of dopamine , burst firing is lost , suggesting that this type of feedback tuning may have general relevance for how the discharge pattern is controlled in dopamine neurons . this ultra - short loop impacts on tida neurons in a manner less dramatic than that for the feedback relayed via prolactin ; while prolactin shifts discharge into tonic firing ( lyons et al . , 2012 ) , maintains tida neurons in an oscillatory configuration but restructures phase relationships . it may be speculated that , as the distance between the feedback signal ( i.e. , effector hormone versus inherent transmitter ) and the controlled system increases , more radical correcting actions may be required , as deviations from the set point have lasted for a longer time . curiously , some degree of continuous d2r - mediated feedback appears necessary for the system to discharge . gradual diminishment of this signal , as mimicked with low doses of eticlopride and haloperidol , results in changes generally opposite to those induced by d2r stimulation combined with blunting of spikes . higher doses and longer application of the antagonists results in depolarization block and collapse of the oscillation , rendering the cell incapable of repetitive spiking . the db that we demonstrate in tida neurons is , in vital ways , similar to what can be observed in midbrain dopamine neurons ( grace and bunney , 1986 ) and has been proposed to provide a physiological mechanism that constrains a cell s firing frequency within a limited range , adding low - pass filter properties to a system ( wong et al . , 2013 ) . haloperidol and other antipsychotic drugs with d2r blocking properties at concentrations below the effective therapeutic dose cause hyperprolactinemia ( gruen et al . , 1978 ) , and associated reproductive dysfunctions such as impotence , menstrual disturbances , and infertility , commonly resulting in drug withdrawal ( serretti and chiesa , 2011 ) . this effect is usually attributed to haloperidol relieving pituitary lactotrophs of the tonic hyperpolarizing influence of d2r activation ( macleod and lehmeyer , 1974 ) . the present results indicate , however , that dmarc tida autoreceptors may also contribute to such hyperprolactinemia by mediating d2r - antagonist - induced db , causing the cessation of discharge and attenuated or abolished dopamine release in the portal vessels and removing inhibition from the lactotrophs . thus , parallel actions of haloperidol at the somatodendritic level and at the endocrine target in the pituitary may underlie the reproductive side effects of d2r - active antipsychotic drugs . thus , db may be a mechanism that underlies not only the therapeutic actions of antidopaminergic antipsychotics ( grace et al . , 1997 ) but also the adverse sexual effects commonly caused by these drugs . in conclusion , our results using a spontaneously active neuroendocrine dopaminergic preparation reveal an ultra - short feedback pathway . these data suggest a rapid and continuous tuning of membrane rhythm to the circuit s own activity echoed in fluctuations of ambient somatodendritic levels of transmitter , and mediated by a combination of pre- and post - synaptic actions . this arrangement may be important both for maintaining physiologically appropriate prolactin levels and for the development of neuroleptic - induced reproductive side effects . acute slices of the mediobasal hypothalamus were prepared from 21- to 31-day - old male sprague - dawley rats ( charles river laboratories ) . all animal experiments had received previous approval from the local ethical board , stockholm s norra djurfrsksetiska nmnd , and were performed in accordance with the european communities council directive of november 24 , 1986 ( 86/609/eec ) . slices were cut on a vibratome to 250-m thickness and continuously perfused with oxygenated acsf containing ( in millimolar ) : 127 nacl , 2.0 kcl , 1.2 nah2po4 , 26 nahco3 , 1.3 mgcl2 , 2.4 cacl2 , and 10 d - glucose , at room temperature during recording , unless stated otherwise . to identify the role of ca on oscillation parameters , low - ca extracellular recording solution was used ( in millimolar ) : 127 nacl , 1.9 kcl , 1.2 nah2po4 , 26 nahco3 , 4.5 mgso4 , 0.15 cacl2 , and 10 d - glucose . each slice was exposed only to a single bath application of pharmacological compounds and was used for a single experiment . whole - cell current- and voltage - clamp recordings were performed with micropipettes filled with intracellular solution containing ( in millimolar ) , 140 k - gluconate , 10 kcl , 10 hepes , 10 egta , and 2 na2atp ( ph 7.3 ) ( with koh ) , unless stated otherwise . recordings were performed using a multiclamp 700b amplifier , a digidata 1440 digitizer , and pclamp10.2 software ( molecular devices ) . access resistance was monitored throughout the experiments , and neurons in which the series resistance exceeded 15 m or changed 20% were excluded from the statistics . recordings were performed using a multiclamp 700b amplifier , a digidata 1440 digitizer , and pclamp10.2 software ( molecular devices ) . to control for any effect on the oscillation due to long - time recording intervals , we performed recordings of up to 100 min without adding any drug ( figure s1 ) ; rhythmic behavior was maintained , and cycle duration did not change significantly over time . for data analysis was performed with graphpad prism 6 , clampfit software ( molecular devices ) , axograph , originpro 8 ( originlab ) , and custom written matlab scripts . phase analysis for each recording was based on data obtained over five consecutive oscillation cycles . control traces were collected after 10 min of baseline and immediately followed by drug application . detection threshold was set at 3-fold the root - mean - square ( rms ) noise level , which typically was 36 pa . frequency , inter - event interval , and amplitudes were calculated as a mean of the values obtained during a 60-s recording period . for postsynaptic current phase analysis , 3-s samples of phase 1 and 1-s samples of phase 3 of three consecutive oscillation cycles the two - sample kolmogorov - smirnov ( ks-2 ) test was used to compare pooled cumulative frequency distributions of each component in the absence versus the presence of quinpirole . statistical significance was set at p < 0.05 and was determined using the appropriate two - tailed student s t test unless otherwise stated . where it is stated that anova was used , a one - way anova was performed with the bonferroni test in post hoc analysis . 0.05 ; = p < 0.01 ; and = p < 0.001 .

summaryhow autoreceptors contribute to maintaining a stable output of rhythmically active neuronal circuits is poorly understood . here , we examine this issue in a dopamine population , spontaneously oscillating hypothalamic rat ( tida ) neurons , that underlie neuroendocrine control of reproduction and neuroleptic side effects . activation of dopamine receptors of the type 2 family ( d2rs ) at the cell - body level slowed tida oscillations through two mechanisms . first , they prolonged the depolarizing phase through a combination of presynaptic increases in inhibition and postsynaptic hyperpolarization . second , they extended the discharge phase through presynaptic attenuation of calcium currents and decreased synaptic inhibition . dopamine reuptake blockade similarly reconfigured the oscillation , indicating that ambient somatodendritic transmitter concentration determines electrical behavior . in the absence of d2r feedback , however , discharge was abolished by depolarization block . these results indicate the existence of an ultra - short feedback loop whereby neuroendocrine dopamine neurons tune network behavior to echoes of their own activity , reflected in ambient somatodendritic dopamine , and also suggest a mechanism for antipsychotic side effects .

Introduction Results Discussion Experimental Procedures Author Contributions

dopamine neurons play a pivotal role in several brain functions , including cognition , reward , and motor output ( jentsch et al . dopamine is also an important signaling molecule in the hypothalamus , where it inhibits prolactin release from the anterior pituitary ( freeman et al . the main source of neuroendocrine dopamine is the tuberoinfundibular dopamine ( tida ) neurons , located in the dorsomedial hypothalamic arcuate nucleus ( dmarc ) . however , the actions of dopamine on tida neurons have been the subject of only a few studies and remain poorly understood . biochemical studies have indicated that tida neurons may be under inhibitory influence by dopamine receptors of the type 2 family ( d2r ) ( berry and gudelsky , 1991 , lin et al . in the ventral tegmental area ( vta ) and the substantia nigra ( sn ) of the midbrain , where this issue has been studied in more depth , autoinhibition appears to protect dopamine neurons from runaway excitation ( bjrklund and lindvall , 1975 , aghajanian and bunney , 1977 , paladini et al . this has been shown to involve d2r - mediated activation of hyperpolarizing k conductances ( silva and bunney , 1988 ) . , 1983 ) , and the ability to exhibit both tonic and phasic discharge configurations . it is not known , however , whether mechanisms of autoregulation are similar or different in tida cells . intriguingly , recent in vitro studies have revealed that tida neurons in the male rat discharge in an oscillating pattern , which can be switched to tonic discharge by hormones and neurotransmitters ( lyons et al . the important question of how an oscillating circuit tunes its activity to internal feedback is poorly understood and ideally studied in a spontaneously active preparation . furthermore , understanding the homeostatic regulation of tida neurons is clinically important , as several of the most troubling side effects associated with antipsychotic , as well as antidepressant , drugs derive from their ability to raise serum prolactin ( madhusoodanan et al . , 2010 , the results we present here suggest the existence of a ultra - short autoinhibitory loop in tida neurons encompassing both pre - and postsynaptic mechanisms that is continuously adjusted to ambient somatodendritic dopamine levels . the tida neurons in the male rat exhibit stereotyped electrophysiological properties , including anomalous inward rectification , an a - like current conductance , and a slow ( 0.050.07 hz ) oscillation . using these criteria , we first sought to determine how tida network behavior is affected by agonists of the dopamine receptors , using whole - cell recordings . application of dopamine ( 20 m ) induced a reversible increase of the oscillation cycle duration ( + 7.66 1.47 s ; n = 5 ; p < 0.05 versus control ; anova ; figures 1ba1bd ; table s1 ) . to identify the different components of the cycle , a custom written analysis program was developed ( figure 1a ; supplemental experimental procedures ) . thus , the dopamine - induced slowing of the oscillation was found to result primarily from an increase in phase 1 ( i.e. the duration of phases 2 ( fast depolarization ) and 4 ( repolarization ) were not affected by the application of dopamine . in the presence of the d1-type - receptor ( d1r)-specific agonist , skf-81,297 ( 10 m ) ( reavill et al . , 1993 ) , however , oscillation parameters were not significantly altered ( n = 5/5 ; figure 1d ; table s1 ) . last , we tested whether d2r activation alters rhythmicity in the presence of fast - ionotropic blockade ( fib ) ( 100 m picrotoxin [ ptx ] , 10 m cnqx , and 25 m ap-5 ) . application of the selective d2r antagonist eticlopride ( 1 m ) resulted in a depolarization of the tida oscillation nadir ( + 2.46 0.77 mv ; n = 5 ; p < 0.05 versus control ; figures 2aa and 2ab ; table s1 ) concomitant with a decrease in phase 1 duration ( 1.88 0.65 s ; n = 5 ; p < 0.05 versus control ; figure 2ab ; table s1 ) and an increase of phase 3 duration ( + 1.51 0.37 s ; n = 5 ; p < 0.05 versus control ; figure 2ab ; table s1 ) . the effect on the waveform of the fifth ap of a cycle is demonstrated in figures 2ac and 2bc . similar changes in ap waveform have previously been described in midbrain dopamine neurons as a prodrome of the depolarization block ( db ) ( bikson et al . this similarity , and the depolarized nadir induced by eticlopride , prompted us to examine the effects of higher concentrations of the d2r antagonists . application of eticlopride ( 10 m , n = 5/5 ) or haloperidol ( 10 m , n = 11/11 ) resulted in a gradual depolarization and ultimate collapse of the oscillation ( figures 2ca and 2cb ) . ap amplitude gradually decreased and was eventually followed by a complete loss of aps concurrent with the collapse of the oscillation ( figures 2ca and 2cb ) . injection of depolarizing square current pulses resulted only in a single , low - amplitude ap , verifying that the capacity for regenerative firing was compromised ( figure 2cc ) . collectively , these results suggest that tida neurons respond to d2r antagonists by excitation , leading to db in a manner highly similar to what has been described in midbrain dopamine neurons following chronic application of dopamine antagonist antipsychotics ( grace and bunney , 1986 ) . the partly opposite effects of d2r agonists and antagonists suggest that the baseline behavior of tida neurons may depend on continuous dopaminergic feedback . evidence has been presented for both the absence ( demarest and moore , 1979b , annunziato et al . , 1997 ) of functional dopamine reuptake in these cells . we addressed this issue directly by applying a dat blocker , either gbr-12783 or methylphenidate , and recorded the electrophysiological response . in the presence of gbr-12783 ( 1 m ) and methylphenidate ( 50 m ) , tida neurons exhibited changes similar to those elicited by d2r agonists , i.e. , dopamine , apomorphine , and qp , and slowed down oscillation frequency ( gbr-12783 , n = 8/8 ; methylphenidate , n = 5/5 ; figures 2da and 2db ; table s1 ) . while both phases 1 and 3 were significantly prolonged in the presence of methylphenidate , the application of gbr-12783 only resulted in a significantly increased duration of phase 1 . these data suggest the existence of endogenous release of dopamine from tida neurons and the presence of functional dopamine reuptake at the somatic level in these cells . next , we sought to identify the membrane actions that underlie the d2r - mediated modulation of tida neurons . under these conditions , and with membrane potential set to 60 mv via negative current injection , application of qp ( 20 m ) resulted in a hyperpolarization of 4.19 0.78 mv ( n = 6 ; p < 0.01 versus control ; figures 3a and 3c ) . these findings suggest a d2r - mediated decrease of ih . to characterize the ionic currents modulated by d2r agonism , tida neurons were recorded in voltage - clamp mode in the presence of ttx ( 0.5 m ) at a holding potential ( vhold ) of 60 mv , i.e. to determine current changes that might occur throughout the membrane potential spectrum , a voltage - clamp ramp protocol of 500-ms durations was used , allowing the identification of d2r - mediated currents throughout the voltage spectrum of 115 mv to 0 mv ( figure 3ga ) . digital subtraction of the ramp performed with control versus qp ( figure 3 gb ) revealed a qp - induced current ( iqp ) that was a non - reversing net outward current , active only at membrane potentials positive of approximately 45 mv ( n = 5 ; figure 3gc ) , increasing with depolarization within the range tested ( vhold = 0 mv ; + 124.83 34.97 pa ) . the iqp may reflect the activation of an outward current and/or the inhibition of an inward current . , 1985 ) , we tested whether d2r stimulation decreased ca flux in tida neurons , using a voltage ramp protocol . application of the l - type - specific blocker nimodipine ( 10 m ) , however , attenuated the ca current ( 62.8 19.04 pa ; n = 5 ; p < 0.05 versus control , anova ; figure 4b ) . application of the n - type - specific ca channel blocker -conotoxin gvia ( 1 m ) decreased the ca currents ( 71.4 22.72 pa ; n = 5 ; p < 0.05 versus control , anova ; figure 4c ) . under n - type ca channel blockade , application of qp ( 20 m ) resulted in a small , but significant , decrease of the remaining ca current ( 7.8 1.91 pa ; n = 5 ; p < 0.05 versus control , anova ; figure 4c ) . in these conditions , application of qp ( 20 m ) failed to induce a significant decrease of the remaining ca current ( 10.6 5.32 pa ; n = 5 ; p > 0.05 versus control , anova ; figure 4d ) . as ca influx through l - type channels has commonly been implicated in the activation of ca - dependent k channels ( e.g. if so , the effect of qp on phase 1 should be diminished or abolished in the absence of gabaa - mediated inhibition . the combined blockade of gabaa ( by ptx ) and gabab receptors ( by the antagonist , cgp-55845 ; 10 m ) did not produce any additional attenuation of the qp - mediated increase in cycle period or phase 1 or 3 duration , compared to ptx alone ( n = 5 ; figures 6b6e ; table s1 ) . these data demonstrate that increased gabaa transmission contributes to dopaminergic autoregulation of the tida oscillation frequency , acting within the same membrane potential spectrum as , and likely parallel to , a cs - sensitive hyperpolarizing postsynaptic current ( figures 3a3c ) . such regulation has generally been studied in the context of multicomponent feedback loops , involving actions of the corresponding pituitary hormone or a peripheral target hormone on the hypothalamic master , prolactin can stimulate both dopamine production ( clemens and meites , 1968 , hkfelt and fuxe , 1972 , demarest et al . however , the possibility that parvocellular neurons can autoregulate their own activity , as a means of immediate homeostatic tuning , has received little attention . in tida neurons , the literature offers conflicting evidence for ( berry and gudelsky , 1991 , liang and pan , 2001 ) and against ( demarest and moore , 1979a , durham et al . our experiments reveal that increasing d2r activation causes two coincident changes in the tida duty cycle : a prolonged duration of phase 1 , resulting in a slower oscillation , followed by a minor prolongation of phase 3 . the similar reconfiguration of the oscillation following the exogenous application of qp , as when ambient endogenous dopamine was increased by gbr-12783 and methylphenidate induced dat blockade ( although the former prolonged only phase 1 ) , strongly indicates that these effects are not simply pharmacological . however , given that the slice preparation used here likely includes little of dopamine populations outside of the a12 group , the results using receptor and dat blockers favor the interpretation that tida neurons are capable of autoregulation . , where the cell bodies of tidas are located . , in the me ) . our finding in which somatic whole - cell electrophysiology responds to dat blockade in the same manner as following d2r stimulation indicates that dat may be present and functional also on release sites that target the tida cell - body region . this anatomical organization is thus consistent with local release of dopamine from dendrites and/or non - synapse forming axon collaterals in the service of autoregulation , analogous to what has been observed in the midbrain dopamine neurons ( bjrklund and lindvall , 1975 , geffen et al . the current underlying the relatively modest ( albeit with an impact at the network level ) hyperpolarization is difficult to extract in voltage - clamp gap - free or ramp recordings ; in tida neurons , this has yet only been reported with currents inducing several - fold larger changes in membrane potential ( lyons et al . pharmacological experiments , however , showed that the hyperpolarization is diminished by intracellular cs , implicating an increased k conductance and/or an inhibition of the hyperpolarization - activated cation current ( ih ) . evidence for each has been reported from midbrain dopamine neurons ( lacey et al . here , the increase in phase 1 duration by d2r activation was abolished during ih blockade , suggesting ih as a mediator of the effect of d2r activation during phase 1 . notably , the increase in the duration of phase 1 was significantly attenuated when qp was applied during gabaa blockade ( by ptx ) . thus , simultaneous increases in presynaptic inhibition and postsynaptic k or ih - mediated outward current underlie the changes in phase 1 that primarily account for the decreased frequency resulting from an increased dopaminergic tone . previous work has suggested the existence of l - type ca currents in these cells ( lyons et al . , our results confirm this and reveal the presence of n - type ca channels in tida neurons ; the existence of p / q and r - type ca channels remains to be investigated . we show that reductions in both l- and n - type ca currents contribute to the hva iqp . however , tida neurons also exhibit ca - dependent k currents ( lyons et al . , 2012 ) , and pharmacological inhibition of ik(ca ) results in an increased duration of phase 3 , as shown in figure 4 . thus , the attenuation of hva ca currents accounts for the prolonged depolarization and discharge during d2r autoreceptor activation in tida neurons , possibly due to a decreased activation the ik(ca ) that ultimately causes repolarization to the nadir . the decreased synaptic inhibition during phase 3 ( figure 5ca ) , though it does not in itself significantly contribute to prolonging the depolarized state , may add a synergistic net excitatory influence . the mechanisms we identify here suggest an additional level of control in the neuroendocrine system where tida neurons homeostatically tune their activity to variations in the ambient extracellular levels of dopamine at the somatodendritic compartment . , 2010 ) may provide efficient transduction of the d2r - induced changes throughout the network . ( 2003 ) have shown that when midbrain dopamine neurons are depleted of dopamine , burst firing is lost , suggesting that this type of feedback tuning may have general relevance for how the discharge pattern is controlled in dopamine neurons . this ultra - short loop impacts on tida neurons in a manner less dramatic than that for the feedback relayed via prolactin ; while prolactin shifts discharge into tonic firing ( lyons et al . higher doses and longer application of the antagonists results in depolarization block and collapse of the oscillation , rendering the cell incapable of repetitive spiking . , 1978 ) , and associated reproductive dysfunctions such as impotence , menstrual disturbances , and infertility , commonly resulting in drug withdrawal ( serretti and chiesa , 2011 ) . this effect is usually attributed to haloperidol relieving pituitary lactotrophs of the tonic hyperpolarizing influence of d2r activation ( macleod and lehmeyer , 1974 ) . the present results indicate , however , that dmarc tida autoreceptors may also contribute to such hyperprolactinemia by mediating d2r - antagonist - induced db , causing the cessation of discharge and attenuated or abolished dopamine release in the portal vessels and removing inhibition from the lactotrophs . thus , parallel actions of haloperidol at the somatodendritic level and at the endocrine target in the pituitary may underlie the reproductive side effects of d2r - active antipsychotic drugs . in conclusion , our results using a spontaneously active neuroendocrine dopaminergic preparation reveal an ultra - short feedback pathway . these data suggest a rapid and continuous tuning of membrane rhythm to the circuit s own activity echoed in fluctuations of ambient somatodendritic levels of transmitter , and mediated by a combination of pre- and post - synaptic actions . this arrangement may be important both for maintaining physiologically appropriate prolactin levels and for the development of neuroleptic - induced reproductive side effects . acute slices of the mediobasal hypothalamus were prepared from 21- to 31-day - old male sprague - dawley rats ( charles river laboratories ) . all animal experiments had received previous approval from the local ethical board , stockholm s norra djurfrsksetiska nmnd , and were performed in accordance with the european communities council directive of november 24 , 1986 ( 86/609/eec ) . to identify the role of ca on oscillation parameters , low - ca extracellular recording solution was used ( in millimolar ) : 127 nacl , 1.9 kcl , 1.2 nah2po4 , 26 nahco3 , 4.5 mgso4 , 0.15 cacl2 , and 10 d - glucose . access resistance was monitored throughout the experiments , and neurons in which the series resistance exceeded 15 m or changed 20% were excluded from the statistics . to control for any effect on the oscillation due to long - time recording intervals , we performed recordings of up to 100 min without adding any drug ( figure s1 ) ; rhythmic behavior was maintained , and cycle duration did not change significantly over time . frequency , inter - event interval , and amplitudes were calculated as a mean of the values obtained during a 60-s recording period . for postsynaptic current phase analysis , 3-s samples of phase 1 and 1-s samples of phase 3 of three consecutive oscillation cycles the two - sample kolmogorov - smirnov ( ks-2 ) test was used to compare pooled cumulative frequency distributions of each component in the absence versus the presence of quinpirole .

the sustained profitability of an organization depends on its workforce job satisfaction and organizational commitment ( 1 ) . employees job satisfaction enhances their motivation , performance and reduces absenteeism and turnover ( 1- 4 ) . job satisfaction is an employee s attitude about his or her job and the organization in which s / he performs the job . employee job satisfaction is correlated with received salaries , benefits , recognition , promotion , coworkers and management support , working conditions , type of work , job security , leadership style of managers , and demographic characteristics such as gender , marital status , educational level , age , work tenure , and number of children ( 5 - 8 ) . organizational commitment shows the psychological attachment of an employee to the organization ( 9 ) . according to meyer and colleagues ( 2002 ) there are three types of organizational commitment : affective , continuance and normative commitment . affective commitment relates to an employee s emotional attachment to the organization and its goals . continuance commitment shows cognitive attachment between an employee and his or her organization because of the costs associated with leaving the organization . finally , normative commitment refers to typical feelings of obligation to remain with an organization ( 10 ) . leadership behavior of managers plays a critical role in employees job satisfaction and commitment ( 5 , 11 , 12 ) . it is the process of influencing a group of people towards achieving organizational goals ( 13 ) . leadership is the ability of a manager to influence , motivate , and enable employees to contribute toward organizational success ( 14 ) . managers can utilize various leadership styles to lead and direct their employees including autocratic , bureaucratic , laissez - faire , charismatic , democratic , participative , transactional , and transformational leadership styles . good human resource management drives employee satisfaction and loyalty ( 4 , 15 , 16 ) . satisfied and committed employees deliver better care , which results in better outcomes and higher patient satisfaction ( 17 - 19 ) . very little research in the literature is available on the links between managers leadership behavior and employees job satisfaction and organizational commitment . these studies were mostly conducted in western countries and limited to health care organizations . however , where job satisfaction and organizational commitment were investigated , leadership behavior of managers was not analyzed . this study aimed to overcome this gap by investigating these variables in a group of hospitals in iran . research has been conducted to identify how leadership behaviors can be used to influence employees to achieve better organizational outcomes ( 20 ) . however , there are no known studies related to the links between these subjects in the health care organizations of the country . the results of this research provide a better understanding of the relationship between leadership styles of managers and employees job satisfaction and commitment . figure 1 presents a conceptual model of relationships between leadership , job satisfaction and organizational commitment . the model proposes that leadership is positively related to job satisfaction , which is positively related to organizational commitment . therefore , i propose the following hypotheses : hypothesis 1 : the greater the employees job satisfaction , the greater their commitment . hypothesis 2 : there is a positive relationship between managers leadership style and employee s job satisfaction and commitment . this research investigates the relationship between perceptions of hospital managers and employees regarding the leadership behavior of hospital managers , and how this is related to the job satisfaction and organizational commitment of employees in isfahan university hospitals ( iuhs ) , isfahan , iran . employee questionnaire package contained a cover letter , and questionnaires related to employees job satisfaction , organizational commitment and the leadership style of hospital manager . managers questionnaire package included a cover letter , their leadership styles , job satisfaction and organizational commitment . the job satisfaction scale ( specter 1997 ) utilized a likerttype scale with six response alternatives ranging from strongly disagree ( weighted 1 ) tostrongly agree ( weighted 6 ) for each of the 36 items to measure job satisfaction ( 21 ) . aspects of job satisfaction addressed are with : ( a ) pay , ( b ) promotion , ( c ) supervision , ( d ) fringe benefits , ( e ) contingent rewards , ( f ) operating conditions , ( g ) co workers , ( h ) nature of work , and ( i ) communication ( 4 items in each domain ) . organizational commitment scale ( meyer et al . 1991 ) contained three eight - item components which were rated on a 6-point likert type scale ( from strongly disagree=1 to strongly agree=6 ) . aspects of organizational commitment addressed were affective , continuance , and normative commitment ( 22 ) . mangers leadership scale ( likert , 1967 ) had 35 items of which 15 items determined a manager s employee oriented dimension ( consideration ) and 20 items determined the task oriented dimension ( initiating structure ) of leadership style ( 23 ) . each statement included a five - point likert scale ( from very rarely = 1 to often = 5 ) . according to likert ( 1967 ) , the four distinct practices that leaders use to affect employee and organizational performance include : exploitative authoritative , benevolent authoritative , consultative and participative management . the author developed a persian translation of these questionnaires by applying a sequential forward and backward translation approach . the final test version questionnaires were then pilot tested , using a random sample of 40 hospital employees ( not included in the sample ) and found to be well accepted and easy to fill in . the translated questionnaires were found to be understandable and could be completed in about 20 min . the reliability coefficient was .8749 for job satisfaction , .7784 for organizational commitment , .8767 for leadership style questionnaire from the view point of employees and .8139 for leadership style from the view point of managers questionnaires . 950 persons were selected for this survey ( n=6405 , d=0.03 , z= 1.96 and s= 0.51 ) . finally , 832 questionnaires were returned and from those , 814 questionnaires were completely filled ( 85.68 % return rate ) . all data were analyzed using spss ( the statistical package for the social sciences ) software . in order to normalize the likert scale on 1- 6 scales for each domain of job satisfaction and commitment questionnaires , the sum of raw scores of items in each domain was divided by the numbers of items in each domain and for overall job satisfaction and commitment , sum of raw scores of items were divided by 36 and 24 respectively . scores of 2 or lower on the total scale indicate very low , scores between 2 and 2.99 indicate low , scores between 3 and 3.99 indicate moderate , scores between 4 and 4.99 indicate high and scores of 5 or higher indicate very high job satisfaction or organizational commitment . the scores of employee oriented and task oriented dimensions of leadership were varied between 15 - 75 and 20- 100 . this research investigates the relationship between perceptions of hospital managers and employees regarding the leadership behavior of hospital managers , and how this is related to the job satisfaction and organizational commitment of employees in isfahan university hospitals ( iuhs ) , isfahan , iran . separate questionnaires were sent to managers and employees . employee questionnaire package contained a cover letter , and questionnaires related to employees job satisfaction , organizational commitment and the leadership style of hospital manager . managers questionnaire package included a cover letter , their leadership styles , job satisfaction and organizational commitment . the job satisfaction scale ( specter 1997 ) utilized a likerttype scale with six response alternatives ranging from strongly disagree ( weighted 1 ) tostrongly agree ( weighted 6 ) for each of the 36 items to measure job satisfaction ( 21 ) . aspects of job satisfaction addressed are with : ( a ) pay , ( b ) promotion , ( c ) supervision , ( d ) fringe benefits , ( e ) contingent rewards , ( f ) operating conditions , ( g ) co workers , ( h ) nature of work , and ( i ) communication ( 4 items in each domain ) . organizational commitment scale ( meyer et al . 1991 ) contained three eight - item components which were rated on a 6-point likert type scale ( from strongly disagree=1 to strongly agree=6 ) . aspects of organizational commitment addressed were affective , continuance , and normative commitment ( 22 ) . mangers leadership scale ( likert , 1967 ) had 35 items of which 15 items determined a manager s employee oriented dimension ( consideration ) and 20 items determined the task oriented dimension ( initiating structure ) of leadership style ( 23 ) . each statement included a five - point likert scale ( from very rarely = 1 to often = 5 ) . according to likert ( 1967 ) , the four distinct practices that leaders use to affect employee and organizational performance include : exploitative authoritative , benevolent authoritative , consultative and participative management . the author developed a persian translation of these questionnaires by applying a sequential forward and backward translation approach . the final test version questionnaires were then pilot tested , using a random sample of 40 hospital employees ( not included in the sample ) and found to be well accepted and easy to fill in . the translated questionnaires were found to be understandable and could be completed in about 20 min . the reliability coefficient was .8749 for job satisfaction , .7784 for organizational commitment , .8767 for leadership style questionnaire from the view point of employees and .8139 for leadership style from the view point of managers questionnaires . 950 persons were selected for this survey ( n=6405 , d=0.03 , z= 1.96 and s= 0.51 ) . finally , 832 questionnaires were returned and from those , 814 questionnaires were completely filled ( 85.68 % return rate ) . all data were analyzed using spss ( the statistical package for the social sciences ) software . in order to normalize the likert scale on 1- 6 scales for each domain of job satisfaction and commitment questionnaires , the sum of raw scores of items in each domain was divided by the numbers of items in each domain and for overall job satisfaction and commitment , sum of raw scores of items scores of 2 or lower on the total scale indicate very low , scores between 2 and 2.99 indicate low , scores between 3 and 3.99 indicate moderate , scores between 4 and 4.99 indicate high and scores of 5 or higher indicate very high job satisfaction or organizational commitment . the scores of employee oriented and task oriented dimensions of leadership were varied between 15 - 75 and 20- 100 . data were collected from 814 persons including 665 employees , 127 first line managers ( head of departments ) , 11 middle managers ( hospital managers ) and 11 senior managers ( hospital presidents ) . as table 1 shows about half of the respondents were males ( 48.4% ) . the majority ( n=340 ) of participants ranged between 31 and 40 years of age . approximately 82% ( n=667 ) of the participants were married and 39.9% ( n=325 ) had earned bachelor s degrees . additionally , 51.5% , 87.4 % , 90.9% and 90.9% of employees , first line , middle and senior managers had permanent employment . the mean age for employees , front line , middle and senior managers were 34 , 42 , 46 and 45 years respectively . employees , front line , middle and senior managers on the average , had 11 , 19 , 20 and 18 years of working experiences respectively . front line , middle and senior managers on the average had 9 , 12 and 9 years of managerial experiences respectively . hospital employees were satisfied overall with their job with a mean score of 3.260.56 on a 6 scale ( moderate satisfaction ) compared with the possible range from 1.52 to 5.61 . employees , first line , middle and senior managers scored a mean of job satisfaction of 3.21 , 3.40 , 3.97 and 3.73 respectively . within the nine items of the job satisfaction scale , the three dimensions of the job with which respondents were most satisfied were : supervision , nature of the job and co workers . respondents were least satisfied with the benefits , contingent rewards , communication , salaries , working conditions , and promotion ( see table 3 ) . a difference existed in perceptions between employees and managers pertaining to what aspects of the job are important to employees job satisfaction . employees reported that managers loyalty to employees , job security , good pay and good working conditions were the most important motivators for them . however , managers thought that sufficient salaries , recognition and job security were most important to employees . the mean score of employees job satisfaction in general and specialized hospitals was 3.19 and 3.37 respectively . the mean score of employees job satisfaction in hospitals with the specialty in burn ( 3.08 ) , psychiatry ( 3.29 ) , and cancer ( 3.31 ) were low and in hospitals with the specialty in ophthalmology ( 3.42 ) and cardiology ( 3.52 ) was high . employees in specialized hospitals receive more monetary benefits than those employees in general hospitals because of the type of expensive services provided to patients . employee s job satisfaction in therapeutic and diagnostic departments was statistically lower than administrative and ancillary departments ( p<0.01 ) . the mean score of employees job satisfaction in the central storage department ( 4.21 ) , secretarial unit ( 4.05 ) , public relations office ( 3.91 ) , social worker office ( 3.90 ) and material supply department ( 3.66 ) were high and in the psychiatry ward ( 2.55 ) , pediatrics ward ( 2.63 ) , dialysis ward ( 2.75 ) , urology ward ( 2.85 ) and medical records department , ( 2.88 ) were low . there was strong correlation between the job satisfaction of employees and their gender , marital status , age , tenure , organizational position and received salaries ( p<0.01 ) . there was no statistically significant correlation between job satisfaction of employees and their graduation levels and type of employment : permanent or contract employment ( p>0.05 ) . in order to determine the main factors that cause satisfaction and/or dissatisfaction with work , the relationship between total job satisfaction and job satisfy factors was analyzed . calculations of spearmen s ratios revealed the strongest correlation between total job satisfaction and such characteristics as salaries , .687 ; fringe benefits , .685 ; promotion , .673 and communication , .637 . on the other hand work conditions , .468 ; nature of the job , .502 ; supervision , .536 ; and co workers , .554 had less effect on employees job satisfaction respectively . the mean score of employees satisfaction of job factors and organizational factors related to job satisfaction was 4.39 and 3.05 on a 6 scale respectively . organizational , job and individual factors overall explained 99.3 % of the variance in employees job satisfaction . organizational factors explained the largest amount of the variance ( 94% ) , followed by job factors and individual factors . regards to organizational factors , pay explained the largest amount of the variance , followed by coworkers , promotion , communication , supervision and benefit . the mean score of respondents organizational commitment was 3.980.49 on a 6 scale ( moderate ) . the mean score of organizational commitment of employees , first line , middle and senior managers was 3.97 , 4.07 , 4.12 and 4.03 from 6 credits respectively . the mean score of affective , continuance , and normative commitment were 3.880.69 , 3.740.61 , and 4.320.38 respectively . the mean score of organizational commitment in employees with relevant educational background towards their job was higher than those without any relevance . a negative association was seen between employees organizational commitment and their educational levels ( r = -0.126 , p=0.001 ) . significant differences were obtained between age , tenure , organizational position , type of employment , received salaries and organizational commitment ( p<0.05 ) . temporary employment and amount of salaries the employee s organizational commitment in therapeutic and diagnostic departments was higher than administrative and ancillary departments . significant differences were not obtained between employees organizational commitment and their gender , and marital status . the kruskal wallis test revealed that the total organizational commitment scores was not differed among twelve hospitals ( p=0.61 ) . in correlation analysis between organizational commitment and its three dimensions , affective commitment , 0.809 ; continuance commitment , 0.741 ; and normative commitment , 0.417 respectively had positive and the highest effect on employees organizational commitment . the results of the simultaneous multiple regression model indicate that organizational , job and individual factors overall explained 54.5% of the variance in employees organizational commitment . organizational factors explained the largest amount of the variance ( 46% ) , followed by job factors and individual factors . this is primarily the result of the effect of the employee s education , the more educated employee reporting less commitment . the mean score of employee - oriented dimension of leadership style in first line , middle and senior managers were 52 6.35 , 54 3.89 , and 54 5.00 ( from 75 credit ) respectively . the mean score of task - oriented dimension of leadership style in first line , middle and senior managers were 68 9.25 , 69 6.70 , and 70 7.20 ( from 100 credit ) respectively . 0.78% , 4.74% and 94.48% of first line managers had exploitative - authoritative , benevolent - authoritative and participative leadership styles . from the viewpoint of employees the mean score of hospital managers employee - oriented and task - oriented credits were 46 ( out of 75 credits ) and 65 ( out of 100 credits ) . from the viewpoint of hospital managers the mean score of their employee - oriented and task - oriented credits were 54 and 69 . in other words , from the view point of employees , hospital managers were more task - oriented and from the view point of the hospital managers themselves , they were more employee - oriented . there was no correlation between leadership style of managers and their demographic variables except age and managerial experience years . pearson correlation coefficients indicate a significant statistically relationship between hospital managers management experience years and their employee oriented ( p= 0.024 and r=0.736 ) and task oriented ( p= 0.023 and r=0.706 ) dimensions of leadership style . there was a statistically significant relationship between employees job satisfaction and their organizational commitment ( p<0.001 and r=0.623 ) indicating that the employees who are more satisfied with their job are also more committed to the health care service . this study observed an asymmetric relationship where satisfaction had a stronger effect on commitment than the reverse . satisfaction with job identity , .589 ; supervision , .463 ; communications , .442 ; contingent rewards , .367 ; promotion , .344 ; fringe benefit , .242 ; salaries , .235 and work conditions , .215 were found to have a significant relationship to organizational commitment . on the other hand , there was a statistically significant correlation between the job satisfaction of employees and the leadership style of managers . this correlation between employees job satisfaction and employee - oriented and task - oriented dimensions of leadership style of hospital managers was at p<0.001 and p<0.01 levels . the correlation co efficient between employee oriented and task oriented dimensions of leadership style and employees satisfaction factors showed that the most positive co - efficiency was between supervision and employee oriented dimension and the most negative co - efficiency was between fringe benefits and task oriented dimension of leadership style of managers . a positive , significant correlation was shown among managers leadership style and employees commitment ( p<0.01 ) . leadership behaviors were positively correlated with affective commitment ( strongest relationship ) and normative commitment . the employee - oriented dimension of leadership had positive , statistically significant ( p < .01 ) correlations with affective commitment and normative commitment . leadership behaviors explained 28% variance in employees job satisfaction and 20 % in their commitment . employees oriented dimension of leadership explained the largest amount of the variance in these two variables . this finding is consistent with the findings of other previous studies in health care settings ( 24 - 25 ) . however , draper et al.s ( 2004 ) study showed a negative correlation between job satisfaction and dimensions of organizational commitment which is contrary to with the finding from the present study ( 26 ) . these differing results may be due to the differences in culture and the use of different instruments to measure commitment . this can be related to having more control over the job , more decision - making latitude , higher salaries and benefits linked to seniority and more social recognition . this study has also shown that there is a significant negative relationship between the education level of employees and their commitment with their organization . employees who work with patients reported less job satisfaction but more commitment in this study . job rotation could possibly be a good strategy for improving job satisfaction of these employees . job enrichment can also be used as a motivational strategy to satisfy these employees through providing opportunities for personal achievement , challenge and recognitions . higher commitment in employees in these wards could be because of investigation of this concept in an islamic country . recognition and respect are highly important especially for employees who are in direct contact with patients , families , and peers . managers recognition for good performance boosts employees morale and increases their satisfaction . a supportive management style , demonstrated through open communication , respect and recognition it has been noted in this study that leadership , job satisfaction and commitment are closely interrelated . these findings are consistent with earlier studies in health care organizations that demonstrate the connection between job satisfaction ( 27 , 28 , 29 ) and organizational commitment ( 30 , 31 ) with leadership . these findings suggest that there is a positive relationship between the employee - oriented leadership behaviors and both affective commitment and normative commitment . therefore , it can be said that participative leadership was not successful in iranian public hospitals . mosadeghrad and tahery ( 2004 ) in their research in iranian public hospitals concluded that managers knowledge about leadership styles was low ( 32 ) . providing more information about leadership theories help managers understand the importance of applying the right leadership style in their organizations . for instance , the effectiveness of participatory leadership can be examined from a cultural perspective . in low power distance cultures , managers in high power distance culture do not provide job enrichment and empowerment and employees do not necessarily want the responsibilities ( 35 ) . managers in such a culture may use a more directive leadership style to communicate with their subordinates . therefore , in high power distance cultures , participative leadership style could be viewed as weak and ineffective leadership style . they prefer the managers to develop a vision and communicate it to them . since charismatic leaders help reduce uncertainty , there is a strong preference for visionary , honest , cooperative , generous , concerned , and modest leaders ( 36 ) . organizational culture influences employees sense of engagement , identification and belonging and subsequently impact on their commitment . in an innovative , corporate and supportive culture , according to mosadeghrad and malek pour ( 2004 ) , 75% of isfahan university hospitals had mechanistic and bureaucratic structures . hospital managers should consider the structure and culture of the organization in choosing the appropriate leadership style ( 37 , 38 , 39 , 40 , 41 ) . maslow s ( 1954 ) believes that human needs form a five - level hierarchy ranging from physiological needs , safety , love , and esteem to self - actualization ( 42 ) . according to maslow s theory of hierarchy of needs , once individuals have satisfied one need in the hierarchy , it ceases to motivate their behavior and they are motivated by the need at the next level up the hierarchy . in this study , employees job satisfaction in relation to their salaries and benefits and working conditions was low . once these primary and basic needs are met , they would think about participating in management decision making processes . the participative management is not a good leadership style for these hospitals at the moment , unless managers meet their employees basic needs , improve employees organizational maturity , promote a culture of teamwork , cooperation and participation and upgrade organizational structure accordingly . hospital managers should understand the impact of varying leadership styles on employees job satisfaction and commitment level . thus , education and training should be provided to develop effective leadership behaviors to have a more positive effect on their employees job satisfaction and organizational commitment . research regarding the impact of leadership style of hospital managers on employees job satisfaction and commitment is relatively new in iranian health care organizations . the purpose of this study was to contribute to a better understanding of the relationship between leadership behaviors of managers and these two employees work - related attitudes . this research documented the level of job satisfaction and organizational commitment among employees and the type of leadership style of managers in iranian hospitals . this research also contributed to the knowledge of factors influencing job satisfaction and organizational commitment . the findings showed that hospital employees were moderately satisfied with their jobs and committed to their organization . employees were mostly dissatisfied with salaries , benefits , rewards , work conditions , and communication . these include demographic variables of age , years of work experiences , marital status , gender and organizational position , monthly salary , type of hospital , employees organizational commitment , leadership style of managers and the nine subscales of job satisfy factors , as indicted in table 3 . factors that may influence the level of employees commitment are demographic variables of age , years of work experiences , educational levels , organizational position , and type of employment , monthly salary , leadership style of managers and the nine subscales of job satisfier factors , as indicted in table 3 . in this study , employee oriented leadership explained significant variance in employees job satisfaction and commitment . the current research was limited to a group of public hospitals in iran . more studies of the iranian health care employees especially within hospitals that are not as hierarchical as the public hospitals surveyed in the current study are needed . more studies which involve hospital employees from other countries would enrich the literature on hospital employees job satisfaction and commitment . the results of such studies can be very helpful in developing strategies to improve the global retention of hospital personnel .

conflict of interest : none declared.introductionemployees job satisfaction and commitment depends upon the leadership style of managers . this study clarifies further the relationships between leadership behaviors of managers and two employees work - related attitudes - job satisfaction and organizational at public hospitals in iran . a better understanding of these issues and their relationships can pinpoint better strategies for recruiting , promotion , and training of future hospital managers and employees , particularly in iran but perhaps in other societies as well.methodsthis cross - sectional study was conducted using self - administered questionnaires distributed among 814 hospital employees and managers through a stratified random sampling.results and discussionthe dominant leadership style of hospital managers was participative style . hospital employees were moderately satisfied with their jobs and committed to their organization . salaries , benefits , promotion , contingent rewards , interpersonal relationships and working conditions were the best predictors of job satisfaction among hospitals employees . leadership , job satisfaction and commitment were closely interrelated . the leadership behavior of managers explained 28% and 20% of the variations in job satisfaction and organizational commitment respectively.conclusionthis study clarifies the causal relations of job satisfaction and commitment , and highlights the crucial role of leadership in employees job satisfaction and commitment . nevertheless , participative management is not always a good leadership style . managers should select the best leadership style according to the organizational culture and employees organizational maturity .

1. INTRODUCTION 2. METHODOLOGY Purpose and objectives The empirical setting Instruments Validation of research instruments Reliability of research instruments Data Collection Analysis of Data 3. RESULTS 4. DISCUSSION 5. CONCLUSION

the sustained profitability of an organization depends on its workforce job satisfaction and organizational commitment ( 1 ) . employees job satisfaction enhances their motivation , performance and reduces absenteeism and turnover ( 1- 4 ) . job satisfaction is an employee s attitude about his or her job and the organization in which s / he performs the job . employee job satisfaction is correlated with received salaries , benefits , recognition , promotion , coworkers and management support , working conditions , type of work , job security , leadership style of managers , and demographic characteristics such as gender , marital status , educational level , age , work tenure , and number of children ( 5 - 8 ) . organizational commitment shows the psychological attachment of an employee to the organization ( 9 ) . according to meyer and colleagues ( 2002 ) there are three types of organizational commitment : affective , continuance and normative commitment . continuance commitment shows cognitive attachment between an employee and his or her organization because of the costs associated with leaving the organization . leadership behavior of managers plays a critical role in employees job satisfaction and commitment ( 5 , 11 , 12 ) . managers can utilize various leadership styles to lead and direct their employees including autocratic , bureaucratic , laissez - faire , charismatic , democratic , participative , transactional , and transformational leadership styles . good human resource management drives employee satisfaction and loyalty ( 4 , 15 , 16 ) . very little research in the literature is available on the links between managers leadership behavior and employees job satisfaction and organizational commitment . however , where job satisfaction and organizational commitment were investigated , leadership behavior of managers was not analyzed . this study aimed to overcome this gap by investigating these variables in a group of hospitals in iran . however , there are no known studies related to the links between these subjects in the health care organizations of the country . the results of this research provide a better understanding of the relationship between leadership styles of managers and employees job satisfaction and commitment . figure 1 presents a conceptual model of relationships between leadership , job satisfaction and organizational commitment . the model proposes that leadership is positively related to job satisfaction , which is positively related to organizational commitment . therefore , i propose the following hypotheses : hypothesis 1 : the greater the employees job satisfaction , the greater their commitment . hypothesis 2 : there is a positive relationship between managers leadership style and employee s job satisfaction and commitment . this research investigates the relationship between perceptions of hospital managers and employees regarding the leadership behavior of hospital managers , and how this is related to the job satisfaction and organizational commitment of employees in isfahan university hospitals ( iuhs ) , isfahan , iran . employee questionnaire package contained a cover letter , and questionnaires related to employees job satisfaction , organizational commitment and the leadership style of hospital manager . managers questionnaire package included a cover letter , their leadership styles , job satisfaction and organizational commitment . the job satisfaction scale ( specter 1997 ) utilized a likerttype scale with six response alternatives ranging from strongly disagree ( weighted 1 ) tostrongly agree ( weighted 6 ) for each of the 36 items to measure job satisfaction ( 21 ) . aspects of job satisfaction addressed are with : ( a ) pay , ( b ) promotion , ( c ) supervision , ( d ) fringe benefits , ( e ) contingent rewards , ( f ) operating conditions , ( g ) co workers , ( h ) nature of work , and ( i ) communication ( 4 items in each domain ) . organizational commitment scale ( meyer et al . aspects of organizational commitment addressed were affective , continuance , and normative commitment ( 22 ) . mangers leadership scale ( likert , 1967 ) had 35 items of which 15 items determined a manager s employee oriented dimension ( consideration ) and 20 items determined the task oriented dimension ( initiating structure ) of leadership style ( 23 ) . according to likert ( 1967 ) , the four distinct practices that leaders use to affect employee and organizational performance include : exploitative authoritative , benevolent authoritative , consultative and participative management . the author developed a persian translation of these questionnaires by applying a sequential forward and backward translation approach . the final test version questionnaires were then pilot tested , using a random sample of 40 hospital employees ( not included in the sample ) and found to be well accepted and easy to fill in . the reliability coefficient was .8749 for job satisfaction , .7784 for organizational commitment , .8767 for leadership style questionnaire from the view point of employees and .8139 for leadership style from the view point of managers questionnaires . in order to normalize the likert scale on 1- 6 scales for each domain of job satisfaction and commitment questionnaires , the sum of raw scores of items in each domain was divided by the numbers of items in each domain and for overall job satisfaction and commitment , sum of raw scores of items were divided by 36 and 24 respectively . scores of 2 or lower on the total scale indicate very low , scores between 2 and 2.99 indicate low , scores between 3 and 3.99 indicate moderate , scores between 4 and 4.99 indicate high and scores of 5 or higher indicate very high job satisfaction or organizational commitment . this research investigates the relationship between perceptions of hospital managers and employees regarding the leadership behavior of hospital managers , and how this is related to the job satisfaction and organizational commitment of employees in isfahan university hospitals ( iuhs ) , isfahan , iran . separate questionnaires were sent to managers and employees . employee questionnaire package contained a cover letter , and questionnaires related to employees job satisfaction , organizational commitment and the leadership style of hospital manager . managers questionnaire package included a cover letter , their leadership styles , job satisfaction and organizational commitment . the job satisfaction scale ( specter 1997 ) utilized a likerttype scale with six response alternatives ranging from strongly disagree ( weighted 1 ) tostrongly agree ( weighted 6 ) for each of the 36 items to measure job satisfaction ( 21 ) . aspects of job satisfaction addressed are with : ( a ) pay , ( b ) promotion , ( c ) supervision , ( d ) fringe benefits , ( e ) contingent rewards , ( f ) operating conditions , ( g ) co workers , ( h ) nature of work , and ( i ) communication ( 4 items in each domain ) . aspects of organizational commitment addressed were affective , continuance , and normative commitment ( 22 ) . mangers leadership scale ( likert , 1967 ) had 35 items of which 15 items determined a manager s employee oriented dimension ( consideration ) and 20 items determined the task oriented dimension ( initiating structure ) of leadership style ( 23 ) . according to likert ( 1967 ) , the four distinct practices that leaders use to affect employee and organizational performance include : exploitative authoritative , benevolent authoritative , consultative and participative management . the final test version questionnaires were then pilot tested , using a random sample of 40 hospital employees ( not included in the sample ) and found to be well accepted and easy to fill in . the reliability coefficient was .8749 for job satisfaction , .7784 for organizational commitment , .8767 for leadership style questionnaire from the view point of employees and .8139 for leadership style from the view point of managers questionnaires . in order to normalize the likert scale on 1- 6 scales for each domain of job satisfaction and commitment questionnaires , the sum of raw scores of items in each domain was divided by the numbers of items in each domain and for overall job satisfaction and commitment , sum of raw scores of items scores of 2 or lower on the total scale indicate very low , scores between 2 and 2.99 indicate low , scores between 3 and 3.99 indicate moderate , scores between 4 and 4.99 indicate high and scores of 5 or higher indicate very high job satisfaction or organizational commitment . data were collected from 814 persons including 665 employees , 127 first line managers ( head of departments ) , 11 middle managers ( hospital managers ) and 11 senior managers ( hospital presidents ) . approximately 82% ( n=667 ) of the participants were married and 39.9% ( n=325 ) had earned bachelor s degrees . employees , front line , middle and senior managers on the average , had 11 , 19 , 20 and 18 years of working experiences respectively . hospital employees were satisfied overall with their job with a mean score of 3.260.56 on a 6 scale ( moderate satisfaction ) compared with the possible range from 1.52 to 5.61 . employees , first line , middle and senior managers scored a mean of job satisfaction of 3.21 , 3.40 , 3.97 and 3.73 respectively . within the nine items of the job satisfaction scale , the three dimensions of the job with which respondents were most satisfied were : supervision , nature of the job and co workers . respondents were least satisfied with the benefits , contingent rewards , communication , salaries , working conditions , and promotion ( see table 3 ) . a difference existed in perceptions between employees and managers pertaining to what aspects of the job are important to employees job satisfaction . employees reported that managers loyalty to employees , job security , good pay and good working conditions were the most important motivators for them . however , managers thought that sufficient salaries , recognition and job security were most important to employees . the mean score of employees job satisfaction in general and specialized hospitals was 3.19 and 3.37 respectively . the mean score of employees job satisfaction in hospitals with the specialty in burn ( 3.08 ) , psychiatry ( 3.29 ) , and cancer ( 3.31 ) were low and in hospitals with the specialty in ophthalmology ( 3.42 ) and cardiology ( 3.52 ) was high . employees in specialized hospitals receive more monetary benefits than those employees in general hospitals because of the type of expensive services provided to patients . the mean score of employees job satisfaction in the central storage department ( 4.21 ) , secretarial unit ( 4.05 ) , public relations office ( 3.91 ) , social worker office ( 3.90 ) and material supply department ( 3.66 ) were high and in the psychiatry ward ( 2.55 ) , pediatrics ward ( 2.63 ) , dialysis ward ( 2.75 ) , urology ward ( 2.85 ) and medical records department , ( 2.88 ) were low . there was strong correlation between the job satisfaction of employees and their gender , marital status , age , tenure , organizational position and received salaries ( p<0.01 ) . there was no statistically significant correlation between job satisfaction of employees and their graduation levels and type of employment : permanent or contract employment ( p>0.05 ) . in order to determine the main factors that cause satisfaction and/or dissatisfaction with work , the relationship between total job satisfaction and job satisfy factors was analyzed . calculations of spearmen s ratios revealed the strongest correlation between total job satisfaction and such characteristics as salaries , .687 ; fringe benefits , .685 ; promotion , .673 and communication , .637 . on the other hand work conditions , .468 ; nature of the job , .502 ; supervision , .536 ; and co workers , .554 had less effect on employees job satisfaction respectively . the mean score of employees satisfaction of job factors and organizational factors related to job satisfaction was 4.39 and 3.05 on a 6 scale respectively . organizational , job and individual factors overall explained 99.3 % of the variance in employees job satisfaction . regards to organizational factors , pay explained the largest amount of the variance , followed by coworkers , promotion , communication , supervision and benefit . the mean score of respondents organizational commitment was 3.980.49 on a 6 scale ( moderate ) . the mean score of organizational commitment of employees , first line , middle and senior managers was 3.97 , 4.07 , 4.12 and 4.03 from 6 credits respectively . the mean score of affective , continuance , and normative commitment were 3.880.69 , 3.740.61 , and 4.320.38 respectively . the mean score of organizational commitment in employees with relevant educational background towards their job was higher than those without any relevance . a negative association was seen between employees organizational commitment and their educational levels ( r = -0.126 , p=0.001 ) . significant differences were obtained between age , tenure , organizational position , type of employment , received salaries and organizational commitment ( p<0.05 ) . temporary employment and amount of salaries the employee s organizational commitment in therapeutic and diagnostic departments was higher than administrative and ancillary departments . significant differences were not obtained between employees organizational commitment and their gender , and marital status . in correlation analysis between organizational commitment and its three dimensions , affective commitment , 0.809 ; continuance commitment , 0.741 ; and normative commitment , 0.417 respectively had positive and the highest effect on employees organizational commitment . the results of the simultaneous multiple regression model indicate that organizational , job and individual factors overall explained 54.5% of the variance in employees organizational commitment . the mean score of employee - oriented dimension of leadership style in first line , middle and senior managers were 52 6.35 , 54 3.89 , and 54 5.00 ( from 75 credit ) respectively . the mean score of task - oriented dimension of leadership style in first line , middle and senior managers were 68 9.25 , 69 6.70 , and 70 7.20 ( from 100 credit ) respectively . from the viewpoint of employees the mean score of hospital managers employee - oriented and task - oriented credits were 46 ( out of 75 credits ) and 65 ( out of 100 credits ) . from the viewpoint of hospital managers the mean score of their employee - oriented and task - oriented credits were 54 and 69 . in other words , from the view point of employees , hospital managers were more task - oriented and from the view point of the hospital managers themselves , they were more employee - oriented . there was no correlation between leadership style of managers and their demographic variables except age and managerial experience years . pearson correlation coefficients indicate a significant statistically relationship between hospital managers management experience years and their employee oriented ( p= 0.024 and r=0.736 ) and task oriented ( p= 0.023 and r=0.706 ) dimensions of leadership style . there was a statistically significant relationship between employees job satisfaction and their organizational commitment ( p<0.001 and r=0.623 ) indicating that the employees who are more satisfied with their job are also more committed to the health care service . satisfaction with job identity , .589 ; supervision , .463 ; communications , .442 ; contingent rewards , .367 ; promotion , .344 ; fringe benefit , .242 ; salaries , .235 and work conditions , .215 were found to have a significant relationship to organizational commitment . on the other hand , there was a statistically significant correlation between the job satisfaction of employees and the leadership style of managers . this correlation between employees job satisfaction and employee - oriented and task - oriented dimensions of leadership style of hospital managers was at p<0.001 and p<0.01 levels . the correlation co efficient between employee oriented and task oriented dimensions of leadership style and employees satisfaction factors showed that the most positive co - efficiency was between supervision and employee oriented dimension and the most negative co - efficiency was between fringe benefits and task oriented dimension of leadership style of managers . a positive , significant correlation was shown among managers leadership style and employees commitment ( p<0.01 ) . leadership behaviors explained 28% variance in employees job satisfaction and 20 % in their commitment . employees oriented dimension of leadership explained the largest amount of the variance in these two variables . however , draper et al.s ( 2004 ) study showed a negative correlation between job satisfaction and dimensions of organizational commitment which is contrary to with the finding from the present study ( 26 ) . these differing results may be due to the differences in culture and the use of different instruments to measure commitment . this study has also shown that there is a significant negative relationship between the education level of employees and their commitment with their organization . employees who work with patients reported less job satisfaction but more commitment in this study . job rotation could possibly be a good strategy for improving job satisfaction of these employees . a supportive management style , demonstrated through open communication , respect and recognition it has been noted in this study that leadership , job satisfaction and commitment are closely interrelated . these findings are consistent with earlier studies in health care organizations that demonstrate the connection between job satisfaction ( 27 , 28 , 29 ) and organizational commitment ( 30 , 31 ) with leadership . mosadeghrad and tahery ( 2004 ) in their research in iranian public hospitals concluded that managers knowledge about leadership styles was low ( 32 ) . providing more information about leadership theories help managers understand the importance of applying the right leadership style in their organizations . managers in such a culture may use a more directive leadership style to communicate with their subordinates . therefore , in high power distance cultures , participative leadership style could be viewed as weak and ineffective leadership style . since charismatic leaders help reduce uncertainty , there is a strong preference for visionary , honest , cooperative , generous , concerned , and modest leaders ( 36 ) . in an innovative , corporate and supportive culture , according to mosadeghrad and malek pour ( 2004 ) , 75% of isfahan university hospitals had mechanistic and bureaucratic structures . hospital managers should consider the structure and culture of the organization in choosing the appropriate leadership style ( 37 , 38 , 39 , 40 , 41 ) . maslow s ( 1954 ) believes that human needs form a five - level hierarchy ranging from physiological needs , safety , love , and esteem to self - actualization ( 42 ) . according to maslow s theory of hierarchy of needs , once individuals have satisfied one need in the hierarchy , it ceases to motivate their behavior and they are motivated by the need at the next level up the hierarchy . in this study , employees job satisfaction in relation to their salaries and benefits and working conditions was low . the participative management is not a good leadership style for these hospitals at the moment , unless managers meet their employees basic needs , improve employees organizational maturity , promote a culture of teamwork , cooperation and participation and upgrade organizational structure accordingly . hospital managers should understand the impact of varying leadership styles on employees job satisfaction and commitment level . thus , education and training should be provided to develop effective leadership behaviors to have a more positive effect on their employees job satisfaction and organizational commitment . research regarding the impact of leadership style of hospital managers on employees job satisfaction and commitment is relatively new in iranian health care organizations . the purpose of this study was to contribute to a better understanding of the relationship between leadership behaviors of managers and these two employees work - related attitudes . this research documented the level of job satisfaction and organizational commitment among employees and the type of leadership style of managers in iranian hospitals . this research also contributed to the knowledge of factors influencing job satisfaction and organizational commitment . the findings showed that hospital employees were moderately satisfied with their jobs and committed to their organization . employees were mostly dissatisfied with salaries , benefits , rewards , work conditions , and communication . these include demographic variables of age , years of work experiences , marital status , gender and organizational position , monthly salary , type of hospital , employees organizational commitment , leadership style of managers and the nine subscales of job satisfy factors , as indicted in table 3 . factors that may influence the level of employees commitment are demographic variables of age , years of work experiences , educational levels , organizational position , and type of employment , monthly salary , leadership style of managers and the nine subscales of job satisfier factors , as indicted in table 3 . in this study , employee oriented leadership explained significant variance in employees job satisfaction and commitment . the current research was limited to a group of public hospitals in iran . more studies of the iranian health care employees especially within hospitals that are not as hierarchical as the public hospitals surveyed in the current study are needed . more studies which involve hospital employees from other countries would enrich the literature on hospital employees job satisfaction and commitment . the results of such studies can be very helpful in developing strategies to improve the global retention of hospital personnel .

we studied 45 patients with chronic hepatitis c obtained from the mayo and scripps clinics . thirtyeight men and seven females , median age 60 years old ( range : 5076 ) . twelve patients had one liver biopsy sample and 33 patients had two biopsy samples ( total samples , 78 ) . fibrosis stages were determined in liver biopsies according to metavir classification 12 and the distribution according to stage was as follows : f0 i ; f1 18 ; f2 15 ; f3 14 , and f4 30 samples . all patients were consented at the mayo and scripps clinics and the institutional review boards of both sites approved the collection of the samples . for liver samples , the mirvana kit ( am1560 ; life technologies ) was used according to the manufacturer 's protocol with a slight modification for two phenol chloroform extractions ( burgos et al . , 2013 ) . rna concentrations were measured using a nanodrop 2000 ( thermo scientific , wilmington , de , usa ) . serum samples ( 12 ml ) were isolated using the same modified mirvana paris protocol ( am1556 ; life technologies ) with sequential phenol the illumina truseq small rna library preparation kit ( rs2000048 ; illumina , san diego , ca , usa ) was used for sequencing all samples . tissue samples were used with the kit at 200 ng , and 15 pcr cycles were used . for serum samples , the total rna isolated from each sample volume ( ~1 ml ) was used for small rna sequencing . fifteen to 20 samples were then pooled and placed on different lanes of a single read illumina v3 flowcell ( gd4013001 ) . one lane of the flowcell was loaded with phix as a reference lane to help with low nucleotide diversity in microrna . reads that passed quality check were trimmed of their adaptor sequence then entered into the analysis pipeline . samples were aligned using mirdeep2 , and version 21 of mirbase ( 13 ) . the mirna read counts identified by mirdeep2 were normalized for compositional bias in sequenced libraries and library size using deseq2 15 ( version : 1.6.1 ) . samples with < 100 000 mapped microrna reads were removed from data , resulting in 43 tissue and 78 serum samples . tissue : stage f1 ( n = 9 ) , stage f2 ( n = 9 ) , stage f3 ( n = 10 ) , stage f4 ( n = 15 ) ; serum : no fibrosis ( n = 3 ) , stage f1 ( n = 14 ) , stage f2 ( n = 16 ) , stage f3 ( n = 15 ) , stage4 ( n = 30 ) . differential expression of mirna read counts was performed using the deseq2 ( v1.6.1 ) package . for the differential expression analyses , mirnas with average ordinal logistic regression and oneway anova were used to assess monotonic relationships between fibrosis stage and normalized mirna counts using the package ordinal ( version : 2014.1114 ) . a graphical method for assessing the parallel slopes assumption parameter confidence intervals were based on the profile likelihood function , and the estimates in the output are given in units of ordered log odds . we fitted a negative binomial generalized linear model for each genotype and carried out a likelihood ratio test in order to test for presence of differentially expressed micrornas between any of the fibrosis stage groups . we found no differentially represented mirnas with relationship to viral genotypes in varying fibrosis stages . taqman microrna reverse transcription kit and primers for mir1835p , mir1825p , mir199a5p and mir148a5p were run according to the manufacturer 's protocol ( life technologies ) using an abi 7900ht fast realtime pcr system ( life technologies ) . 2 was used to calculate the fold change of microrna expression levels between ( f0f2 ) and advanced ( f3f4 ) fibrosis stages . a ttest ( two tailed , type 2 ) was performed to assess statistical significance between the normalized ct values for the two groups . we studied 45 patients with chronic hepatitis c obtained from the mayo and scripps clinics . thirtyeight men and seven females , median age 60 years old ( range : 5076 ) . twelve patients had one liver biopsy sample and 33 patients had two biopsy samples ( total samples , 78 ) . fibrosis stages were determined in liver biopsies according to metavir classification 12 and the distribution according to stage was as follows : f0 i ; f1 18 ; f2 15 ; f3 14 , and f4 30 samples . all patients were consented at the mayo and scripps clinics and the institutional review boards of both sites approved the collection of the samples . for liver samples , the mirvana kit ( am1560 ; life technologies ) was used according to the manufacturer 's protocol with a slight modification for two phenol chloroform extractions ( burgos et al . , 2013 ) . rna concentrations were measured using a nanodrop 2000 ( thermo scientific , wilmington , de , usa ) . serum samples ( 12 ml ) were isolated using the same modified mirvana paris protocol ( am1556 ; life technologies ) with sequential phenol the illumina truseq small rna library preparation kit ( rs2000048 ; illumina , san diego , ca , usa ) was used for sequencing all samples . tissue samples were used with the kit at 200 ng , and 15 pcr cycles were used . for serum samples , the total rna isolated from each sample volume ( ~1 ml ) was used for small rna sequencing . fifteen to 20 samples were then pooled and placed on different lanes of a single read illumina v3 flowcell ( gd4013001 ) . one lane of the flowcell was loaded with phix as a reference lane to help with low nucleotide diversity in microrna . reads that passed quality check were trimmed of their adaptor sequence then entered into the analysis pipeline . samples were aligned using mirdeep2 , and version 21 of mirbase ( 13 ) . the mirna read counts identified by mirdeep2 were normalized for compositional bias in sequenced libraries and library size using deseq2 15 ( version : 1.6.1 ) . samples with < 100 000 mapped microrna reads were removed from data , resulting in 43 tissue and 78 serum samples . tissue : stage f1 ( n = 9 ) , stage f2 ( n = 9 ) , stage f3 ( n = 10 ) , stage f4 ( n = 15 ) ; serum : no fibrosis ( n = 3 ) , stage f1 ( n = 14 ) , stage f2 ( n = 16 ) , stage f3 ( n = 15 ) , stage4 ( n = 30 ) . differential expression of mirna read counts was performed using the deseq2 ( v1.6.1 ) package . for the differential expression analyses , mirnas with average < 5 counts across all samples ordinal logistic regression and oneway anova were used to assess monotonic relationships between fibrosis stage and normalized mirna counts using the package ordinal ( version : 2014.1114 ) . a graphical method for assessing the parallel slopes assumption parameter confidence intervals were based on the profile likelihood function , and the estimates in the output are given in units of ordered log odds . we fitted a negative binomial generalized linear model for each genotype and carried out a likelihood ratio test in order to test for presence of differentially expressed micrornas between any of the fibrosis stage groups . we found no differentially represented mirnas with relationship to viral genotypes in varying fibrosis stages . taqman microrna reverse transcription kit and primers for mir1835p , mir1825p , mir199a5p and mir148a5p were run according to the manufacturer 's protocol ( life technologies ) using an abi 7900ht fast realtime pcr system ( life technologies ) . cycle threshold values for each target microrna were normalized against u6 . 2 was used to calculate the fold change of microrna expression levels between ( f0f2 ) and advanced ( f3f4 ) fibrosis stages . a representative nine samples were chosen from each group for qrtpcr validation . a ttest ( two tailed , type 2 ) was performed to assess statistical significance between the normalized ct values for the two groups . using next generation sequencing technologies , we profiled micrornas in liver biopsy samples from patients with hepatitis c infection . we obtained tissue samples from nine patients with stage f1 liver fibrosis , nine patients with stage f2 , 10 patients with fibrosis stage f3 , and 15 patients with stage f4 . we first tested to see if there was an interaction between mirnas and viral genotype . we did not find micrornas in the tissue samples that were expressed differently with viral genotype , nor in association with viral genotype and fibrosis stage . for tissue samples , the median number of reads mapped to microrna per sample was 504 351 , with a range from 121 504 to 5 708 614 . the total number of micrornas that were detected in liver tissue were 658 , and those with a normalized count of 5 were 301 . we obtained the following serum samples : three patients with stage f0 liver fibrosis , 13 patients with stage f1 liver fibrosis , 17 patients with stage f2 , 15 patients with fibrosis stage f3 and 30 patients with stage f4 . for the serum samples , the median number of reads mapped to microrna was 2 084 067 , with a range from 159 783 to 10 833 093 . the total number of micrornas that were detected in the serum were 549 , and those with a normalized count of 5 were 397 . normalized counts for micrornas detected in serum or tissue samples are displayed in tables s1 and s2 , micrornas overlapping in at least 30% of tissue and serum samples are highlighted . we combined the microrna data from liver biopsies classified as fibrosis f1 and f2 ( early fibrosis ) separately from f3 and f4 ( advanced fibrosis ) . there were 18 samples in the early and 25 samples in the advanced fibrosis groups . we compared the microrna between these two groups and corrected for multiple tests using benjamini there are 37 micrornas differentially expressed between the two groups , table 1 contains 25 significant micrornas with a log2fold change > 0.6 or < 0.6 . we also assessed micrornas differentially expressed between the two most extreme fibrosis stages , f1 and f4 , 44 micrornas were significantly different ( table s4 ) . differentially expressed micrornas in tissue between early ( f1 and f2 ) and advance ( f3 and f4 ) fibrosis . significant micrornas with a smaller fold change are listed in table s2 the volcano plot in fig . 1a displays significant micrornas and their relative fold changes when comparing early ( f1 and f2 ) to advanced ( f3 and f4 ) fibrosis . mir182 and mir183 ( green , log2fold change > 1 and corrected pvalue < 0.05 ) and mir200a5p and mir199a5p ( blue , log2fold change > 0.6 and corrected pvalue < box and whisker plots of normalized count data between early and advanced fibrosis for five micrornas are displayed in fig . ( a ) a volcano plot of samples from f1 and f2 ( early ) liver fibrosis compared with samples from f3 and f4 ( advanced fibrosis ) . micrornas with a log2fold change > 1 and significance < 0.05 are indicated in green , micrornas with fold change < 0.6 and > 0.6 and adjusted pvalue < 0.05 are blue , smaller fold changes with adjusted pvalue < 0.05 are red . ( b ) box and whisker plots for mir1835p , mir1825p , mir200a5p , and mir199a5p , the four micrornas with the smallest pvalues and largest fold changes . we wanted to verify the sequencing results using a second assay platform , qrtpcr . from liver tissue we chose the following micrornas with increased expression in advanced fibrosis : mir1835p , mir1825p , mir199a5p and with decreased expression in advanced fibrosis , mir148a5p . we did not attempt to validate mir200a5p by qrtpcr , the average count across samples was low , ~11 . eighteen samples were reverse transcribed using specific taqman microrna probes for the four selected micrornas . nine representative samples from the f1 and f2 group were compared with nine samples selected from the f3 and f4 group . figure 2a shows the log2fold change between early and advanced fibrosis calculated using 2 and u6 as the reference for normalization . micrornas with an asterisk , mir1835p , mir1485p and mir199a5p , were significantly different between early and advanced fibrosis ( p 0.05 , ttest ) . although the trend was the same as exhibited in the sequencing data , the change in the microrna expression levels between early and advanced fibrosis was not significant for mir1825p in this subset of samples ( p 0.34 ; fig . qrtpcr validation of four micrornas with highest significance and fold change in comparison of f1 and f2 ( early ) with f3 and f4 ( advanced ) fibrosis . ( a ) calculated fold change for micrornas 1835p , 1825p , mir199a5p and mir148a5p . ( b ) average ct values normalized to u6 and the standard error of the mean . * indicates qrtpcr changes that are statistically significant by ttest ( p 0.05 ) . we assessed the data using ordinal logistic regression analysis to determine if microrna expression changes , either up or down , behaved in a progressive manner with increasing fibrosis from f1 to f4 . we assessed all micrornas across each stage and found 35 micrornas that indicated an ordered change in expression after multiple corrections ( table 2 ) . mir1825p , mir1835p , mir199a5p , and mir1485p were among the significant micrornas that showed monotonic trend , continuous change with increasing fibrosis ( fig . ordinal logistic regression was implemented in order to detect mirnas exhibiting monotonic expression trend with fibrosis advancement . we report mirnas with the lowest akaike information criterion value significant at adjusted pvalue < 0.05 we next examined serum samples to determine whether or not we could detect significant microrna changes that indicated fibrosis stage . we assessed micrornas that were detectable in both serum and liver ( number = 495 ) . we compared microrna expression levels in patients with f0 , f1 and f2 ( combined , n = 33 ) to microrna expression levels in patients with f3 and f4 ( n = 45 ) . we found 34 micrornas that had pvalue < 0.05 , however , none of them survived benjaminihochberg correction for multiple testing ( table s5 ) . by using next generation sequencing as our discovery platform , we were able to investigate the number and potential significance of variations in the mature microrna sequence isomirs . we categorized isomirs as the following : 3 length variants ( lv3p ) , 5 length variants ( lv5p ) and nontemplated nucleotide additions ( ntaa , ntat , ntac , ntag ) . to provide a more detailed description of microrna length heterogeneities , we indicated the number of nucleotides added ( + ) or trimmed ( ) from the 3 or 5 end in the isomir categories . of 37 differentially expressed mirnas between early and advanced fibrosis in liver , 10 were differentially expressed for at least one of the isomir categories : 199b3p , 148a3p , 1505p , 1223p , 199a5p , 10a5p , 92a3p , 1955p , 181c5p , 181a5p ( table 3 ) . several mirnas with no evidence of differential expression for the exact canonical sequence were significantly differentially expressed for one or more isomir categories ( table 3 ) . for some micrornas , when we combined all the counts for isomirs and the canonical sequence , they became differentially expressed . while the functional consequences of these nucleotide changes are not yet fully understood , we report our findings in order to increase the growing knowledgebase on these types of microrna modifications . micrornas in tissue differentially expressed solely for one or more isomir categories between early and advanced fibrosis . isomir categories incorporate 3 length variants ( lv3p ) , 5 length variants ( lv5p ) and nontemplated nucleotide additions ( ntaa , ntat , ntac , ntag ) with + / indicating additions and trimming respectively . we identified 37 micrornas , 26 up and 11 downregulated , between early and advanced fibrotic stages in liver biopsies from patients with chronic hepatitis c. early fibrosis were metavir classification f1 and f2 biopsy samples grouped together . samples classified as f3 and f4 were grouped as advanced fibrosis . in serum , we were not able to identify micrornas that were significant after adjusting for multiple comparisons ( table s5 ) . the impact of these single nucleotide changes on microrna function is not well understood . with respect to microrna expression analysis , it is also not clear how these single nucleotide changes might alter microrna measurements using other methods such as microarray and qrtpcr . it might cause some difficulties comparing microrna expression levels across platforms that are sensitive or insensitive to these changes . we focused our attention on four micrornas with the lowest pvalues , mir1835p , mir1825p , mir200a5p and mir199a5p , all with elevated expression levels , as well as mir148a5p that had significantly decreased expression when comparing early and advanced liver fibrosis . each of these selected micrornas demonstrated similar direction and fold changes when assessed with qrtpcr . in addition , we used ordinal logistic regression to examine the progressive increase or decrease in each of these micrornas with advancing fibrosis severity . each of these micrornas becomes increasingly dysregulated in hepatic tissue with progressive fibrosis ( adjusted pvalue < 0.05 ) , except mir200a5p ( fig . very little is known about which micrornas play a role in the pathogenesis of liver fibrosis , or increase the risk for downstream complications , such as hepatic cancer . using pathway analysis to get a better understanding of the function of micrornas in liver fibrosis can be very difficult to interpret . not only can a single microrna regulate many mrna targets , but there is also a large false positive rate for the prediction of micrornas and mrna targets . . ingenuity pathway analysis ( qiagen http://www.ingenuity.com ) , lists 114 human micrornas as having experimentally validated target mrnas , most micrornas are linked to several mrna targets . we chose to focus our discussion on a few micrornas identified in these experiments and the published literature that strengthens their link to liver disease . while this is not an exhaustive list , and there are many studies that found different micrornas to be important in liver fibrosis , the micrornas reported here have been found to be associated with liver damage in several papers . figure 3 is a network diagram describing the relationships for each of the mirnas and the available information from the literature and the association of each one with liver disease , injury or cancer . network analysis for the five mirnas that had the highest fold change and lowest pvalue . using the current literature and what is known about these mirnas with respect to liver disease , fibrosis and hepatocellular carcinoma , we created a network diagram . the diagram indicates experimentally validated biological processes , gene targets and disease pathways affected by these mirnas . several papers have linked high mirna182 expression levels to hepatocellular carcinoma 17 , 18 or liver metastasis 19 , 20 . metastasis suppressor 1 18 , 21 , 22 was identified as a target for mir182 in liver . microrna expression measured in mouse models of alcoholic and nonalcoholic steatohepatitis 23 found mir182 and mir183 ( both identified in our study ) to be downregulated in alcoholic liver and upregulated in nonalcoholic fatty liver . 24 . compared liver samples from patients with chronic hepatitis and cirrhosis and found mir182 to be one of the most differentially expressed micrornas , upregulated in chronic hepatitis . in the current paper , we also found mir182 to increase with fibrosis stage in patients with chronic hepatitis c. wojcicka et al . 5 . examined micrornas in cirrhotic liver and hepatocellular carcinoma . mir1835p and mir199a5p ( both significantly upregulated with advanced fibrosis in our data ) were among the top ten most differentially expressed micrornas when comparing tumour and normal tissue . mir183 was also found to be significantly upregulated in hepatocellular carcinoma samples from patients with chronic hepatitis c 25 . in a larger study examining liver biopsy samples from normal controls and patients with hepatitis c , hepatitis b , cirrhosis , hepatocellular carcinoma , they found that mir183 increased with increasing liver damage , highest in cirrhosis and cancer . 10 . found mir183 to be upregulated in cirrhotic liver and premalignant lesions , and went on to investigate potential mrna targets for mir183 . they found akap12 , a tumour suppressor gene found to be downregulated in cirrhotic liver , premalignant lesions and hepatocellular carcinoma . mir183 was found to be upregulated in hepatocellular carcinoma samples in a study by li et al . 9 , where they also found that programmed cell death 4 , a proapoptotic molecule was targeted by mir183 . in our studies the expression level of mir200a was very low average read counts per sample were 11 . we observed a small significant increase with fibrosis stage ( table 1 , fig . several papers implicate mir200a in liver damage and cancer , but there are discrepancies in the level and direction of expression . perhaps these differences in findings are , in part , a reflection of the overall low abundance we found for this microrna in tissue . in many cases , reduced expression of mir200a predicted prognosis 26 , 30 and expression inversely correlated with tumour size 31 . 32 . found that mir200a was significantly decreased in patients with hepatocellular carcinoma and in cirrhotic tissue from patients with hepatocellular carcinoma compared with patients with cirrhosis . however , other papers found increased levels of mir200a promoting hepatocellular carcinoma progression 33 . in a rat model of nonalcoholic fatty liver disease , researchers found mir200a to be significantly upregulated in rats that received a highfat diet 27 , 34 , 35 . . also showed that human hepatocytes treated with free fatty acids and inflammatory factors had increased mir200a levels . mir200a was found to be associated with epithelialmesenchymal transition and invasion when overexpressed 26 , 36 . identified mrna targets of mir200a were macc1 ( metastasis associated in colon cancer1 ) 27 , ctnnb1 ( catenin , cadherinassociated protein beta1 ) 28 , 29 ; tgf2 ( transforming growth factor beta2 ) 29 and mdm2 ( e3 ubiquitinprotein ligase ) 37 . there are several papers describing the relevance of both mir199a5p and mir199a3p in liver disease and injury , however , there were variable findings with respect to expression levels of these micrornas in the literature . murakami et al . 38 . found that mir199a regulates hepatitis c virus replication . several papers found the expression of mir199a to be down with injury and disease 19 , 21 , 31 , 39 , 40 , 41 , 42 , 43 , and in other papers mir199a was found to be upregulated in hepatocellular carcinoma and liver injury 44 , 45 , 46 and increased with liver fibrosis progression 6 , 47 . experimentally identified targets of mir199a5p in liver disease were smarca4 ( swi / snfrelated , matrixassociated , actindependent regulator of chromatin , subfamily a , member 4 ) and mst1 ( macrophage stimulating 1 ) 48 , mmp9 ( matrix metallopeptidase 9 ) 44 , grp78 ( glucoseregulated protein , 78 kda ) , endoplasmic reticulum to nucleus signalling 1 , activating transcription factor 6 45 , clathrin heavy chain 21 , atg7 ( autophagy related 7 ) 49 , hypoxia inducible factor1 41 and discoidin domain receptor1 43 . in our study , mir148a5p was the most significantly downregulated microrna in advanced fibrosis . . found mir148a5p was downregulated in hepatocellular carcinoma and expression was correlated with tumour stage . several other papers found downregulation of mir148a 51 , 52 , targeting ubiquitin specific protease 4 and sphingosine 1phosphate receptor 1 53 , genes in the met / snail signalling pathway 54 , wnt1 55 , dna methyltransferase 52 , 56 and cmyc 57 . in a large study examining two subtypes of hepatocellular carcinoma 58 , mir148a was downregulated in both cancer types compared with controls , and the more aggressive subtype had significantly less mir148a expression . acvr1 ( activin a receptor , type1 ) was identified as a downstream target in that study . xu et al . 60 . found that mir148a is repressed by hepatitis b virus x protein , and this in turn allows enhanced expression of hematopoietic preb cell leukaemia transcription factorinteracting protein . mir148a was also found to be downregulated in liver injury and rejection with liver transplantation 61 and downregulated in hepatoblastoma 62 . in conclusion , our data identified several micrornas that are altered with worsening liver fibrosis in chronic hepatitis c patients . these data provide further support for the role of specific micrornas in the pathogenesis of fibrosis and focuses attention on identifying micrornas that could be used to predict clinical outcomes and complications . several papers have linked high mirna182 expression levels to hepatocellular carcinoma 17 , 18 or liver metastasis 19 , 20 . metastasis suppressor 1 18 , 21 , 22 was identified as a target for mir182 in liver . microrna expression measured in mouse models of alcoholic and nonalcoholic steatohepatitis 23 found mir182 and mir183 ( both identified in our study ) to be downregulated in alcoholic liver and upregulated in nonalcoholic fatty liver . 24 . compared liver samples from patients with chronic hepatitis and cirrhosis and found mir182 to be one of the most differentially expressed micrornas , upregulated in chronic hepatitis . in the current paper , we also found mir182 to increase with fibrosis stage in patients with chronic hepatitis c. wojcicka et al . 5 . examined micrornas in cirrhotic liver and hepatocellular carcinoma . mir1835p and mir199a5p ( both significantly upregulated with advanced fibrosis in our data ) were among the top ten most differentially expressed micrornas when comparing tumour and normal tissue . mir183 was also found to be significantly upregulated in hepatocellular carcinoma samples from patients with chronic hepatitis c 25 . in a larger study examining liver biopsy samples from normal controls and patients with hepatitis c , hepatitis b , cirrhosis , hepatocellular carcinoma , they found that mir183 increased with increasing liver damage , highest in cirrhosis and cancer . 10 . found mir183 to be upregulated in cirrhotic liver and premalignant lesions , and went on to investigate potential mrna targets for mir183 . they found akap12 , a tumour suppressor gene found to be downregulated in cirrhotic liver , premalignant lesions and hepatocellular carcinoma . mir183 was found to be upregulated in hepatocellular carcinoma samples in a study by li et al . 9 , where they also found that programmed cell death 4 , a proapoptotic molecule was targeted by mir183 . in our studies the expression level of mir200a was very low average read counts per sample were 11 . we observed a small significant increase with fibrosis stage ( table 1 , fig . 1 ) . several papers implicate mir200a in liver damage and cancer , but there are discrepancies in the level and direction of expression . perhaps these differences in findings are , in part , a reflection of the overall low abundance we found for this microrna in tissue . in many cases , reduced expression of mir200a predicted prognosis 26 , 30 and expression inversely correlated with tumour size 31 . 32 . found that mir200a was significantly decreased in patients with hepatocellular carcinoma and in cirrhotic tissue from patients with hepatocellular carcinoma compared with patients with cirrhosis . however , other papers found increased levels of mir200a promoting hepatocellular carcinoma progression 33 . in a rat model of nonalcoholic fatty liver disease , researchers found mir200a to be significantly upregulated in rats that received a highfat diet 27 , 34 , 35 . . also showed that human hepatocytes treated with free fatty acids and inflammatory factors had increased mir200a levels . mir200a was found to be associated with epithelialmesenchymal transition and invasion when overexpressed 26 , 36 . identified mrna targets of mir200a were macc1 ( metastasis associated in colon cancer1 ) 27 , ctnnb1 ( catenin , cadherinassociated protein beta1 ) 28 , 29 ; tgf2 ( transforming growth factor beta2 ) 29 and mdm2 ( e3 ubiquitinprotein ligase ) 37 . there are several papers describing the relevance of both mir199a5p and mir199a3p in liver disease and injury , however , there were variable findings with respect to expression levels of these micrornas in the literature . murakami et al . 38 . found that mir199a regulates hepatitis c virus replication . several papers found the expression of mir199a to be down with injury and disease 19 , 21 , 31 , 39 , 40 , 41 , 42 , 43 , and in other papers mir199a was found to be upregulated in hepatocellular carcinoma and liver injury 44 , 45 , 46 and increased with liver fibrosis progression 6 , 47 . experimentally identified targets of mir199a5p in liver disease were smarca4 ( swi / snfrelated , matrixassociated , actindependent regulator of chromatin , subfamily a , member 4 ) and mst1 ( macrophage stimulating 1 ) 48 , mmp9 ( matrix metallopeptidase 9 ) 44 , grp78 ( glucoseregulated protein , 78 kda ) , endoplasmic reticulum to nucleus signalling 1 , activating transcription factor 6 45 , clathrin heavy chain 21 , atg7 ( autophagy related 7 ) 49 , hypoxia inducible factor1 41 and discoidin domain receptor1 43 . in our study , mir148a5p was the most significantly downregulated microrna in advanced fibrosis . in a recent publication , pan et al . 50 . found mir148a5p was downregulated in hepatocellular carcinoma and expression was correlated with tumour stage . several other papers found downregulation of mir148a 51 , 52 , targeting ubiquitin specific protease 4 and sphingosine 1phosphate receptor 1 53 , genes in the met / snail signalling pathway 54 , wnt1 55 , dna methyltransferase 52 , 56 and cmyc 57 . in a large study examining two subtypes of hepatocellular carcinoma 58 , mir148a was downregulated in both cancer types compared with controls , and the more aggressive subtype had significantly less mir148a expression . acvr1 ( activin a receptor , type1 ) was identified as a downstream target in that study . 60 . found that mir148a is repressed by hepatitis b virus x protein , and this in turn allows enhanced expression of hematopoietic preb cell leukaemia transcription factorinteracting protein . mir148a was also found to be downregulated in liver injury and rejection with liver transplantation 61 and downregulated in hepatoblastoma 62 . in conclusion , our data identified several micrornas that are altered with worsening liver fibrosis in chronic hepatitis c patients . these data provide further support for the role of specific micrornas in the pathogenesis of fibrosis and focuses attention on identifying micrornas that could be used to predict clinical outcomes and complications . additional supporting information may be found at onlinelibrary.wiley.com/doi/10.1111/liv.12919/suppinfo click here for additional data file .

abstractbackground & aimsaccumulating evidence indicates that micrornas play a role in a number of disease processes including the pathogenesis of liver fibrosis in hepatitis c infection . our goal is to add to the accruing information regarding microrna deregulation in liver fibrosis to increase our understanding of the underlying mechanisms of pathology and progression.methodswe used next generation sequencing to profile all detectable micrornas in liver tissue and serum from patients with hepatitis c , stages f1f4 of fibrosis.resultswe found altered expression of several micrornas , in particular , mir182 , mir199a5p , mir200a5p and mir183 were found to be significantly upregulated in tissue from liver biopsies of hepatitis c patients with advanced fibrosis , stage f3 and f4 , when compared with liver biopsies from patients with early fibrosis , stages f1 and f2 . we also found mir1485p , mir1260b , mir1223p and mir378i among the micrornas most significantly downregulated from early to advanced fibrosis of the liver . we also sequenced the serum micrornas ; however , we were not able to detect significant changes in circulating micrornas associated with fibrosis stage after adjusting for multiple tests.conclusionsadding measurements of tissue micrornas acquired during routine biopsies will continue to increase our knowledge of underlying mechanisms of fibrosis . our goal is that these data , in combination with studies from other researchers and future longterm studies , could be used to enhance the staging accuracy of liver biopsies and expand the surveillance of patients at increased risk for cancer and progression to advanced fibrosis .

Patients and methods Subjects Approval RNA isolation Sequencing Sequencing and statistical analysis qPCR Results Discussion miR182 miR1835p miR200a5p miR199a5p miR148a5p Supporting information

we studied 45 patients with chronic hepatitis c obtained from the mayo and scripps clinics . fibrosis stages were determined in liver biopsies according to metavir classification 12 and the distribution according to stage was as follows : f0 i ; f1 18 ; f2 15 ; f3 14 , and f4 30 samples . all patients were consented at the mayo and scripps clinics and the institutional review boards of both sites approved the collection of the samples . one lane of the flowcell was loaded with phix as a reference lane to help with low nucleotide diversity in microrna . samples with < 100 000 mapped microrna reads were removed from data , resulting in 43 tissue and 78 serum samples . tissue : stage f1 ( n = 9 ) , stage f2 ( n = 9 ) , stage f3 ( n = 10 ) , stage f4 ( n = 15 ) ; serum : no fibrosis ( n = 3 ) , stage f1 ( n = 14 ) , stage f2 ( n = 16 ) , stage f3 ( n = 15 ) , stage4 ( n = 30 ) . for the differential expression analyses , mirnas with average ordinal logistic regression and oneway anova were used to assess monotonic relationships between fibrosis stage and normalized mirna counts using the package ordinal ( version : 2014.1114 ) . we fitted a negative binomial generalized linear model for each genotype and carried out a likelihood ratio test in order to test for presence of differentially expressed micrornas between any of the fibrosis stage groups . taqman microrna reverse transcription kit and primers for mir1835p , mir1825p , mir199a5p and mir148a5p were run according to the manufacturer 's protocol ( life technologies ) using an abi 7900ht fast realtime pcr system ( life technologies ) . we studied 45 patients with chronic hepatitis c obtained from the mayo and scripps clinics . fibrosis stages were determined in liver biopsies according to metavir classification 12 and the distribution according to stage was as follows : f0 i ; f1 18 ; f2 15 ; f3 14 , and f4 30 samples . one lane of the flowcell was loaded with phix as a reference lane to help with low nucleotide diversity in microrna . samples with < 100 000 mapped microrna reads were removed from data , resulting in 43 tissue and 78 serum samples . tissue : stage f1 ( n = 9 ) , stage f2 ( n = 9 ) , stage f3 ( n = 10 ) , stage f4 ( n = 15 ) ; serum : no fibrosis ( n = 3 ) , stage f1 ( n = 14 ) , stage f2 ( n = 16 ) , stage f3 ( n = 15 ) , stage4 ( n = 30 ) . for the differential expression analyses , mirnas with average < 5 counts across all samples ordinal logistic regression and oneway anova were used to assess monotonic relationships between fibrosis stage and normalized mirna counts using the package ordinal ( version : 2014.1114 ) . we fitted a negative binomial generalized linear model for each genotype and carried out a likelihood ratio test in order to test for presence of differentially expressed micrornas between any of the fibrosis stage groups . taqman microrna reverse transcription kit and primers for mir1835p , mir1825p , mir199a5p and mir148a5p were run according to the manufacturer 's protocol ( life technologies ) using an abi 7900ht fast realtime pcr system ( life technologies ) . using next generation sequencing technologies , we profiled micrornas in liver biopsy samples from patients with hepatitis c infection . we obtained tissue samples from nine patients with stage f1 liver fibrosis , nine patients with stage f2 , 10 patients with fibrosis stage f3 , and 15 patients with stage f4 . we did not find micrornas in the tissue samples that were expressed differently with viral genotype , nor in association with viral genotype and fibrosis stage . the total number of micrornas that were detected in liver tissue were 658 , and those with a normalized count of 5 were 301 . we obtained the following serum samples : three patients with stage f0 liver fibrosis , 13 patients with stage f1 liver fibrosis , 17 patients with stage f2 , 15 patients with fibrosis stage f3 and 30 patients with stage f4 . for the serum samples , the median number of reads mapped to microrna was 2 084 067 , with a range from 159 783 to 10 833 093 . the total number of micrornas that were detected in the serum were 549 , and those with a normalized count of 5 were 397 . normalized counts for micrornas detected in serum or tissue samples are displayed in tables s1 and s2 , micrornas overlapping in at least 30% of tissue and serum samples are highlighted . we combined the microrna data from liver biopsies classified as fibrosis f1 and f2 ( early fibrosis ) separately from f3 and f4 ( advanced fibrosis ) . we compared the microrna between these two groups and corrected for multiple tests using benjamini there are 37 micrornas differentially expressed between the two groups , table 1 contains 25 significant micrornas with a log2fold change > 0.6 or < 0.6 . we also assessed micrornas differentially expressed between the two most extreme fibrosis stages , f1 and f4 , 44 micrornas were significantly different ( table s4 ) . differentially expressed micrornas in tissue between early ( f1 and f2 ) and advance ( f3 and f4 ) fibrosis . 1a displays significant micrornas and their relative fold changes when comparing early ( f1 and f2 ) to advanced ( f3 and f4 ) fibrosis . mir182 and mir183 ( green , log2fold change > 1 and corrected pvalue < 0.05 ) and mir200a5p and mir199a5p ( blue , log2fold change > 0.6 and corrected pvalue < box and whisker plots of normalized count data between early and advanced fibrosis for five micrornas are displayed in fig . ( a ) a volcano plot of samples from f1 and f2 ( early ) liver fibrosis compared with samples from f3 and f4 ( advanced fibrosis ) . ( b ) box and whisker plots for mir1835p , mir1825p , mir200a5p , and mir199a5p , the four micrornas with the smallest pvalues and largest fold changes . from liver tissue we chose the following micrornas with increased expression in advanced fibrosis : mir1835p , mir1825p , mir199a5p and with decreased expression in advanced fibrosis , mir148a5p . nine representative samples from the f1 and f2 group were compared with nine samples selected from the f3 and f4 group . micrornas with an asterisk , mir1835p , mir1485p and mir199a5p , were significantly different between early and advanced fibrosis ( p 0.05 , ttest ) . although the trend was the same as exhibited in the sequencing data , the change in the microrna expression levels between early and advanced fibrosis was not significant for mir1825p in this subset of samples ( p 0.34 ; fig . qrtpcr validation of four micrornas with highest significance and fold change in comparison of f1 and f2 ( early ) with f3 and f4 ( advanced ) fibrosis . mir1825p , mir1835p , mir199a5p , and mir1485p were among the significant micrornas that showed monotonic trend , continuous change with increasing fibrosis ( fig . ordinal logistic regression was implemented in order to detect mirnas exhibiting monotonic expression trend with fibrosis advancement . we report mirnas with the lowest akaike information criterion value significant at adjusted pvalue < 0.05 we next examined serum samples to determine whether or not we could detect significant microrna changes that indicated fibrosis stage . we compared microrna expression levels in patients with f0 , f1 and f2 ( combined , n = 33 ) to microrna expression levels in patients with f3 and f4 ( n = 45 ) . we found 34 micrornas that had pvalue < 0.05 , however , none of them survived benjaminihochberg correction for multiple testing ( table s5 ) . by using next generation sequencing as our discovery platform , we were able to investigate the number and potential significance of variations in the mature microrna sequence isomirs . to provide a more detailed description of microrna length heterogeneities , we indicated the number of nucleotides added ( + ) or trimmed ( ) from the 3 or 5 end in the isomir categories . of 37 differentially expressed mirnas between early and advanced fibrosis in liver , 10 were differentially expressed for at least one of the isomir categories : 199b3p , 148a3p , 1505p , 1223p , 199a5p , 10a5p , 92a3p , 1955p , 181c5p , 181a5p ( table 3 ) . for some micrornas , when we combined all the counts for isomirs and the canonical sequence , they became differentially expressed . while the functional consequences of these nucleotide changes are not yet fully understood , we report our findings in order to increase the growing knowledgebase on these types of microrna modifications . micrornas in tissue differentially expressed solely for one or more isomir categories between early and advanced fibrosis . we identified 37 micrornas , 26 up and 11 downregulated , between early and advanced fibrotic stages in liver biopsies from patients with chronic hepatitis c. early fibrosis were metavir classification f1 and f2 biopsy samples grouped together . samples classified as f3 and f4 were grouped as advanced fibrosis . in serum , we were not able to identify micrornas that were significant after adjusting for multiple comparisons ( table s5 ) . we focused our attention on four micrornas with the lowest pvalues , mir1835p , mir1825p , mir200a5p and mir199a5p , all with elevated expression levels , as well as mir148a5p that had significantly decreased expression when comparing early and advanced liver fibrosis . very little is known about which micrornas play a role in the pathogenesis of liver fibrosis , or increase the risk for downstream complications , such as hepatic cancer . using pathway analysis to get a better understanding of the function of micrornas in liver fibrosis can be very difficult to interpret . while this is not an exhaustive list , and there are many studies that found different micrornas to be important in liver fibrosis , the micrornas reported here have been found to be associated with liver damage in several papers . figure 3 is a network diagram describing the relationships for each of the mirnas and the available information from the literature and the association of each one with liver disease , injury or cancer . microrna expression measured in mouse models of alcoholic and nonalcoholic steatohepatitis 23 found mir182 and mir183 ( both identified in our study ) to be downregulated in alcoholic liver and upregulated in nonalcoholic fatty liver . compared liver samples from patients with chronic hepatitis and cirrhosis and found mir182 to be one of the most differentially expressed micrornas , upregulated in chronic hepatitis . in the current paper , we also found mir182 to increase with fibrosis stage in patients with chronic hepatitis c. wojcicka et al . mir1835p and mir199a5p ( both significantly upregulated with advanced fibrosis in our data ) were among the top ten most differentially expressed micrornas when comparing tumour and normal tissue . mir183 was also found to be significantly upregulated in hepatocellular carcinoma samples from patients with chronic hepatitis c 25 . in a larger study examining liver biopsy samples from normal controls and patients with hepatitis c , hepatitis b , cirrhosis , hepatocellular carcinoma , they found that mir183 increased with increasing liver damage , highest in cirrhosis and cancer . found mir183 to be upregulated in cirrhotic liver and premalignant lesions , and went on to investigate potential mrna targets for mir183 . they found akap12 , a tumour suppressor gene found to be downregulated in cirrhotic liver , premalignant lesions and hepatocellular carcinoma . mir183 was found to be upregulated in hepatocellular carcinoma samples in a study by li et al . we observed a small significant increase with fibrosis stage ( table 1 , fig . perhaps these differences in findings are , in part , a reflection of the overall low abundance we found for this microrna in tissue . found that mir200a was significantly decreased in patients with hepatocellular carcinoma and in cirrhotic tissue from patients with hepatocellular carcinoma compared with patients with cirrhosis . in a rat model of nonalcoholic fatty liver disease , researchers found mir200a to be significantly upregulated in rats that received a highfat diet 27 , 34 , 35 . mir200a was found to be associated with epithelialmesenchymal transition and invasion when overexpressed 26 , 36 . there are several papers describing the relevance of both mir199a5p and mir199a3p in liver disease and injury , however , there were variable findings with respect to expression levels of these micrornas in the literature . several papers found the expression of mir199a to be down with injury and disease 19 , 21 , 31 , 39 , 40 , 41 , 42 , 43 , and in other papers mir199a was found to be upregulated in hepatocellular carcinoma and liver injury 44 , 45 , 46 and increased with liver fibrosis progression 6 , 47 . in our study , mir148a5p was the most significantly downregulated microrna in advanced fibrosis . in a large study examining two subtypes of hepatocellular carcinoma 58 , mir148a was downregulated in both cancer types compared with controls , and the more aggressive subtype had significantly less mir148a expression . mir148a was also found to be downregulated in liver injury and rejection with liver transplantation 61 and downregulated in hepatoblastoma 62 . in conclusion , our data identified several micrornas that are altered with worsening liver fibrosis in chronic hepatitis c patients . these data provide further support for the role of specific micrornas in the pathogenesis of fibrosis and focuses attention on identifying micrornas that could be used to predict clinical outcomes and complications . microrna expression measured in mouse models of alcoholic and nonalcoholic steatohepatitis 23 found mir182 and mir183 ( both identified in our study ) to be downregulated in alcoholic liver and upregulated in nonalcoholic fatty liver . compared liver samples from patients with chronic hepatitis and cirrhosis and found mir182 to be one of the most differentially expressed micrornas , upregulated in chronic hepatitis . in the current paper , we also found mir182 to increase with fibrosis stage in patients with chronic hepatitis c. wojcicka et al . mir1835p and mir199a5p ( both significantly upregulated with advanced fibrosis in our data ) were among the top ten most differentially expressed micrornas when comparing tumour and normal tissue . mir183 was also found to be significantly upregulated in hepatocellular carcinoma samples from patients with chronic hepatitis c 25 . in a larger study examining liver biopsy samples from normal controls and patients with hepatitis c , hepatitis b , cirrhosis , hepatocellular carcinoma , they found that mir183 increased with increasing liver damage , highest in cirrhosis and cancer . found mir183 to be upregulated in cirrhotic liver and premalignant lesions , and went on to investigate potential mrna targets for mir183 . they found akap12 , a tumour suppressor gene found to be downregulated in cirrhotic liver , premalignant lesions and hepatocellular carcinoma . mir183 was found to be upregulated in hepatocellular carcinoma samples in a study by li et al . we observed a small significant increase with fibrosis stage ( table 1 , fig . perhaps these differences in findings are , in part , a reflection of the overall low abundance we found for this microrna in tissue . found that mir200a was significantly decreased in patients with hepatocellular carcinoma and in cirrhotic tissue from patients with hepatocellular carcinoma compared with patients with cirrhosis . in a rat model of nonalcoholic fatty liver disease , researchers found mir200a to be significantly upregulated in rats that received a highfat diet 27 , 34 , 35 . mir200a was found to be associated with epithelialmesenchymal transition and invasion when overexpressed 26 , 36 . there are several papers describing the relevance of both mir199a5p and mir199a3p in liver disease and injury , however , there were variable findings with respect to expression levels of these micrornas in the literature . found that mir199a regulates hepatitis c virus replication . several papers found the expression of mir199a to be down with injury and disease 19 , 21 , 31 , 39 , 40 , 41 , 42 , 43 , and in other papers mir199a was found to be upregulated in hepatocellular carcinoma and liver injury 44 , 45 , 46 and increased with liver fibrosis progression 6 , 47 . experimentally identified targets of mir199a5p in liver disease were smarca4 ( swi / snfrelated , matrixassociated , actindependent regulator of chromatin , subfamily a , member 4 ) and mst1 ( macrophage stimulating 1 ) 48 , mmp9 ( matrix metallopeptidase 9 ) 44 , grp78 ( glucoseregulated protein , 78 kda ) , endoplasmic reticulum to nucleus signalling 1 , activating transcription factor 6 45 , clathrin heavy chain 21 , atg7 ( autophagy related 7 ) 49 , hypoxia inducible factor1 41 and discoidin domain receptor1 43 . in our study , mir148a5p was the most significantly downregulated microrna in advanced fibrosis . in a large study examining two subtypes of hepatocellular carcinoma 58 , mir148a was downregulated in both cancer types compared with controls , and the more aggressive subtype had significantly less mir148a expression . mir148a was also found to be downregulated in liver injury and rejection with liver transplantation 61 and downregulated in hepatoblastoma 62 . in conclusion , our data identified several micrornas that are altered with worsening liver fibrosis in chronic hepatitis c patients . these data provide further support for the role of specific micrornas in the pathogenesis of fibrosis and focuses attention on identifying micrornas that could be used to predict clinical outcomes and complications .

it has long been known that a subgroup of patients with ms show positive mood and optimism that is incongruous with their circumstances , as well as unawareness of increasing impairment . a critical review of the historical development of these constructs has revealed a number of changes in the conceptual definitions since their first appearances in the literature , regarding the number of symptoms that constitute euphoria as well as the definitions of those types . however , a change in incidence of these symptoms also appears to have occurred . charcot and sigerson , circa 1877 , is well - known for his description of the cognitive and affective sequelae and the stupid indifference of ms patients , which he said referred to most of the patients ( p. 194 ) . in 1878 , wilk 's [ 3 , 4 ] characterisation of ms patients as happy and more likely to be found laughing than crying applied to many patients , and in the 1880s the accounts of moxon and gowers of the positive mood and optimism of ms patients were as a rule and especially frequent ( p. 244 , 245 ) . in 1904 , hoffman stated that euphoria was a these early reports gave the impression that pathological euphoria was the dominant mood state of patients with ms , and although they gave examples from particular cases , the claims made were often based on anecdotal observations , the total numbers of which were not readily reported . the 1920s saw the beginning of larger sample sizes being reported ; however , the earlier suppositions concerning high rates of pathological euphoria continued to be confirmed . in 1922 , brown and davis found 71% ( 10/14 ) of their ms patients with mental symptoms to be euphoric ( p. 629 ) . in 1926 , ( i.e. , a sense of physical well - being or an unawareness of physical deficit ) in 84% , and spes sclerotica ( i.e. , optimism for the future ) in 84% of their 100 ms patients . even as late as 1943 sugar and nadell , who attempted to replicate the earlier findings of cottrell and wilson , found euphoria sclerotica in 53.6% , eutonia sclerotica in 50% , and spes sclerotica in 50% of their 28 ms patients . a shift in research focus appeared to occur in the latter half of the 20th century , and when interest in euphoria in ms resumed at the turn of the 21st century , a change appeared to have occurred . today the symptom is considered to be dramatically less common than in the classical literature . with specific reference to the main contemporary studies investigating the frequency of euphoria in ms , figved et al . demonstrated pathological euphoria in only 4.7% ( n = 86 ) and diaz - olavarrieta et al . in 13% ( n = 44 ) of their samples . fishman et al . demonstrated pathological euphoria in 14.6% of their sample ( n = 75 ) ; however , they considered their euphoria / disinhibition factor , which they demonstrated in only 9% of their sample , to be more representative of the classic euphoria sclerotica . it therefore becomes apparent that discrepancies exist regarding the incidence rates of pathological euphoria reported throughout the history of this phenomenon . although reasons , such as differences in disease duration across samples [ 12 , 13 ] , inadequate screening among early reports to rule out other diseases such as neurosyphilis [ 3 , 14 ] , and differences in definitions and measurement instruments [ 3 , 1216 ] , have been postulated , no study has yet specifically addressed the issue of differing incidence rates or empirically investigated factors which may directly impact this question . the current study aimed to address this perplexing situation . we hypothesised that the change in incidence could be related to the demonstrated change in conceptual definition and also to a change in the operational definitions of the symptoms ( i.e. , the instruments used to measure these symptoms ) rather than a change in the ms population itself and that high rates of pathological euphoria could be replicated by using a classical measure and low rates could be replicated by using a contemporary measure . such an investigation is important in better understanding euphoria as well as the frequencies with which it can be found in ms patients . the study obtained ethical approval from the research ethics committee of the faculty of health sciences , university of cape town . one hundred patients with a diagnosis of ms and an informant known to each patient as well as 100 matched healthy controls ( hc ) and an informant known to each control were recruited for voluntary participation . the characteristics of the participants are presented in table 1 . the original interview schedule published by cottrell and wilson ( see appendix ) was included as a representation of the classical operational definition and measurement instrument . it can be found in the public domain and assesses three types of euphoria : euphoria sclerotica ( positive mood ) , eutonia sclerotica ( physical well - being and unawareness of physical deficit ) , and spes sclerotica ( optimism ) . the questions , which appear to elicit subtle , mild symptoms , include , for example , do you feel consistently cheerful or happy ? , are you conscious of any pleasant or unpleasant sensation in your body as a whole or a part ? , is the feeling one of bodily ease ? , and are you naturally optimistic ? . yes / no answers are given by the participant . in their article , cottrell and wilson listed their questions ; however , they did not specify the rating criteria imposed to determine the frequencies found for each of the three types . rating criteria , used by 3 independent raters , were therefore created for this study . initially , a present / absent criterion was used , but after confusion was voiced by the raters regarding a mixed picture in some answers , each of the 3 raters were rather asked to score each type of euphoria a 2 if all answers pertaining to the specific type of euphoria were affirmative and the raters therefore considered the symptom to be definitely present ; a 1 if the answers were mixed with only some answers being affirmative and the raters were of the opinion that the symptom was possibly present ; and a 0 if no answers were affirmative and the raters therefore considered the symptom to be absent . the average of the three raters was then calculated and rounded up or down to the nearest whole number ( 2 , 1 , or 0 ) , as per the rating criteria . interrater reliability was calculated for each of the three types : ( a ) icc = .82 ( euphoria sclerotica ) , ( b ) icc = .60 ( eutonia sclerotica ) , and ( c ) icc = .90 ( spes sclerotica ) . the question referring to euphoria in the neuropsychiatric inventory ( npi ) was included as a representation of the measure ( i.e. , operational definition ) most often used in contemporary euphoria research [ 911 ] . in contrast to the classical measure , the npi only refers to positive mood ( i.e. , one instead of three types of euphoria ) and does not address aspects relating to physical well - being / unawareness of physical deficit or optimism . further , it appears to focus more on extreme symptoms : does the patient seem too cheerful or too happy for no reason ? i do not mean the normal happiness that comes from seeing friends , receiving presents , or spending time with family members . i am asking if the patient has a persistent and abnormally good mood or finds humour where others do not . standard administration requires an informant known to the patient to answer the euphoria question by providing a yes / no response about the patient . however , in this study , participants were also asked to self - report euphoria by answering the same question about themselves . the frequencies of the three types of euphoria , according to the classical measure , within the two groups , as well as a comparison between ms and hc groups for the total presence of these variables are depicted in tables 2 and 3 . in terms of the contemporary measure , 11% of informants reported their ms loved one as being pathologically euphoric , and 16% of participants self - reported the experience of pathological euphoria . with regard to the healthy control group , 4% of informants reported their healthy loved one as being pathologically euphoric , and 4% of healthy controls self - reported the experience of pathological euphoria . a summary of the current results , along with previously reported frequencies using the same measures , are presented in table 5 . as other studies using the npi only use the standard administration method , only informant reported euphoria is included . the aim of this study was to address the discrepancies noted in the literature regarding incidence rates of pathological euphoria in ms patients . it was the first study of its kind to specifically investigate these discrepancies and the impact of different measures of euphoria , by using different operationalisations on a single sample of ms patients . since no guidance was given regarding the interpretation of the classical measure , if one presumes that the current study 's criteria employed for a definite presence of the symptom were too strict and one includes the possibly present cases , where some of the answers were affirmative a the particular type of euphoria , euphoria sclerotica was found in 63% , eutonia sclerotica in 48% , and spes sclerotica in 70% of the current sample of 100 ms patients . this is comparable to the 63% euphoria sclerotica , 84% eutonia sclerotica , and 84% spes sclerotica found by cottrell and wilson and with the 53.6% euphoria sclerotica , 50% eutonia sclerotica , and 50% spes sclerotica reported by sugar and nadell , who later attempted to replicate the original study . thus , high frequencies of the euphoria types , albeit with slight interpretation , were found by the current study when using the more subtle classical operational definition and measure . even when one only considers the definite cases , the frequencies remain higher than those which are considered the norm today . by contrast , low rates of pathological euphoria ( in terms of only abnormal positive mood ) were demonstrated by this study when using the npi ; that is , only 11% of informants rated their ms loved ones as euphoric according to the npi definition . this is similar to the 13% found by diaz - olavarrieta et al . and the 14.6% found by fishman et al . thus , low incidences of pathological euphoria were found when using the more severe contemporary measure ( and operational definition ) . as the same ms patients ( and their informants ) were tested on both measures at the same time and a dramatic change in the patients ' mood is unlikely to have occurred to account for the discrepancy seen , the current results appear to demonstrate that pathological euphoric symptoms can be found in relatively high frequencies when described in more mild , subtle terms , but in relatively low frequencies when defined more severely . furthermore , it is important to note that the classical measure was based on a self - report questionnaire / interview , while the npi was informant - based . however , even when patients were asked to rate themselves according to the npi euphoria question , only 16% ( a similarly low rate ) self - reported pathological euphoria according to the contemporary definition , negating the possible confounding influence of self- versus informant - reporting on the inconsistency between classical versus contemporary incidence rates . in our previous work , we established that a change in the conceptual definition of pathological euphoria appears to have occurred over the last 100 years . in this paper , we broaden that notion to include that a change in operational definition appears to have also taken place . the classical instrument used in this study measures pathological euphoric mood , for example , by asking , do you feel consistently cheerful or happy ? , while the modern npi refers to pathological euphoric mood by asking if the patient has a persistent and abnormally good mood . these clearly represent different mood states and the findings of the current study therefore imply that different measuring instruments ( based on operational definitions which also appear to have changed ) have influenced the rates of pathological euphoria reported throughout the ms literature and that discrepancies between high classical and low contemporary rates could be the result of measurements artefacts [ 18 , 19 ] , rather than any change in reality . thus , not only is pathological euphoria described differently today but the new definitions have also influenced the rates at which it is found . what caused the change in definition and incidence is beyond the scope of this paper . however , one possible contributing factor could have been experimenter bias , as classical authors appeared to be biased towards detecting the presence of pathological euphoria , believing it to be the dominant mood state of ms patients , while contemporary authors appear to be biased against it , believing it to be rare . as their operational definition is considerably more subtle and their rating criteria were not objectively stated , the early authors cottrell and wilson may represent a prime example of this bias by being too inclusive and classifying patients whose answers were mixed as euphoric . however , it is important to note that although benedict et al . and fishman et al . justify their use of the npi in euphoria research based on the fact that it is a standardised measurement instrument [ 11 , 20 ] , no literature exists criticising the more subtle classical operational definition in favour of a more severe one , thereby supporting a need for the change . other possibilities , relating to bias , also exist which may have influenced incidence rates . ms was not a treatable condition during the period in which higher rates of pathological euphoria were found . a selection bias may have occurred with the researchers and neurologists investigating pathological euphoria in that perhaps those ms patients coming to their attention were more hopeful about their prognosis and more likely to seek out care than those ms patients who had accepted their fate . conversely , it may not have been those ms patients who sought out treatment but rather those who required treatment who were brought to the attention of the early researchers . we now know that pathological euphoria in ms has been found to correlate with dementia [ 11 , 13 ] . it may be possible that the ms patients who came into contact with the neurologists and other researchers in early times did so because they required psychiatric attention due to dementia . this seems unlikely in relation to the study by cottrell and wilson , due to the relatively short disease duration among their sample ( i.e. , 51% of their sample had had ms for 5 years or less ) and the findings that both euphoria and dementia correlate with advanced disease [ 9 , 13 ] . however , the possibility of ms patients coming to the attention of neurologists due to their needing psychiatric care may be a possible factor leading to higher rates of pathological euphoria in other early ms samples . regardless of what caused the change , these findings leave open the question as to which rates and associated definitions are correct . the answer , however , may not be a clear cut choice between the two . since pathological euphoria in ms has been demonstrated to occur in patients with cerebral and not spinal cord involvement , it is typically regarded as an organic symptom and not a psychological reaction to the disease . furthermore , pathological euphoria ( in terms of the npi definition ) has been found to correlate with both lesion load and atrophy , grey matter atrophy , and frontotemporal changes on mri ; and some have proposed it may be due either to a disconnection of the frontal cortex and limbic structures by white matter lesions or to grey matter atrophy of the prefrontal cortex [ 11 , 23 ] . thus , the way in which euphoria is defined and measured and the question of what represents a pathological symptom are of vital importance when considering which definition best represents this symptom . with regard to the classical measure , how can we be sure that the subtle questions included and the symptom measured truly represent a pathological symptom ? equally , when considering the more severe modern operational definition , can we be sure we are not excluding some patients experiencing slightly milder yet still pathological euphoria ? in order to address this issue , the frequency of euphoria was compared among ms patients and a matched sample of healthy controls in this study . unlike other modern studies , a control group equal in size to the patient group was used and 4% of hcs were reported as having or self - reported the experience of ( contemporary npi ) euphoria . this is in contrast to the 0% found by diaz - olavarrieta et al . and fishman et al . who used more limited control samples of 25 each in comparison to their patient samples that were at least double that [ 10 , 11 ] significant differences between ms patients and hcs were demonstrated only for self - reported euphoria ( in terms of the modern npi ) with higher frequencies of ms patients experiencing euphoria ( p = .005 ) . although higher frequencies of ms patients than hc were identified as euphoric using the traditional informant - based administration , this comparison did not yield significant results ( p = .060 ) . since pathology can be defined implicitly as a significant difference between patients and controls , one therefore can not assume that the npi is effectively identifying a pathological symptom , although the results imply that the measure is measuring something close to pathological as more ms patients than hcs identified themselves and were identified by their loved ones as being euphoric . the earlier studies of cottrell and wilson and sugar and nadell , by contrast , did not include a control group of any kind . however , the current study identified significantly fewer ms patients than matched hcs as having euphoria as defined and measured by the classical measure ( all p values < .0001 ) . one might take this to mean that the classical operational definition ( and measure ) is even less likely than the npi to represent a pathological symptom . although both measures use a present / absent approach , it may be that the method of analysis for the classical measure is too inclusive and that those that were identified as definitely euphoric represent a more pathological representation of the symptom . however , the discrepancies between ms and hc participants for definite euphoria were also significant , with fewer ms patients demonstrating definite euphoria than hc ( euphoria sclerotica : p = .008 ; eutonia , and spes sclerotica : p < .0001 ) . thus , when compared to the contemporary measure , the classical measure certainly appears to be ill - representative of pathological euphoria . this may mean that the interview of cottrell and wilson is not valid and that it simply does not elicit and measure pathological euphoria . issues around the interpretability of this measure and the lack of direction in the original paper regarding rating criteria have already been raised . further , the subtle nature of the questions , such as do you feel consistently cheerful or happy ? , has also been noted . while questions that are slightly more subtle than the persistent and abnormally good mood of the npi may be appropriate , the questions of cottrell and wilson may simply be too general , reflective of basic personality traits rather than anything pathological . indeed , in research with ms patients using the five factor model and the revised version of the neo personality inventory ( which both measure the same five factors of personality : neuroticism , extraversion , openness to experience , agreeableness , and conscientiousness ) , a subset of ms patients have been found to demonstrate elevated neuroticism , as well as reduced agreeableness and conscientiousness . cottrell and wilson 's question , are you naturally optimistic ? , is not reflective of psychopathology , but the item would likely load on the neuroticism factor in a personality trait study . thus , the potential invalidity of cottrell and wilson 's interview as a measure of pathological euphoria appears to be a more likely reason for the higher rates of euphoria among hcs than ms patients . however , of interest is that the elevated neuroticism and reduced agreeableness and conscientiousness within this subset of ms patients have been found to correlate with a euphoria / disinhibition factor identified using the npi . thus , while the cottrell and wilson interview may not measure pathological euphoria , it may nevertheless measure personality traits consistent with euphoric ms patients . although the npi may be more appropriate than mild , subtle definitions , significant differences were not identified between ms and hc groups for informant reported euphoria , the standard way in which this test is administered . this may imply that the modern npi definition is not quite as severe ( or exclusive ) as we , the current authors , might have believed and that even more severe definitions are required to determine a cut - off rate that is deemed pathological ( when comparing patients and controls ) . although it may have been favoured due to its being standardised , other measures may exist that better encompass euphoria , and the popularity of this measure may simply be due to a number of papers being published using it to represent euphoria , which has , in turn , led to the perpetuation of perhaps erroneous incidence rates of euphoria . there may still , however , be use for more subtle definitions , somewhere between mild and harshly defined euphoria . since modern research has identified that npi euphoria tends to occur later in the disease along with significant disability [ 9 , 11 , 13 ] , the identification of more subtle forms may allow for the prediction of which patients will develop more severe forms as the disease progresses and the representation of pathological euphoria in ms on a continuum from more subtle to severe forms may be of clinical significance . further , although abnormality may be defined as a significant difference between patients and controls , pathology can also be defined in terms of the extent to which a symptom impacts the patient 's ability to function , and even subtle forms of euphoria may impact the care and treatment of patients and affect their families . in our review of the change in conceptual definition of euphoria , we noted the need for clear , reliable , and widely recognised conceptual definitions of euphoria and we extend this here to consistent and accepted means of measuring these complex constructs as well as a comprehensive understanding of what characterises a pathological representation of this symptom . without standardised measurement instruments we will continue to face unreliable incidence rates that can not be compared and will continue to either under- or overreport the incidence of this interesting cluster of neuropsychiatric symptoms . the discrepancies in incidence rates of pathological euphoria noted between the historic and contemporary literatures do not reflect a change in the incidence of euphoria in ms , but rather in the definition and operationalisation of the term . furthermore , measures often used in euphoric literature may be ill - representative of a symptom that could be considered pathological .

background . a subgroup of ms patients present with euphoria . classical authors describe this symptom as the predominant mood state of these patients , while contemporary authors regard it as rare . objective . this study aimed to address these discrepancies and investigate the contributions made by varying operational definitions and measurement instruments . methods . one hundred ms patients and 100 matched controls completed the classical interview of cottrell and wilson and the modern neuropsychiatric inventory in a once - off interview . results . the ms group demonstrated high frequencies of euphoria using the classical measure but low frequencies using the contemporary measure and definition . the matched control group demonstrated significantly higher rates than the ms group using the classical measure and lower rates than the ms group using the contemporary measure . conclusion . the discrepancies in incidence rates of euphoria noted in the literature do not reflect a change in the incidence of euphoria in ms , but rather in the definition and operationalisation of euphoria . furthermore , these results highlight the importance of characterising what represents pathological euphoria as well as the need for better definitions and instruments of measure .

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

it has long been known that a subgroup of patients with ms show positive mood and optimism that is incongruous with their circumstances , as well as unawareness of increasing impairment . a critical review of the historical development of these constructs has revealed a number of changes in the conceptual definitions since their first appearances in the literature , regarding the number of symptoms that constitute euphoria as well as the definitions of those types . however , a change in incidence of these symptoms also appears to have occurred . charcot and sigerson , circa 1877 , is well - known for his description of the cognitive and affective sequelae and the stupid indifference of ms patients , which he said referred to most of the patients ( p. 194 ) . in 1878 , wilk 's [ 3 , 4 ] characterisation of ms patients as happy and more likely to be found laughing than crying applied to many patients , and in the 1880s the accounts of moxon and gowers of the positive mood and optimism of ms patients were as a rule and especially frequent ( p. 244 , 245 ) . in 1904 , hoffman stated that euphoria was a these early reports gave the impression that pathological euphoria was the dominant mood state of patients with ms , and although they gave examples from particular cases , the claims made were often based on anecdotal observations , the total numbers of which were not readily reported . the 1920s saw the beginning of larger sample sizes being reported ; however , the earlier suppositions concerning high rates of pathological euphoria continued to be confirmed . in 1922 , brown and davis found 71% ( 10/14 ) of their ms patients with mental symptoms to be euphoric ( p. 629 ) . even as late as 1943 sugar and nadell , who attempted to replicate the earlier findings of cottrell and wilson , found euphoria sclerotica in 53.6% , eutonia sclerotica in 50% , and spes sclerotica in 50% of their 28 ms patients . a shift in research focus appeared to occur in the latter half of the 20th century , and when interest in euphoria in ms resumed at the turn of the 21st century , a change appeared to have occurred . today the symptom is considered to be dramatically less common than in the classical literature . with specific reference to the main contemporary studies investigating the frequency of euphoria in ms , figved et al . demonstrated pathological euphoria in only 4.7% ( n = 86 ) and diaz - olavarrieta et al . demonstrated pathological euphoria in 14.6% of their sample ( n = 75 ) ; however , they considered their euphoria / disinhibition factor , which they demonstrated in only 9% of their sample , to be more representative of the classic euphoria sclerotica . it therefore becomes apparent that discrepancies exist regarding the incidence rates of pathological euphoria reported throughout the history of this phenomenon . although reasons , such as differences in disease duration across samples [ 12 , 13 ] , inadequate screening among early reports to rule out other diseases such as neurosyphilis [ 3 , 14 ] , and differences in definitions and measurement instruments [ 3 , 1216 ] , have been postulated , no study has yet specifically addressed the issue of differing incidence rates or empirically investigated factors which may directly impact this question . the current study aimed to address this perplexing situation . we hypothesised that the change in incidence could be related to the demonstrated change in conceptual definition and also to a change in the operational definitions of the symptoms ( i.e. , the instruments used to measure these symptoms ) rather than a change in the ms population itself and that high rates of pathological euphoria could be replicated by using a classical measure and low rates could be replicated by using a contemporary measure . such an investigation is important in better understanding euphoria as well as the frequencies with which it can be found in ms patients . one hundred patients with a diagnosis of ms and an informant known to each patient as well as 100 matched healthy controls ( hc ) and an informant known to each control were recruited for voluntary participation . the original interview schedule published by cottrell and wilson ( see appendix ) was included as a representation of the classical operational definition and measurement instrument . it can be found in the public domain and assesses three types of euphoria : euphoria sclerotica ( positive mood ) , eutonia sclerotica ( physical well - being and unawareness of physical deficit ) , and spes sclerotica ( optimism ) . in their article , cottrell and wilson listed their questions ; however , they did not specify the rating criteria imposed to determine the frequencies found for each of the three types . rating criteria , used by 3 independent raters , were therefore created for this study . initially , a present / absent criterion was used , but after confusion was voiced by the raters regarding a mixed picture in some answers , each of the 3 raters were rather asked to score each type of euphoria a 2 if all answers pertaining to the specific type of euphoria were affirmative and the raters therefore considered the symptom to be definitely present ; a 1 if the answers were mixed with only some answers being affirmative and the raters were of the opinion that the symptom was possibly present ; and a 0 if no answers were affirmative and the raters therefore considered the symptom to be absent . the question referring to euphoria in the neuropsychiatric inventory ( npi ) was included as a representation of the measure ( i.e. in contrast to the classical measure , the npi only refers to positive mood ( i.e. , one instead of three types of euphoria ) and does not address aspects relating to physical well - being / unawareness of physical deficit or optimism . i do not mean the normal happiness that comes from seeing friends , receiving presents , or spending time with family members . i am asking if the patient has a persistent and abnormally good mood or finds humour where others do not . the frequencies of the three types of euphoria , according to the classical measure , within the two groups , as well as a comparison between ms and hc groups for the total presence of these variables are depicted in tables 2 and 3 . in terms of the contemporary measure , 11% of informants reported their ms loved one as being pathologically euphoric , and 16% of participants self - reported the experience of pathological euphoria . with regard to the healthy control group , 4% of informants reported their healthy loved one as being pathologically euphoric , and 4% of healthy controls self - reported the experience of pathological euphoria . a summary of the current results , along with previously reported frequencies using the same measures , are presented in table 5 . the aim of this study was to address the discrepancies noted in the literature regarding incidence rates of pathological euphoria in ms patients . it was the first study of its kind to specifically investigate these discrepancies and the impact of different measures of euphoria , by using different operationalisations on a single sample of ms patients . since no guidance was given regarding the interpretation of the classical measure , if one presumes that the current study 's criteria employed for a definite presence of the symptom were too strict and one includes the possibly present cases , where some of the answers were affirmative a the particular type of euphoria , euphoria sclerotica was found in 63% , eutonia sclerotica in 48% , and spes sclerotica in 70% of the current sample of 100 ms patients . this is comparable to the 63% euphoria sclerotica , 84% eutonia sclerotica , and 84% spes sclerotica found by cottrell and wilson and with the 53.6% euphoria sclerotica , 50% eutonia sclerotica , and 50% spes sclerotica reported by sugar and nadell , who later attempted to replicate the original study . thus , high frequencies of the euphoria types , albeit with slight interpretation , were found by the current study when using the more subtle classical operational definition and measure . by contrast , low rates of pathological euphoria ( in terms of only abnormal positive mood ) were demonstrated by this study when using the npi ; that is , only 11% of informants rated their ms loved ones as euphoric according to the npi definition . thus , low incidences of pathological euphoria were found when using the more severe contemporary measure ( and operational definition ) . as the same ms patients ( and their informants ) were tested on both measures at the same time and a dramatic change in the patients ' mood is unlikely to have occurred to account for the discrepancy seen , the current results appear to demonstrate that pathological euphoric symptoms can be found in relatively high frequencies when described in more mild , subtle terms , but in relatively low frequencies when defined more severely . furthermore , it is important to note that the classical measure was based on a self - report questionnaire / interview , while the npi was informant - based . however , even when patients were asked to rate themselves according to the npi euphoria question , only 16% ( a similarly low rate ) self - reported pathological euphoria according to the contemporary definition , negating the possible confounding influence of self- versus informant - reporting on the inconsistency between classical versus contemporary incidence rates . in our previous work , we established that a change in the conceptual definition of pathological euphoria appears to have occurred over the last 100 years . in this paper , we broaden that notion to include that a change in operational definition appears to have also taken place . the classical instrument used in this study measures pathological euphoric mood , for example , by asking , do you feel consistently cheerful or happy ? , while the modern npi refers to pathological euphoric mood by asking if the patient has a persistent and abnormally good mood . these clearly represent different mood states and the findings of the current study therefore imply that different measuring instruments ( based on operational definitions which also appear to have changed ) have influenced the rates of pathological euphoria reported throughout the ms literature and that discrepancies between high classical and low contemporary rates could be the result of measurements artefacts [ 18 , 19 ] , rather than any change in reality . thus , not only is pathological euphoria described differently today but the new definitions have also influenced the rates at which it is found . what caused the change in definition and incidence is beyond the scope of this paper . however , one possible contributing factor could have been experimenter bias , as classical authors appeared to be biased towards detecting the presence of pathological euphoria , believing it to be the dominant mood state of ms patients , while contemporary authors appear to be biased against it , believing it to be rare . as their operational definition is considerably more subtle and their rating criteria were not objectively stated , the early authors cottrell and wilson may represent a prime example of this bias by being too inclusive and classifying patients whose answers were mixed as euphoric . justify their use of the npi in euphoria research based on the fact that it is a standardised measurement instrument [ 11 , 20 ] , no literature exists criticising the more subtle classical operational definition in favour of a more severe one , thereby supporting a need for the change . other possibilities , relating to bias , also exist which may have influenced incidence rates . ms was not a treatable condition during the period in which higher rates of pathological euphoria were found . a selection bias may have occurred with the researchers and neurologists investigating pathological euphoria in that perhaps those ms patients coming to their attention were more hopeful about their prognosis and more likely to seek out care than those ms patients who had accepted their fate . conversely , it may not have been those ms patients who sought out treatment but rather those who required treatment who were brought to the attention of the early researchers . we now know that pathological euphoria in ms has been found to correlate with dementia [ 11 , 13 ] . it may be possible that the ms patients who came into contact with the neurologists and other researchers in early times did so because they required psychiatric attention due to dementia . this seems unlikely in relation to the study by cottrell and wilson , due to the relatively short disease duration among their sample ( i.e. , 51% of their sample had had ms for 5 years or less ) and the findings that both euphoria and dementia correlate with advanced disease [ 9 , 13 ] . however , the possibility of ms patients coming to the attention of neurologists due to their needing psychiatric care may be a possible factor leading to higher rates of pathological euphoria in other early ms samples . regardless of what caused the change , these findings leave open the question as to which rates and associated definitions are correct . since pathological euphoria in ms has been demonstrated to occur in patients with cerebral and not spinal cord involvement , it is typically regarded as an organic symptom and not a psychological reaction to the disease . furthermore , pathological euphoria ( in terms of the npi definition ) has been found to correlate with both lesion load and atrophy , grey matter atrophy , and frontotemporal changes on mri ; and some have proposed it may be due either to a disconnection of the frontal cortex and limbic structures by white matter lesions or to grey matter atrophy of the prefrontal cortex [ 11 , 23 ] . thus , the way in which euphoria is defined and measured and the question of what represents a pathological symptom are of vital importance when considering which definition best represents this symptom . with regard to the classical measure , how can we be sure that the subtle questions included and the symptom measured truly represent a pathological symptom ? equally , when considering the more severe modern operational definition , can we be sure we are not excluding some patients experiencing slightly milder yet still pathological euphoria ? in order to address this issue , the frequency of euphoria was compared among ms patients and a matched sample of healthy controls in this study . unlike other modern studies , a control group equal in size to the patient group was used and 4% of hcs were reported as having or self - reported the experience of ( contemporary npi ) euphoria . who used more limited control samples of 25 each in comparison to their patient samples that were at least double that [ 10 , 11 ] significant differences between ms patients and hcs were demonstrated only for self - reported euphoria ( in terms of the modern npi ) with higher frequencies of ms patients experiencing euphoria ( p = .005 ) . although higher frequencies of ms patients than hc were identified as euphoric using the traditional informant - based administration , this comparison did not yield significant results ( p = .060 ) . since pathology can be defined implicitly as a significant difference between patients and controls , one therefore can not assume that the npi is effectively identifying a pathological symptom , although the results imply that the measure is measuring something close to pathological as more ms patients than hcs identified themselves and were identified by their loved ones as being euphoric . the earlier studies of cottrell and wilson and sugar and nadell , by contrast , did not include a control group of any kind . however , the current study identified significantly fewer ms patients than matched hcs as having euphoria as defined and measured by the classical measure ( all p values < .0001 ) . one might take this to mean that the classical operational definition ( and measure ) is even less likely than the npi to represent a pathological symptom . although both measures use a present / absent approach , it may be that the method of analysis for the classical measure is too inclusive and that those that were identified as definitely euphoric represent a more pathological representation of the symptom . however , the discrepancies between ms and hc participants for definite euphoria were also significant , with fewer ms patients demonstrating definite euphoria than hc ( euphoria sclerotica : p = .008 ; eutonia , and spes sclerotica : p < .0001 ) . thus , when compared to the contemporary measure , the classical measure certainly appears to be ill - representative of pathological euphoria . this may mean that the interview of cottrell and wilson is not valid and that it simply does not elicit and measure pathological euphoria . issues around the interpretability of this measure and the lack of direction in the original paper regarding rating criteria have already been raised . while questions that are slightly more subtle than the persistent and abnormally good mood of the npi may be appropriate , the questions of cottrell and wilson may simply be too general , reflective of basic personality traits rather than anything pathological . indeed , in research with ms patients using the five factor model and the revised version of the neo personality inventory ( which both measure the same five factors of personality : neuroticism , extraversion , openness to experience , agreeableness , and conscientiousness ) , a subset of ms patients have been found to demonstrate elevated neuroticism , as well as reduced agreeableness and conscientiousness . cottrell and wilson 's question , are you naturally optimistic ? , is not reflective of psychopathology , but the item would likely load on the neuroticism factor in a personality trait study . thus , the potential invalidity of cottrell and wilson 's interview as a measure of pathological euphoria appears to be a more likely reason for the higher rates of euphoria among hcs than ms patients . however , of interest is that the elevated neuroticism and reduced agreeableness and conscientiousness within this subset of ms patients have been found to correlate with a euphoria / disinhibition factor identified using the npi . thus , while the cottrell and wilson interview may not measure pathological euphoria , it may nevertheless measure personality traits consistent with euphoric ms patients . this may imply that the modern npi definition is not quite as severe ( or exclusive ) as we , the current authors , might have believed and that even more severe definitions are required to determine a cut - off rate that is deemed pathological ( when comparing patients and controls ) . although it may have been favoured due to its being standardised , other measures may exist that better encompass euphoria , and the popularity of this measure may simply be due to a number of papers being published using it to represent euphoria , which has , in turn , led to the perpetuation of perhaps erroneous incidence rates of euphoria . since modern research has identified that npi euphoria tends to occur later in the disease along with significant disability [ 9 , 11 , 13 ] , the identification of more subtle forms may allow for the prediction of which patients will develop more severe forms as the disease progresses and the representation of pathological euphoria in ms on a continuum from more subtle to severe forms may be of clinical significance . further , although abnormality may be defined as a significant difference between patients and controls , pathology can also be defined in terms of the extent to which a symptom impacts the patient 's ability to function , and even subtle forms of euphoria may impact the care and treatment of patients and affect their families . in our review of the change in conceptual definition of euphoria , we noted the need for clear , reliable , and widely recognised conceptual definitions of euphoria and we extend this here to consistent and accepted means of measuring these complex constructs as well as a comprehensive understanding of what characterises a pathological representation of this symptom . without standardised measurement instruments we will continue to face unreliable incidence rates that can not be compared and will continue to either under- or overreport the incidence of this interesting cluster of neuropsychiatric symptoms . the discrepancies in incidence rates of pathological euphoria noted between the historic and contemporary literatures do not reflect a change in the incidence of euphoria in ms , but rather in the definition and operationalisation of the term . furthermore , measures often used in euphoric literature may be ill - representative of a symptom that could be considered pathological .

the molecular formula was determined as c13h16n2o4 according to its hresims peak at m / z 265.1183 [ m + h ] . there were multiple ir stretches at 1740 cm indicative of a lactam or ester moiety ( five - membered - ring lactams absorb in the 17501700 cm region ; four - membered - ring lactams absorb at 17601730 cm ; esters absorb at 17501735 cm ; ,-unsaturated esters appears at 17301715 cm ) . the c nmr spectrum showed 13 carbon signals that were classified by hsqc as three methyl carbons , one sp methylene carbon , four methine groups ( two olefinic carbons , one carbon connected with oxygen , and one carbon connected with nitrogen ) , and five nonprotonated carbons ( three carbonyls and two olefinic carbons ) ( table 1 ) . the h nmr spectrum showed five coupled signals at h 1.23 ( h-8 , d , j = 6.5 ) , 4.55 ( h-4 , qd , j = 6.7 , 6.6 ) , 4.43 ( h-5 , ddd , j = 8.8 , 8.0 , 6.7 ) , 2.67 ( h-6 , dd , j = 16.9 , 8.0 ) , and 2.63 ( h-6 , dd , j = 16.9 , 8.8 ) . these data combined with cosy correlations of h-8/h-4/h-5/h-6 and the key hmbc correlations of h-8 to c-4/c-5 and h-4/h-5 to c-6/c-7 ( figure 1 ) constituted the 4-hydroxy-5-methyl--lactam moiety . two vinyl methyl groups at h 2.25 ( h-5 , d , j = 1.2 hz ) and 2.03 ( h-4 , d , j = 1.2 hz ) and one olefinic proton signal at h 5.98 ( m ) , along with the hmbc correlations of h-2 to c-4/c-5 and h-4 to c-5 ( figure 1 ) , indicated the presence of an isobutenyl fragment . the isobutenyl fragment should be connected with c-1 ( c 163.7 ) , which was determined by the hmbc correlation of h-2 to c-1 ( figure 1 ) . the remaining h and c nmr signals and the hmbc correlations of h-2 ( h / c 6.73/89.0 ) to c-1 ( c 159.8 ) and c-3 ( c 155.3 ) ( figure 1 ) , along with the molecular formula , provided two possible structures that could not be easily distinguished : a 1,3-oxazin-6-one ( 1 ) or an unsaturated -lactam unit ( 1a ) ( figure 2 ) . the proposed unsaturated -lactam in 1a would be unprecedented in a natural product . only one natural product possessing an unsaturated -lactam moiety was reported , but it was later revised to be a six - membered - ring metabolite . synthetic efforts to produce similar unsaturated -lactams have been reported ; however , literature reports suggest that such -lactams rearrange to form the corresponding 1,3-oxazin-6-ones ( scheme 1 ) . in general , the h and c nmr data for the corresponding -lactam and 1,3-oxazin-6-one structures for this natural product would be nearly identical . predicted c nmr shifts of both 1,3-oxazin-6-one ( 1 ) and -lactam ( 1a ) , which were computed at the b3lyp/6 - 311++g(2d , p)//b3lyp/6 - 31g(d ) level ( full details on the calculation can be found in the supporting information ) , strongly support that the chemical shift values of 1 are consistent with a 1,3-oxazin-6-one skeleton ( table s1 ) , particularly the c shifts of c-1 , c-2 , and c-3 . the linkage between the -lactam and the 1,3-oxazin-6-one could be confirmed by the hmbc correlation of h-4 to c-3 . two structures , 1 and 1a , in general agreement with the nmr data . noe correlations between h-8 and h-5/h-6 , between h-4 and h-5 , and between h-5 and h-6/h-6 were observed in the 2d noesy spectrum , suggesting the relative configuration of 1 as shown ( figure 3 ) . noe correlations combined with the coupling constants between h-4 and h-5 ( j = 6.7 hz ) , between h-5 and h-6 ( j = 8.8 hz ) , and between h-5 and h-6 ( j = 8.0 hz ) also indicated the conformation of 1 as shown in figure 4 . we utilized the empirical electronic circular dichrosim ( ecd ) rule of -lactams to determine the absolute configuration of 1 . the ecd sign of -lactams can be determined by addition of two effects : configuration of c and ring chirality . if the -lactams have no lone pair electrons on the c substituent , the ecd signs are determined by the ring chirality ( conformation a gives a negative sign , and b gives a positive sign ) ( figure 4 ) . the conformation of ( 4s , 5s)-1 , suggested by noe correlations and chemical calculation ( supporting information ) , is consistent with a , which was responsible for the negative cotton effect ( * transition ) in accordance with the measured negative ecd cotton effect at max 254 nm of 1 ( figure 4 ) . as the 1,3-oxazin-6-one group connected with the nitrogen nearly lay on the same plane of the amide , it made a very small contribution to the lactam cotton effect . moreover , the predicted ecd spectrum was obtained by the tddft [ b3lyp/6 - 31g(d ) ] method ( supporting information ) , which was subsequently compared with the experimental data . the measured ecd curve of 1 showed a cotton effect at max ( ) 254 ( 4.7 ) nm , matching with the calculated ecd curve of ( 4s,5s)-1 ( figure 5 ) . ( b ) ring chirality and ecd sign of -lactams and the conformation of 1 . measured and calculated ecd spectra for 1 . the molecular formula of spinoxazine b ( 2 ) was assigned as c13h16n2o3 based on the hresims , which was only one oxygen less than that of 1 . careful comparison of its h and c nmr spectra ( table 1 ) with those of 1 showed that methylene signals at c / h 24.7/2.21 and 1.71 in 2 replaced the corresponding oxygenated methine signals at c / h 64.1/4.43 in 1 . the cosy correlations of h-8/h-4/h-5/h-6 and the key hmbc correlations of h-8 to c-4/c-5 and h-4/h-5 to c-6/c-7 also supported this change in 2 ( figure 1 ) . the ecd cotton effect at max ( ) 267 ( 2.6 ) was nearly identical to 1 ( figure 4 ) , indicating the same absolute configuration . the uv spectrum showed similar absorptions to bohemamines at 252 , 283 , and 332 nm . comparison of its nmr spectra with those of known bohemamine analogues indicated that the h and c nmr data of 3 ( table 2 ) were similar to np25302 ( 9 ) . the signal of the c-7 methyl group in np25302 was no longer present , and c at c-7 was shifted deshielded to 95.3 , leading to the assignment of a c-7 hydroxy group . the molecular formula and the key hmbc correlations of h-8 to c-4/c-5 and h-6 to c-4/c-5/c-7/c-1 ( figure 1 ) also confirmed the structure of 3 . in order to determine its relative configuration , a methylation reaction was carried out using iodomethane and sodium hydride to generate 3a ( figure s2 ) . in the noesy spectrum of compound 3a , the signal between h-8 and 7-och3 could be observed , which indicated the relative configuration of 3 as shown . the octant rule for cyclopentenone was used to determine the absolute configuration ( figure 6 ) . the conformation of ( 4s,7s)-3 as shown in figure 6 was placed in accordance with the octant rule model . the functional group at c-7 lying in the back lower left area was responsible for the negative cotton effect ( n * ) , which was consistent with the cotton effect at 335 nm ( 19.0 ) . moreover , the ecd helicity rule of ,-unsaturated ketones was used to further confirm the absolute configuration of compound 3 . the p - helicity of the o = c c=c torsion angle correlates the positive cotton effect ( * ) at 297 nm ( figure 6 ) . ecd spectra and the representation of the octant rule and helicity rule for the cyclopentenone moiety of 3 ( a ) and 9 ( b ) . bohemamine e ( 4 ) was also thought to be an analogue of bohemamines due to the similar uv absorptions at 252 , 280 , and 335 nm . the molecular formula was determined as c14h20n2o4 by hresims , with a h2o unit more than bohemamine ( 12 ) . the 1d nmr spectrum was similar to 12 except for the shifts of c-5 and c-6 . so , we deduced that the epoxide ring in 12 was hydrolyzed to yield compound 4 . noe correlations between h-5 and h-9 , between h-4 and h-8 , and between h-6 and h-8 were observed in the 2d noesy , suggesting the relative configuration of 4 as shown ( figure 3 ) . the molecular formula of bohemamine f ( 5 ) was determined to be c14h18n2o2 from the protonated molecule peak at m / z 247.1441 [ m + h ] in the positive - ion hresims spectrum , which was two hydrogen atoms less than that of np25302 ( 9 ) . comparison of its h and c nmr spectra ( table 2 ) with those of 9 revealed that the signals of two methylene groups ( c-5 and c-6 ) were replaced by two coupled olefinic methine signals ( c-5 : c / h 128.0/5.74 , dd , j = 6.0 , 2.2 ; c-6 : c / h 133.9/5.67 , dd , j = 6.0 , 1.4 ) . this structure can be confirmed by cosy correlations of h-9/h-4/h-5/h-6 and the key hmbc correlations from h-9 to c-4/c-5 , h-6 to c-4/c-5/c-7/c-1 , and h-8 to c-6/c-7 . the relative configurations of c-4 and c-7 could be determined by the noe correlation between h-8 and h-4 . i ( 68 ) were determined as c15h22n2o2 , c14h20n2o2 , and c16h18n2o2 based on their hresims spectra at m / z 263.1754 [ m + h ] , 249.1596 [ m + h ] , and 271.1443 [ m + h ] , respectively . examination of their h and c nmr spectra ( table 3 ) showed that they had the same pyrrolizidine skeleton as 9 , with variation only in the amide moiety . for compound 6 , the cosy correlations of h-2/h-3/h-4 and h-5/h-4/h-6 and the key hmbc correlations from h-2 to c-1 , h-3 to c-1/c-4 , h-4 to c-6 , h-5 to c-6 , and h-6 to c-3 suggested the presence of a 4-methylpent-2-enamide side chain ( figure 1 ) . the large coupling constant between h-2 and h-3 ( j = 15.5 hz ) indicated the e- configuration . compound 7 was determined to be an isomer of 9 ( table 3 ) , where the amide moiety is a 2-methylbut-2-enamide group . this structure was supported by the cosy correlation of h-3/h-4 and the hmbc correlations from h-3 to c-1/c-5 , h-4 to c-2/c-3 , and h-5 to c-1 ( figure 1 ) . the e - configuration for this side chain was determined by the noe correlation between h-3 and 3-nh ( figure 3 ) . the nmr data were also consistent with those reported in the literature . in the 1d nmr spectrum of compound 8 ( table 3 ) , the signals of a monosubstituted phenyl group were observed , which were further supported by 2d correlations ( figure 1 ) . the noe correlations between h-9 and h-5/h-6 and between h-8 and h-4/h-5/h-6 could be observed in the 2d noesy spectra of compounds 68 , which revealed that they had the same relative configuration as shown ( figure 3 ) . the absolute configuration of compound 9 has been determined as ( 4s,7s ) by total synthesis , which can be confirmed with the octant rule for cyclopentenone ( figure 6 ) . the ecd helicity rule of ,-unsaturated ketones can also be used to confirm this conclusion . m - helicity of the o = c c=c torsion angle of compound 9 was consistent with the negative cotton effect ( * ) at 283 nm ( figure 6 ) , which suggested the absolute configuration of 9 as ( 4s,7s ) . the ecd spectra of compounds 48 showed the same cotton effects as those of 9 ( figure 7 ) , indicating the same core configurations . a plausible biogenetic pathway for spinoxazines a and b ( 1 and 2 ) and bohemamine d ( 3 ) is postulated ( scheme 2 ) . l - glutamine undergoes decarboxylation and oxidation to generate the primary amine intermediate a , followed by condensation with two acetyl - coa units and reduction to yield intermediate b. dual nucleophilic addition of the amine to the amide and methyl ketone carbonyls , respectively , followed by elimination and reduction would provide the pyrrolizidine core d. oxidation of the bridgehead carbon would result in the hemiaminal intermediate e , which undergoes n - acylation to yield compound 3 . dehydrogenation of the pyrrolizidine core would give the ring cleavage intermediate f , which could undergo a pericyclic reaction to form the 1,3-oxazin-6-one compound spinoxazine b ( 2 ) . compounds 18 did not show cytotoxicity to non - small - cell lung cancer cell lines hcc366 , a549 , hcc44 , and hcc1171 . in addition , they showed no antibacterial activities against pseudomonas aeruginosa and bacillus subtilis . optical rotations were recorded with an autopol ap iv-6w polarimeter equipped with a halogen lamp ( 589 nm ) . h and 2d nmr spectroscopic data were recorded at 600 mhz in dmso - d6 or cdcl3 solution on a varian system spectrometer , and chemical shifts were referenced to the corresponding residual solvent signal ( h / c 2.50/39.52 for dmso - d6 and h / c 7.26/77.16 for cdcl3 ) . high - resolution esi - tof mass spectra were provided by the scripps research institute ( la jolla , ca , usa ) . low - resolution lc / esims data were measured using an agilent 1200 series lc / ms system with a reversed - phase c18 column ( phenomenex luna , 150 mm 4.6 mm , 5 m ) at a flow rate of 0.7 ml / min . preparative hplc was performed on an agilent 1200 series instrument with a dad detector , using a reversed - phase c18 column ( phenomenex luna , 250 10.0 mm , 5 m ) , a phenyl - hexyl column ( phenomenex luna , 250 10.0 mm , 5 m ) , or a cn column ( phenomenex luna , 250 10.0 mm , 5 m ) . sephadex lh-20 ( ge healthcare ) and ods ( 50 mm , merck ) were used for column chromatography . strain snb-048 was isolated from a sand sample collected from a bahamian tidal flat . the sediment was desiccated and stamped onto agar plates using gauze 1 acidic media ( 10 g starch , 1 g nano3 , 0.5 g k2hpo4 , 0.5 g mgso4 , 0.5 g nacl , 0.01 g feso4 , 1 l seawater , 15 g agar , ph adjusted to 5.3 with phosphate buffer ) . bacterium snb-048 was cultured in 20 2.8 l fernbach flasks each containing 1 l of a seawater - based medium ( 10 g starch , 4 g yeast extract , 2 g peptone , 1 g caco3 , 40 mg fe2(so4)34h2o , 100 mg kbr ) and shaken at 200 rpm at 27 c . g / l ) was added to adsorb the organic products , and the culture and resin were shaken at 200 rpm for 2 h. the resin was filtered through cheesecloth , washed with deionized h2o , and eluted with acetone . the acetone - soluble fraction was dried in vacuo to yield 1.2 g of extract . in order to get more material , another cultivation of 10 the extract ( 1.2 g ) of the first cultivation was fractionated by flash column chromatography on ods ( 50 m , 30 g ) , eluting with a step gradient of meoh and h2o ( 10:90100:0 ) , and 12 fractions ( fr1.1fr1.12 ) were collected . fractions 7 and 8 ( 403.7 mg ) were combined and separated into six fractions ( fr1.7.1fr1.7.6 ) on sephadex lh-20 , eluting with meoh . fr1.7.3 ( 151.2 mg ) was further separated into 16 fractions ( fr1.7.3.1fr1.7.3.16 ) by flash column chromatography on ods ( 50 m , 30 g ) , eluting with a step gradient of meoh and h2o ( 10:90100:0 ) . fr1.7.3.6 ( 6.3 mg ) was purified by hplc on a phenyl - hexyl column ( phenomenex luna , 250 10.0 mm , 5 m , 2.5 ml / min ) using a gradient solvent system from 20% to 100% ch3cn ( 0.1% formic acid ) over 20 min to yield compound 4 ( 1.4 mg , tr = 9.5 min ) . fr.7.3.8 ( 17.5 mg ) was purified by hplc on a phenyl - hexyl column ( phenomenex luna , 250 10.0 mm , 5 m , 2.5 ml / min ) using a gradient solvent system from 20% to 85% ch3cn ( 0.1% formic acid ) over 20 min to afford compound 3 ( 1.9 mg , tr = 11.1 min ) . fr1.7.3.9 ( 8.1 mg ) was also separated by hplc on a phenyl - hexyl column ( phenomenex luna , 250 10.0 mm , 5 m , 2.5 ml / min ) using a gradient solvent system from 20% to 85% ch3cn ( 0.1% formic acid ) over 20 min to afford compound 7 ( 0.9 mg , tr = 12.5 min ) . fr1.7.3.10 ( 31.7 mg ) was separated by a reversed - phase hplc ( phenomenex luna , c18 , 250 10.0 mm , 5 m , 2.5 ml / min ) using a gradient solvent system from 20% to 80% ch3cn ( 0.1% formic acid ) over 20 min to yield compound 9 ( 3.8 mg , tr = 13.4 min ) and fr1.7.3.10.4 ( 12.6 mg , tr = 14.3 min ) . fr1.7.3.10.4 ( 12.6 mg ) was further purified by hplc on a cn column ( phenomenex luna , 250 10.0 mm , 5 m , 2.5 ml / min ) using a gradient solvent system from 20% to 100% ch3cn ( 0.1% formic acid ) over 20 min to afford compounds 5 ( 3.3 mg , tr = 11.5 min ) and 8 ( 2.3 mg , tr = 12.0 min ) . fr1.7.3.11 ( 9.1 mg ) was purified by hplc on a phenyl - hexyl column ( phenomenex luna , 250 10.0 mm , 5 m , 2.5 ml / min ) using a gradient solvent system from 20% to 100% ch3cn ( 0.1% formic acid ) over 20 min to afford compound 6 ( 1.8 mg , tr = 13.6 min ) . fr1.7.5 ( 21.7 mg ) was purified by reversed - phase hplc ( phenomenex luna , c18 , 250 10.0 mm , 5 m , 2.5 ml / min ) using a gradient solvent system from 20% to 90% ch3cn ( 0.1% formic acid ) over 15 min to afford fr1.7.5.1 ( 1.2 mg , tr = 12.2 min ) . fr1.7.5.1 ( 1.2 mg ) was further purified by hplc on a phenyl - hexyl column ( phenomenex luna , 250 10.0 mm , 5 m , 2.5 ml / min ) using a gradient solvent system from 20% to 100% ch3cn ( 0.1% formic acid ) over 20 min to yield compound 1 ( 0.8 mg , tr = 13.4 min ) . the extract ( 460 mg ) of the second cultivation was fractionated by flash column chromatography on ods ( 50 m , 30 g ) , eluting with a step gradient of meoh and h2o ( 10:90100:0 ) , and eight fractions ( fr2.1fr2.8 ) were collected . fr2.4 ( 21.1 mg ) separated into nine fractions ( fr2.4.1fr2.4.9 ) on sephadex lh-20 , eluting with meoh . fr2.4.6 ( 3.5 mg ) was purified by reversed - phase hplc ( phenomenex luna , c18 , 250 10.0 mm , 5 m , 2.5 ml / min ) using a gradient solvent system from 30% to 100% ch3cn ( 0.1% formic acid ) over 20 min to yield compound 2 ( 0.6 mg , tr = 15.7 min ) . white powder , [ ]d + 17 ( c 0.05 , meoh ) ; uv ( meoh ) max ( log ) 254 ( 4.04 ) , 318 ( 3.79 ) nm ; ecd ( c = 0.87 mm , meoh ) max ( ) 291 ( + 0.5 ) , 254 ( 4.7 ) nm ; ir ( nacl disk ) max 3398 , 2982 , 2925 , 1740 , 1594 , 1541 , 1394 , 1272 , 1258 , 1208 , 1187 , 1102 , 1071 , 830 cm ; h and c nmr , table 1 ; hresims m / z 265.1183 [ m + h ] ( calcd for c13h17n2o4 , 265.1183 ) . white powder , [ ]d + 6 ( c 0.03 , meoh ) ; uv ( meoh ) max ( log ) 255 ( 4.10 ) , 320 ( 3.82 ) nm ; ecd ( c = 1.33 mm , meoh ) max ( ) 333 ( + 0.5 ) , 267 ( 2.6 ) nm ; ir ( nacl disk ) max 2918 , 2850 , 1739 , 1734 , 1593 , 1540 , 1387 , 1254 , 1184 , 830 cm ; h and c nmr , table 1 ; hresims m / z 249.1235 [ m + h ] ( calcd for c13h17n2o3 , 249.1234 ) . colorless oil , [ ]d 34 ( c 0.05 , meoh ) ; uv ( meoh ) max ( log ) 252 ( 4.05 ) , 283 ( 3.79 ) , 332 ( 3.69 ) nm ; ecd ( c = 0.40 mm , meoh ) max ( ) 336 ( 19.0 ) , 297 ( + 24.5 ) nm ; ir ( nacl disk ) max 3430 , 2100 , 1645 , 1506 , 1394 , 1206 , 1135 , 1058 , 793 , 733 cm ; h and c nmr , table 2 ; hresims m / z 251.1391 [ m + h ] ( calcd for c13h19n2o3 , 251.1390 ) . colorless oil , [ ]d 7 ( c 0.09 , meoh ) ; uv ( meoh ) max ( log ) 252 ( 4.01 ) , 280 ( 3.78 ) , 335 ( 3.70 ) nm ; ecd ( c = 0.25 mm , meoh ) max ( ) 335 ( + 45.8 ) , 282 ( 106.8 ) , 248 ( + 29.9 ) , 207 ( 10.5 ) nm ; ir ( nacl disk ) max 3422 , 2092 , 1638 , 1201 , 1130 , 734 cm ; h and c nmr , table 2 ; hresims m / z 281.1496 [ m + h ] ( calcd for c14h21n2o4 , 281.1496 ) . colorless oil , [ ]d + 422 ( c 0.16 , meoh ) ; uv ( meoh ) max ( log ) 246 ( 4.02 ) , 288 ( 3.77 ) , 338 ( 3.66 ) nm ; ecd ( c = 0.41 mm , meoh ) max ( ) 339 ( + 57.6 ) , 290 ( 93.7 ) , 248 ( + 28.7 ) , 207 ( 26.2 ) nm ; ir ( nacl disk ) max : 3432 , 2101 , 1643 , 1506 , 1361 , 1228 , 1134 , 1099 , 756 cm ; h and c nmr , table 2 ; hresims m / z 247.1441 [ m + h ] ( calcd for c14h19n2o2 , 247.1441 ) . colorless oil , [ ]d + 17 ( c 0.05 , meoh ) ; uv ( meoh ) max ( log ) 245 ( 4.09 ) , 284 ( 3.81 ) , 337 ( 3.71 ) nm ; ecd ( c = 0.38 mm , meoh ) max ( ) 335 ( + 34.9 ) , 284 ( 72.3 ) , 244 ( + 16.8 ) , 209 ( 7.1 ) nm ; ir ( nacl disk ) max 3428 , 2100 , 1504 , 1132 , 677 cm ; h and c nmr , table 3 ; hresims m / z 263.1754 [ m + h ] ( calcd for c15h23n2o2 , 263.1754 ) . colorless oil , [ ]d + 37 ( c 0.03 , meoh ) ; uv ( meoh ) max ( log ) 244 ( 4.09 ) , 284 ( 3.81 ) , 337 ( 3.71 ) nm ; ecd ( c = 0.40 mm , meoh ) max ( ) 331 ( + 63.2 ) , 281 ( 129.0 ) , 244 ( + 25.7 ) , 207 ( 8.2 ) nm ; ir ( nacl disk ) max 3429 , 2099 , 1638 , 1506 , 1260 , 1174 , 1121 , 734 cm ; h and c nmr , table 3 ; hresims m / z 249.1596 [ m + h ] ( calcd for c14h21n2o2 , 249.1598 ) . colorless oil , [ ]d + 39 ( c 0.11 , meoh ) ; uv ( meoh ) max ( log ) 248 ( 4.02 ) , 282 ( 3.78 ) , 336 ( 3.69 ) nm ; ecd ( c = 0.37 mm , meoh ) max ( ) 335 ( + 18.7 ) , 284 ( 38.4 ) , 246 ( + 10.7 ) , 214 ( 4.9 ) nm ; ir ( nacl disk ) max 3431 , 2100 , 1645 , 1506 , 1258 , 1137 , 794 , 723 , 704 cm ; h and c nmr , table 3 ; hresims m / z 271.1443 [ m + h ] ( calcd for c16h19n2o2 , 271.1441 ) . [ ]d + 104 ( c 0.10 , meoh ) , literature [ ]d + 116 ( c 0.11 , meoh ) ; ecd ( c = 0.40 mm , meoh ) max ( ) 335 ( + 54.3 ) , 283 ( 115.9 ) , 248 ( + 27.4 ) , 210 ( 12.2 ) nm . to a solution of 3 ( 1.5 mg ) in dmf ( anhydrous , 0.5 ml ) was added 2.0 mg of nah . after allowing it to stir at 0 c for 0.5 h it was stirred at room temperature for another 0.5 h. then , a saturated solution of nh4cl ( 2.0 ml ) was added to quench the reaction . the product was extracted with etoac ( 3 3.0 ml ) and purified by reversed - phase hplc ( phenomenex luna , c18 , 250 10.0 mm , 2.5 ml / min , 5 m ) using a gradient solvent system from 20% to 100% ch3cn ( 0.1% formic acid ) over 20 min to afford compound 3a ( 0.5 mg , tr = 14.4 min ) . compound 3a : white powder ; h nmr ( 600 mhz , cdcl3 ) 5.99 ( m , 1h , h-2 ) , 5.05 ( s , 1h , h-2 ) , 3.62 ( m , 1h , h-4 ) , 3.29 ( s , 3h , 3-nch3 ) , 3.27 ( s , 3h , 7-och3 ) , 2.17 ( s , 3h , h-5 ) , 2.17 ( m , 1h , h-5 ) , 2.09 ( m , 1h , h-6 ) , 2.00 ( m , 1h , h-5 ) , 1.90 ( s , 3h , h-4 ) , 1.88 ( m , 1h , h-6 ) , 1.28 ( d , j = 6.8 , 3h , h-8 ) ; c and 2d nmr , figure s2 ; hresims m / z 279.1704 [ m + h ] ( calcd for c15h23n2o3 , 279.1703 ) .

two new 1,3-oxazin-6-one derivatives ( 1 and 2 ) and six new bohemamine - type pyrrolizidine alkaloids ( 38 ) were isolated from the marine - derived streptomyces spinoverrucosus strain snb-048 . their structures including the absolute configurations were fully elucidated on the basis of spectroscopic analysis , ecd spectra , quantum chemical calculations , and chemical methods . compounds 1 and 2 possess a -lactam moiety and a 1,3-oxazin-6-one system .

Results and Discussion Experimental Section

the c nmr spectrum showed 13 carbon signals that were classified by hsqc as three methyl carbons , one sp methylene carbon , four methine groups ( two olefinic carbons , one carbon connected with oxygen , and one carbon connected with nitrogen ) , and five nonprotonated carbons ( three carbonyls and two olefinic carbons ) ( table 1 ) . the h nmr spectrum showed five coupled signals at h 1.23 ( h-8 , d , j = 6.5 ) , 4.55 ( h-4 , qd , j = 6.7 , 6.6 ) , 4.43 ( h-5 , ddd , j = 8.8 , 8.0 , 6.7 ) , 2.67 ( h-6 , dd , j = 16.9 , 8.0 ) , and 2.63 ( h-6 , dd , j = 16.9 , 8.8 ) . two vinyl methyl groups at h 2.25 ( h-5 , d , j = 1.2 hz ) and 2.03 ( h-4 , d , j = 1.2 hz ) and one olefinic proton signal at h 5.98 ( m ) , along with the hmbc correlations of h-2 to c-4/c-5 and h-4 to c-5 ( figure 1 ) , indicated the presence of an isobutenyl fragment . the remaining h and c nmr signals and the hmbc correlations of h-2 ( h / c 6.73/89.0 ) to c-1 ( c 159.8 ) and c-3 ( c 155.3 ) ( figure 1 ) , along with the molecular formula , provided two possible structures that could not be easily distinguished : a 1,3-oxazin-6-one ( 1 ) or an unsaturated -lactam unit ( 1a ) ( figure 2 ) . only one natural product possessing an unsaturated -lactam moiety was reported , but it was later revised to be a six - membered - ring metabolite . predicted c nmr shifts of both 1,3-oxazin-6-one ( 1 ) and -lactam ( 1a ) , which were computed at the b3lyp/6 - 311++g(2d , p)//b3lyp/6 - 31g(d ) level ( full details on the calculation can be found in the supporting information ) , strongly support that the chemical shift values of 1 are consistent with a 1,3-oxazin-6-one skeleton ( table s1 ) , particularly the c shifts of c-1 , c-2 , and c-3 . two structures , 1 and 1a , in general agreement with the nmr data . noe correlations between h-8 and h-5/h-6 , between h-4 and h-5 , and between h-5 and h-6/h-6 were observed in the 2d noesy spectrum , suggesting the relative configuration of 1 as shown ( figure 3 ) . noe correlations combined with the coupling constants between h-4 and h-5 ( j = 6.7 hz ) , between h-5 and h-6 ( j = 8.8 hz ) , and between h-5 and h-6 ( j = 8.0 hz ) also indicated the conformation of 1 as shown in figure 4 . we utilized the empirical electronic circular dichrosim ( ecd ) rule of -lactams to determine the absolute configuration of 1 . if the -lactams have no lone pair electrons on the c substituent , the ecd signs are determined by the ring chirality ( conformation a gives a negative sign , and b gives a positive sign ) ( figure 4 ) . as the 1,3-oxazin-6-one group connected with the nitrogen nearly lay on the same plane of the amide , it made a very small contribution to the lactam cotton effect . measured and calculated ecd spectra for 1 . the molecular formula of spinoxazine b ( 2 ) was assigned as c13h16n2o3 based on the hresims , which was only one oxygen less than that of 1 . the uv spectrum showed similar absorptions to bohemamines at 252 , 283 , and 332 nm . comparison of its nmr spectra with those of known bohemamine analogues indicated that the h and c nmr data of 3 ( table 2 ) were similar to np25302 ( 9 ) . the signal of the c-7 methyl group in np25302 was no longer present , and c at c-7 was shifted deshielded to 95.3 , leading to the assignment of a c-7 hydroxy group . the octant rule for cyclopentenone was used to determine the absolute configuration ( figure 6 ) . moreover , the ecd helicity rule of ,-unsaturated ketones was used to further confirm the absolute configuration of compound 3 . ecd spectra and the representation of the octant rule and helicity rule for the cyclopentenone moiety of 3 ( a ) and 9 ( b ) . bohemamine e ( 4 ) was also thought to be an analogue of bohemamines due to the similar uv absorptions at 252 , 280 , and 335 nm . noe correlations between h-5 and h-9 , between h-4 and h-8 , and between h-6 and h-8 were observed in the 2d noesy , suggesting the relative configuration of 4 as shown ( figure 3 ) . the molecular formula of bohemamine f ( 5 ) was determined to be c14h18n2o2 from the protonated molecule peak at m / z 247.1441 [ m + h ] in the positive - ion hresims spectrum , which was two hydrogen atoms less than that of np25302 ( 9 ) . comparison of its h and c nmr spectra ( table 2 ) with those of 9 revealed that the signals of two methylene groups ( c-5 and c-6 ) were replaced by two coupled olefinic methine signals ( c-5 : c / h 128.0/5.74 , dd , j = 6.0 , 2.2 ; c-6 : c / h 133.9/5.67 , dd , j = 6.0 , 1.4 ) . this structure can be confirmed by cosy correlations of h-9/h-4/h-5/h-6 and the key hmbc correlations from h-9 to c-4/c-5 , h-6 to c-4/c-5/c-7/c-1 , and h-8 to c-6/c-7 . i ( 68 ) were determined as c15h22n2o2 , c14h20n2o2 , and c16h18n2o2 based on their hresims spectra at m / z 263.1754 [ m + h ] , 249.1596 [ m + h ] , and 271.1443 [ m + h ] , respectively . for compound 6 , the cosy correlations of h-2/h-3/h-4 and h-5/h-4/h-6 and the key hmbc correlations from h-2 to c-1 , h-3 to c-1/c-4 , h-4 to c-6 , h-5 to c-6 , and h-6 to c-3 suggested the presence of a 4-methylpent-2-enamide side chain ( figure 1 ) . this structure was supported by the cosy correlation of h-3/h-4 and the hmbc correlations from h-3 to c-1/c-5 , h-4 to c-2/c-3 , and h-5 to c-1 ( figure 1 ) . the absolute configuration of compound 9 has been determined as ( 4s,7s ) by total synthesis , which can be confirmed with the octant rule for cyclopentenone ( figure 6 ) . m - helicity of the o = c c=c torsion angle of compound 9 was consistent with the negative cotton effect ( * ) at 283 nm ( figure 6 ) , which suggested the absolute configuration of 9 as ( 4s,7s ) . the ecd spectra of compounds 48 showed the same cotton effects as those of 9 ( figure 7 ) , indicating the same core configurations . a plausible biogenetic pathway for spinoxazines a and b ( 1 and 2 ) and bohemamine d ( 3 ) is postulated ( scheme 2 ) . dehydrogenation of the pyrrolizidine core would give the ring cleavage intermediate f , which could undergo a pericyclic reaction to form the 1,3-oxazin-6-one compound spinoxazine b ( 2 ) . compounds 18 did not show cytotoxicity to non - small - cell lung cancer cell lines hcc366 , a549 , hcc44 , and hcc1171 . h and 2d nmr spectroscopic data were recorded at 600 mhz in dmso - d6 or cdcl3 solution on a varian system spectrometer , and chemical shifts were referenced to the corresponding residual solvent signal ( h / c 2.50/39.52 for dmso - d6 and h / c 7.26/77.16 for cdcl3 ) . sephadex lh-20 ( ge healthcare ) and ods ( 50 mm , merck ) were used for column chromatography . strain snb-048 was isolated from a sand sample collected from a bahamian tidal flat . bacterium snb-048 was cultured in 20 2.8 l fernbach flasks each containing 1 l of a seawater - based medium ( 10 g starch , 4 g yeast extract , 2 g peptone , 1 g caco3 , 40 mg fe2(so4)34h2o , 100 mg kbr ) and shaken at 200 rpm at 27 c . g / l ) was added to adsorb the organic products , and the culture and resin were shaken at 200 rpm for 2 h. the resin was filtered through cheesecloth , washed with deionized h2o , and eluted with acetone . in order to get more material , another cultivation of 10 the extract ( 1.2 g ) of the first cultivation was fractionated by flash column chromatography on ods ( 50 m , 30 g ) , eluting with a step gradient of meoh and h2o ( 10:90100:0 ) , and 12 fractions ( fr1.1fr1.12 ) were collected . fractions 7 and 8 ( 403.7 mg ) were combined and separated into six fractions ( fr1.7.1fr1.7.6 ) on sephadex lh-20 , eluting with meoh . fr1.7.3.6 ( 6.3 mg ) was purified by hplc on a phenyl - hexyl column ( phenomenex luna , 250 10.0 mm , 5 m , 2.5 ml / min ) using a gradient solvent system from 20% to 100% ch3cn ( 0.1% formic acid ) over 20 min to yield compound 4 ( 1.4 mg , tr = 9.5 min ) . fr.7.3.8 ( 17.5 mg ) was purified by hplc on a phenyl - hexyl column ( phenomenex luna , 250 10.0 mm , 5 m , 2.5 ml / min ) using a gradient solvent system from 20% to 85% ch3cn ( 0.1% formic acid ) over 20 min to afford compound 3 ( 1.9 mg , tr = 11.1 min ) . fr1.7.3.10 ( 31.7 mg ) was separated by a reversed - phase hplc ( phenomenex luna , c18 , 250 10.0 mm , 5 m , 2.5 ml / min ) using a gradient solvent system from 20% to 80% ch3cn ( 0.1% formic acid ) over 20 min to yield compound 9 ( 3.8 mg , tr = 13.4 min ) and fr1.7.3.10.4 ( 12.6 mg , tr = 14.3 min ) . fr1.7.3.10.4 ( 12.6 mg ) was further purified by hplc on a cn column ( phenomenex luna , 250 10.0 mm , 5 m , 2.5 ml / min ) using a gradient solvent system from 20% to 100% ch3cn ( 0.1% formic acid ) over 20 min to afford compounds 5 ( 3.3 mg , tr = 11.5 min ) and 8 ( 2.3 mg , tr = 12.0 min ) . fr1.7.3.11 ( 9.1 mg ) was purified by hplc on a phenyl - hexyl column ( phenomenex luna , 250 10.0 mm , 5 m , 2.5 ml / min ) using a gradient solvent system from 20% to 100% ch3cn ( 0.1% formic acid ) over 20 min to afford compound 6 ( 1.8 mg , tr = 13.6 min ) . fr1.7.5 ( 21.7 mg ) was purified by reversed - phase hplc ( phenomenex luna , c18 , 250 10.0 mm , 5 m , 2.5 ml / min ) using a gradient solvent system from 20% to 90% ch3cn ( 0.1% formic acid ) over 15 min to afford fr1.7.5.1 ( 1.2 mg , tr = 12.2 min ) . fr1.7.5.1 ( 1.2 mg ) was further purified by hplc on a phenyl - hexyl column ( phenomenex luna , 250 10.0 mm , 5 m , 2.5 ml / min ) using a gradient solvent system from 20% to 100% ch3cn ( 0.1% formic acid ) over 20 min to yield compound 1 ( 0.8 mg , tr = 13.4 min ) . the extract ( 460 mg ) of the second cultivation was fractionated by flash column chromatography on ods ( 50 m , 30 g ) , eluting with a step gradient of meoh and h2o ( 10:90100:0 ) , and eight fractions ( fr2.1fr2.8 ) were collected . colorless oil , [ ]d + 422 ( c 0.16 , meoh ) ; uv ( meoh ) max ( log ) 246 ( 4.02 ) , 288 ( 3.77 ) , 338 ( 3.66 ) nm ; ecd ( c = 0.41 mm , meoh ) max ( ) 339 ( + 57.6 ) , 290 ( 93.7 ) , 248 ( + 28.7 ) , 207 ( 26.2 ) nm ; ir ( nacl disk ) max : 3432 , 2101 , 1643 , 1506 , 1361 , 1228 , 1134 , 1099 , 756 cm ; h and c nmr , table 2 ; hresims m / z 247.1441 [ m + h ] ( calcd for c14h19n2o2 , 247.1441 ) . colorless oil , [ ]d + 37 ( c 0.03 , meoh ) ; uv ( meoh ) max ( log ) 244 ( 4.09 ) , 284 ( 3.81 ) , 337 ( 3.71 ) nm ; ecd ( c = 0.40 mm , meoh ) max ( ) 331 ( + 63.2 ) , 281 ( 129.0 ) , 244 ( + 25.7 ) , 207 ( 8.2 ) nm ; ir ( nacl disk ) max 3429 , 2099 , 1638 , 1506 , 1260 , 1174 , 1121 , 734 cm ; h and c nmr , table 3 ; hresims m / z 249.1596 [ m + h ] ( calcd for c14h21n2o2 , 249.1598 ) . colorless oil , [ ]d + 39 ( c 0.11 , meoh ) ; uv ( meoh ) max ( log ) 248 ( 4.02 ) , 282 ( 3.78 ) , 336 ( 3.69 ) nm ; ecd ( c = 0.37 mm , meoh ) max ( ) 335 ( + 18.7 ) , 284 ( 38.4 ) , 246 ( + 10.7 ) , 214 ( 4.9 ) nm ; ir ( nacl disk ) max 3431 , 2100 , 1645 , 1506 , 1258 , 1137 , 794 , 723 , 704 cm ; h and c nmr , table 3 ; hresims m / z 271.1443 [ m + h ] ( calcd for c16h19n2o2 , 271.1441 ) . [ ]d + 104 ( c 0.10 , meoh ) , literature [ ]d + 116 ( c 0.11 , meoh ) ; ecd ( c = 0.40 mm , meoh ) max ( ) 335 ( + 54.3 ) , 283 ( 115.9 ) , 248 ( + 27.4 ) , 210 ( 12.2 ) nm . to a solution of 3 ( 1.5 mg ) in dmf ( anhydrous , 0.5 ml ) was added 2.0 mg of nah . the product was extracted with etoac ( 3 3.0 ml ) and purified by reversed - phase hplc ( phenomenex luna , c18 , 250 10.0 mm , 2.5 ml / min , 5 m ) using a gradient solvent system from 20% to 100% ch3cn ( 0.1% formic acid ) over 20 min to afford compound 3a ( 0.5 mg , tr = 14.4 min ) .

since quality has an abstract concept , presenting a practical method for determining it is not easy . however , this is the conceptual quality that can be felt , perceived , and judged and when a product or good does not have quality , lack of quality can easily be sensed . therefore , to improve the reception and use of a product or service , the way quality is determined should be defined , and its evaluation and control procedure should be understood ( 1 ) , so that during the evaluation process , it is ensured that the delegated tasks and responsibilities are done well ( 2 ) . one of the services that has had a considerable development in recent years is the world wide web , providing the world with a phenomenon called websites . websites involve connection points and communication of users with electronic information and considering nowadays all institutes try to present their services to different classes , the world wide web has been successful in meeting this need in generating information and making them accessible with an ever - increasing speed ( 3 ) . in the health sector , many hospitals have designed individual websites for themselves presenting various services . given the sensitivity of hospitals , the services they are seeking to introduce , in response to the increasing development of hospital websites to allow the access of various people to these websites , the issue of website quality has arisen . this is because the richness of websites and the number of referrals to them , contribute to improving the status of the hospital in the specialized area and in offering services to patients . if the performance of a hospital website is weaker than the expected level , redesigning the website or substantial enhancement of the volume and quality of electronic journal may be required ( 3 ) . to evaluate the quality of websites , various models and criteria the first one is the iso-9126 model whose criteria are usability , reliability , efficiency , and functionality . the mile model , focusing on the usability of the website , measures the quality of websites based on content , services , navigation , cognitive features of the interface , aesthetics , and technology . and finally , the minerva model , proposed to evaluate the quality of cultural websites ( 1 , 4 , 5 ) . unfortunately , despite the various models presented for evaluating websites , determination of proper models and criteria for evaluating the quality of hospital websites has been understudied . thus , this research tried to identify the criteria required for a hospital website by investigating the evaluation models of the quality of websites . it is hoped that the results of this research are found useful in presenting a model for evaluation of the quality of hospital websites to promote their quality . the studies either in english or persian , that have been related to evaluation of website quality and whose title and abstract included keywords such as evaluation , website quality , and website model , were included in this research . the international database including science direct , google scholar , pubmed , proquest , ovid , elsevier , springer , ebsco , and regional databases including magiran , scientific information database and persian journal citation report ( pjcr ) , were searched using the keywords of website , evaluation , and website quality , according to table 1 . all papers , regardless of the methodology including systematic review , experimental , and cross - sectional studies that were related to the research subject and were accessible in their full text were selected . the models were then reviewed and out of the selected papers , the criteria and sub criteria of website quality were extracted and then classified . the papers that were in line with the subject whose full text version was accessible , were included , while the papers that had no conceptual relationship with the subject or their full text was not available , were excluded from the study . the results extracted and investigated in this research were the criteria required for evaluating the quality of hospital websites . following specialist discussion and opinion exchange , 22 papers were selected ( table 2 ) . the international database including science direct , google scholar , pubmed , proquest , ovid , elsevier , springer , ebsco , and regional databases including magiran , scientific information database and persian journal citation report ( pjcr ) , were searched using the keywords of website , evaluation , and website quality , according to table 1 . all papers , regardless of the methodology including systematic review , experimental , and cross - sectional studies that were related to the research subject and were accessible in their full text were selected . the models were then reviewed and out of the selected papers , the criteria and sub criteria of website quality were extracted and then classified . the papers that were in line with the subject whose full text version was accessible , were included , while the papers that had no conceptual relationship with the subject or their full text was not available , were excluded from the study . the results extracted and investigated in this research were the criteria required for evaluating the quality of hospital websites . to evaluate the papers qualitatively , the extracted articles were studied by two relevant experts ( figure 1 ) . following specialist discussion and opinion exchange , to evaluate the quality of hospital websites , various models had been presented , the most famous of which were : the quality criteria in this model had been developed based on iso-9126 model , including usability , reliability , efficiency , and functionality . the specific feature of this model was determination of a certain domain of evaluation and conductance of the evaluation step by step ( 1 , 6 ) . this model focuses on usability of the website and measures the quality by combining the inspection by both an evaluator and users ( 1 ) . the specified criteria in this model are : content : content means the quality of the information inside the website , and its communicative level . services : services are all the functionalities that the website allows its users to do . navigation : this criterion encompasses 1 ) various means of access by users to a certain part of the information in the website and 2 ) the logical structure of information to move across one part of information to another . cognitive features of the interface : it takes the perception and feeling of the user into consideration and deals with how the users memorize the structure of the website . aesthetics : it addresses the graphic design and template of the website , the type of font , color , size , images , and other graphic features in a website . technology : it represents the adaption of the website for proper function in a variety of explorers , the security level of the host server , and the interaction between the website and remote database ( 1 , 5 ) . this model was suggested for evaluation of the quality of cultural websites ( seminaries , archives , libraries , and other cultural institutions ) ( 1 ) . in this model , the aim of quality indices in this model is to present indices for evaluation of the quality of websites and support the design and development of the cultural websites ( 5 ) . this model involves the following axes : clarity : the clarity of objectives , mission , and the identity of the website for users . effectiveness : the content of the website is related to the validity of the website content and presentation of sound information to users . maintenance : it focuses on the technical and content support , fresh content , and improvement of the website s technical functions . accessibility : it represents easy access of all users to the website,(for example , access for blind users , those with relative vision , or users with hearing problems ) , the possibility of using a variety of technologies to present information to users , and the functionality with a variety of explorers , operating systems , and devices . user - centeredness : this means that the website should meet the needs of users . further , the users should regard the website as useful , attractive , and easy to use . responsibility : it refers to the ability of the website owners in responding to the questions of users , helping users to participate in generating content and answering the questions posed in a forum . if a website has been based on standard technologies , techniques , and models , interaction and adaption with other websites and institutions would be easy . managed : it denotes the legal issues related to protecting copyright and privacy ( 1 , 6 ) . this model involves entity ( who ) , content ( what ) , services ( why ) , location ( where ) , maintenance ( when ) , usability ( how ) , and possibility ( by what means ) which are defined as follow : entity : includes the sub features of identification of characteristics . facilities : encompasses resources , information and communication technology ( 5 ) this instrument has 12 dimensions including information suited to users , interaction , reliability , responsibility time , appearance , design , guesswork , innovativeness , stream , coherent connections , business process , and the ability for substitution ( 5 ) ( table 3 ) . some of them focused on the structure , the homepage content , page content , and subject content ( other links ) of websites for evaluation purposes ( 7 , 11 , 13 , 15 , 1921 , 2325 ) . other studies mentioned characteristics of audience , website objective , loading , and structural stability among websites as important factors in the success of a website ( 8 , 12 , 14 , 26 ) . also quality , size , language , background , and inclusiveness as the determinants of the final success of a website were referred ( 9 , 12 ) . another study found that attention to the update rate of pages ( the duration between the last update and the date the website is used ) is essential ( 10 ) . a technical framework presented seven criteria including : content quality , design quality , organization quality , user - friendliness , functional quality , service quality , and website quality from a technical point of view ( 11 ) . moreover the evaluation criteria as the accessibility and updating were mentioned ( 10 , 12 , 1518 , 22 , 27 ) . furthermore , entertainment and fun , and website speed was presented as evaluation criteria for websites ( 1315 ) . another study stated the security , and confidentiality of personal information as the most important factors affecting the willingness of customers to purchase from a website ( 2325 ) as well as multilinguality ( 27 ) ( table 4 ) . to evaluate the quality of hospital websites , various models had been presented , the most famous of which were : the quality criteria in this model had been developed based on iso-9126 model , including usability , reliability , efficiency , and functionality . the specific feature of this model was determination of a certain domain of evaluation and conductance of the evaluation step by step ( 1 , 6 ) . this model focuses on usability of the website and measures the quality by combining the inspection by both an evaluator and users ( 1 ) . the specified criteria in this model are : content : content means the quality of the information inside the website , and its communicative level . services : services are all the functionalities that the website allows its users to do . navigation : this criterion encompasses 1 ) various means of access by users to a certain part of the information in the website and 2 ) the logical structure of information to move across one part of information to another . cognitive features of the interface : it takes the perception and feeling of the user into consideration and deals with how the users memorize the structure of the website . aesthetics : it addresses the graphic design and template of the website , the type of font , color , size , images , and other graphic features in a website . technology : it represents the adaption of the website for proper function in a variety of explorers , the security level of the host server , and the interaction between the website and remote database ( 1 , 5 ) . this model was suggested for evaluation of the quality of cultural websites ( seminaries , archives , libraries , and other cultural institutions ) ( 1 ) . in this model , the aim of quality indices in this model is to present indices for evaluation of the quality of websites and support the design and development of the cultural websites ( 5 ) . this model involves the following axes : clarity : the clarity of objectives , mission , and the identity of the website for users . effectiveness : the content of the website is related to the validity of the website content and presentation of sound information to users . maintenance : it focuses on the technical and content support , fresh content , and improvement of the website s technical functions . accessibility : it represents easy access of all users to the website,(for example , access for blind users , those with relative vision , or users with hearing problems ) , the possibility of using a variety of technologies to present information to users , and the functionality with a variety of explorers , operating systems , and devices . user - centeredness : this means that the website should meet the needs of users . further , the users should regard the website as useful , attractive , and easy to use . responsibility : it refers to the ability of the website owners in responding to the questions of users , helping users to participate in generating content and answering the questions posed in a forum . if a website has been based on standard technologies , techniques , and models , interaction and adaption with other websites and institutions would be easy . managed : it denotes the legal issues related to protecting copyright and privacy ( 1 , 6 ) . this model involves entity ( who ) , content ( what ) , services ( why ) , location ( where ) , maintenance ( when ) , usability ( how ) , and possibility ( by what means ) which are defined as follow : entity : includes the sub features of identification of characteristics . facilities : encompasses resources , information and communication technology ( 5 ) this instrument has 12 dimensions including information suited to users , interaction , reliability , responsibility time , appearance , design , guesswork , innovativeness , stream , coherent connections , business process , and the ability for substitution ( 5 ) ( table 3 ) . the quality criteria in this model had been developed based on iso-9126 model , including usability , reliability , efficiency , and functionality . the specific feature of this model was determination of a certain domain of evaluation and conductance of the evaluation step by step ( 1 , 6 ) . this model focuses on usability of the website and measures the quality by combining the inspection by both an evaluator and users ( 1 ) . the specified criteria in this model are : content : content means the quality of the information inside the website , and its communicative level . services : services are all the functionalities that the website allows its users to do . navigation : this criterion encompasses 1 ) various means of access by users to a certain part of the information in the website and 2 ) the logical structure of information to move across one part of information to another . cognitive features of the interface : it takes the perception and feeling of the user into consideration and deals with how the users memorize the structure of the website . aesthetics : it addresses the graphic design and template of the website , the type of font , color , size , images , and other graphic features in a website . technology : it represents the adaption of the website for proper function in a variety of explorers , the security level of the host server , and the interaction between the website and remote database ( 1 , 5 ) . this model was suggested for evaluation of the quality of cultural websites ( seminaries , archives , libraries , and other cultural institutions ) ( 1 ) . in this model , the aim of quality indices in this model is to present indices for evaluation of the quality of websites and support the design and development of the cultural websites ( 5 ) . this model involves the following axes : clarity : the clarity of objectives , mission , and the identity of the website for users . effectiveness : the content of the website is related to the validity of the website content and presentation of sound information to users . maintenance : it focuses on the technical and content support , fresh content , and improvement of the website s technical functions . accessibility : it represents easy access of all users to the website,(for example , access for blind users , those with relative vision , or users with hearing problems ) , the possibility of using a variety of technologies to present information to users , and the functionality with a variety of explorers , operating systems , and devices . user - centeredness : this means that the website should meet the needs of users . further , the users should regard the website as useful , attractive , and easy to use . responsibility : it refers to the ability of the website owners in responding to the questions of users , helping users to participate in generating content and answering the questions posed in a forum . if a website has been based on standard technologies , techniques , and models , interaction and adaption with other websites and institutions would be easy . managed : it denotes the legal issues related to protecting copyright and privacy ( 1 , 6 ) . this model involves entity ( who ) , content ( what ) , services ( why ) , location ( where ) , maintenance ( when ) , usability ( how ) , and possibility ( by what means ) which are defined as follow : entity : includes the sub features of identification of characteristics . this instrument has 12 dimensions including information suited to users , interaction , reliability , responsibility time , appearance , design , guesswork , innovativeness , stream , coherent connections , business process , and the ability for substitution ( 5 ) ( table 3 ) . some of them focused on the structure , the homepage content , page content , and subject content ( other links ) of websites for evaluation purposes ( 7 , 11 , 13 , 15 , 1921 , 2325 ) . other studies mentioned characteristics of audience , website objective , loading , and structural stability among websites as important factors in the success of a website ( 8 , 12 , 14 , 26 ) . also quality , size , language , background , and inclusiveness as the determinants of the final success of a website were referred ( 9 , 12 ) . another study found that attention to the update rate of pages ( the duration between the last update and the date the website is used ) is essential ( 10 ) . a technical framework presented seven criteria including : content quality , design quality , organization quality , user - friendliness , functional quality , service quality , and website quality from a technical point of view ( 11 ) . moreover the evaluation criteria as the accessibility and updating were mentioned ( 10 , 12 , 1518 , 22 , 27 ) . furthermore , entertainment and fun , and website speed was presented as evaluation criteria for websites ( 1315 ) . another study stated the security , and confidentiality of personal information as the most important factors affecting the willingness of customers to purchase from a website ( 2325 ) as well as multilinguality ( 27 ) ( table 4 ) . one of the most useful applications of the web is the web portals or website . the aim of this study was to identify the models for evaluating the quality of hospital websites . the results indicated that a great variety and diversity exists among the criteria presented for evaluating the quality of websites . on the other hand , many of these criteria can be considered as the sub criteria of a more general criterion . a study investigated the factors associated with the success of websites in the area of electronic business in the us . the study introduced four factors of web success in electronic business as information , the quality of services , use of systems , entertainment and fun , and the quality of system design ( 19 ) . another study experimentally examined the relationship between the quality of websites and integration of investment of website customers in kuwait . the researcher categorized the quality of services in four aspects of technical , general and specific content , and the apparent quality . he believes that these dimensions influence the attitude of website users , where the positive attitude of users in turn affects their tendency to buy through the internet ( 22 ) . a paper called the government in the digital era : concept , practice , and development in thailand , stated 10 characteristics of an electronic government , arguing that an electronic government should be comprehensive , interactive , inclusive , convenient , accessible , secure , customizable , multifunctional , and flexible ( 16 ) . in spain experimentally and quantitatively investigated websites of electronic banking with the aim of finding an index for web evaluation , reported that the evaluation index for analyzing the website of commercial , educational , and nonprofit organizations is speed , navigation , and content ( 21 ) . a paper called the qualitative framework of evaluation of hospital / medical websites presented a qualitative framework consisting of seven criteria : the quality of content , design , organization , user - friendliness , performance , services , and technical quality ( 11 ) . a research entitled electronic government in cyprus proposed a framework for evaluating the development of electronic government . this framework comprised content , design , and typical features of electronic government websites ( 20 ) . results of a research introduced six important factors for success of websites as design , content , consumer education , security , customer support , online communities , and the market situation ( 24 ) . a model for the quality of websites with the criteria of correctness , presentation , navigation , content , and the final success of websites , associated with factors such as quality , size , language , background , inclusion , etc . and it was stated that one can not mention one or two factors as the only reasons for the success of a website ( 9 ) . the result of investigation of the selected studies revealed that the criteria of accessibility , content , and apparent features of websites from user perspective such as design , the graphics used in the website , and attractiveness of the page have been mentioned in the majority of studies . the results also showed that the important point in determining the criteria for evaluating the quality of websites is attention to the major differences in the special features of any website ; features considered during the design and selection of content for groups with various aims , so that a website becomes successful in responding to an objective for which it has been designed . these features are especially important during the evaluation of websites , since neglecting these features causes many websites to fail to reach the aims for which they have been designed , thus requiring revision . the results also indicated that the features of the information content , website design , security , and confidentiality of personal information are among the most important factors affecting the tendency of clients to use a website , thus paying attention to it in the design of any website and any evaluation model as a common criterion is essential . minimum quality criteria for a website are usability , efficiency , ease of use , user friendliness , service , reliability and interaction . the criteria of accessibility , content , design , security , and confidentially of personal information , as the essential criteria , should be taken into account when evaluating a website . it is suggested that the criteria such as ease of use , playing a role in bringing about satisfaction of users , the graphics used in the website , attractiveness of the website , and other apparent characteristics of websites are considered as sub criteria for the criterion of user - friendliness . it is also proposed that the criteria of speed and accessibility of websites , which indicate the efficiency of a website , are included in all models of website evaluation as the sub criterion of efficiency .

introductionhospital websites are important tools in establishing communication and exchanging information between patients and staff , and thus should enjoy an acceptable level of quality . the aim of this study was to identify proper models and criteria to evaluate the quality of hospital websites.methodsthis research was a systematic review study . the international databases such as science direct , google scholar , pubmed , proquest , ovid , elsevier , springer , and ebsco together with regional database such as magiran , scientific information database , persian journal citation report ( pjcr ) and iranmedex were searched . suitable keywords including website , evaluation , and quality of website were used . full text papers related to the research were included . the criteria and sub criteria of the evaluation of website quality were extracted and classified.resultsto evaluate the quality of the websites , various models and criteria were presented . the web - q - im , mile , minerva , seruni luci , and web - qual models were the designed models . the criteria of accessibility , content and apparent features of the websites , the design procedure , the graphics applied in the website , and the page s attractions have been mentioned in the majority of studies.conclusionthe criteria of accessibility , content , design method , security , and confidentiality of personal information are the essential criteria in the evaluation of all websites . it is suggested that the ease of use , graphics , attractiveness and other apparent properties of websites are considered as the user - friendliness sub criteria . further , the criteria of speed and accessibility of the website should be considered as sub criterion of efficiency . when determining the evaluation criteria of the quality of websites , attention to major differences in the specific features of any website is essential .

1. Introduction 2. Material and Methods 2.1. The search strategy 2.2. The inclusion and exclusion criteria 2.3. Quality assessment 3. Results 3.1. Results resulted from the identified models 3.1.1. Web-Q-I-M Model 3.1.2. Mile Model 3.1.3. Minerva Model 3.1.4. Seruni Lucci Model 3.1.5. Web-Qual Model 3.2. The results obtained from the reviewed studies 4. Discussion 5. Conclusions

however , this is the conceptual quality that can be felt , perceived , and judged and when a product or good does not have quality , lack of quality can easily be sensed . therefore , to improve the reception and use of a product or service , the way quality is determined should be defined , and its evaluation and control procedure should be understood ( 1 ) , so that during the evaluation process , it is ensured that the delegated tasks and responsibilities are done well ( 2 ) . given the sensitivity of hospitals , the services they are seeking to introduce , in response to the increasing development of hospital websites to allow the access of various people to these websites , the issue of website quality has arisen . this is because the richness of websites and the number of referrals to them , contribute to improving the status of the hospital in the specialized area and in offering services to patients . if the performance of a hospital website is weaker than the expected level , redesigning the website or substantial enhancement of the volume and quality of electronic journal may be required ( 3 ) . to evaluate the quality of websites , various models and criteria the first one is the iso-9126 model whose criteria are usability , reliability , efficiency , and functionality . the mile model , focusing on the usability of the website , measures the quality of websites based on content , services , navigation , cognitive features of the interface , aesthetics , and technology . and finally , the minerva model , proposed to evaluate the quality of cultural websites ( 1 , 4 , 5 ) . unfortunately , despite the various models presented for evaluating websites , determination of proper models and criteria for evaluating the quality of hospital websites has been understudied . thus , this research tried to identify the criteria required for a hospital website by investigating the evaluation models of the quality of websites . it is hoped that the results of this research are found useful in presenting a model for evaluation of the quality of hospital websites to promote their quality . the studies either in english or persian , that have been related to evaluation of website quality and whose title and abstract included keywords such as evaluation , website quality , and website model , were included in this research . the international database including science direct , google scholar , pubmed , proquest , ovid , elsevier , springer , ebsco , and regional databases including magiran , scientific information database and persian journal citation report ( pjcr ) , were searched using the keywords of website , evaluation , and website quality , according to table 1 . all papers , regardless of the methodology including systematic review , experimental , and cross - sectional studies that were related to the research subject and were accessible in their full text were selected . the models were then reviewed and out of the selected papers , the criteria and sub criteria of website quality were extracted and then classified . the papers that were in line with the subject whose full text version was accessible , were included , while the papers that had no conceptual relationship with the subject or their full text was not available , were excluded from the study . the results extracted and investigated in this research were the criteria required for evaluating the quality of hospital websites . the international database including science direct , google scholar , pubmed , proquest , ovid , elsevier , springer , ebsco , and regional databases including magiran , scientific information database and persian journal citation report ( pjcr ) , were searched using the keywords of website , evaluation , and website quality , according to table 1 . all papers , regardless of the methodology including systematic review , experimental , and cross - sectional studies that were related to the research subject and were accessible in their full text were selected . the models were then reviewed and out of the selected papers , the criteria and sub criteria of website quality were extracted and then classified . the results extracted and investigated in this research were the criteria required for evaluating the quality of hospital websites . to evaluate the papers qualitatively , the extracted articles were studied by two relevant experts ( figure 1 ) . following specialist discussion and opinion exchange , to evaluate the quality of hospital websites , various models had been presented , the most famous of which were : the quality criteria in this model had been developed based on iso-9126 model , including usability , reliability , efficiency , and functionality . the specific feature of this model was determination of a certain domain of evaluation and conductance of the evaluation step by step ( 1 , 6 ) . this model focuses on usability of the website and measures the quality by combining the inspection by both an evaluator and users ( 1 ) . the specified criteria in this model are : content : content means the quality of the information inside the website , and its communicative level . navigation : this criterion encompasses 1 ) various means of access by users to a certain part of the information in the website and 2 ) the logical structure of information to move across one part of information to another . cognitive features of the interface : it takes the perception and feeling of the user into consideration and deals with how the users memorize the structure of the website . aesthetics : it addresses the graphic design and template of the website , the type of font , color , size , images , and other graphic features in a website . technology : it represents the adaption of the website for proper function in a variety of explorers , the security level of the host server , and the interaction between the website and remote database ( 1 , 5 ) . this model was suggested for evaluation of the quality of cultural websites ( seminaries , archives , libraries , and other cultural institutions ) ( 1 ) . in this model , the aim of quality indices in this model is to present indices for evaluation of the quality of websites and support the design and development of the cultural websites ( 5 ) . this model involves the following axes : clarity : the clarity of objectives , mission , and the identity of the website for users . effectiveness : the content of the website is related to the validity of the website content and presentation of sound information to users . maintenance : it focuses on the technical and content support , fresh content , and improvement of the website s technical functions . accessibility : it represents easy access of all users to the website,(for example , access for blind users , those with relative vision , or users with hearing problems ) , the possibility of using a variety of technologies to present information to users , and the functionality with a variety of explorers , operating systems , and devices . user - centeredness : this means that the website should meet the needs of users . further , the users should regard the website as useful , attractive , and easy to use . responsibility : it refers to the ability of the website owners in responding to the questions of users , helping users to participate in generating content and answering the questions posed in a forum . this model involves entity ( who ) , content ( what ) , services ( why ) , location ( where ) , maintenance ( when ) , usability ( how ) , and possibility ( by what means ) which are defined as follow : entity : includes the sub features of identification of characteristics . facilities : encompasses resources , information and communication technology ( 5 ) this instrument has 12 dimensions including information suited to users , interaction , reliability , responsibility time , appearance , design , guesswork , innovativeness , stream , coherent connections , business process , and the ability for substitution ( 5 ) ( table 3 ) . some of them focused on the structure , the homepage content , page content , and subject content ( other links ) of websites for evaluation purposes ( 7 , 11 , 13 , 15 , 1921 , 2325 ) . also quality , size , language , background , and inclusiveness as the determinants of the final success of a website were referred ( 9 , 12 ) . another study found that attention to the update rate of pages ( the duration between the last update and the date the website is used ) is essential ( 10 ) . a technical framework presented seven criteria including : content quality , design quality , organization quality , user - friendliness , functional quality , service quality , and website quality from a technical point of view ( 11 ) . moreover the evaluation criteria as the accessibility and updating were mentioned ( 10 , 12 , 1518 , 22 , 27 ) . another study stated the security , and confidentiality of personal information as the most important factors affecting the willingness of customers to purchase from a website ( 2325 ) as well as multilinguality ( 27 ) ( table 4 ) . to evaluate the quality of hospital websites , various models had been presented , the most famous of which were : the quality criteria in this model had been developed based on iso-9126 model , including usability , reliability , efficiency , and functionality . the specific feature of this model was determination of a certain domain of evaluation and conductance of the evaluation step by step ( 1 , 6 ) . this model focuses on usability of the website and measures the quality by combining the inspection by both an evaluator and users ( 1 ) . the specified criteria in this model are : content : content means the quality of the information inside the website , and its communicative level . services : services are all the functionalities that the website allows its users to do . navigation : this criterion encompasses 1 ) various means of access by users to a certain part of the information in the website and 2 ) the logical structure of information to move across one part of information to another . cognitive features of the interface : it takes the perception and feeling of the user into consideration and deals with how the users memorize the structure of the website . aesthetics : it addresses the graphic design and template of the website , the type of font , color , size , images , and other graphic features in a website . technology : it represents the adaption of the website for proper function in a variety of explorers , the security level of the host server , and the interaction between the website and remote database ( 1 , 5 ) . this model was suggested for evaluation of the quality of cultural websites ( seminaries , archives , libraries , and other cultural institutions ) ( 1 ) . in this model , the aim of quality indices in this model is to present indices for evaluation of the quality of websites and support the design and development of the cultural websites ( 5 ) . this model involves the following axes : clarity : the clarity of objectives , mission , and the identity of the website for users . effectiveness : the content of the website is related to the validity of the website content and presentation of sound information to users . maintenance : it focuses on the technical and content support , fresh content , and improvement of the website s technical functions . accessibility : it represents easy access of all users to the website,(for example , access for blind users , those with relative vision , or users with hearing problems ) , the possibility of using a variety of technologies to present information to users , and the functionality with a variety of explorers , operating systems , and devices . user - centeredness : this means that the website should meet the needs of users . further , the users should regard the website as useful , attractive , and easy to use . responsibility : it refers to the ability of the website owners in responding to the questions of users , helping users to participate in generating content and answering the questions posed in a forum . this model involves entity ( who ) , content ( what ) , services ( why ) , location ( where ) , maintenance ( when ) , usability ( how ) , and possibility ( by what means ) which are defined as follow : entity : includes the sub features of identification of characteristics . facilities : encompasses resources , information and communication technology ( 5 ) this instrument has 12 dimensions including information suited to users , interaction , reliability , responsibility time , appearance , design , guesswork , innovativeness , stream , coherent connections , business process , and the ability for substitution ( 5 ) ( table 3 ) . the quality criteria in this model had been developed based on iso-9126 model , including usability , reliability , efficiency , and functionality . the specific feature of this model was determination of a certain domain of evaluation and conductance of the evaluation step by step ( 1 , 6 ) . this model focuses on usability of the website and measures the quality by combining the inspection by both an evaluator and users ( 1 ) . the specified criteria in this model are : content : content means the quality of the information inside the website , and its communicative level . services : services are all the functionalities that the website allows its users to do . navigation : this criterion encompasses 1 ) various means of access by users to a certain part of the information in the website and 2 ) the logical structure of information to move across one part of information to another . cognitive features of the interface : it takes the perception and feeling of the user into consideration and deals with how the users memorize the structure of the website . aesthetics : it addresses the graphic design and template of the website , the type of font , color , size , images , and other graphic features in a website . technology : it represents the adaption of the website for proper function in a variety of explorers , the security level of the host server , and the interaction between the website and remote database ( 1 , 5 ) . this model was suggested for evaluation of the quality of cultural websites ( seminaries , archives , libraries , and other cultural institutions ) ( 1 ) . in this model , the aim of quality indices in this model is to present indices for evaluation of the quality of websites and support the design and development of the cultural websites ( 5 ) . this model involves the following axes : clarity : the clarity of objectives , mission , and the identity of the website for users . effectiveness : the content of the website is related to the validity of the website content and presentation of sound information to users . maintenance : it focuses on the technical and content support , fresh content , and improvement of the website s technical functions . accessibility : it represents easy access of all users to the website,(for example , access for blind users , those with relative vision , or users with hearing problems ) , the possibility of using a variety of technologies to present information to users , and the functionality with a variety of explorers , operating systems , and devices . user - centeredness : this means that the website should meet the needs of users . further , the users should regard the website as useful , attractive , and easy to use . responsibility : it refers to the ability of the website owners in responding to the questions of users , helping users to participate in generating content and answering the questions posed in a forum . this model involves entity ( who ) , content ( what ) , services ( why ) , location ( where ) , maintenance ( when ) , usability ( how ) , and possibility ( by what means ) which are defined as follow : entity : includes the sub features of identification of characteristics . this instrument has 12 dimensions including information suited to users , interaction , reliability , responsibility time , appearance , design , guesswork , innovativeness , stream , coherent connections , business process , and the ability for substitution ( 5 ) ( table 3 ) . some of them focused on the structure , the homepage content , page content , and subject content ( other links ) of websites for evaluation purposes ( 7 , 11 , 13 , 15 , 1921 , 2325 ) . other studies mentioned characteristics of audience , website objective , loading , and structural stability among websites as important factors in the success of a website ( 8 , 12 , 14 , 26 ) . also quality , size , language , background , and inclusiveness as the determinants of the final success of a website were referred ( 9 , 12 ) . another study found that attention to the update rate of pages ( the duration between the last update and the date the website is used ) is essential ( 10 ) . a technical framework presented seven criteria including : content quality , design quality , organization quality , user - friendliness , functional quality , service quality , and website quality from a technical point of view ( 11 ) . moreover the evaluation criteria as the accessibility and updating were mentioned ( 10 , 12 , 1518 , 22 , 27 ) . furthermore , entertainment and fun , and website speed was presented as evaluation criteria for websites ( 1315 ) . another study stated the security , and confidentiality of personal information as the most important factors affecting the willingness of customers to purchase from a website ( 2325 ) as well as multilinguality ( 27 ) ( table 4 ) . the aim of this study was to identify the models for evaluating the quality of hospital websites . the results indicated that a great variety and diversity exists among the criteria presented for evaluating the quality of websites . on the other hand , many of these criteria can be considered as the sub criteria of a more general criterion . the study introduced four factors of web success in electronic business as information , the quality of services , use of systems , entertainment and fun , and the quality of system design ( 19 ) . another study experimentally examined the relationship between the quality of websites and integration of investment of website customers in kuwait . the researcher categorized the quality of services in four aspects of technical , general and specific content , and the apparent quality . a paper called the government in the digital era : concept , practice , and development in thailand , stated 10 characteristics of an electronic government , arguing that an electronic government should be comprehensive , interactive , inclusive , convenient , accessible , secure , customizable , multifunctional , and flexible ( 16 ) . in spain experimentally and quantitatively investigated websites of electronic banking with the aim of finding an index for web evaluation , reported that the evaluation index for analyzing the website of commercial , educational , and nonprofit organizations is speed , navigation , and content ( 21 ) . a paper called the qualitative framework of evaluation of hospital / medical websites presented a qualitative framework consisting of seven criteria : the quality of content , design , organization , user - friendliness , performance , services , and technical quality ( 11 ) . this framework comprised content , design , and typical features of electronic government websites ( 20 ) . results of a research introduced six important factors for success of websites as design , content , consumer education , security , customer support , online communities , and the market situation ( 24 ) . a model for the quality of websites with the criteria of correctness , presentation , navigation , content , and the final success of websites , associated with factors such as quality , size , language , background , inclusion , etc . the result of investigation of the selected studies revealed that the criteria of accessibility , content , and apparent features of websites from user perspective such as design , the graphics used in the website , and attractiveness of the page have been mentioned in the majority of studies . the results also showed that the important point in determining the criteria for evaluating the quality of websites is attention to the major differences in the special features of any website ; features considered during the design and selection of content for groups with various aims , so that a website becomes successful in responding to an objective for which it has been designed . these features are especially important during the evaluation of websites , since neglecting these features causes many websites to fail to reach the aims for which they have been designed , thus requiring revision . the results also indicated that the features of the information content , website design , security , and confidentiality of personal information are among the most important factors affecting the tendency of clients to use a website , thus paying attention to it in the design of any website and any evaluation model as a common criterion is essential . minimum quality criteria for a website are usability , efficiency , ease of use , user friendliness , service , reliability and interaction . the criteria of accessibility , content , design , security , and confidentially of personal information , as the essential criteria , should be taken into account when evaluating a website . it is suggested that the criteria such as ease of use , playing a role in bringing about satisfaction of users , the graphics used in the website , attractiveness of the website , and other apparent characteristics of websites are considered as sub criteria for the criterion of user - friendliness . it is also proposed that the criteria of speed and accessibility of websites , which indicate the efficiency of a website , are included in all models of website evaluation as the sub criterion of efficiency .

though kidney transplant ( ktx ) might induce a regression of most metabolic derangements characterizing end - stage renal disease ( esrd ) , electrolyte disturbances are not uncommonly encountered in patients with an even well - functioning renal graft . limiting ourselves to mentioning only the most frequent electrolyte disturbances , we would like to list : metabolic acidosis , hypo- and hyperkaliemia , hypomagnesiemia , hypophosphatemia , and hypercalcemia [ 16 ] . over the last decade , hypercalcemia ( hc ) has received much attention from the renal transplant community since it has been claimed to have a negative impact on both the graft and patient outcomes . in the following paragraphs , we will deal with the main pathogenic aspects , the most relevant clinical consequences , and the possible therapeutic approach of the hc in ktx patients . hc has been reported to occur with an extremely variable incidence after ktx ( from < 5% up to > 50% ) [ 3 , 612 ] . table 1 shows the main characteristics of some of the most relevant studies , reporting on hc prevalence after ktx . the high variability of prevalence of hc might be explained at least in part by a number of factors . first , it is worth noting that the older the study was the higher the prevalence of hc was evident , probably due to a less efficient control of shp in earlier studies . second , it is also worth stressing that most studies had a cross - sectional and retrospective design and included a relatively low number of patients or only part of the global cohort of transplanted patients . this might have introduced a selection bias , where only patients with previously recognized severe hyperparathyroidism were included . third , the time elapsed from the time of transplantation to the time when serum calcium levels were evaluated was consistently variable in the different studies . in fact , an increase in hc prevalence would be expected during the first year after ktx , with a reduction thereafter . however , the changes in the prevalence of hc over time was also consistently different among the various studies . in fact , some aa reported that calcium levels had a trend toward an early reduction ( first 3 months ) and then increased up to the sixth month , stabilizing thereafter over all the first year [ 11 , 13 ] , while others found an almost constant prevalence of hc over all the first year after ktx . a fourth possible confounding factor might be a possible different use of vitamin d and/or calcium supplements in the ktx patients . in fact , most of the quoted studies do not report on this aspect . another possible confounder in the evaluation of the real prevalence of hc in transplanted patients is the different methodology used for calcium assessment . in fact , it has been reported that elevated ca levels might be often overlooked if evaluated as total serum ca rather than ionized serum ca , since the former underestimates hc in ktx patients . this effect can be only in part explained by the reduction of serum albumin levels , which often characterize the early period after ktx , since the total serum calcium corrected by albumin does not predict ionized calcium levels substantially better than the uncorrected ones . finally , these different results might also be explained by differences among the various transplant centres in the wait - listing policy for the patients with severe shp . summarizing , though hc after ktx has been reported to occur with an extremely high variability , when calcium levels are evaluated by the appropriate methodology ( ionized calcium ) , its prevalence is relatively high , particularly during the first year . the first - well recognized factor in causing the occurrence of hc after ktx is the persistence of shp after transplantation [ 11 , 13 ] . pt - hpt after ktx is much more likely to occur when a severe form of shp was present during the uraemic state preceding ktx . it is also well acknowledged that the most severe degree of shp is almost invariably associated with the nodular form of parathyroid gland hyperplasia which is much less prone to undergo spontaneous regression even after the uraemic state has been corrected [ 11 , 14 ] . this also explains why , when the uraemic milieu has been corrected , the phosphate levels have been reduced , and the bone cell sensitivity to both pth and vitamin d has been recovered by a functioning renal graft , the exceedingly high pth levels are more effective in inducing hc due to enhanced bone response to its calcemic action . however , the increased pth - mediated bone resorption does not seem to be the only mechanism responsible for the hc in renal transplanted patients . one of the few studies which explored the skeletal status in ktx by bone biopsy found that hc was indifferently associated to either the increased bone turnover disease or to the adynamic bone disease . another potential factor causing hc after ktx is the recovered circulating levels of calcitriol , secondary to its increased renal tubular synthesis , further stimulated by the inappropriately high pth and low phosphorus levels . the recovered calcitriol levels might concur in inducing hc by both its intestinal and bone effects . this potential hypercalcemic condition might be in part counteracted by two opposing factors : the persistence of elevated fgf-23 levels , which is a strong inhibitor of 1--ohase activity , and the very frequent occurrence of the deficiency / insufficiency of the calcitriol precursor , calcifediol in ktx patients [ 18 , 19 ] . this point might have some therapeutic consequences , as will be discussed more in depth later on . in fact , with the progressive reduction of fgf23 , which is often observed after a few months from ktx , and particularly when patients are supplemented with vitamin d , overt hc might develop . another factor which has been cited as a potential cause of hc after ktx is the supposed reabsorption of vascular calcifications ( vc ) . however , there are no data in the literature supporting the above hypothesis , and on the contrary some aa have reported a trend toward an increase of vc after ktx [ 21 , 22 ] . furthermore , some preliminary data of ours seem to suggest that the higher the serum calcium levels , the higher the progression of vc after ktx . from all these data , it seems quite unlikely that vc reabsorption might play any major role among the causes of hc of ktx patients . whether different immune - suppressive drugs might be in some way related to hc is not completely clear . steroids have been recognized to carry with them a number of effects potentially affecting calcium metabolism ( antivitamin d effect , calciuretic activity , induction of osteoblast apoptosis , etc . ) . however , most of these effects are expected to induce more hypocalcemia rather than hypercalcemia . there is some evidence that calcineurin inhibitors ( cnis ) , in particular cyclosporine , might increase bone resorption and thus potentially promote the development of hc . however , there is no clear data supporting this putative hypercalcemic effect of cnis . a recent paper reported that ktx patients on rapamycin therapy would be more prone to develop persistent shp , frequently associated with hc . finally , the possibility that some polymorphic variations in genes coding for some calcium controlling factors might increase the propensity to develop hc after ktx can not be excluded . in fact , some years ago we found that the bb polymorphic variant of vitamin d receptor ( vdr ) gene was associated with lower prevalence of hc pt - hpt . more recently , a genome - wide association study ( gwas ) has clearly demonstrated that a single nucleotide polymorphism in the gene coding for the calcium sensing receptor ( casr ) is strongly associated with the levels of serum calcium in the general population . whether this genetic background might contribute also to the occurrence of hc after ktx taken together all these data strongly suggest that the occurrence of hc after ktx is mostly sustained by the persistence of a severe form of pt - hpt which is mainly secondary to the previous state of mineral metabolism derangement developed throughout the often long story of chronic kidney disease . figure 1 summarizes the main hypothesized pathogenic mechanisms through which hc might develop after ktx . there is some evidence that hc may have a negative clinical impact on either the graft and/or the patient outcome . though it is not always easy to dissect the two clinical fields from each other , we will deal with each one separately so as to treat more clearly the topic . a number of experimental studies have clearly demonstrated that hc can induce renal damage by different mechanisms . in animal models , the first type is represented by macroscopic nephrocalcinosis , which is characterized by coarse deposits of calcium salts both in renal papillae and the urinary tract which can be detected also by ultra - sound and/or radiological examination . the second one is defined microscopic nephrocalcinosis , characterized by smaller calcium salt depositions at the tubular cell and/or the interstitial space level and can be detected only by microscopic examination . this type of kidney damage can be defined as unspecific morphologic and/or functional cell changes which occur in the presence of hc and can regress or reduce after calcium level correction . the pathogenesis of this last type of kidney damage is mostly speculative and has been ascribed to some hemodynamic and biochemical effects of hc ( vasoconstriction ; increased natriuresis and diuresis ; activation of a huge number of enzymes , cytokines , growth factors ; etc . ) , even in the absence of any observable calcium salt deposition [ 29 , 30 ] . the data present in the literature establishing a clear relationship between the presence of high calcium levels and kidney damage in humans and particularly in ktx patients are scanty and not fully conclusive . some years ago , nankivell et al . pointed out that microcalcifications were a common finding in protocol biopsies performed in transplanted kidneys , since they were observed in 43% and 79% of biopsies at 1 year and 10 years after ktx , respectively . later studies demonstrated that the presence of hc is associated with a higher prevalence of calcium salt deposits within the transplanted kidney and that this nephrocalcinosis can adversely affect the graft survival [ 9 , 3235 ] . at variance with these results , other aa , though confirming that calcium deposits are frequently found ( 44% ) in transplanted kidney biopsies , were not able to find any relationship between nephrocalcinosis and serum calcium levels . on the other hand , in the experience of the above aa it was the presence of low citrate and high oxalate urinary excretion which were more associated with calcium deposition within renal graft . independently of the presence or not of clear calcium deposits , it is worth remembering that hc can induce an indirect kidney damage , secondary to its well - recognized sodiuretic and aquaretic effects which , in addition to its vasoconstrictor action , can consistently reduce renal perfusion , inducing a fall in glomerular filtration rate , which can progress in the long term from an initial functional form to a stabilized organic kidney injury condition . summarizing , though we have no conclusive evidence that hc can be responsible for the nephrocalcinosis , which is frequently observed in kidney graft biopsies , there are plausible reasons for thinking that persistently high serum calcium levels might adversely affect kidney graft outcome . a number of potential negative effects of hc on different tissues , organs , and systems have been extensively reported in the literature . in the following paragraphs , we will just limit ourselves to briefly mention the most relevant ones which can more frequently occur in ktx patients . it has been well acknowledged for some time that acute changes in extracellular calcium concentration might induce consensual changes of intracellular calcium levels which are the ultimate controller of cardiomyocyte and vascular smooth muscle cell contraction and relaxation [ 3739 ] . on the other hand , there is not much evidence that chronic changes of serum calcium levels , in particular chronic hc , might cause any sustained cardiovascular effect , either in healthy people or in pathological conditions , such as in ktx patients . however , there are some plausible reasons for believing that sustained high calcium levels might have some potential negative effect at least on the vascular calcification ( vc ) process . as previously mentioned , vcs not infrequently tend to progress after ktx , even though at a lower rate as compared with dialysis patients [ 21 , 22 , 40 , 41 ] . some of our preliminary results , produced as yet only in abstract form , suggested that aortic calcifications , evaluated according to kauppila et al . , increased in 30% of patients over the first year after ktx . furthermore , patients heading for a progression of their vc process had consistently higher serum calcium levels as compared with patients who did not have any vc worsening . however , these data alone can not be considered to be definite proof of a casual role of hc in inducing negative cardiovascular effects . randomized after ktx ; increases in red blood cell number and in hemoglobin levels are not infrequently observed . though the prominent causal factor of the erythrocytosis in ktx patients can be recognized in the recovered synthesis of erythropoietin ( epo ) by the functioning kidney graft , in the presence of a well - responding bone marrow , after the correction of the uraemic state , other potential players might be also involved in this process . among them , an increased local and/or systemic activity of renin - angiotensin - system ( ras ) has been advocated as a potential pathogenic factor , since ras has been shown to increase epo synthesis , and furthermore the use of either ace - inhibitors or angiotensin - receptor blockers has been demonstrated to be effective in controlling posttransplant erythrocytosis [ 44 , 45 ] . recently , it has been suggested that calcium may contribute to the ras - mediated epo stimulation . furthermore , recently some aa reported that posttransplant erythrocytosis is 2 - 3 times more frequent in ktx patients who have higher serum calcium levels as compared with normocalcemic patients [ 47 , 48 ] . however , there are as yet no data demonstrating that the correction of hc might translate into a correction of posttransplant erythrocytosis . severe hc , particularly encountered in the most advanced stages of neoplasias , can be complicated by a relevant pancreatic damage . if this is also true for the degree of hc usually observed in ktx patients is not clear . two decades ago , frick et al . , studying 224 consecutive ktx patients , reported 8 patients who experienced an acute pancreatitis episode and 20 patients who had an asymptomatic increase of serum pancreatic enzymes . the aa did not observe any association between the pancreatitis episodes and the well - known specific risk factors , such as immune suppressive therapy doses , viral infections , alcohol consumption , or biliary tract lithiasis . the only factor which was significantly associated with the pancreatitis episodes was the presence of hc . however , no further paper , as far as we know , has been published in the following decades proving or disproving these findings . in conclusion , it has been suggested that hc occurring after a ktx can negatively impact both graft and patient clinical outcomes . however , there is no evidence which clearly demonstrates the mechanisms by which these negative effects might act , nor can we exclude that the high pth levels , which are almost invariably associated with hc , more than the elevated calcium level per se , might be the possible primary adverse factor . furthermore , it is still to be demonstrated which is the threshold calcium concentration , if any , over which these negative effects can happen . the presence of persistently elevated serum calcium concentrations , associated with elevated pth levels , has for some time been considered a possible indication to parathyroidectomy ( ptx ) in ktx patients . however , it is worth underlining that there is no guide - line for establishing which are the pth and the serum calcium levels over which the indication to ptx should be given in the ktx clinical set . therefore , the indication for the ptx intervention is quite variable from one transplant centre to another . furthermore , there is no general agreement on which type of ptx should be preferred : total , subtotal with or without the auto - transplantation of a parathyroid gland . it has long been considered that ptx might negatively affect the graft outcome , possibly due to either the haemodynamic and/or immune - mediated changes triggered by the acute changes in pth and/or electrolyte concentrations after the parathyroid gland removal [ 51 , 52 ] . on the other hand , recent data lessened this conviction , showing that no difference in the long - term graft outcome was evident between ktx patients who were submitted to ptx as compared with a comparable group of patients who were not . however , the need to face a ptx intervention is still a matter of concern for many nephrologists in the early phase after a ktx , given the burden of clinical problems which can be present during this period . furthermore , some of these patients have already undergone such an intervention and for this or any other reason they may not be prepared to undergo a new intervention . for this reason , many transplant centers warmly recommend ptx in waiting - listed patients , before ktx when even a moderate form of shp is present . conflicting with this trend , other nephrologists advocate some grounds for limiting the indication to ptx before ktx to only the most severe forms of shp . the main reason is that there is some evidence which suggests that a regression of parathyroid gland hyperplasia could be expected after a well - functioning ktx . some years ago , it was first reported that the maximal pth response to a hypocalcemic challenge , which is considered a good marker of the functional parathyroid gland mass , substantially reduced from the 1st to the 6th month after renal transplantation . more recently it has been also reported that in the parathyroid glands removed from ktx recipients , the cellular proliferative events are reduced and apoptotic figures increased as compared to the glands removed from uraemic patients on dialysis , even though in both groups the parathyroid hyperplasia was in the most advanced form characterized by nodular hyperplasia . these observed histological changes might suggest that in the long run even the most severe form of shp might be expected to regress after ktx . reinforcing this view , recent papers observed a regression of parathyroid gland volume , as assessed by us methodology , in ktx patients treated with a calcimimetic drug [ 56 , 57 ] . it is widely recognized that the introduction of cinacalcet , as yet the only calcimimetic drug in clinical use , has consistently increased the possibility for controlling , in particular , the most severe forms of shp in dialysis patients , reducing the indications for ptx in this clinical set . this fact enhances the complexity of the indication for ptx for the patients on a transplant waiting - list who suffer from a severe form of shp which is well controlled by cinacalcet . to further complicate this issue , it is worth remembering that , at the present time , this drug has not been registered for use also in transplanted patients . on the other hand , a number of small studies have been published in recent years on the use of cinacalcet in ktx recipients which can give some indicative instruction on this topic [ 8 , 5868 ] . the main results of these studies have been extensively discussed in a recent review . all these studies homogeneously demonstrated that cinacalcet is effective in reducing serum calcium concentration . another constant finding of these studies was a consistent increase in serum phosphorus levels . this last result can be considered a positive result , since it is well known that ktx recipients are prone to develop hypophosphatemia , which might contribute to the skeletal problem frequently observed in transplanted patients . in fact , though pth levels significantly reduced in most studies , the extent of these changes was quite variable , since some authors described no significant change at all while others found up to a 4050% reduction in pth levels [ 8 , 60 , 61 ] . it is also worth - underlining that only a part of these studies reported on the effect of cinacalcet on urinary calcium excretion . furthermore , the results were very contradictory , with unchanged , increased , or reduced calcium excretion having been reported . this variability might be at least in part dependent on the time when the assessment was performed . in fact , in the early treatment phase , when serum calcium levels ( and consequently the glomerular filtered load of calcium ) are still high , the reduction in renal tubular calcium absorption induced by cinacalcet , due to both the reduced pth levels and to the cinacalcet direct calciuretic effects , is expected to induce high urinary calcium output . with ongoing treatment , these effects can be counteracted by the reduced glomerular filtered calcium load , secondary to the reduction of serum calcium levels which eventually bring its urinary excretion back to the pretreatment levels . on the other hand , we can not rule out that some genetic variability in the casr gene might play some role in the possible variable calciuretic effect of casr activation by cinacalcet . in fact a recent paper demonstrated that some polymorphic variants of the casr gene might play some role in the different urinary calcium excretion found in primary hyperparathyroid patients with or without nephrolithiasis . whatever the extent of the potential calciuretic effect of cinacalcet , this is a potentially negative effect of the drug , since , as previously mentioned , ktx patients are already prone to develop nephrocalcinosis . so , any metabolic change which might potentially increase this risk should be considered with great caution . to reinforce this concern , a case of nephrocalcinosis which occurred in a ktx patient treated with cinacalcet the problem of nephrocalcinosis in ktx patients indirectly introduces another linked therapeutic issue in this clinical set . in fact , though ktx patients often present a more or less severe vitamin d deficiency , there is much controversy on whether this deficit should be replaced even in the presence of elevated serum calcium levels . as previously mentioned , it is also to be expected that also in nonhypercalcemic ktx patients overt hc might ensue after starting the replacement therapy with vitamin d metabolites . however , some recent studies seem to minimize this concern , since no relevant adverse event related to hc episodes have been reported in ktx patients treated with cholecalciferol [ 71 , 72 ] . in any case , it seems to be advisable to carefully check serum calcium levels when vitamin d therapy is started . whether the new less calcemic vitamin d analogues would represent a clinical advantage also in this field deserves specific prospective studies . although the potential effects of cinacalcet treatment on bone metabolism in ktx patients is an important issue , only two papers have dealt with this problem . the first study reported on a significant increase of bone mineral density at the radial level in 9 patients treated with cinacalcet . to the best of our knowledge , the second study was the only one that prospectively examined bone histomorphometric parameters in 10 transplant recipients before and after 1824-month treatment with cinacalcet . the main results were a decrease of bone formation rate in 7 , an increase in 2 , and no change in 1 patient . these conflicting results , far from being conclusive , underline the need for gaining further information on this critical issue . another point of potential concern is the possible interference of cinacalcet with the immune - suppressive ( is ) drugs and its potential effect on the graft function . the first reports on the topic exploring the possible interference between cinacalcet and the is therapy demonstrated no or only marginal effects of cinacalcet on the blood levels of the most frequently used immune - suppressive drugs [ 73 , 74 ] however , following studies , though performed in small groups of patients , provided results which suggest that cinacalcet might reduce tacrolimus auc024 by approximately 14% and might also promote the accumulation of a nephrotoxic metabolite of cyclosporine ( am19 ) [ 75 , 76 ] . as far as the effects of cinacalcet on renal function are concerned , some of the quoted studies reported on the change in serum creatinine levels and/or on the variably evaluated glomerular filtration rate . the results were quite variable with some studies showing a slight worsening [ 63 , 73 , 74 ] , others a slight improvement [ 60 , 64 , 67 ] , and the other studies [ 58 , 59 , 65 ] no change of renal function at all . it is worth stressing that the results of all the quoted studies are flawed in many critical aspects , since most of them were performed in relatively small groups of patients , had a retrospective design , and a lack of a control group . furthermore , the criteria for choosing the treated patients were not homogeneous , and many treated patients had normal or slightly increased ca levels before treatment , with pth levels only moderately increased . in fact , in our opinion it is questionable to treat ktx patients with normocalcemic pt - hpt with cinacalcet . hc after ktx is mainly the terminal event of an evolved and uncontrolled shp which maybe began long before transplantation . even though not completely clear , it is plausible that hc might negatively impact both graft and patient outcome . the best way for avoiding hc after ktx is to optimally treat shp before ktx . the first question concerns the criteria for choosing the patient on the transplant waiting - list who should be referred to ptx or maintained on medical therapy . given the complete lack of evidence on this issue , in our opinion an absolute indication to ptx for a patient on the ktx waiting list can be the presence of a severe shp , defined by contemporary high pth levels ( intact pth > 800 pg / ml ) and hc ( tot s - ca > 10.4 mg / dl ) , which can not be controlled by the available medical therapy . a strong , though not absolute , further indication to the surgical intervention before renal transplantation might be also the presence of a shp controlled only by maximal doses of medical therapy . in all the other cases , we do not believe that there is any compelling indication to ptx . a second critical point is the management of pt - hpt associated with hc . . however , independently of its possible negative impact on graft function , which moreover still remains unproven , ptx entails a number of problems such as the type and the timing of the intervention to be performed , the need for a continuous treatment for the correction of the residual hypoparathyroidism after a successful intervention , the problems related to some comorbid clinical conditions which make the surgical intervention a risk procedure and more . for all these reasons , the recent availability of a medical approach to hypercalcemic pt - hpt based on the use of cinacalcet challenged the concept of considering ptx the best choice for this clinical condition . however , it should be also considered that many unsolved issues bring into question the prolonged use of cinacalcet . these areas of uncertainty should be clarified by a randomized controlled trial before a widespread use of the drug can be suggested in ktx patients . in this critical scenario , it is our opinion that cinacalcet should be reserved to some specific cases such as the ktx patients with pt - hpt associated with overt hc ( > 12 mg / dl ) and have been already submitted to a previous ptx and/or have clinical conditions which make the intervention a risk procedure and/or refuse the intervention for any reason . at the present time , the clinical evaluation of each single patient still remains the best guide - line for choosing the best treatment of hc in ktx patients .

hypercalcemia ( hc ) has been variably reported in kidney transplanted ( ktx ) recipients ( 515% ) . calcium levels peak around the 3rd month after ktx and thereafter slightly reduce and stabilize . though many factors have been claimed to induce hc after ktx , the persistence of posttransplant hyperparathyroidism ( pt - hpt ) of moderate - severe degree is universally considered the first causal factor . though not proven , there are experimental and clinical suggestions that hc can adversely affect either the graft ( nephrocalcinosis ) and other organs or systems ( vascular calcifications , erythrocytosis , pancreatitis , etc . ) . however , there is no conclusive evidence that correction of serum calcium levels might avoid the occurrence of these claimed clinical effects of hc . the best way to reduce the occurrence of hc after ktx is to treat as best we can the secondary hyperparathyroidism ( shp ) during the uraemic stages . the indication to parathyroidectomy ( ptx ) , either before or after ktx , in order to prevent or to treat , respectively , hc after ktx , is still a matter of debate which has been revived by the availability of the calcimimetic cinacalcet for the treatment of pt - hpt . however , we still need to better clarify many points as regards the potential adverse effects related to either ptx or cinacalcet use in this clinical set , and we are waiting for the results of future randomized controlled trials to achieve some more definite conclusions on this topic .

1. Introduction 2. Prevalence of HC after KTx 3. Pathogenic Mechanisms of HC in KTx Patients 4. Clinical Consequences of HC in KTx 5. Therapeutic Intervention 6. Conclusions and Opinions

though kidney transplant ( ktx ) might induce a regression of most metabolic derangements characterizing end - stage renal disease ( esrd ) , electrolyte disturbances are not uncommonly encountered in patients with an even well - functioning renal graft . over the last decade , hypercalcemia ( hc ) has received much attention from the renal transplant community since it has been claimed to have a negative impact on both the graft and patient outcomes . in the following paragraphs , we will deal with the main pathogenic aspects , the most relevant clinical consequences , and the possible therapeutic approach of the hc in ktx patients . third , the time elapsed from the time of transplantation to the time when serum calcium levels were evaluated was consistently variable in the different studies . in fact , an increase in hc prevalence would be expected during the first year after ktx , with a reduction thereafter . however , the changes in the prevalence of hc over time was also consistently different among the various studies . in fact , some aa reported that calcium levels had a trend toward an early reduction ( first 3 months ) and then increased up to the sixth month , stabilizing thereafter over all the first year [ 11 , 13 ] , while others found an almost constant prevalence of hc over all the first year after ktx . in fact , most of the quoted studies do not report on this aspect . another possible confounder in the evaluation of the real prevalence of hc in transplanted patients is the different methodology used for calcium assessment . this effect can be only in part explained by the reduction of serum albumin levels , which often characterize the early period after ktx , since the total serum calcium corrected by albumin does not predict ionized calcium levels substantially better than the uncorrected ones . summarizing , though hc after ktx has been reported to occur with an extremely high variability , when calcium levels are evaluated by the appropriate methodology ( ionized calcium ) , its prevalence is relatively high , particularly during the first year . the first - well recognized factor in causing the occurrence of hc after ktx is the persistence of shp after transplantation [ 11 , 13 ] . pt - hpt after ktx is much more likely to occur when a severe form of shp was present during the uraemic state preceding ktx . it is also well acknowledged that the most severe degree of shp is almost invariably associated with the nodular form of parathyroid gland hyperplasia which is much less prone to undergo spontaneous regression even after the uraemic state has been corrected [ 11 , 14 ] . this also explains why , when the uraemic milieu has been corrected , the phosphate levels have been reduced , and the bone cell sensitivity to both pth and vitamin d has been recovered by a functioning renal graft , the exceedingly high pth levels are more effective in inducing hc due to enhanced bone response to its calcemic action . however , the increased pth - mediated bone resorption does not seem to be the only mechanism responsible for the hc in renal transplanted patients . one of the few studies which explored the skeletal status in ktx by bone biopsy found that hc was indifferently associated to either the increased bone turnover disease or to the adynamic bone disease . another potential factor causing hc after ktx is the recovered circulating levels of calcitriol , secondary to its increased renal tubular synthesis , further stimulated by the inappropriately high pth and low phosphorus levels . this potential hypercalcemic condition might be in part counteracted by two opposing factors : the persistence of elevated fgf-23 levels , which is a strong inhibitor of 1--ohase activity , and the very frequent occurrence of the deficiency / insufficiency of the calcitriol precursor , calcifediol in ktx patients [ 18 , 19 ] . another factor which has been cited as a potential cause of hc after ktx is the supposed reabsorption of vascular calcifications ( vc ) . however , there are no data in the literature supporting the above hypothesis , and on the contrary some aa have reported a trend toward an increase of vc after ktx [ 21 , 22 ] . furthermore , some preliminary data of ours seem to suggest that the higher the serum calcium levels , the higher the progression of vc after ktx . steroids have been recognized to carry with them a number of effects potentially affecting calcium metabolism ( antivitamin d effect , calciuretic activity , induction of osteoblast apoptosis , etc . ) however , most of these effects are expected to induce more hypocalcemia rather than hypercalcemia . there is some evidence that calcineurin inhibitors ( cnis ) , in particular cyclosporine , might increase bone resorption and thus potentially promote the development of hc . however , there is no clear data supporting this putative hypercalcemic effect of cnis . finally , the possibility that some polymorphic variations in genes coding for some calcium controlling factors might increase the propensity to develop hc after ktx can not be excluded . in fact , some years ago we found that the bb polymorphic variant of vitamin d receptor ( vdr ) gene was associated with lower prevalence of hc pt - hpt . more recently , a genome - wide association study ( gwas ) has clearly demonstrated that a single nucleotide polymorphism in the gene coding for the calcium sensing receptor ( casr ) is strongly associated with the levels of serum calcium in the general population . whether this genetic background might contribute also to the occurrence of hc after ktx taken together all these data strongly suggest that the occurrence of hc after ktx is mostly sustained by the persistence of a severe form of pt - hpt which is mainly secondary to the previous state of mineral metabolism derangement developed throughout the often long story of chronic kidney disease . there is some evidence that hc may have a negative clinical impact on either the graft and/or the patient outcome . in animal models , the first type is represented by macroscopic nephrocalcinosis , which is characterized by coarse deposits of calcium salts both in renal papillae and the urinary tract which can be detected also by ultra - sound and/or radiological examination . the pathogenesis of this last type of kidney damage is mostly speculative and has been ascribed to some hemodynamic and biochemical effects of hc ( vasoconstriction ; increased natriuresis and diuresis ; activation of a huge number of enzymes , cytokines , growth factors ; etc . ) pointed out that microcalcifications were a common finding in protocol biopsies performed in transplanted kidneys , since they were observed in 43% and 79% of biopsies at 1 year and 10 years after ktx , respectively . later studies demonstrated that the presence of hc is associated with a higher prevalence of calcium salt deposits within the transplanted kidney and that this nephrocalcinosis can adversely affect the graft survival [ 9 , 3235 ] . at variance with these results , other aa , though confirming that calcium deposits are frequently found ( 44% ) in transplanted kidney biopsies , were not able to find any relationship between nephrocalcinosis and serum calcium levels . independently of the presence or not of clear calcium deposits , it is worth remembering that hc can induce an indirect kidney damage , secondary to its well - recognized sodiuretic and aquaretic effects which , in addition to its vasoconstrictor action , can consistently reduce renal perfusion , inducing a fall in glomerular filtration rate , which can progress in the long term from an initial functional form to a stabilized organic kidney injury condition . summarizing , though we have no conclusive evidence that hc can be responsible for the nephrocalcinosis , which is frequently observed in kidney graft biopsies , there are plausible reasons for thinking that persistently high serum calcium levels might adversely affect kidney graft outcome . a number of potential negative effects of hc on different tissues , organs , and systems have been extensively reported in the literature . it has been well acknowledged for some time that acute changes in extracellular calcium concentration might induce consensual changes of intracellular calcium levels which are the ultimate controller of cardiomyocyte and vascular smooth muscle cell contraction and relaxation [ 3739 ] . on the other hand , there is not much evidence that chronic changes of serum calcium levels , in particular chronic hc , might cause any sustained cardiovascular effect , either in healthy people or in pathological conditions , such as in ktx patients . however , there are some plausible reasons for believing that sustained high calcium levels might have some potential negative effect at least on the vascular calcification ( vc ) process . as previously mentioned , vcs not infrequently tend to progress after ktx , even though at a lower rate as compared with dialysis patients [ 21 , 22 , 40 , 41 ] . furthermore , patients heading for a progression of their vc process had consistently higher serum calcium levels as compared with patients who did not have any vc worsening . though the prominent causal factor of the erythrocytosis in ktx patients can be recognized in the recovered synthesis of erythropoietin ( epo ) by the functioning kidney graft , in the presence of a well - responding bone marrow , after the correction of the uraemic state , other potential players might be also involved in this process . among them , an increased local and/or systemic activity of renin - angiotensin - system ( ras ) has been advocated as a potential pathogenic factor , since ras has been shown to increase epo synthesis , and furthermore the use of either ace - inhibitors or angiotensin - receptor blockers has been demonstrated to be effective in controlling posttransplant erythrocytosis [ 44 , 45 ] . however , there are as yet no data demonstrating that the correction of hc might translate into a correction of posttransplant erythrocytosis . however , there is no evidence which clearly demonstrates the mechanisms by which these negative effects might act , nor can we exclude that the high pth levels , which are almost invariably associated with hc , more than the elevated calcium level per se , might be the possible primary adverse factor . the presence of persistently elevated serum calcium concentrations , associated with elevated pth levels , has for some time been considered a possible indication to parathyroidectomy ( ptx ) in ktx patients . however , it is worth underlining that there is no guide - line for establishing which are the pth and the serum calcium levels over which the indication to ptx should be given in the ktx clinical set . therefore , the indication for the ptx intervention is quite variable from one transplant centre to another . furthermore , there is no general agreement on which type of ptx should be preferred : total , subtotal with or without the auto - transplantation of a parathyroid gland . it has long been considered that ptx might negatively affect the graft outcome , possibly due to either the haemodynamic and/or immune - mediated changes triggered by the acute changes in pth and/or electrolyte concentrations after the parathyroid gland removal [ 51 , 52 ] . however , the need to face a ptx intervention is still a matter of concern for many nephrologists in the early phase after a ktx , given the burden of clinical problems which can be present during this period . conflicting with this trend , other nephrologists advocate some grounds for limiting the indication to ptx before ktx to only the most severe forms of shp . more recently it has been also reported that in the parathyroid glands removed from ktx recipients , the cellular proliferative events are reduced and apoptotic figures increased as compared to the glands removed from uraemic patients on dialysis , even though in both groups the parathyroid hyperplasia was in the most advanced form characterized by nodular hyperplasia . it is widely recognized that the introduction of cinacalcet , as yet the only calcimimetic drug in clinical use , has consistently increased the possibility for controlling , in particular , the most severe forms of shp in dialysis patients , reducing the indications for ptx in this clinical set . this fact enhances the complexity of the indication for ptx for the patients on a transplant waiting - list who suffer from a severe form of shp which is well controlled by cinacalcet . on the other hand , a number of small studies have been published in recent years on the use of cinacalcet in ktx recipients which can give some indicative instruction on this topic [ 8 , 5868 ] . the main results of these studies have been extensively discussed in a recent review . furthermore , the results were very contradictory , with unchanged , increased , or reduced calcium excretion having been reported . in fact , in the early treatment phase , when serum calcium levels ( and consequently the glomerular filtered load of calcium ) are still high , the reduction in renal tubular calcium absorption induced by cinacalcet , due to both the reduced pth levels and to the cinacalcet direct calciuretic effects , is expected to induce high urinary calcium output . with ongoing treatment , these effects can be counteracted by the reduced glomerular filtered calcium load , secondary to the reduction of serum calcium levels which eventually bring its urinary excretion back to the pretreatment levels . whatever the extent of the potential calciuretic effect of cinacalcet , this is a potentially negative effect of the drug , since , as previously mentioned , ktx patients are already prone to develop nephrocalcinosis . to reinforce this concern , a case of nephrocalcinosis which occurred in a ktx patient treated with cinacalcet the problem of nephrocalcinosis in ktx patients indirectly introduces another linked therapeutic issue in this clinical set . in fact , though ktx patients often present a more or less severe vitamin d deficiency , there is much controversy on whether this deficit should be replaced even in the presence of elevated serum calcium levels . however , some recent studies seem to minimize this concern , since no relevant adverse event related to hc episodes have been reported in ktx patients treated with cholecalciferol [ 71 , 72 ] . in any case , it seems to be advisable to carefully check serum calcium levels when vitamin d therapy is started . although the potential effects of cinacalcet treatment on bone metabolism in ktx patients is an important issue , only two papers have dealt with this problem . the first reports on the topic exploring the possible interference between cinacalcet and the is therapy demonstrated no or only marginal effects of cinacalcet on the blood levels of the most frequently used immune - suppressive drugs [ 73 , 74 ] however , following studies , though performed in small groups of patients , provided results which suggest that cinacalcet might reduce tacrolimus auc024 by approximately 14% and might also promote the accumulation of a nephrotoxic metabolite of cyclosporine ( am19 ) [ 75 , 76 ] . as far as the effects of cinacalcet on renal function are concerned , some of the quoted studies reported on the change in serum creatinine levels and/or on the variably evaluated glomerular filtration rate . the results were quite variable with some studies showing a slight worsening [ 63 , 73 , 74 ] , others a slight improvement [ 60 , 64 , 67 ] , and the other studies [ 58 , 59 , 65 ] no change of renal function at all . it is worth stressing that the results of all the quoted studies are flawed in many critical aspects , since most of them were performed in relatively small groups of patients , had a retrospective design , and a lack of a control group . furthermore , the criteria for choosing the treated patients were not homogeneous , and many treated patients had normal or slightly increased ca levels before treatment , with pth levels only moderately increased . in fact , in our opinion it is questionable to treat ktx patients with normocalcemic pt - hpt with cinacalcet . hc after ktx is mainly the terminal event of an evolved and uncontrolled shp which maybe began long before transplantation . the best way for avoiding hc after ktx is to optimally treat shp before ktx . the first question concerns the criteria for choosing the patient on the transplant waiting - list who should be referred to ptx or maintained on medical therapy . given the complete lack of evidence on this issue , in our opinion an absolute indication to ptx for a patient on the ktx waiting list can be the presence of a severe shp , defined by contemporary high pth levels ( intact pth > 800 pg / ml ) and hc ( tot s - ca > 10.4 mg / dl ) , which can not be controlled by the available medical therapy . in all the other cases , we do not believe that there is any compelling indication to ptx . a second critical point is the management of pt - hpt associated with hc . however , independently of its possible negative impact on graft function , which moreover still remains unproven , ptx entails a number of problems such as the type and the timing of the intervention to be performed , the need for a continuous treatment for the correction of the residual hypoparathyroidism after a successful intervention , the problems related to some comorbid clinical conditions which make the surgical intervention a risk procedure and more . for all these reasons , the recent availability of a medical approach to hypercalcemic pt - hpt based on the use of cinacalcet challenged the concept of considering ptx the best choice for this clinical condition . in this critical scenario , it is our opinion that cinacalcet should be reserved to some specific cases such as the ktx patients with pt - hpt associated with overt hc ( > 12 mg / dl ) and have been already submitted to a previous ptx and/or have clinical conditions which make the intervention a risk procedure and/or refuse the intervention for any reason . at the present time , the clinical evaluation of each single patient still remains the best guide - line for choosing the best treatment of hc in ktx patients .

reactions in which a cyano ( cn ) radical abstracts a hydrogen atom from an organic molecule present an unusual opportunity to contrast chemical reaction dynamics under isolated conditions and in the presence of a liquid solvent . these reactions are exothermic , typically releasing more than 100 kj mol ; in contrast to the liquid phase where this energy rapidly transfers to the surrounding solvent , the energy in low - pressure gas - phase reactions can only be distributed among the translational and internal degrees of freedom of the two products . the potential energy surfaces for reactions of the type summarized by eq 1 , with r = h , ch3 , or other alkyl groups,1have early and low energy barriers , with a flat angular dependence at the transition state . consequently , the energy released by the reaction excites the hcn product mode - specifically in the c h stretching ( v3 ) and bending ( v2 ) vibrations , as confirmed by infrared ( ir ) emission and absorption spectroscopy measurements , as well as trajectory calculations . the rates and mechanisms of reactions of cyano radicals with hydrocarbons have been extensively studied at low pressures and temperatures because of their importance in the chemistry of the atmospheres of titan , triton , and pluto . crossed molecular beam studies of cn + alkane reactions , in which the alkyl radical products were ionized without quantum - state specificity , showed that approximately 8085% of the energy of reaction is deposited in internal modes of the products , which are scattered with angular distributions that indicate direct dynamics . the energy deposited in hcn bending and c h stretching vibrations persists for reactions in liquid solvents such as chloroform and dichloromethane , although the degree of vibrational excitation is somewhat reduced . the reaction mechanism in solution may be modified by factors such as formation of complexes between the cn radical and a solvent molecule , or coupling of the reaction degrees of freedom with the solvent bath . nevertheless , any shifts in the position of the transition state must be modest , and any solute solvent couplings must be weak for the gas - phase - like dynamics to persist to such an extent in solution at least , for these types of organic solvent . velocity map imaging studies of various exothermic bimolecular reactions in the gas phase have shown that measurement of the energy disposal to both products of reaction carries additional insights beyond what can be learned by studying only one of the products . these experiments used molecular beam techniques to ensure isolated collisions , and under favorable circumstances could map out the correlated vibrational energy content of both reaction products . the correlations were deduced from measurement of kinetic energy release distributions of one product in a specific vibrational quantum state , and application of momentum and energy conservation arguments . no corresponding methodology has yet been developed to observe correlated product energies for reactions in solution , but both products can be independently probed to determine their internal energy content . we illustrate this idea in the current report for the case of the reaction of cn radicals with acetone , eq 2,2 in deuterated chloroform ( cdcl3 ) solutions . we used time - resolved vibrational absorption spectroscopy ( tvas ) in the infrared region with picosecond time resolution to observe absorption features of the hcn and 2-oxopropyl ( or acetonyl ) radical ( ch3c(o)ch2 ) products and present evidence that both are vibrationally excited at their point of formation . this excess vibrational energy couples to the solvent bath , flowing from the product molecules on time scales of tens to hundreds of picoseconds . time - resolved electronic absorption spectroscopy ( teas ) provides complementary information on the interactions of the cn radicals with the solvent and their rates of reactive loss . building on pioneering work by hochstrasser and co - workers , crowther et al . studied the rates of cn radical reactions in solution by teas and tvas . their experiments were conducted in ch2cl2 and chcl3 solutions , and the proposal was made that cn forms linear and bridged complexes with these chlorinated solvents . in these complexes , the maximum in the cn ( b x ) absorption band shifts to shorter wavelength and the band broadens compared to the gas phase . the reactions of cn radicals with organic cosolutes were determined to have bimolecular rate coefficients smaller than the corresponding gas - phase reactions because of the stabilizing effect of the cn we introduce acetone to an icn / cdcl3 solution , and advances in vibrational probing highlight additional intermolecular interactions between the cn radical and the carbonyl group in the ketone . the results presented here provide evidence for contributions to the observed reactions in solution from both cn cdcl3 and cn acetone complexes . the time - resolved transient - absorption experiments were performed using the ultra facility at the rutherford appleton laboratory . the instrumentation has been described previously , and a brief summary is provided here of the details of particular relevance to the current study . samples were prepared with 0.36 m icn ( acros scientific , > 97.5% ) in anhydrous cdcl3 ( sigma - aldrich , 99.96 atom % ) or chcl3 ( sigma - aldrich , > 99.8% ) with acetone ( sigma - aldrich , 99.9% ) concentrations up to 1.5 m. solutions were kept at room temperature ( 294 k ) in brown - glass volumetric flasks . all glassware was stored in a drying oven when not in use , and water contamination was minimized by drying the acetone and chloroform over 3 molecular sieves . the flow cells used for the experiment were assembled with 0.2 mm ptfe spacers between caf2 windows and sealed using kalrez o - rings , which are chemically compatible with acetone and chloroform . using deuterated chloroform helps isolate any observed hcn as a product of hydrogen abstraction by cn from acetone . the ir spectrum of cdcl3 does not contain any strong spectral features in the c = o and c = n stretching regions that might interfere with transient ir spectroscopy measurements . icn was photodissociated at a wavelength of 267 nm by a 1 j laser pulse of 50 fs duration and products were probed over time delays of 12500 ps by teas using a white light continuum ( 310700 nm ) and by tvas using tunable broadband ( 500 cm bandwidth ) mid - ir light pulses . in both cases , the transmitted probe pulses were dispersed by a grating onto an array detector ( 512 pixel for teas , 128 pixel for tvas ) to measure changes in optical density induced by the uv excitation pulse . experiments were also conducted on solutions of acetone in cdcl3 without addition of icn to identify transient features associated with acetone photochemistry that we attribute to triplet states . samples of cdcl3 without icn and acetone showed only a weak response in teas under our experimental conditions . irradiation of a solution of icn and acetone in cdcl3 at 267 nm induces both icn dissociation and acetone photochemistry , followed by various possible radical reactions . multiple intermediate and product species might therefore contribute to the teas and tvas spectra . hence , calculations were carried out to characterize the harmonic vibrational frequencies and electronic excitations of possible reaction intermediates , reaction products , and acetone photofragments to guide the assignments of bands in the teas and tvas spectra . dft calculations of harmonic vibrational frequencies were performed in gaussian 09 using the b3lyp functional with the 6 - 311++g(d , p ) basis set . this method was chosen for its computational efficiency and because of its reliability of calculation of energetics of first row compounds . the diffuse functions were necessary to capture the interactions between -orbitals in cn radicals and acetone . a polarizable continuum model was applied to mimic chloroform solvent effects , as implemented in gaussian 09 . the calculated vibrational band frequencies were offset from their experimental values , so an empirical linear correction factor , fitted to be 0.948 from comparison with an ftir spectrum of 1.0 m acetone in cdcl3 , was applied to all computed frequencies . the calculated wavenumbers of bands that might contribute to the tvas spectra in the carbonyl stretching region are used as a guide to assignment of spectral features , with the caveat that the continuum model may not capture all solvent effects , such as formation of solute the enthalpy changes ( rh0k ) associated with h - abstraction from acetone reaction 2 were also computed in gaussian using various theoretical methods to quantify the reaction exothermicity and to benchmark more computationally efficient techniques against cbs - qb3 calculations . the cbs - qb3 calculations predict rh0k = 125.0 kj mol for the gas - phase reaction , and mimicking the effects of a chloroform solvent using a polarizable continuum model modifies this computed enthalpy change to 134.7 kj mol . for comparison , dft calculations at the b3lyp/6 - 31++g(d , p ) level incorporating the same treatment of the chloroform give rh0k = 153.5 kj mol for reaction 2 . the similarly exothermic h atom abstraction reactions of cn with propane and cyclohexane were previously shown to be barrierless , and we therefore expect facile reaction of cn with acetone to make hcn . our group has recently shown that molecular dynamics simulations of cn reactions are helpful as we build atomistic interpretations of the liquid - phase reaction mechanisms . in a computational study of the dynamics of cn radical reactions with propane and cyclohexane in the gas phase and in solution , glowacki et al . computed structures and energies using dft with the bb1k functional , modified to have 56% hartree fock exchange . trajectory calculations presented in the current study for the cn + acetone reaction , however , sought only qualitative mechanistic insights , so a simpler treatment of the energetics was used . direct dynamics trajectory calculations for isolated collisions were performed using the venus program with local gradients extracted from nwchem electronic structure calculations . trajectories were propagated using potential energy gradients computed by the dft / b3lyp method with a 6 - 31++g(d , p ) basis set . the cn radicals were initially provided with 2.4 kj mol of translational energy ( corresponding to kbt ) and were directed at the acetone with randomly selected initial orientations and with zero - point vibrational energy but no rotational energy . impact parameters were fixed to values of 0 , 1 , 2 , 4 , and 5 . time - step sizes of 0.2 fs were used , and propagation was maintained for up to 1 ps . the focus of this study is the reaction of cn radicals with acetone in solution in cdcl3 . acetone was chosen as the coreagent because the strong infrared absorption from the c = o stretching mode facilitates detection of possible organic radical reaction products such as 2-oxopropyl , ch3c(o)ch2 . ultraviolet photolysis of icn dissolved in a solution of acetone in cdcl3 was used to initiate the reaction . the 267 nm wavelength photolysis liberates cn radicals in less than 50 fs , and teas in the near - uv and visible regions revealed the production of cn radicals , their association with solvent molecules , and their reactive removal . the hcn and 2-oxopropyl products were monitored by trvs using broadband ultrafast ir pulses . acetone absorbs weakly at 267 nm ( contributing an absorbance a 1 for most of our samples ) and the photoexcited acetone can undergo norrish type i photochemistry by cleavage of a c c bond , or vibrational quenching of the photoexcited state can populate low vibrational levels of the s1 and t1 states of acetone with long lifetimes . teas and tvas of acetone / cdcl3 solutions were therefore used to characterize any contributions by acetone photochemistry to the spectra obtained for icn / acetone / cdcl3 solutions . ultraviolet photolysis of icn in organic solvents and geminate recombination to icn and inc have been the subject of several previous experimental and computational studies ; these processes are not discussed here , but both icn and inc bands appear in our tvas spectra , as shown below . this section considers the information obtained from the teas experiments and then turns to the tvas results . spectral assignments and dynamical deductions are guided by electronic structure calculations of electronic and vibrational band frequencies . we turn first , however , to discuss the outcomes of the trajectory calculations because the insights they provide influence the interpretation of the teas and tvas data . the results of our trajectory simulations , which do not include solvent effects , can be separated into the two classes of reaction pathways represented pictorially in figure 1 . direct abstraction of a hydrogen atom ( figure 1a ) appears to be favored by approach of the cn from the side opposite to the carbonyl group . this orientation prevents the cn radical from experiencing an attractive interaction with the carbonyl orbitals . on the other hand , approach of the cn toward the carbonyl group ( figure 1b ) results in three types of trajectories ; in all cases , the cn is first attracted toward a potential well corresponding to a cn the trajectory may then redistribute the energy of the collision among the internal modes of acetone so that the complex survives longer than the 1 ps simulation time . alternatively , the cn radical can experience large - amplitude motions on a flat area of the potential surrounding the carbonyl group , after which the two species separate or the cn abstracts a hydrogen atom . the competition between direct abstraction and this addition elimination pathways is reminiscent of the mechanisms for reactions of gas - phase cl atoms with propene and other alkenes . the direct - abstraction mechanism generates simulated hcn products that are internally hot , with up to 3 quanta of excitation in the c h stretching mode and 8 quanta in the bending vibration . in our measurements , the solvent may quench the energy required to escape from potential energy wells associated with structures like the cn acetone complex , trapping these solute solvent adducts and slowing , or preventing further reaction . schematic diagram of the classes of trajectories observed in simulations of isolated collisions of cn radicals with acetone . ( b ) pathways initiated by c = n carbonyl group interactions , leading to a cn acetone complex , or large amplitude cn radical motion followed by cn separation from acetone or h atom abstraction . the b x electronic band of the cn radical centered at 389 nm provides a distinctive signature of this species in teas spectra . this band evolves with time as the cn radicals form complexes with solvent molecules , and the intensities of transient bands associated with these solvent complexes decay with increasing time delay because of reactive loss of cn radicals . figure 2 illustrates this behavior following 267 nm excitation of a 0.36 m icn/1.0 m acetone solution in cdcl3 . decomposition of one illustrative transient spectrum , at a time delay of 2.5 ps , shows the constituent absorptions assigned to uncomplexed ( free ) cn radicals and solvent - complexed ( solvated ) cn radicals , and a long - wavelength feature attributed to i*(p1/2)-solvent charge transfer ( ct ) . the corresponding ct band of ground - state i(p3/2 ) atoms underlies the cn radical features and can not be distinguished from them but will grow as i * atoms electronically quench to ground - state i. teas spectra of separate solutions of acetone in cdcl3 and icn in cdcl3 are shown in supporting information . the spectra support these assignments and identify other contributors ; in particular , a transient absorption band assigned to the t1 state of acetone is observed at wavelengths below 400 nm . this triplet state has a lifetime > 2 ns in solution in cdcl3 but may be more rapidly quenched in the presence of iodine atoms . under the conditions of our experiment , the acetone t1 band contributes about 20% of the intensity of spectral features attributed to cn radicals and can not reliably be separated from the solvated - cn bands . inspection of figure 2a shows a weak absorption that remains at the longest measurement time of 1.75 ns . this feature is absent without acetone , but the band shape is inconsistent with both the t1 absorption band of acetone , and with cn cdcl3 complexes . instead , it may correspond to surviving cn radicals bound to cdcl3 or the carbonyl group of acetone . the latter interpretation is encouraged by teas spectroscopy of an icn solution in acetone , in which a band similar in shape to the long - time feature in figure 2a was observed . this band was not separately fitted in our spectral decomposition because of its similarity to the free cn band ; instead , it contributes an apparent growth to the free cn band with a time constant of 263 22 ps . transient electronic absorption spectra of icn/1.0 m acetone / cdcl3 solutions and the spectral decomposition into component absorptions . ( a ) teas spectra following 267 nm excitation for times delays from 0 to 1750 ps . ( b ) example of decomposition of the spectrum obtained at a time delay of 2.5 ps into its constituent parts . ( c ) time dependence of the free and complexed cn and the i * absorption bands obtained from decomposition of teas spectra . the solid lines are biexponential fits with time constants and amplitudes reported in table 1 . spectral decomposition produced the time - dependent band intensities for free cn , solvated cn , and i * of the type shown in figure 2 . this decomposition and others presented in this paper all used the koala software package . the faster decay time scale 1 of the free cn radicals matches the rise in the solvated cn signal ; the time constants for both these processes are controlled by a combination of the cooling of initially rotationally hot cn photofragments , complexation with cdcl3 or acetone , geminate recombination to icn and inc , and reaction with acetone . the time constant 2 , and the relative amplitude of the slower free cn decay component indicate that up to about one - third of these cn radicals avoid complexation , reaction and geminate recombination on an approximately 12 ps time scale . the decay of the solvated cn signal is attributed to reaction with acetone or solvent . the time dependence of the feature assigned to i * is not considered in detail here , but the rapid initial decay is consistent with geminate recombination with cn radicals and is similar to time constants reported by rivera et al . for i * decay in water and ethanol . the longer - time decay may be associated with spin orbit quenching of i * that has separated from its geminate cn photoproduct or is an artifact of a contribution from cn similar measurements were made for other solutions with acetone concentrations in the range 01.5 m. the second time constant , 2 , from biexponential fits of the type shown in figure 2 for both free and solvated forms of cn shows a dependence on acetone concentration because this decay component is associated with the bimolecular reaction between cn and acetone . under conditions in which the acetone is in excess over cn radicals , the reciprocals of the 2 time constants correspond to pseudo - first - order rate coefficients for h atom abstraction from acetone reaction 2 by free and solvated cn radicals . reaction with the cdcl3 may also contribute to cn - radical loss , but it is known to be slow and is unimportant to this analysis.figure 3 shows pseudo - first - order kinetic plots from which bimolecular rate coefficients for h atom abstraction of k2f = ( 8.3 0.6 ) 10 m s ( for the free cn + acetone reaction ) and k2c = ( 2.5 1.1 ) 10 m s ( for the solvent - complexed cn + acetone reaction ) are determined . uncertainties are 2 sd from the linear fits . the greater uncertainty in the data for the solvated cn loss may be a consequence of overlapping absorption by the i(p3/2)-solvent ct band or by acetone ( t1 ) produced by the 267 nm excitation . fitting of the sharper free - cn absorption band is relatively immune to these interferences from broad underlying features . the solvent - complexed cn reaction rate coefficient value must be treated with some caution because of possible spectral interferences but is similar in value to bimolecular rate coefficients reported by crowther et al . for cn radical reactions with various organic solutes in chlorinated solvents . the lower reaction rate for solvated compared to free cn is consistent with both its stabilization by complexation , and unfavorable orientation of the cn radical for h - abstraction from neighboring molecules . the large value of k2f for the facile bimolecular reaction of free cn with acetone can exceed the diffusion limit expected for a homogeneous solution if the acetone solvates the icn precursor preferentially over cdcl3 . the estimated rate of growth of the weak , broad absorption band centered just below 400 nm ( figure 2a ) , and most clearly observable at longer time delays , is similar to the rate of loss of cn cdcl3 complexes . the band is not observed in the absence of acetone , but a similar feature appears following uv photolysis of icn in neat acetone ( supporting information ) . this evidence suggest assignment to a cn acetone complex , which can form by transfer of a cn radical from cn cdcl3 . dependence of the pseudo - first - order rate coefficients on acetone concentration for loss of ( a ) free cn and ( b ) solvated and complexed cn radicals . pseudo - first - order rate coefficients were obtained as the reciprocals of the time constants 2 from biexponential fits to time - dependent band intensities . nonzero intercept values indicate reactive loss of cn radicals with species other than acetone , such as the solvent . the teas data provide quantitative information on the rates of removal of cn radicals , and distinguish solvated and unsolvated forms . consequently , tvas spectra were obtained and analyzed for spectroscopic signatures of reaction products . transient ir absorption spectra were obtained with the probe laser spanning 14501850 cm to observe carbonyl ( c = o ) , and 20002200 cm to observe nitrile ( c = n ) stretching modes . in the nitrile region , the spectral resolution was improved to 1.5 cm per pixel by changing the order of the spectrometer grating to resolve better some overlapping spectroscopic features . in the carbonyl region , the acetone band centered near 1690 cm strongly attenuated the probe light and masked the region from 16701720 cm . the tvas spectra measured in the 20002200 cm region showed numerous , partially overlapping bands , which were assigned on the basis of previous studies of icn photolysis and cn a sample set of spectra is shown in figure 4 together with the band assignments . typical data sets contained spectra at 50 or more time delays from 0 to 2500 ps , only a few of which are shown in figure 4 for clarity . the band centered at 2162 cm is assigned to the c = n stretching mode of icn and appears in the transient spectra as a negative - going bleach feature because the icn is depleted by the 267 nm photolysis laser . spectral decomposition suggests there is a broad positive - going feature with a center displaced to higher wavenumber than the icn bleach ; this feature decays rapidly and is tentatively attributed to vibrationally hot icn formed by i + cn geminate recombination on the ground electronic state . on the basis of this assignment , its decay ( with a time constant of 5.5 0.6 ps ) is indicative of vibrational cooling by coupling to the solvent bath . our caution in the assignment stems from the absence of similar signatures of vibrationally hot icn from i + cn geminate recombination in other solvents . the interpretation of this feature is not critical to the main themes of this paper . a band centered at 2067 cm is assigned to the inc product of in - cage geminate recombination ; although a minor channel , it is observed in our spectra because of the large transition dipole moment of the isocyanide group . evidence from our laboratory indicates that the inc also forms vibrationally excited and that the growth rate of the band at 2067 cm is controlled by vibrational relaxation . time - resolved vibrational spectra of 267 nm excited icn / acetone / cdcl3 solutions in the 20202180 cm range . we attributed the weak feature at 2040 cm , discernible to the low - wavenumber side of the inc band , to solvated cn radicals in previous work . it grows with an initial ( few picoseconds ) spurt followed by a slower component with a time constant of 27 7 ps ( for a solution with 1.0 m acetone ) . the initial rise can be accounted for by direct complexation of free cn radicals with either cdcl3 or acetone because it is consistent with the 2.3 ps rise of the solvated cn band in the teas spectra . the slower time constant for growth of the 2040 cm band depends linearly on the concentration of acetone , and a pseudo - first - order kinetic analysis gives a bimolecular rate coefficient of ( 2.6 0.8 ) 10 m s ( 2 sd uncertainties ) . acetone complexes of the type discussed earlier , and the observed time constants indicate an indirect route in addition to the direct formation from free cn . we suggest that some fraction of the cn first complexes to the more abundant cdcl3 molecules , then transforms into the more strongly bound acetone complexes . if this interpretation is correct , we must invoke an intermediate denoted cn cdcl3 * to reconcile the rate coefficient value above with the k2c = ( 2.5 1.1 ) 10 m s value for the loss of cn cdcl3 such as an isomer or an internally hot complex , in which case it will also contribute to the observed teas band . acetone complex , or by vibrational energy transfer to the solvent to form thermalized cn the proposed assignment of the broad uv / visible band centered below 400 nm in teas data ( figure 2a ) to a cn cdcl3 complexes by cdcl3/acetone exchange on a time scale of a few hundred picoseconds . these various processes are summarized in scheme 1 , with time constants appropriate for 1.0 m acetone solutions and the associated up and down arrows indicating growth and decay , respectively . acetone complexes comes from the carbonyl region tvas data discussed in section iv.c.ii , where some of the time constants incorporated in scheme 1 are explained . however , we must consider a second possible explanation based on recent work from our group on icn photolysis in various solvents : as was noted earlier , some fraction of the in - cage geminate recombination of cn + i produces inc , and the nascent molecules are vibrationally excited . hot bands of the inc are shifted to lower wavenumber than the fundamental n = c stretching band at 2067 cm , and their decay rates may be enhanced by vibrational energy transfer to acetone , giving the observed concentration dependence . because this interpretation requires population to feed from higher vibrational levels into the levels observed via the 2040 cm absorption band to maintain a spectroscopic feature over several hundred picoseconds , we consider it unlikely . ( a ) time dependence of the hcn band shown in figure 4 , with the inset showing an expanded view of the first 500 ps . ( b ) a pseudo - first - order plot of the dependence of first - order rate coefficients ( obtained as reciprocals of exponential time constants for hcn growth ) on acetone concentration . the band of most interest to the current study is centered at 2097 cm and corresponds to the fundamental c = n stretching v1 = 1 v1 = 0 absorption of hcn reaction products . the time dependence of the growth of this feature is shown in figure 5a . an exponential rise in intensity is preceded by an initial decay with time constant of 5.9 0.6 ps that is associated with imperfect separation of the hcn feature from the broad band observed at early times to higher wavenumber ( tentatively icn * ) . we therefore concentrate on times after 10 ps when this interfering feature has a negligible influence . the spectral decomposition indicates that the hcn band has a negative intensity from 10 to 100 ps . the precise zero of intensity is difficult to identify because of neighboring features , but various different fitting procedures consistently returned negative intensities at early times and a delayed growth of the hcn fundamental band . we have observed these characteristics before : our prior studies of cn radical reactions in solution showed them to be signatures of an initial population inversion in the vibrational levels of hcn . greater population in vibrationally excited levels than in the vibrational ground state is a consequence of the dynamics of these exothermic reactions with early transition states and has also been reported for gas - phase reactions of cn radicals . we therefore conclude that the cn + acetone reaction produces vibrationally hot hcn , and past precedent suggests that this excitation will be localized in the c h stretching and bending modes . the rate of rise in the intensity of the hcn fundamental band at 2097 cm depends linearly on acetone concentration ( figure 5b ) and a pseudo - first - order analysis gives a bimolecular rate coefficient of khcn = ( 2.0 0.9 ) 10 m s ( 2 sd uncertainties ) that is consistent with the rate coefficient for loss of solvent - complexed cn of k2c = ( 2.5 1.1 ) 10 m s deduced from the teas spectra presented in figure 2 . this correspondence indicates that the chemical reaction step is rate determining for production of hcn from solvated cn radicals . in some of our prior studies of cn radical reactions in chlorinated solvents , vibrational relaxation of the internally excited hcn instead controlled the rate of increasing intensity of the fundamental band . the two different limiting behaviors depend on whether this vibrational relaxation is faster or slower than the chemical production of hcn . in the current case , the coupling of vibrationally hot hcn to cdcl3 is weak , but acetone appears to be an efficient alternative quencher . vibrationally hot hcn from reactions of free cn radicals should give rise to transient absorption bands observable at short time delays to lower wavenumber than the 2097 cm fundamental band . however , we are unable to observe these hot bands directly in the current experiments because the small anharmonicity of the c = n stretch ( x11 = 10 cm ) and weak anharmonic couplings between the c = n stretching mode and the bending ( x12 = 3 cm ) and c h stretching ( x13 = 15 cm ) vibrations , shift the absorptions to regions overlapped by the inc band and the low - wavenumber side of the hcn fundamental band . there are two important features in the c = o stretching region following icn photodissociation in acetone / cdcl3 solutions . a representative set of tvas spectra is shown in figure 6 . in this section , we describe our assignment of the weaker feature at 1640 cm to the cn acetone complex and the stronger feature at 1550 cm to the 2-oxopropyl reaction product . both bands are only present in the transient spectra when both acetone and icn are dissolved in cdcl3 , indicating both features involve cn interaction with acetone . calculated vibrational frequencies and steady - state infrared spectra guided the interpretations we make , and relevant computed frequencies and relative band intensities of candidate species are presented in table 2 alongside the measured frequencies of the observed absorption bands . our trajectory calculations indicate that the cn radical can form transiently stable complexes with acetone . ( an addition reaction at the carbonyl group may also be possible but was not observed in the trajectory calculations and would lead to a product with a significantly shifted c o stretching frequency . ) the transient complexes were suggested to contribute a band centered just below 400 nm in the teas spectra of figure 2 and a weak feature at 2040 cm that is evident in the c = n stretching region spectra shown in figure 4 . they are stabilized by approximately 3600 cm compared to separated cn and acetone , according to our calculations . furthermore , these stabilized structures are dynamically decoupled from the h - abstraction pathway , which inhibits further reaction . the cn shifts the acetone c = o stretch down in wavenumber in both our calculations and the spectrum . the calculations predict the band to occur at 16081618 cm , with the range encompassing two isomers in which the c or n end of the cyano radical is oriented toward the acetone , as shown in figure 6b . other potential absorbers in this region include acetaldehyde , ch3cho , a stable product of the possible radical chemistry . its fundamental c = o stretch , calculated to be at 1717 cm , lies to the higher wavenumber side of the acetone carbonyl band . similarly , acetyl radical photoproducts of acetone fragmentation have computed ir bands at 1833 cm and are not responsible for the features of interest in figure 6a . the other features in the spectrum that lie close to the strong acetone fundamental band must be interpreted with caution , but there are indications of time - dependent bands at 1740 and 1770 cm ; both are observed with and without icn . we suspect they are associated with the uv photochemistry of acetone and with different solvation environments of acetone in cdcl3 . ( a ) time - resolved vibrational spectra spanning 14701755 cm after 267 nm excitation of icn / acetone / cdcl3 solutions . ( b ) time dependence of the weak feature at 1640 cm and computed structures of the cn acetone complexes negative intensities are a consequence of baseline fluctuations at early times because of the neighboring acetone band . the weak , time - dependent feature at 1640 cm assigned to the cn acetone complex sits upon a shifting baseline because of proximity to the strong acetone band . nevertheless , it can be fitted to extract time - dependent band intensities , as shown in figure 6b . its growth fits to a biexponential rise with time constants of 1 = 3.0 0.3 ps and 2 = 55 25 ps ( both for 0.5 m acetone solutions ; 2 sd uncertainties ) . it then undergoes a slow decay ( with 3 = 800 400 ps ) . the two rise times are consistent with the formation of the complexes directly from unsolvated free cn , which teas data show decays on a 2.3 0.2 ps time scale , and from cn a time constant of 27 7 ps was suggested from the c = n stretching region tvas feature at 2040 cm , which is approximately half that for the 0.5 m solution but was measured for twice the acetone concentration . cdcl3 complexes because the former are more strongly bound ( we estimate 3600 cm versus 1800 cm for cn acetone and cn cdcl3 from our calculations ) . the remainder of this analysis describes our assignment and interpretation of the temporal evolution of the stronger transient feature at 15051575 cm . we attribute this band to the 2-oxopropyl product of the h - abstraction reaction of cn with acetone , on the basis of our calculations that place the band center at 1545 cm . several alternative assignments can be quickly discounted because the band lies 150 cm below the carbonyl band of acetone . for example , the c = o stretching band of 1-iodoacetone from recombination of i atoms with 2-oxopropyl is expected near 1700 cm ( on the basis of the known gas - phase ir spectrum of 1-chloroacetone , and our observed solvent shifts in chloroform solutions ) and will be masked by the acetone feature . the band assigned to 2-oxopropyl develops in the tvas spectra with a center at 1552 cm at late time delays . however , at early times , this feature is broadened to lower wavenumber and coalesces into the sharp 2-oxopropyl feature over a time scale of 50 ps . this behavior is suggestive of vibrational cooling of an initially internally hot radical , with broadening and shifting of the absorption to lower wavenumber , because of either vibrational anharmonicity in the c = o stretch or anharmonic coupling to other excited modes . it is less likely to be a consequence of an equilibrating solvent environment around the newly formed radical , which we expect to shift the vibrational frequencies from higher to lower wavenumber with time . energy flow from the hot hcn to the 2-oxopropyl radical , an effect we first noted in the reaction of cn radicals with cyclohexane , can be ruled out as the cause of the narrowing feature because the vibrationally hot 2-oxopropyl radicals are formed more promptly than the likely time scale for vibrational relaxation of the hcn . the time dependence of the 2-oxopropyl band was analyzed by fitting to two gaussian functions with centers x0i and widths i ( both specified in cm ) : x0 g = 1552 and g = 14 for absorptions by ground - state molecules ( as determined from fitting the late - time spectra ) , and x0e = 1533 and e = 35 for vibrationally excited molecules . the areas of the two gaussian components were free parameters in the fits , and figure 7 illustrates the outcomes . the temporal evolutions of the two bands are both satisfactorily modeled with exponential time constants of 1 = 2.3 ps ( fixed to match the faster time constant for loss of free cn radicals from teas data ) and 2 = 34 10 ps ( determined by simultaneous fits to the time - dependent intensities of both bands ; 2 sd uncertainty ) . the analysis is therefore consistent with production of vibrationally hot 2-oxopropyl radicals from cn reaction with acetone with a time constant of 2.3 ps , and vibrational relaxation with a time constant of 34 ps . this cooling may correspond to loss of multiple quanta of vibrational excitation in different modes and is an average time scale . more quantitative information on the vibrational energy content of the nascent 2-oxopropyl radical can not be deduced from our tvas data alone . ( b ) tvas spectra ( dots ) and illustrative fits to two gaussian functions ( lines ) for time delays of 2.0 ps ( dark blue ) , 11 ps ( light blue ) , and 1200 ps ( orange ) . ( c ) time dependences of the feature attributed to vibrationally excited ( red ) and ground - state ( blue ) 2-oxopropyl radicals . the 2-oxopropyl radical and its coproduct hcn have different long - time kinetics . the band attributed to the c = o stretch of ground - state 2-oxopropyl radicals reaches a steady - state absorbance at 50 ps but the hcn absorption continues to grow for time scales up to our experimental limit of 2 ns . the differences in longer time behavior of these two populations hint toward subsequent 2-oxopropyl reactions . this radical is therefore an intermediate in a sequence of steps , which result in a balance between its production and loss . a steady - state ( but low ) concentration will occur if the rate of removal of the 2-oxopropyl radicals is larger than their rate of production . in addition to the fast reactive production by free cn radicals , a slower contribution to 2-oxopropyl growth will derive from reaction of solvated cn radicals , for which the rate coefficient is 2 10 m s as determined from hcn growth and cn the time constants for production of the 2-oxopropyl radicals are therefore several hundred picoseconds under our experimental conditions , and assuming that steady - state behavior leads to the effects we see , indicates a decay lifetime of 100 ps . the analysis of the teas and tvas data obtained for uv - excited icn / acetone / cdcl3 solutions reveals distinguishable reactivity of free and solvent - complexed cn radicals . the free cn quickly ( cn = 2.3 ps ) decays by geminate recombination ( to icn and inc ) , association with a cdcl3 solvent molecule or acetone cosolute molecule to form complexes , and reaction with acetone to produce hcn . the bimolecular rate coefficient for reaction of acetone with free cn radicals , i.e. , for those cn that survive initial loss or complexation to the solvent , is deduced to be k2f = ( 8.0 0.5 ) 10 m s. we suspect that this large reaction rate coefficient results in part from microscopic inhomogeneity that develops in these solutions . although the reaction itself is facile , preferential solvation of icn by acetone over cdcl3 will augment the reaction rate . cdcl3 complexes with acetone are more than an order of magnitude slower and are controlled by both diffusion and activation : we extracted values of k2c = ( 2.5 1.1 ) 10 m s from the loss of the solvated cn band in the teas spectra and k2c = ( 2.0 0.9 ) 10 m s from the rise in the hcn band in tvas experiments ( see sections iv.b and iv.c ) . reactions of cn acetone complexes are slower still because of the greater stabilization deriving from cn carbonyl interactions . acetone and 1800 cm for cn cdcl3 complexes , which support this idea . acetone complexes form both directly from free cn radicals that happen to have a neighboring acetone molecule when photolytically generated , and by exchange of partner following initial complexation to cdcl3 solvent molecules . the driving force for the cn figure 8 summarizes the various processes that follow uv excitation of icn in an acetone / cdcl3 solution and the associated time constants ( specified for a 0.5 m acetone solution where dependent on the acetone concentration ) . it extends the initial kinetics of solvation of the free cn radicals with cdcl3 and acetone summarized in scheme 1 to include the abstraction reaction pathways . the figure excludes the photochemistry that results from 267 nm excitation of acetone molecules . under the conditions of our experiments , we estimate that fewer than 0.02% of the acetone molecules within the volume of the 266 nm laser beam are photoexcited . hence excited - state ( s1 and t1 ) acetone molecules and acetone photoproducts ( e.g. , acetyl radical ) are unlikely to react with cn radicals in sufficient numbers to contribute to our observed time - resolved spectra . summary of the photochemical processes involving cn radicals and acetone following uv photolysis of icn in an icn / acetone / cdcl3 solution . the rate coefficient values deduced from this work are k2f = ( 8.0 0.5 ) 10 m s for the slower component of the reaction of the free cn radical with acetone , k2c = ( 2.5 1.1 ) 10 m s for the solvent - complexed cn reaction , and kcomplex = ( 2.6 0.8 ) 10 m s for formation of the cn acetone complex . lifetimes ( from decay time constants ) are 1,cn = 2.3 ps for the initial fast - component of the loss of free cn , cn acetone = 800 ps for the complex , and r * = 34 ps for the cooling of vibrationally excited 2-oxypropyl radicals . both the hcn and the 2-oxopropyl radical products form with some degree of vibrational excitation greater than expected for thermalized products ( see sections iv.c.i and iv.c.ii ) . in the case of the hcn product , the evidence from the current and our previous studies of cn radical reactions in solution is that there is a vibrational population inversion , with the majority of the hcn formed both c h stretching excitation in the hcn corresponds to an energy of 39 kj mol , and each bending quantum adds approximately 8 kj mol more internal energy . in this regard , the reaction dynamics are similar to those for h atom abstraction reactions by cn in the gas phase . the extent of vibrational excitation of the 2-oxopropyl radical is less clear - cut , but the evolution from its initially broad to its final , narrow c = o stretching spectrum in figure 7 shows that a large fraction of the 2-oxopropyl radicals are vibrationally excited . a franck condon type model can be applied to predict a limiting case for the internal excitation of the 2-oxopropyl radical . sudden abstraction of a hydrogen atom may be a reasonable approximation for the direct dynamics with an early transition state and will leave the 2-oxopropyl radical in a geometry otherwise identical to that of acetone . electronic structure calculations indicate that relaxation to the equilibrium structure of the 2-oxopropyl radical most significantly involves changes to the c h bond length in the ch2 group from 1.098 ( the c h bond length in acetone ) to 1.083 , and a change from tetrahedral to trigonal planar coordination . we estimate from these calculations that the gain in internal energy associated with this structural reorganization is 46 kj mol . the structural changes identified might excite c h stretching and ch2 wagging and scissoring modes . these arguments suggest that approximately 70% of the available energy may be channeled into product internal motions , which is similar to the fraction reported for cn reactions in the gas phase . the fast rise time of a few picoseconds of the vibrationally hot 2-oxopropyl radicals , and the concomitant development of the population inversion of vibrational levels of hcn intimate that these products result from reactions of the free cn radicals before they complex to solvent molecules . acetone complexes react to form hcn more slowly , with time constants on the order of a few hundred picoseconds ( depending on acetone concentration ) . we are unable to detect vibrationally excited hcn and 2-oxopropyl radicals from the slower reactions of the complexed forms of cn . this absence may indicate that the complexed reactions exhibit different reaction dynamics , perhaps with later transition states along the reaction pathway , so that the vibrational excitation does not develop in the products . more likely , given the large exothermicity of the reactions ( 140 kj mol ) and the relatively small stabilization energies of the complexes ( < 45 kj mol ) , is that this product vibrational excitation does develop , but that it relaxes by solvent coupling on faster time scales than the overall buildup of products , so can not be detected in our experiments . we have previously shown that exothermic reactions in solution have a propensity to form one vibrationally hot product despite the coupling of the solvent bath to nuclear motions along and orthogonal to the reaction coordinate . this vibrational excitation mostly appears in the new bond formed from the reaction as a consequence of an early transition state , as predicted by the polanyi rules for gas - phase reactions . the current study demonstrates that the coproduct of a bimolecular reaction in solution can also be formed with significant amounts of internal vibrational excitation . similar behavior is known from gas - phase studies of exothermic reactions of f atoms with methane and may also occur for cn ( anti)correlations between the degrees of vibrational excitation of the two products can be established from velocity map imaging measurements . methodology is lacking to make the corresponding correlated measurements in solution , but our current efforts show that dynamical insights can still be forthcoming from the uncorrelated observations . time - resolved absorption spectroscopy of the exothermic reaction of cn radicals with acetone in cdcl3 solutions demonstrates that a significant fraction of the energy released enters vibrational modes of the hcn and the 2-oxopropyl radical coproduct . transfer of this excess energy to the solvent bath occurs on time scales on the order of 10 ps , leading to thermally equilibrated reaction products . we have previously reported substantial vibrational excitation of one product of a reaction in solution ( for exothermic reactions of cn radicals , and of f and cl atoms ) , but this is our first observation that both nascent products can be vibrationally hot . the cn radicals were generated photolytically from icn , and take 2.3 ps to equilibrate with the solvent and form cn cdcl3 complexes . distinct spectroscopic signatures of complexed and uncomplexed ( free ) cn radicals allow us to distinguish their reactions . in the reactions with acetone , the complexed cn radicals react more than an order of magnitude more slowly , but the kinetics of reactions of the free cn radicals are partly controlled by in - cage geminate recombination with i atoms , and solvent - complex formation . the cn radicals also complex to acetone molecules , and c = n carbonyl interactions thermodynamically favor these species over cn evidence from our measurements suggests that the cn radicals complexed with acetone have significantly reduced reactivities . the combination of time - resolved electronic and vibrational spectroscopies allows us to unravel the rates and dynamics of several competing processes that occur following photoinitiation of radical chemistry in solution .

transient electronic and vibrational absorption spectroscopy unravel the mechanisms and dynamics of bimolecular reactions of cn radicals with acetone in deuterated chloroform solutions . the cn radicals are produced by ultrafast ultraviolet photolysis of dissolved icn . two reactive forms of cn radicals are distinguished by their electronic absorption bands : free ( uncomplexed ) cn radicals , and solvated cn radicals that are complexed with solvent molecules . the lifetimes of the free cn radicals are limited to a few picoseconds following their photolytic production because of geminate recombination to icn and inc , complexation with cdcl3 molecules , and reaction with acetone . the acetone reaction occurs with a rate coefficient of ( 8.0 0.5 ) 1010 m1 s1 and transient vibrational spectra in the c = n and c = o stretching regions reveal that both the nascent hcn and 2-oxopropyl ( ch3c(o)ch2 ) radical products are vibrationally excited . the rate coefficient for the reaction of solvated cn with acetone is 40 times slower than for free cn , with a rate coefficient of ( 2.0 0.9 ) 109 m1 s1 obtained from the rise in the hcn product v1(c = n stretch ) ir absorption band . evidence is also presented for cn complexes with acetone that are more strongly bound than the cn cdcl3 complexes because of cn interactions with the carbonyl group . the rates of reactions of these more strongly associated radicals are slower still .

Introduction Experimental Details Computational Details Results Discussion Conclusions

consequently , the energy released by the reaction excites the hcn product mode - specifically in the c h stretching ( v3 ) and bending ( v2 ) vibrations , as confirmed by infrared ( ir ) emission and absorption spectroscopy measurements , as well as trajectory calculations . the rates and mechanisms of reactions of cyano radicals with hydrocarbons have been extensively studied at low pressures and temperatures because of their importance in the chemistry of the atmospheres of titan , triton , and pluto . we illustrate this idea in the current report for the case of the reaction of cn radicals with acetone , eq 2,2 in deuterated chloroform ( cdcl3 ) solutions . we used time - resolved vibrational absorption spectroscopy ( tvas ) in the infrared region with picosecond time resolution to observe absorption features of the hcn and 2-oxopropyl ( or acetonyl ) radical ( ch3c(o)ch2 ) products and present evidence that both are vibrationally excited at their point of formation . time - resolved electronic absorption spectroscopy ( teas ) provides complementary information on the interactions of the cn radicals with the solvent and their rates of reactive loss . the reactions of cn radicals with organic cosolutes were determined to have bimolecular rate coefficients smaller than the corresponding gas - phase reactions because of the stabilizing effect of the cn we introduce acetone to an icn / cdcl3 solution , and advances in vibrational probing highlight additional intermolecular interactions between the cn radical and the carbonyl group in the ketone . the ir spectrum of cdcl3 does not contain any strong spectral features in the c = o and c = n stretching regions that might interfere with transient ir spectroscopy measurements . the calculated wavenumbers of bands that might contribute to the tvas spectra in the carbonyl stretching region are used as a guide to assignment of spectral features , with the caveat that the continuum model may not capture all solvent effects , such as formation of solute the enthalpy changes ( rh0k ) associated with h - abstraction from acetone reaction 2 were also computed in gaussian using various theoretical methods to quantify the reaction exothermicity and to benchmark more computationally efficient techniques against cbs - qb3 calculations . the similarly exothermic h atom abstraction reactions of cn with propane and cyclohexane were previously shown to be barrierless , and we therefore expect facile reaction of cn with acetone to make hcn . the focus of this study is the reaction of cn radicals with acetone in solution in cdcl3 . the 267 nm wavelength photolysis liberates cn radicals in less than 50 fs , and teas in the near - uv and visible regions revealed the production of cn radicals , their association with solvent molecules , and their reactive removal . ultraviolet photolysis of icn in organic solvents and geminate recombination to icn and inc have been the subject of several previous experimental and computational studies ; these processes are not discussed here , but both icn and inc bands appear in our tvas spectra , as shown below . direct abstraction of a hydrogen atom ( figure 1a ) appears to be favored by approach of the cn from the side opposite to the carbonyl group . on the other hand , approach of the cn toward the carbonyl group ( figure 1b ) results in three types of trajectories ; in all cases , the cn is first attracted toward a potential well corresponding to a cn the trajectory may then redistribute the energy of the collision among the internal modes of acetone so that the complex survives longer than the 1 ps simulation time . this band evolves with time as the cn radicals form complexes with solvent molecules , and the intensities of transient bands associated with these solvent complexes decay with increasing time delay because of reactive loss of cn radicals . this feature is absent without acetone , but the band shape is inconsistent with both the t1 absorption band of acetone , and with cn cdcl3 complexes . spectral decomposition produced the time - dependent band intensities for free cn , solvated cn , and i * of the type shown in figure 2 . the faster decay time scale 1 of the free cn radicals matches the rise in the solvated cn signal ; the time constants for both these processes are controlled by a combination of the cooling of initially rotationally hot cn photofragments , complexation with cdcl3 or acetone , geminate recombination to icn and inc , and reaction with acetone . the time constant 2 , and the relative amplitude of the slower free cn decay component indicate that up to about one - third of these cn radicals avoid complexation , reaction and geminate recombination on an approximately 12 ps time scale . the longer - time decay may be associated with spin orbit quenching of i * that has separated from its geminate cn photoproduct or is an artifact of a contribution from cn similar measurements were made for other solutions with acetone concentrations in the range 01.5 m. the second time constant , 2 , from biexponential fits of the type shown in figure 2 for both free and solvated forms of cn shows a dependence on acetone concentration because this decay component is associated with the bimolecular reaction between cn and acetone . under conditions in which the acetone is in excess over cn radicals , the reciprocals of the 2 time constants correspond to pseudo - first - order rate coefficients for h atom abstraction from acetone reaction 2 by free and solvated cn radicals . reaction with the cdcl3 may also contribute to cn - radical loss , but it is known to be slow and is unimportant to this analysis.figure 3 shows pseudo - first - order kinetic plots from which bimolecular rate coefficients for h atom abstraction of k2f = ( 8.3 0.6 ) 10 m s ( for the free cn + acetone reaction ) and k2c = ( 2.5 1.1 ) 10 m s ( for the solvent - complexed cn + acetone reaction ) are determined . the large value of k2f for the facile bimolecular reaction of free cn with acetone can exceed the diffusion limit expected for a homogeneous solution if the acetone solvates the icn precursor preferentially over cdcl3 . the estimated rate of growth of the weak , broad absorption band centered just below 400 nm ( figure 2a ) , and most clearly observable at longer time delays , is similar to the rate of loss of cn cdcl3 complexes . the teas data provide quantitative information on the rates of removal of cn radicals , and distinguish solvated and unsolvated forms . transient ir absorption spectra were obtained with the probe laser spanning 14501850 cm to observe carbonyl ( c = o ) , and 20002200 cm to observe nitrile ( c = n ) stretching modes . the initial rise can be accounted for by direct complexation of free cn radicals with either cdcl3 or acetone because it is consistent with the 2.3 ps rise of the solvated cn band in the teas spectra . the slower time constant for growth of the 2040 cm band depends linearly on the concentration of acetone , and a pseudo - first - order kinetic analysis gives a bimolecular rate coefficient of ( 2.6 0.8 ) 10 m s ( 2 sd uncertainties ) . we suggest that some fraction of the cn first complexes to the more abundant cdcl3 molecules , then transforms into the more strongly bound acetone complexes . if this interpretation is correct , we must invoke an intermediate denoted cn cdcl3 * to reconcile the rate coefficient value above with the k2c = ( 2.5 1.1 ) 10 m s value for the loss of cn cdcl3 such as an isomer or an internally hot complex , in which case it will also contribute to the observed teas band . however , we must consider a second possible explanation based on recent work from our group on icn photolysis in various solvents : as was noted earlier , some fraction of the in - cage geminate recombination of cn + i produces inc , and the nascent molecules are vibrationally excited . greater population in vibrationally excited levels than in the vibrational ground state is a consequence of the dynamics of these exothermic reactions with early transition states and has also been reported for gas - phase reactions of cn radicals . the rate of rise in the intensity of the hcn fundamental band at 2097 cm depends linearly on acetone concentration ( figure 5b ) and a pseudo - first - order analysis gives a bimolecular rate coefficient of khcn = ( 2.0 0.9 ) 10 m s ( 2 sd uncertainties ) that is consistent with the rate coefficient for loss of solvent - complexed cn of k2c = ( 2.5 1.1 ) 10 m s deduced from the teas spectra presented in figure 2 . however , we are unable to observe these hot bands directly in the current experiments because the small anharmonicity of the c = n stretch ( x11 = 10 cm ) and weak anharmonic couplings between the c = n stretching mode and the bending ( x12 = 3 cm ) and c h stretching ( x13 = 15 cm ) vibrations , shift the absorptions to regions overlapped by the inc band and the low - wavenumber side of the hcn fundamental band . ( an addition reaction at the carbonyl group may also be possible but was not observed in the trajectory calculations and would lead to a product with a significantly shifted c o stretching frequency . ) the calculations predict the band to occur at 16081618 cm , with the range encompassing two isomers in which the c or n end of the cyano radical is oriented toward the acetone , as shown in figure 6b . the two rise times are consistent with the formation of the complexes directly from unsolvated free cn , which teas data show decays on a 2.3 0.2 ps time scale , and from cn a time constant of 27 7 ps was suggested from the c = n stretching region tvas feature at 2040 cm , which is approximately half that for the 0.5 m solution but was measured for twice the acetone concentration . cdcl3 complexes because the former are more strongly bound ( we estimate 3600 cm versus 1800 cm for cn acetone and cn cdcl3 from our calculations ) . we attribute this band to the 2-oxopropyl product of the h - abstraction reaction of cn with acetone , on the basis of our calculations that place the band center at 1545 cm . for example , the c = o stretching band of 1-iodoacetone from recombination of i atoms with 2-oxopropyl is expected near 1700 cm ( on the basis of the known gas - phase ir spectrum of 1-chloroacetone , and our observed solvent shifts in chloroform solutions ) and will be masked by the acetone feature . this behavior is suggestive of vibrational cooling of an initially internally hot radical , with broadening and shifting of the absorption to lower wavenumber , because of either vibrational anharmonicity in the c = o stretch or anharmonic coupling to other excited modes . energy flow from the hot hcn to the 2-oxopropyl radical , an effect we first noted in the reaction of cn radicals with cyclohexane , can be ruled out as the cause of the narrowing feature because the vibrationally hot 2-oxopropyl radicals are formed more promptly than the likely time scale for vibrational relaxation of the hcn . the analysis is therefore consistent with production of vibrationally hot 2-oxopropyl radicals from cn reaction with acetone with a time constant of 2.3 ps , and vibrational relaxation with a time constant of 34 ps . in addition to the fast reactive production by free cn radicals , a slower contribution to 2-oxopropyl growth will derive from reaction of solvated cn radicals , for which the rate coefficient is 2 10 m s as determined from hcn growth and cn the time constants for production of the 2-oxopropyl radicals are therefore several hundred picoseconds under our experimental conditions , and assuming that steady - state behavior leads to the effects we see , indicates a decay lifetime of 100 ps . the free cn quickly ( cn = 2.3 ps ) decays by geminate recombination ( to icn and inc ) , association with a cdcl3 solvent molecule or acetone cosolute molecule to form complexes , and reaction with acetone to produce hcn . the bimolecular rate coefficient for reaction of acetone with free cn radicals , i.e. , for those cn that survive initial loss or complexation to the solvent , is deduced to be k2f = ( 8.0 0.5 ) 10 m s. we suspect that this large reaction rate coefficient results in part from microscopic inhomogeneity that develops in these solutions . cdcl3 complexes with acetone are more than an order of magnitude slower and are controlled by both diffusion and activation : we extracted values of k2c = ( 2.5 1.1 ) 10 m s from the loss of the solvated cn band in the teas spectra and k2c = ( 2.0 0.9 ) 10 m s from the rise in the hcn band in tvas experiments ( see sections iv.b and iv.c ) . reactions of cn acetone complexes are slower still because of the greater stabilization deriving from cn carbonyl interactions . acetone complexes form both directly from free cn radicals that happen to have a neighboring acetone molecule when photolytically generated , and by exchange of partner following initial complexation to cdcl3 solvent molecules . it extends the initial kinetics of solvation of the free cn radicals with cdcl3 and acetone summarized in scheme 1 to include the abstraction reaction pathways . the rate coefficient values deduced from this work are k2f = ( 8.0 0.5 ) 10 m s for the slower component of the reaction of the free cn radical with acetone , k2c = ( 2.5 1.1 ) 10 m s for the solvent - complexed cn reaction , and kcomplex = ( 2.6 0.8 ) 10 m s for formation of the cn acetone complex . lifetimes ( from decay time constants ) are 1,cn = 2.3 ps for the initial fast - component of the loss of free cn , cn acetone = 800 ps for the complex , and r * = 34 ps for the cooling of vibrationally excited 2-oxypropyl radicals . in the case of the hcn product , the evidence from the current and our previous studies of cn radical reactions in solution is that there is a vibrational population inversion , with the majority of the hcn formed both c h stretching excitation in the hcn corresponds to an energy of 39 kj mol , and each bending quantum adds approximately 8 kj mol more internal energy . the extent of vibrational excitation of the 2-oxopropyl radical is less clear - cut , but the evolution from its initially broad to its final , narrow c = o stretching spectrum in figure 7 shows that a large fraction of the 2-oxopropyl radicals are vibrationally excited . the fast rise time of a few picoseconds of the vibrationally hot 2-oxopropyl radicals , and the concomitant development of the population inversion of vibrational levels of hcn intimate that these products result from reactions of the free cn radicals before they complex to solvent molecules . we are unable to detect vibrationally excited hcn and 2-oxopropyl radicals from the slower reactions of the complexed forms of cn . time - resolved absorption spectroscopy of the exothermic reaction of cn radicals with acetone in cdcl3 solutions demonstrates that a significant fraction of the energy released enters vibrational modes of the hcn and the 2-oxopropyl radical coproduct . we have previously reported substantial vibrational excitation of one product of a reaction in solution ( for exothermic reactions of cn radicals , and of f and cl atoms ) , but this is our first observation that both nascent products can be vibrationally hot . the cn radicals were generated photolytically from icn , and take 2.3 ps to equilibrate with the solvent and form cn cdcl3 complexes . in the reactions with acetone , the complexed cn radicals react more than an order of magnitude more slowly , but the kinetics of reactions of the free cn radicals are partly controlled by in - cage geminate recombination with i atoms , and solvent - complex formation . the cn radicals also complex to acetone molecules , and c = n carbonyl interactions thermodynamically favor these species over cn evidence from our measurements suggests that the cn radicals complexed with acetone have significantly reduced reactivities . the combination of time - resolved electronic and vibrational spectroscopies allows us to unravel the rates and dynamics of several competing processes that occur following photoinitiation of radical chemistry in solution .

a large number of animal models , primarily in mice , currently are available for the study of experimental ibd ( table 1 ) . however , the majority of these models are generated by either chemical or immunologic manipulation , or gene targeting , and therefore do not fully resemble the multifactorial nature of the human condition . in 1985 , warren strober proposed the characteristics of an ideal animal model for ibd , stating that the model should develop disease that is identical to human ibd , with the same casual factors , pathology , and clinical spectrum . it should occur in an animal that is accessible and inexpensive , with a defined genetic background , and a similar immune system to that in human beings . in addition , the disease should be able to be manipulated by diet , immunologic status , infection , and various forms of treatment . although not all available models meet these standards , each has specific utility for different types of investigations and research questions . in this context , however , the samp1/yitfc mouse model of cd - like ileitis fulfills most of the desired criteria and represents an ideal model to study ibd.table 1mouse models for the study of experimental ibdmode of generation and modelinflammation phenotype / relevance to human ibdchemical induction advantages : easy to induce , inexpensive disadvantages : lack of reproducibility owing to different protocols , diminished pathogenic relevance to the human condition dss78 , 79tissue / epithelial injury and repair dnbs and tnbs81 , 82acute and chronic colitis ( delayed hypersensitivity ) acetic acidacute colitis / motility oxazoloneacute th2-mediated colitis pg - psacute and chronic colitiscell transfer advantages : chronic features of intestinal inflammation , ability to investigate the role of specific t - cell subsets disadvantages : use of immunodeficient mice cd45rbscid cd8scid , samp1/yitfc cd4 scidchronic ileitis and colitis / t - cell homing ecovascidsubacute colitis bone marrow chimera / ragisolation of inflammatory defects to hematopoietic / nonhematopoietic compartmentsgenetically engineered advantages : ability to determine the role of specific genetic mutations , ability to target specific cell types disadvantages : diminished pathogenic relevance owing to lack of single - gene deletion in human disease conventional knock - out il10 , il2 , jak3 , wasp , a20 , tlr5 , tcrchronic colitis mdr1a and il2ruc - like colitis ship-1cd - like granulocyte and monocyte drive ileitis , lung inflammation tgfchronic colitis and multi - organ failure runx3chronic colitis , gastric lesions keratin 8th2-driven colitis muc2epithelial - driven colitis conditional knock - out myeloid / stat-3 and cd4/pdk1chronic colitis epi / c1galt1epithelial - driven uc - like colitis epi / xbp1epithelial - driven acute ileitis epi / faddepithelial - driven colitis epi / casp8terminal ileitis epi / n - cadherincd - like ileitis conventional transgenic il7 transgenicchronic colitis t - bet / rag2uc - like colitis , dendritic cell , and tnf- driven anti - cd40/ragacute innate immune - mediated colitis tnfacute and chronic cd - like ileitis , extra - intestinal manifestations ( skin rashes , arthralgia)congenic advantages : spontaneous multifactorial disease with increased pathogenic relevance to the human condition disadvantages : poor breeding ability , increased cost of colony maintenance , and specific etiology unknown samp1/yitspontaneous acute and chronic cd - like ileitis , extra - intestinal manifestations ( skin lesions ) samp1/yitfcspontaneous acute and chronic cd - like ileitis , extra - intestinal manifestations ( skin lesions ) , early onset , perianal disease ( 5% ) , stricturing c3h / hejbirspontaneous colitiscasp8 , caspase 8 ; c1 galt1 , core 1 synthase glycoprotein - n - acetylgalactosyltransferase 1 ; dnbs , dinitrobenzene sulfonic acid ; ecova , e. coli - expressing ova peptide ; epi , epithelial cell ; fadd , fas - associated via death domain ; iec , intestinal epithelial cell ; jak3 , janus kinase 3 ; mdr1a , multi - drug resistance protein 1a ; muc2 , mucin 2 ; pdk1 , phosphoinositide dependent protein kinase-1 ; pg - ps , peptidoglycan - polysaccharide ; rag , recombination - activating gene ; runx3 , runt related transcription factor 3 ; scid , severe combined immunodeficient ; ship-1 , sh2-containing inositol phosphatase-1 ; stat3 , signal transducer and activator of transcription 3 ; tcr , t - cell receptor ; tgf , transforming growth factor ; tlr , toll - like receptor ; wasp , wiskott - aldrich syndrome protein ; xbp1 , x - box binding protein 1 . mouse models for the study of experimental ibd casp8 , caspase 8 ; c1 galt1 , core 1 synthase glycoprotein - n - acetylgalactosyltransferase 1 ; dnbs , dinitrobenzene sulfonic acid ; ecova , e. coli - expressing ova peptide ; epi , epithelial cell ; fadd , fas - associated via death domain ; iec , intestinal epithelial cell ; jak3 , janus kinase 3 ; mdr1a , multi - drug resistance protein 1a ; muc2 , mucin 2 ; pdk1 , phosphoinositide dependent protein kinase-1 ; pg - ps , peptidoglycan - polysaccharide ; rag , recombination - activating gene ; runx3 , runt related transcription factor 3 ; scid , severe combined immunodeficient ; ship-1 , sh2-containing inositol phosphatase-1 ; stat3 , signal transducer and activator of transcription 3 ; tcr , t - cell receptor ; tgf , transforming growth factor ; tlr , toll - like receptor ; wasp , wiskott - aldrich syndrome protein ; xbp1 , x - box binding protein 1 . animal models that use noxious chemicals to induce colitis ( ie , dextran sodium sulfate [ dss ] , trinitrobenzene sulfate [ tnbs ] , acetic acid , oxazolone , and so forth ) are used most commonly because they are accessible , inexpensive , and relatively easy to induce . however , the resulting inflammation is generally less representative of the specific immunohistopathology present in the inflamed colons of patients with uc or crohn s colitis . chemically induced animal models of colitis can be quite useful , however , for the study of acute colonic tissue injury and repair mechanisms , or when paired with genetically engineered mice , to study the role of a targeted gene , or gene product , in mediating colitis . ibd is a t - cell mediated disease that involves the recruitment of lymphocytes to the inflamed gut mucosa , evidenced by the clinical effectiveness of vedolizumab , a recently approved drug for the treatment of ibd that specifically targets gut - homing lymphocytes by blocking their migration to the intestine.12 , 13 animal models that involve adoptive transfer of immune cells to induce colitis in recipient immunodeficient mice ( eg , severe combined immunodeficient or recombination - activating gene ) , which lack b and t cells , are powerful tools for understanding the role of specific lymphocyte subpopulations in either promoting or preventing colitis . bone marrow chimeric models similarly pair irradiated mice with adoptive transfer experiments to localize the cellular compartments ( hematopoietic vs nonhematopoietic ) responsible for conferring disease . by their nature , genetically engineered animal models ( the majority of which artificially alter only a single gene or locus ) do not likely fully represent the underlying biological conditions that lead to ibd pathogenesis in human beings . however , such models can be extremely useful for understanding the functional role of a specific gene product in contributing to , or protecting against , experimental ibd . for example , introduction of a deletion for the gene encoding fibroblast growth factor inducible-14 , the receptor for tnf - like weak inducer of apoptosis , a tnf superfamily member involved in intestinal tissue repair and innate and adaptive immunity , into c57bl/6 mice enhances their susceptibility to dss - induced colitis , suggesting that the tnf - like weak inducer of apoptosis / fibroblast growth factor inducible-14 signaling pathway may play a protective role in mitigating acute colonic tissue injury and repair . the vast majority of animal models of ibd only develop inflammation within the colon , however , active cd most commonly occurs in the terminal ileum and genome - wide association studies now provide evidence that crohn s ileitis may , in fact , represent a genetically distinct form of ibd . two mouse models have been reported to develop crohns - like ileitis : the inbred samp1/yitfc strain and genetically engineered tnf mice . both strains show striking similarities to active crohn s ileitis , making them particularly relevant to the human condition and valuable resources for furthering cd research . the spontaneous , inbred samp1/yitfc mouse strain originally was derived from selective brother sister mating of parental akr / j mice ( the jackson laboratory , bar harbor , me ) . after 24 generations , the mice developed a senescence - accelerated phenotype , yielding 10 lines of senescence - prone mice ( ie , samp 110 ) that showed accelerated aging , skin lesions , and autoimmune dysfunctions . sister mating based on the presence of skin lesions and ileitis . by the 20th generation , the strain had lost its senescence - accelerated phenotype , and consistently showed both acute and chronic transmural small intestinal inflammation with nearly 100% penetrance by 3040 weeks of age , and persisting up to 80 weeks of age . the disease was concentrated within the terminal ileum and cecum and manifested in a discontinuous pattern , similar to that seen in human cd . continuous inbreeding of a samp1/yit colony led to the development of a unique samp1/yitfc substrain , with a novel phenotype that included an earlier emergence of ileitis by 10 weeks of age , preceded by increased levels of intestinal interferon ( ifn) production by 4 weeks of age , and the development of perianal fistulae in 5%10% of the colony , as well as intestinal strictures in nearly 50% of mice older than 40 weeks of age.17 , 18 of note , perianal disease and fibrostenotic / stricturing are both hallmark features of human cd that are not observed commonly in animal models of ibd . samp1/yitfc ileitis is characterized by areas of discontinuous cobble - stoning with transmural inflammation dispersed between areas of normal gut mucosa within the terminal ileum and cecum , similar to the patchy nature of cd ( figure 1 ) . early alterations in epithelial architecture and defective barrier function are present by 3 weeks of age , preceding the onset of overt ileitis , and include increased numbers of secretory paneth , goblet , and intermediate cells , with a simultaneous reduction in absorptive enterocytes . this trend increases at sites of active inflammation as the animals age and as the disease becomes more severe.20 , 21 ileal epithelial barrier dysfunction leads to infiltration of active and chronic immune cells that populate the intestinal lamina propria , and include both polymorphonuclear and mononuclear cells.17 , 18 in fact , chronic samp1/yitfc ileitis shows large numbers of activated th1-type polarized cd4 cells within the lamina propria that produce increased levels of intestinal tnf and ifn , as well as increased numbers of cd8 t - cell receptor ( tcr) t cells . likewise , the intraepithelial lymphocytic compartment also is shifted toward cd8tcr cells and away from cd8tcr cells . the majority of samp1/yitfc mice with advanced disease ( age , > 40 wk ) show thickening of the bowel wall that often leads to collagen deposition and terminal ileal stricture formation ( figure 1 ) , as well as focal granulomatous inflammation , basal plasmacytosis , and neural hyperplasia . interestingly , samp1/yitfc mice also show extraintestinal manifestations of cd ( eg , skin rashes and inflammation of the eye ) , as well as pathologies similar to human perianal cd , including anal fissures , rectal prolapse , and perianal fistulae . samp1/yitfc mice also have been reported to develop crohns - like gastritis ( in the absence of helicobacter infection ) , autoimmune hepatitis , and periodontitis.figure 1characteristics of chronic intestinal inflammation in samp1/yitfc mice . ( a ) normal endoscopic appearance of colonic mucosa in akr control mice showing a normal vascular pattern . ( b ) endoscopic appearance of severe colitis in samp1/yitfc mice showing friable mucosa with hemorrhage and ulcerations . ( c ) normal stereomicroscopic ileal appearance of a 20-week - old akr control mouse with intact mucosa . ( d ) higher magnification shows normal structure of intestinal villi with no visible lesions . ( e ) histologic image of d ( black dot ) shows normal histologic morphology of intestinal villi and mucosa . ( f ) abnormal stereomicroscopic ileal appearance of age - matched samp1/yitfc mouse showing the presence of cobblestone lesions . ( h ) histologic image of panel g ( red dot ) shows distorted and blunted villi with severe inflammatory cell infiltrate in mucosa . ( a ) normal endoscopic appearance of colonic mucosa in akr control mice showing a normal vascular pattern . ( b ) endoscopic appearance of severe colitis in samp1/yitfc mice showing friable mucosa with hemorrhage and ulcerations . ( c ) normal stereomicroscopic ileal appearance of a 20-week - old akr control mouse with intact mucosa . ( d ) higher magnification shows normal structure of intestinal villi with no visible lesions . ( e ) histologic image of d ( black dot ) shows normal histologic morphology of intestinal villi and mucosa . ( f ) abnormal stereomicroscopic ileal appearance of age - matched samp1/yitfc mouse showing the presence of cobblestone lesions . ( h ) histologic image of panel g ( red dot ) shows distorted and blunted villi with severe inflammatory cell infiltrate in mucosa . samp1/yitfc ileitis can be considered a truly spontaneous model of crohn s ileitis because the disease presents independent of any need for genetic , chemical , or immunologic manipulation , and closely resembles the human condition with regard to location , histologic features , and extra - intestinal manifestations . the disease occurs in virtually 100% of mice by 10 weeks of age and persists for up to 80 weeks . unknown host - microbial interactions amplify the severity of disease because samp1/yitfc mice reared under germ - free conditions still develop an attenuated ileitis , but exposure to the natural flora is necessary for its full manifestation . adoptive transfer experiments and cytokine blockade studies have shown that the disease also requires the presence of t cells and proinflammatory cytokines.17 , 26 finally , the disease is responsive to standard cd therapies , such as anti - tnf drugs and steroids , and also responds to probiotics for disease prevention , further supporting its use as a highly relevant model for understanding the pathogenic mechanisms that underlie the development and perpetuation of cd . because of the spontaneous and multifactorial nature of this model , samp1/yitfc mice can be used to characterize specific new biomarkers , including microbiome and metabolomic markers , which may be important for monitoring drug response and management . one disadvantage of this model is the poor breeding ability of this strain , requiring a large breeding colony for yielding experimental mice and associated costs compared with other ibd models . tnf is a central mediator of intestinal inflammation and ibd , most notably evidenced by the clinical effectiveness of anti - tnf monoclonal antibodies in treating both cd and uc.29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 the tnf mouse model of crohns - like ileitis originally was developed to understand the mechanisms of tnf - driven crohns - like ibd and chronic inflammatory arthritis . mice were generated by introducing a 69-bp targeted deletion of the are of tnf into sv/129-c57bl/6 mice . the au - rich element is an area of adenosine - uracil nucleotide repeats ( auuua ) located in the 3 untranslated region of a gene , and is responsible for messenger rna destabilization and translational silencing . interestingly , mice with intestinal - specific deletion of fas - associated via death domain and caspase-8 , which are important regulators of tnf , develop small intestinal inflammation and terminal ileitis,38 , 39 highlighting the critical role of tnf in mediating chronic small intestinal inflammation . mice homozygous for the deleted au - rich element ( tnf ) overexpress tnf messenger rna and protein , and develop early severe inflammatory disease , limiting their lifespan to only 512 weeks ( figure 1 ) . heterozygous tnf mice develop chronic arthritis and crohns - like ileitis , with a normal lifespan and sporadic evidence of mild liver and lung inflammation , as well as occasional proximal colitis . the intestinal disease in tnf heterozygotes includes early villous blunting , with severe patchy terminal ileitis by 8 weeks of age and acute and chronic transmural inflammation by 16 weeks of age ; older mice ( age , 57 mo ) show a loss of villous architecture and the development of granulomas , similar to that seen in patients with severe active cd ( figure 2 ) . unlike the associated arthritis , tnf ileitis is dependent on the presence of mature b and t cells , suggesting differential mechanisms of disease within the 2 organ systems . moreover , the intestinal disease is driven by t - helper ( th)1 cytokines ( il12 , ifn ) and cytotoxic cd8 effector t cells.figure 2characteristics of chronic intestinal inflammation in tnfmice . ( a ) phenotypic appearance of 8-week - old tnf littermates . tnf mice ( a ) have severe wasting syndrome , ruffled coat , alopecia - like lesions , impairment in mobility , and increased mortality as compared with tnf ( b ) or tnf mice ( c ) . ( b ) total inflammatory scores ( calculated as the sum of panels c f ) show severe ileitis in tnf mice and moderate ileitis in tnf compared with tnf littermates . ( c ) villous distortion index , ( d ) active inflammation index , ( e ) chronic inflammation index , and ( f ) transmural inflammatory index are increased significantly in tnf and tnf vs tnf littermates ( * p < .001 ) ; n = 12/group . h&e - stained sections of the terminal ileum from 8-week - old mice show increased intestinal inflammation in ( i ) tnf and ( h ) tnf as compared with ( g ) tnf mice . tnf mice ( a ) have severe wasting syndrome , ruffled coat , alopecia - like lesions , impairment in mobility , and increased mortality as compared with tnf ( b ) or tnf mice ( c ) . ( b ) total inflammatory scores ( calculated as the sum of panels c f ) show severe ileitis in tnf mice and moderate ileitis in tnf compared with tnf littermates . ( c ) villous distortion index , ( d ) active inflammation index , ( e ) chronic inflammation index , and ( f ) transmural inflammatory index are increased significantly in tnf and tnf vs tnf littermates ( * p < .001 ) ; n = 12/group . h&e - stained sections of the terminal ileum from 8-week - old mice show increased intestinal inflammation in ( i ) tnf and ( h ) tnf as compared with ( g ) tnf mice . the samp1/yitfc and tnf mouse models are particularly useful for understanding mechanisms of ibd pathogenesis that may uniquely lead to the development of crohn s ileitis . we summarize some of the important findings from studies performed using these 2 models , and how they have contributed to our general understanding of experimental crohn s ileitis and its application to the human condition . cd traditionally was regarded as an adaptive immune disease , primarily driven by th1-type immune responses . by using samp1/yitfc mice , bamias et al tested the hypothesis that the natural course of ileitis may be characterized instead by multiple immunologically distinct phases , and that the th2 pathway also may be important during the maintenance phase of ileitis . histologic analysis showed that a distinct natural course of samp1/yitfc ileitis exists with an early inductive phase of acute ileitis present from 4 to 8 weeks of age that correlates with increased production of the th1-type cytokines , tnf and ifn , followed by a chronic phase that begins after 8 weeks of age that is associated with continued increased production of th1 cytokines , but also increased levels of the th2 cytokines il4 and il5 by 23 weeks of age . recent studies have investigated the role of il12/il23 and il17 blockade as a novel therapy for ibd , leading to the recent approval of a monoclonal antibody against p40 ( ustekinumab ) for the treatment of cd . interestingly , blockade of il17 had no effects and a higher rate of adverse events in a randomized controlled trial of patients with cd . of note , the role of il12/il23 and il17 in samp1/yitfc and tnf mice have not been studied fully , and it would be interesting to see whether blockade of either il23 or il17 in these models correlates with the reported results in patients with cd . the precise regulation of the intestinal barrier allows the maintenance of mucosal immune homeostasis and prevents the onset of uncontrolled inflammation . in support of this concept , several lines of evidence point to defects in components of the epithelium as etiologic factors in the pathogenesis of ibd . for example , early samp1/yitfc ileitis results from an underlying defect in epithelial barrier function . by using bone marrow chimera experiments in samp1/yitfc and akr / j control mice , olson et al discovered a primary defect in samp1/yitfc mice attributed to nonhematopoietic cellular sources , and found decreased epithelial barrier resistance and increased epithelial permeability before the onset of ileitis , suggesting early epithelial barrier dysfunction that precedes the development of disease . moreover , additional bone marrow chimera experiments showed that hematopoietic cells isolated from akr / j control mice were capable of inducing ileitis in the presence of abnormal samp1/yitfc epithelium . the effect occurred in the absence of a commensal flora and was supported by differential expression of tight junction proteins , claudin-2 and occludin , in ileal epithelial cells from samp1/yitfc compared with akr / j mice . lopetuso et al recently showed that samp1/yitfc mice express increased ileal epithelial toll - like receptor 5 and serum antiflagellin igg antibodies , which both were augmented further in the presence of the gut microbiome . toll - like receptor 5 activation in response to bacterial flagellin , in fact , altered expression of tight junction proteins and resulted in an inability to maintain appropriate epithelial barrier integrity of ex vivo stimulated ileal tissues from samp1/yitfc mice . in tnf mice , 2 separate reports clearly show the ability of tnf , specifically overexpressed in the intestinal epithelium , to sufficiently generate chronic ileitis in the absence of extra - intestinal manifestations , and point out the central role of the gut epithelium in this model.45 , 46 this concept is supported further by marini et al , who identified that decreased intestinal epithelial apoptosis was a key mechanism of action of anti - tnf therapy in samp1/yitfc mice . the role of mucosal healing and resolution of inflammation is an active area of investigation in the field of ibd pathogenesis . samp1/yitfc and tnf mice develop crohns - like ileitis in the absence of spontaneous colonic inflammation , making them very useful models to study the contribution of mucosal healing in the presence of immunologic and genetic dysfunction , independent of active colonic inflammation . in fact , corridoni et al recently showed that induction of acute colitis by dss administration in samp1/yitfc mice generated more severe and prolonged colitis compared with akr control mice , suggesting that mucosal healing and impaired resolution of inflammation may be important in this model . further studies are warranted to determine the precise potential dysfunction of mucosal healing in both models and its contribution to the pathogenesis of ibd . one of the major recent advances in the treatment of ibd has been the approval of agents that specifically block leukocyte trafficking to the gut , such as vedolizumab . seminal work using both samp1/yitfc and tnf mice have shown the importance of blocking leukocyte trafficking to the inflamed intestinal mucosa , and the critical role of adhesion molecules , such as endothelial psgl1 , ccl25/ccr9 , 47 , ccl21 , and s1p1 in the pathogenesis of ibd . card15/nod2 ( also known as ibd1 ) is a gene associated with cd in human beings.7 , 8 the gene product , nod2 , is an intracellular pattern recognition receptor and key mediator of intestinal innate immunity through sensing of its ligand , bacterial muramyl dipeptide ( mdp ) , and initiation of nf-b dependent immune responses within the gut . the identification of nod2 generated a fundamental shift in understanding cd pathogenesis , with a new focus on mechanisms of innate immunity and host - microbial interactions within the gut . interestingly , nod2 genetic deletion in samp1/yitfc mice significantly suppresses chronic ileitis by approximately 50% , supporting the concept of a dichotomous role of nod2 in early vs late ( chronic ) phases of intestinal inflammation . of note , only 10%15% of crohn s patients actually possess mutations within the nod2 gene , raising the question of whether the remaining 85% of patients actually experience a functional defect in their mdp / nod2 pathway despite having a normal nod2 gene . samp1/yitfc mice represent a suitable model system to address this question because these mice also lack nod2 mutations.55 , 56 mdp administration is known to prevent the development of dss colitis in normal mice . however , treatment of samp1/yitfc mice with mdp before dss colitis induction has no mitigating effects , and deletion of nod2 in samp1/yitfc mice causes a decrease in the severity of ileitis , supporting the concept that a functional defect related to the nod2/mdp pathway contributes to crohns - like ileitis in samp1/yitfc , despite their wild - type nod2 status . because samp1/yitfc mice are one of the only animal models of ibd that develops intestinal inflammation spontaneously without genetic , chemical , or immunologic manipulation , these mice can provide insights into the possible genetic determinants of cd in human beings . a genome - wide scan of samp1/yitfc mice identified 4 susceptibility loci on chromosomes 9 , 6 , 8 , and x. the loci on chromosome 9 ( ibdq1 ) was linked strongly to epithelial changes in samp1/yitfc mice and contains several potential candidate genes that may play a role in promoting epithelial abnormalities during crohns - like ileitis . interestingly , the ibdq1 region overlaps with 2 chemically induced colitis susceptibility loci ( dss and tnbs1 ) , as well as the genes that encode il10-receptor antagonist and il18 , an epithelial - derived th1-polarizing cytokine that has protective effects in tnbs - induced murine colitis . the loci on chromosome 6 ( ibdq2 ) also was linked to ileitis and includes a homolog for the human chr3 ( p21-p26 ) region in which several genome - wide association studies have identified cd susceptibility loci.60 , 61 , 62 , 63 , 64 the remaining 2 loci on chromosomes 8 and x ( ibdq3 and ibdq4 , respectively ) appear to associate with epithelial changes in samp1/yitfc ileitis . linkage with ibdq3 only occurs in young mice ( age , 1012 wk ) , suggesting that this region may include susceptibility loci involved in the early / acute phase of disease . the ibdq2 region also contains the gene that encodes peroxisome proliferation - activated receptor ( ppar) , a nuclear receptor that is highly expressed by intestinal epithelial cells and inhibits innate immune responses through suppression of nf-b dependent signaling . sugawara et al reported that ppar expression levels were reduced in the ilea of samp1/yitfc mice compared with akr / j controls , and the magnitude of this difference correlated with age and the severity of ileitis . a mutation in the gene encoding ppar could provide an explanatory mechanism for the observed functional abnormality in the nod2 pathway in samp1/yitfc mice despite the fact that these mice lack any nod2 mutations because ppar and nod2 may cooperatively regulate nf-b dependent signaling and innate intestinal immune responses . after the advent of new methodologies , genome - wide sequencing of samp1/yitfc mice has been performed by our group and the results will be available in the near future . the development of advanced murine imaging technologies and validated scoring systems for quantifying the extent of intestinal inflammation has the potential to greatly enhance the quality and generalizability of data obtained from animal studies in ibd . kodani et al used samp1/yitfc mice to develop and validate murine endoscopy as a useful technology for assessing intestinal inflammation without killing the animal . this nonlethal procedure allows comparative assessments of endoscopic inflammation within a single animal ( figure 1 ) . an accompanying validated scoring system allows investigators to systematically quantify the severity of inflammation and the presence of tumors within the murine intestine in a manner that is generalizable across laboratories and studies , as well as within a particular experiment . another newly developed technology for assessing intestinal inflammation in mice uses stereomicroscopic analysis of the 3-dimensional intestinal architecture in conjunction with localized myeloperoxidase activity levels to characterize the microscopic features and quantify levels of intestinal inflammation . by using this method , rodriguez - palacios et al found samp1/yitfc mice showed unique 3-dimensional stereomicroscopic features ( cobble - stoning ) within their inflamed intestines that was not seen in tnf mice ; cobblestone lesions are also a signature pathologic feature reported in the inflamed intestinal tissues of patients with cd . mucosal addressin cellular adhesion molecule-1 antibodies conjugated to encapsulated gaseous microbubbles have been used successfully to detect and quantify ileal inflammation in both the samp1/yitfc and tnf mice . in addition , our laboratories also are involved in developing novel noninvasive magnetic resonance imaging techniques to quantify intestinal inflammation in both samp1/yitfc and tnf mice . initial studies of il1 blockade in rabbit formalin - induced colitis clearly showed the power of animal models to decipher the role of cytokines in mediating intestinal inflammation.9 , 68 given their spontaneous nature and marked similarities to the human condition , both the samp1/yitfc and tnf models have proven highly useful for evaluating the role of novel cytokines in experimental crohns - like ileitis . tnf - like ligand 1a ( tl1a ) is a member of the tnf superfamily of proteins that binds to death receptor 3 ( dr3 ) and provides co - stimulatory signals to activated t lymphocytes . inflamed intestinal tissues from patients with cd produce increased tl1a levels , specifically antigen - presenting cells within the intestinal lamina propria and cd4 and cd8 lymphocytes ; in vitro studies have shown that tl1a induces ifn production by activated lamina propria mononuclear cells isolated from cd patients . to better understand the pathogenic mechanisms related to this novel cytokine and its receptor in cd , bamias et al used samp1yit / fc and tnf mice to study the role of the tl1a / dr3 signaling complex during spontaneous experimental crohns - like ileitis . increased levels of both tl1a and the active transmembrane form of dr3 were found in the inflamed mucosa of both strains . specifically , tl1a was expressed by cd11c dendritic cells within the lamina propria and induced proliferation of memory cd4cd45rb t cells , but not cd4cd45rb cells . the tl1a / dr3 signaling complex synergized with il12/il18 signaling to induce ifn production by activated t lymphocytes , promoting th1 immune responses and ileitis . samp1/yitfc ileitis shows a dichotomous immune profile , with th1 immune responses driving the early acute phase and th2 immune responses present during chronic ileitis ( after 8 weeks of age ) . il33 is a th2-polarizing cytokine that is increased in the intestinal epithelium of patients with uc , and recently was found to be increased in a subpopulation of pediatric patients with stricturing crohn s ileitis . to determine whether il33 plays a protective or pathogenic role in ibd , de salvo et al used samp1yit / fc mice to study the role of il33 within the context of chronic intestinal inflammation . samp1/yitfc ileitis was associated with a large infiltration of eosinophils by 12 weeks of age , which persisted through 20 weeks of age and correlated with disease severity and production of th2 cytokines ( ie , il33 and il5 ) and eotaxins . il33 levels correlated positively with the level of inflammation and the number of invading eosinophils , and blockade of il33 resulted in decreased inflammation and eosinophils , as well as reduced th2 cytokine production , suggesting a proinflammatory role for il33 within the context of chronic experimental ibd . interestingly , induction of il33 was dependent on interaction with the gut microbiome , and fecal transplantation from specific pathogen - free samp1/yitfc mice donors into germ - free raised samp1/yitfc recipients restored increased il33 , as well as eosinophilia , in these mice . the vast majority of animal models of ibd do not develop inflammation when reared under germ - free conditions , strongly implicating the gut microbiome as a contributor to ibd pathogenesis . however , bamias et al showed that germ - free samp1/yitfc mice develop ileitis with varying degrees of severity , the majority showing a milder form of the disease , confirming the importance of the gut microbiome as a modulating factor of chronic intestinal inflammation . the absence of a commensal flora appears preferentially to impact the th2-driven chronic phase of samp1/yitfc ileitis , with significantly reduced levels of il33 , and downstream il5 and il13 at 13 weeks of age , and decreased chronic inflammatory scores through 30 weeks of age , providing further evidence that interactions with the gut microbiome drive production of th2 cytokine production during the chronic phase of samp1/yitfc ileitis.26 , 73 the gut microbiome also appears to influence the function of regulatory t cells during samp1/yitfc ileitis . mesenteric lymph nodes from germ - free samp1/yitfc mice have decreased numbers of cd4cd25foxp3 regulatory t cells . moreover , cd4 lymphocytes from germ - free mice are able to confer disease to severe combined immunodeficient recipients , unlike cd4 cells derived from spf samp1/yitfc mice , suggesting that absence of exposure to the gut microbiome compromises normal development of the regulatory component of the cd4 t - cell population in samp1/yitfc mice . this finding is supported further by studies in samp1/yitfc mice showing dysfunction within their mesenteric lymph node regulatory t - cell compartment . studies performed in tnf mice also confirm the importance of host - gut microbiome interactions and dysbiosis in the pathogenesis of cd - like ileitis.75 , 76 schaubeck et al recently showed that germ - free tnf mice were free of intestinal inflammation . in addition , 16s analysis and metaproteomics showed specific compositional and functional alterations of bacterial communities in inflamed mice . finally , transplantation of disease - associated , but not healthy , microbiota transmitted cd - like ileitis to germ - free tnf recipients . in comparative studies , roulis et al showed that defective expression of antimicrobials and dysbiosis are characteristic of tnf - driven cd - like ileitis . in addition , they showed that indigenous microbiota is sufficient to drive tnf overexpression and cd - like ileitis in this model . mouse models of ibd are extremely useful tools for exploring pathogenic mechanisms of chronic intestinal inflammation . the many available models vary by the type of inflammation that they produce ( acute vs chronic ) , their mode of generation ( chemical , genetic , immunologic , or spontaneous ) , and whether they show colitis or ileitis . however , to understand pathogenic mechanisms that are relevant to the human condition and preclinical testing of novel treatments , spontaneous models , such as samp1/yitfc mice , offer a unique advantage and strong scientific premise . with continuing advances in targeted drug technologies , important mechanistic findings from animal studies now can be translated readily into clinical development of novel therapies to combat these devastating diseases .

crohn s disease and ulcerative colitis , together known as inflammatory bowel disease , are debilitating chronic disorders of unknown cause and cure . our evolving understanding of these pathologies is enhanced greatly by the use of animal models of intestinal inflammation that allow in vivo mechanistic studies , preclinical evaluation of new therapies , and investigation into the causative factors that underlie disease pathogenesis . several animal models , most commonly generated in mice , exist for the study of colitis . the appropriateness of their use often can be determined by their mode of generation ( ie , chemical induction , t - cell transfer , targeted genetic manipulation , spontaneously occurring , and so forth ) , the type of investigation ( mechanistic studies , pathogenic experiments , preclinical evaluations , and so forth ) , and the type of inflammation that occurs in the model ( acute vs chronic colitis , tissue injury / repair , and so forth ) . although most murine models of inflammatory bowel disease develop inflammation in the colon , crohn s disease most commonly occurs in the terminal ileum , where a very limited number of mouse models manifest disease . this review discusses appropriate experimental applications for different mouse models of colitis , and highlights the particular utility of 2 highly relevant models of crohns - like ileitis the spontaneous samp1/yitfc inbred mouse strain and the genetically engineered tnfau - rich element/+ mouse model of tumor necrosis factor overexpression , both of which bear strong resemblance to the human condition . similar to patients with crohn s disease , samp1/yitfc ileitis develops spontaneously , without chemical , genetic , or immunologic manipulation , making this model particularly relevant for studies aimed at identifying the primary defect underlying the occurrence of crohn s ileitis , as well as preclinical testing of novel treatment modalities .

Animal Models of IBD Mouse Models of CD-Like Ileitis Pathogenic Mechanisms of Crohns-Like Ileitis Conclusions

a large number of animal models , primarily in mice , currently are available for the study of experimental ibd ( table 1 ) . however , the majority of these models are generated by either chemical or immunologic manipulation , or gene targeting , and therefore do not fully resemble the multifactorial nature of the human condition . in this context , however , the samp1/yitfc mouse model of cd - like ileitis fulfills most of the desired criteria and represents an ideal model to study ibd.table 1mouse models for the study of experimental ibdmode of generation and modelinflammation phenotype / relevance to human ibdchemical induction advantages : easy to induce , inexpensive disadvantages : lack of reproducibility owing to different protocols , diminished pathogenic relevance to the human condition dss78 , 79tissue / epithelial injury and repair dnbs and tnbs81 , 82acute and chronic colitis ( delayed hypersensitivity ) acetic acidacute colitis / motility oxazoloneacute th2-mediated colitis pg - psacute and chronic colitiscell transfer advantages : chronic features of intestinal inflammation , ability to investigate the role of specific t - cell subsets disadvantages : use of immunodeficient mice cd45rbscid cd8scid , samp1/yitfc cd4 scidchronic ileitis and colitis / t - cell homing ecovascidsubacute colitis bone marrow chimera / ragisolation of inflammatory defects to hematopoietic / nonhematopoietic compartmentsgenetically engineered advantages : ability to determine the role of specific genetic mutations , ability to target specific cell types disadvantages : diminished pathogenic relevance owing to lack of single - gene deletion in human disease conventional knock - out il10 , il2 , jak3 , wasp , a20 , tlr5 , tcrchronic colitis mdr1a and il2ruc - like colitis ship-1cd - like granulocyte and monocyte drive ileitis , lung inflammation tgfchronic colitis and multi - organ failure runx3chronic colitis , gastric lesions keratin 8th2-driven colitis muc2epithelial - driven colitis conditional knock - out myeloid / stat-3 and cd4/pdk1chronic colitis epi / c1galt1epithelial - driven uc - like colitis epi / xbp1epithelial - driven acute ileitis epi / faddepithelial - driven colitis epi / casp8terminal ileitis epi / n - cadherincd - like ileitis conventional transgenic il7 transgenicchronic colitis t - bet / rag2uc - like colitis , dendritic cell , and tnf- driven anti - cd40/ragacute innate immune - mediated colitis tnfacute and chronic cd - like ileitis , extra - intestinal manifestations ( skin rashes , arthralgia)congenic advantages : spontaneous multifactorial disease with increased pathogenic relevance to the human condition disadvantages : poor breeding ability , increased cost of colony maintenance , and specific etiology unknown samp1/yitspontaneous acute and chronic cd - like ileitis , extra - intestinal manifestations ( skin lesions ) samp1/yitfcspontaneous acute and chronic cd - like ileitis , extra - intestinal manifestations ( skin lesions ) , early onset , perianal disease ( 5% ) , stricturing c3h / hejbirspontaneous colitiscasp8 , caspase 8 ; c1 galt1 , core 1 synthase glycoprotein - n - acetylgalactosyltransferase 1 ; dnbs , dinitrobenzene sulfonic acid ; ecova , e. coli - expressing ova peptide ; epi , epithelial cell ; fadd , fas - associated via death domain ; iec , intestinal epithelial cell ; jak3 , janus kinase 3 ; mdr1a , multi - drug resistance protein 1a ; muc2 , mucin 2 ; pdk1 , phosphoinositide dependent protein kinase-1 ; pg - ps , peptidoglycan - polysaccharide ; rag , recombination - activating gene ; runx3 , runt related transcription factor 3 ; scid , severe combined immunodeficient ; ship-1 , sh2-containing inositol phosphatase-1 ; stat3 , signal transducer and activator of transcription 3 ; tcr , t - cell receptor ; tgf , transforming growth factor ; tlr , toll - like receptor ; wasp , wiskott - aldrich syndrome protein ; xbp1 , x - box binding protein 1 . mouse models for the study of experimental ibd casp8 , caspase 8 ; c1 galt1 , core 1 synthase glycoprotein - n - acetylgalactosyltransferase 1 ; dnbs , dinitrobenzene sulfonic acid ; ecova , e. coli - expressing ova peptide ; epi , epithelial cell ; fadd , fas - associated via death domain ; iec , intestinal epithelial cell ; jak3 , janus kinase 3 ; mdr1a , multi - drug resistance protein 1a ; muc2 , mucin 2 ; pdk1 , phosphoinositide dependent protein kinase-1 ; pg - ps , peptidoglycan - polysaccharide ; rag , recombination - activating gene ; runx3 , runt related transcription factor 3 ; scid , severe combined immunodeficient ; ship-1 , sh2-containing inositol phosphatase-1 ; stat3 , signal transducer and activator of transcription 3 ; tcr , t - cell receptor ; tgf , transforming growth factor ; tlr , toll - like receptor ; wasp , wiskott - aldrich syndrome protein ; xbp1 , x - box binding protein 1 . animal models that use noxious chemicals to induce colitis ( ie , dextran sodium sulfate [ dss ] , trinitrobenzene sulfate [ tnbs ] , acetic acid , oxazolone , and so forth ) are used most commonly because they are accessible , inexpensive , and relatively easy to induce . however , the resulting inflammation is generally less representative of the specific immunohistopathology present in the inflamed colons of patients with uc or crohn s colitis . chemically induced animal models of colitis can be quite useful , however , for the study of acute colonic tissue injury and repair mechanisms , or when paired with genetically engineered mice , to study the role of a targeted gene , or gene product , in mediating colitis . ibd is a t - cell mediated disease that involves the recruitment of lymphocytes to the inflamed gut mucosa , evidenced by the clinical effectiveness of vedolizumab , a recently approved drug for the treatment of ibd that specifically targets gut - homing lymphocytes by blocking their migration to the intestine.12 , 13 animal models that involve adoptive transfer of immune cells to induce colitis in recipient immunodeficient mice ( eg , severe combined immunodeficient or recombination - activating gene ) , which lack b and t cells , are powerful tools for understanding the role of specific lymphocyte subpopulations in either promoting or preventing colitis . by their nature , genetically engineered animal models ( the majority of which artificially alter only a single gene or locus ) do not likely fully represent the underlying biological conditions that lead to ibd pathogenesis in human beings . for example , introduction of a deletion for the gene encoding fibroblast growth factor inducible-14 , the receptor for tnf - like weak inducer of apoptosis , a tnf superfamily member involved in intestinal tissue repair and innate and adaptive immunity , into c57bl/6 mice enhances their susceptibility to dss - induced colitis , suggesting that the tnf - like weak inducer of apoptosis / fibroblast growth factor inducible-14 signaling pathway may play a protective role in mitigating acute colonic tissue injury and repair . the vast majority of animal models of ibd only develop inflammation within the colon , however , active cd most commonly occurs in the terminal ileum and genome - wide association studies now provide evidence that crohn s ileitis may , in fact , represent a genetically distinct form of ibd . two mouse models have been reported to develop crohns - like ileitis : the inbred samp1/yitfc strain and genetically engineered tnf mice . both strains show striking similarities to active crohn s ileitis , making them particularly relevant to the human condition and valuable resources for furthering cd research . after 24 generations , the mice developed a senescence - accelerated phenotype , yielding 10 lines of senescence - prone mice ( ie , samp 110 ) that showed accelerated aging , skin lesions , and autoimmune dysfunctions . by the 20th generation , the strain had lost its senescence - accelerated phenotype , and consistently showed both acute and chronic transmural small intestinal inflammation with nearly 100% penetrance by 3040 weeks of age , and persisting up to 80 weeks of age . continuous inbreeding of a samp1/yit colony led to the development of a unique samp1/yitfc substrain , with a novel phenotype that included an earlier emergence of ileitis by 10 weeks of age , preceded by increased levels of intestinal interferon ( ifn) production by 4 weeks of age , and the development of perianal fistulae in 5%10% of the colony , as well as intestinal strictures in nearly 50% of mice older than 40 weeks of age.17 , 18 of note , perianal disease and fibrostenotic / stricturing are both hallmark features of human cd that are not observed commonly in animal models of ibd . samp1/yitfc ileitis is characterized by areas of discontinuous cobble - stoning with transmural inflammation dispersed between areas of normal gut mucosa within the terminal ileum and cecum , similar to the patchy nature of cd ( figure 1 ) . this trend increases at sites of active inflammation as the animals age and as the disease becomes more severe.20 , 21 ileal epithelial barrier dysfunction leads to infiltration of active and chronic immune cells that populate the intestinal lamina propria , and include both polymorphonuclear and mononuclear cells.17 , 18 in fact , chronic samp1/yitfc ileitis shows large numbers of activated th1-type polarized cd4 cells within the lamina propria that produce increased levels of intestinal tnf and ifn , as well as increased numbers of cd8 t - cell receptor ( tcr) t cells . the majority of samp1/yitfc mice with advanced disease ( age , > 40 wk ) show thickening of the bowel wall that often leads to collagen deposition and terminal ileal stricture formation ( figure 1 ) , as well as focal granulomatous inflammation , basal plasmacytosis , and neural hyperplasia . interestingly , samp1/yitfc mice also show extraintestinal manifestations of cd ( eg , skin rashes and inflammation of the eye ) , as well as pathologies similar to human perianal cd , including anal fissures , rectal prolapse , and perianal fistulae . samp1/yitfc mice also have been reported to develop crohns - like gastritis ( in the absence of helicobacter infection ) , autoimmune hepatitis , and periodontitis.figure 1characteristics of chronic intestinal inflammation in samp1/yitfc mice . samp1/yitfc ileitis can be considered a truly spontaneous model of crohn s ileitis because the disease presents independent of any need for genetic , chemical , or immunologic manipulation , and closely resembles the human condition with regard to location , histologic features , and extra - intestinal manifestations . adoptive transfer experiments and cytokine blockade studies have shown that the disease also requires the presence of t cells and proinflammatory cytokines.17 , 26 finally , the disease is responsive to standard cd therapies , such as anti - tnf drugs and steroids , and also responds to probiotics for disease prevention , further supporting its use as a highly relevant model for understanding the pathogenic mechanisms that underlie the development and perpetuation of cd . because of the spontaneous and multifactorial nature of this model , samp1/yitfc mice can be used to characterize specific new biomarkers , including microbiome and metabolomic markers , which may be important for monitoring drug response and management . tnf is a central mediator of intestinal inflammation and ibd , most notably evidenced by the clinical effectiveness of anti - tnf monoclonal antibodies in treating both cd and uc.29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 the tnf mouse model of crohns - like ileitis originally was developed to understand the mechanisms of tnf - driven crohns - like ibd and chronic inflammatory arthritis . mice homozygous for the deleted au - rich element ( tnf ) overexpress tnf messenger rna and protein , and develop early severe inflammatory disease , limiting their lifespan to only 512 weeks ( figure 1 ) . heterozygous tnf mice develop chronic arthritis and crohns - like ileitis , with a normal lifespan and sporadic evidence of mild liver and lung inflammation , as well as occasional proximal colitis . moreover , the intestinal disease is driven by t - helper ( th)1 cytokines ( il12 , ifn ) and cytotoxic cd8 effector t cells.figure 2characteristics of chronic intestinal inflammation in tnfmice . h&e - stained sections of the terminal ileum from 8-week - old mice show increased intestinal inflammation in ( i ) tnf and ( h ) tnf as compared with ( g ) tnf mice . the samp1/yitfc and tnf mouse models are particularly useful for understanding mechanisms of ibd pathogenesis that may uniquely lead to the development of crohn s ileitis . we summarize some of the important findings from studies performed using these 2 models , and how they have contributed to our general understanding of experimental crohn s ileitis and its application to the human condition . samp1/yitfc and tnf mice develop crohns - like ileitis in the absence of spontaneous colonic inflammation , making them very useful models to study the contribution of mucosal healing in the presence of immunologic and genetic dysfunction , independent of active colonic inflammation . card15/nod2 ( also known as ibd1 ) is a gene associated with cd in human beings.7 , 8 the gene product , nod2 , is an intracellular pattern recognition receptor and key mediator of intestinal innate immunity through sensing of its ligand , bacterial muramyl dipeptide ( mdp ) , and initiation of nf-b dependent immune responses within the gut . however , treatment of samp1/yitfc mice with mdp before dss colitis induction has no mitigating effects , and deletion of nod2 in samp1/yitfc mice causes a decrease in the severity of ileitis , supporting the concept that a functional defect related to the nod2/mdp pathway contributes to crohns - like ileitis in samp1/yitfc , despite their wild - type nod2 status . because samp1/yitfc mice are one of the only animal models of ibd that develops intestinal inflammation spontaneously without genetic , chemical , or immunologic manipulation , these mice can provide insights into the possible genetic determinants of cd in human beings . a genome - wide scan of samp1/yitfc mice identified 4 susceptibility loci on chromosomes 9 , 6 , 8 , and x. the loci on chromosome 9 ( ibdq1 ) was linked strongly to epithelial changes in samp1/yitfc mice and contains several potential candidate genes that may play a role in promoting epithelial abnormalities during crohns - like ileitis . interestingly , the ibdq1 region overlaps with 2 chemically induced colitis susceptibility loci ( dss and tnbs1 ) , as well as the genes that encode il10-receptor antagonist and il18 , an epithelial - derived th1-polarizing cytokine that has protective effects in tnbs - induced murine colitis . an accompanying validated scoring system allows investigators to systematically quantify the severity of inflammation and the presence of tumors within the murine intestine in a manner that is generalizable across laboratories and studies , as well as within a particular experiment . initial studies of il1 blockade in rabbit formalin - induced colitis clearly showed the power of animal models to decipher the role of cytokines in mediating intestinal inflammation.9 , 68 given their spontaneous nature and marked similarities to the human condition , both the samp1/yitfc and tnf models have proven highly useful for evaluating the role of novel cytokines in experimental crohns - like ileitis . il33 is a th2-polarizing cytokine that is increased in the intestinal epithelium of patients with uc , and recently was found to be increased in a subpopulation of pediatric patients with stricturing crohn s ileitis . il33 levels correlated positively with the level of inflammation and the number of invading eosinophils , and blockade of il33 resulted in decreased inflammation and eosinophils , as well as reduced th2 cytokine production , suggesting a proinflammatory role for il33 within the context of chronic experimental ibd . interestingly , induction of il33 was dependent on interaction with the gut microbiome , and fecal transplantation from specific pathogen - free samp1/yitfc mice donors into germ - free raised samp1/yitfc recipients restored increased il33 , as well as eosinophilia , in these mice . the vast majority of animal models of ibd do not develop inflammation when reared under germ - free conditions , strongly implicating the gut microbiome as a contributor to ibd pathogenesis . moreover , cd4 lymphocytes from germ - free mice are able to confer disease to severe combined immunodeficient recipients , unlike cd4 cells derived from spf samp1/yitfc mice , suggesting that absence of exposure to the gut microbiome compromises normal development of the regulatory component of the cd4 t - cell population in samp1/yitfc mice . studies performed in tnf mice also confirm the importance of host - gut microbiome interactions and dysbiosis in the pathogenesis of cd - like ileitis.75 , 76 schaubeck et al recently showed that germ - free tnf mice were free of intestinal inflammation . the many available models vary by the type of inflammation that they produce ( acute vs chronic ) , their mode of generation ( chemical , genetic , immunologic , or spontaneous ) , and whether they show colitis or ileitis . however , to understand pathogenic mechanisms that are relevant to the human condition and preclinical testing of novel treatments , spontaneous models , such as samp1/yitfc mice , offer a unique advantage and strong scientific premise .

scientists who address the pathophysiology of disease are well aware of this and often resort to the simplification of human pathology through the development of animal models that eliminate this confounding dimension through generations of inbreeding . the immune system is clearly most profoundly affected by the genetic variation of the human species . this is why jean dausset observed in 1952 that individuals who had received several transfusions from strangers developed antibodies against the donor 's leukocytes . this observation eventually led to the identification of the human leukocyte antigen ( hla ) system , a nomenclature that refers to the human major histocompatibility complex ( mhc ) . it turned out that the mhc complex includes the most polymorphic genes in the human and wild animals ' genomes and the implications of this polymorphism in relation to transplantation , immune response and autoimmune disease had stirred an ongoing debate [ 3 - 6 ] . conservation is generally considered in biology a structural requirement for function : protein domains that are most conserved are also most likely to be those that are most critical to the function of that protein . conversely , polymorphism is regarded as a dispensable component of the human genome where random mutations are not erased by evolutionary pressure . this concept may very well apply to functions that do not require extensive adaptability of the specie to environmental pressure . molecules like insulin which responds to a well defined and invariable stimulus ( blood glucose ) by reducing its circulating levels with almost mathematical predictability do not need much adaptation and are affected by minimal genetic variation across mammalian species . the immune system , on the other hand , has the more complex task of responding to ever evolving environmental components that enter the organism through different routes in the form of pathogens . this adaptation can occur through genetic recombination throughout life as in the case of antibody formation . hla molecules , which have the task of presenting intra - cellular antigens on the surface of cells to cytotoxic and helper t cells , have adopted another strategy to increase their antigen presenting repertoire . this strategy included extensive duplication of genes with redundant function but subtle differences in the way such function is implemented . all classical hla molecules present small portions of antigenic proteins ( epitopes ) to t cells ; however the selection of these epitopic determinants markedly varies across the hla genes and their alleles . thus hla molecules are generally conserved in domains of the protein responsible for interactions with conserved components of the t cell receptors and their co - receptors ( like cd8 molecules ) while displaying extensive polymorphism in domains responsible for antigen binding and interactions in variable regions of the t cell receptors . therefore , a first lesson that the hla system has taught us is that polymorphism can occur preferentially in functional domains of a given molecule with dramatic effects on epitope selection and presentation . a more practical question learned from hla is that the extent of recognized polymorphisms of a given gene is directly proportional to the efforts spent for their identification . not coincidentally , hla , being for practical reasons one of most intensely screened genes for polymorphisms , has experienced an exponential increase in the number of variant alleles during the last decade . for instance , with the introduction of high - resolution molecular typing by polymerase chain reaction ( pcr ) [ 8 - 10 ] and subsequently routine , high - throughput sequence - based typing ( sbt ) the serological hla - a2 family has rapidly grown to include more than 60 members ( figure 1 ) and we have a new one in our hands while preparing this article . the same exponential growth , of course has affected all hla loci to reach for instance for hla class i approximately 300 hla - a , 600 hla - b and more than 100 hla - c alleles ( figure 2 ) . increase in hla - a , -b and -cw alleles in the last few years . it is believed that the major benefit provided by the extensive mhc polymorphism is an increased likelihood that individuals of a given species will be heterozygous and consequently carry two different mhc alleles for each hla locus . since mhc polymorphism(s ) occur in domains responsible for epitope binding , heterozygosity may double the antigen presenting potential of each individual within an ethnic group . most importantly , since individuals within the same ethnic group are likely to express different hla phenotypes , the overall repertoire of the group is exponentially broadened by the presence of extensive polymorphism enhancing the likelihood of the species of surviving a wide variety of pathogens . the evolutionarily success of the human specie in broadening the hla repertoire might explain why it has been difficult to pinpoint associations between a particular hla phenotype and susceptibility to infectious processes . indeed , only few examples of such associations have been reported and even in such cases antigen presenting efficiency does not seem to be the explanation . interestingly , the functional repercussions that polymorphism(s ) carries on antigen presentation could be more easily observed in animal species like chicken that bear a lower number of mhc loci and consequently a restricted antigen presenting repertoire . in spite of the difficulty to demonstrate a clear association between hla phenotype and disease susceptibility , it has been clearly demonstrated at the molecular levels that even one amino acid change in the sequence of hla genes can cause dramatic alterations in antigen binding affinity and consequently efficiency of induction of t cell reactivity in vitro and in vivo . this can in turn modulate the immune dominance of individual antigens according to the hla haplotype bore by different individuals . in addition , it should not be ignored that associations have been observed between hla phenotype and diseases that may or may not be caused by pathogens [ 16 - 23 ] . interestingly , in such cases the importance of the structural changes caused by the polymorphic site on the etiology and pathogenesis remains uncharacterized [ 22 - 29 ] . thus , we can conclude that hla has taught us that polymorphisms can occur in functional regions of molecules , they have functional significance and they may have clinical relevance . the completion of the human genome project has provided a reference sequence of all human chromosomes . however , the challenge remains of characterizing the frequency of deviations from this reference among individuals of similar or divergent ethnic background . it is estimated that 1.42 million single nucleotide polymorphisms ( snp ) are distributed throughout the human genome and about 60,000 snp fall within coding regions . possibly , approximately 25 % of the non - synonymous snp could affect the function of the correspondent gene product [ 32 - 35 ] . it remains unclear whether the prevalence of common diseases can be truly attributed to genetic variation due in part to the incomplete information available and in part to the likely overlap in function of several genes regulating the organism . indeed , most studies testing putative associations between genetic variation and disease have focused on one are few genes at the time . however , it is likely that the analysis of individual loci is too restrictive in complex diseases resulting from the involvement of multiple genes . information from the human genome project can not provide comprehensive knowledge of sequence variations because sequences are based on data compiled from few randomly chosen individuals and only few examples of systematic searches for genetic variants within a specific genomic region are available . however , in the context of clinical research a large number of individuals may need to be screened when investigating associations between genetic variation and disease susceptibility or responsiveness to treatment . in such an endeavor , a tool capable of efficiently identifying known and flagging unknown snp could dramatically increase the efficiency of the study of human pathology through direct application of genome - derived information . although extensive genetic studies have been done predominantly on hla molecules , it is becoming increasingly clear that other molecules related to immune function may be quite polymorphic . non classical mhc loci have demonstrated various degrees of polymorphism . like for classical hla genes , besides the ubiquitously expressed highly polymorphic " classical " hla class i molecules , humans encode three relatively conserved " non - classical " , selectively expressed ( hla - e , f and g ) mhc class i genes ( also known as mhc - ib ) that evolved at different rates in primates reflecting differential involvement in the modulation of immune responses . these molecules are characterized by unique patterns of transcription , protein structure and immunological function . in addition , mhc class i related chain genes ( mic - a and mic - b ) are located within the mhc region and are characterized by high polymorphism ( more than 50 alleles so far identified ) . the molecules encoded by these genes do not appear to bind peptides , nor associate with 2-microglobulin . their polymorphic variants are not concentrated around the peptide binding groove , yet they seem to have functional significance since most of the mutations are non - synonymous suggesting selective pressure as driving force . their tissue distribution is restricted to epithelial and endothelial cells and fibroblasts . it appears that mic genes modulate the function of nk and cd8 + t cells by binding the nkg2d stimulating receptor . mic has also been implicated in transplant rejection as allo - antibodies against them are often found in transplant recipients that may exert complement mediated cytotoxicity against endothelial cells from the graft . other " unusual " mhc - like molecules are present in the genome and have disparate functions including presentation of lipid antigens ( cd1 ) , transport of immunoglobulins ( fc receptor ) and regulation of iron metabolism ( hemochromatosis gene product ) . the extent of polymorphism of these molecules is unknown although it is likely to be minimal . hla - g also is characterized by low polymorphism . because of the minimal polymorphism the repertoire of peptides presented is likely to be limited suggesting that peptide binding is necessary to stabilize the molecule rather than being involved in antigen presentation . this molecule binds hydrophobic peptides from other hla class i leader sequences and interacts with cd94/nkg2 lectin - like receptors present predominately on natural killer and partially on cd8 + t cells which are also minimally polymorphic [ 44 - 48 ] . the peptide binding is highly specific and stabilizes the hla - e protein allowing its migration to the cell surface . thus , surface density of hla - e is an indirect reflection of the number of hla class i alleles expressed by a cell . the interaction of hla - e with cd94/nkg2 protects hla - e expressing cells from killing . cells damaged by viral infection or neoplastic degeneration may loose hla class i expression . since cd94/nkg2 is expressed by most nk cells of most individuals it is likely that this " conserved " hla / inhibitory receptor relationship assures that a constant protection of normal cells is present in most people . another group of genes associated with immune functions that are demonstrating increasing evidence of polymorphism(s ) are killer cell immunoglobulin - like receptors ( kir ) . these molecules are expressed on the surface of natural killer ( nk ) and cd8 + t cells and have strong regulatory hold on their function [ 50 - 53 ] . ligands for inhibitory kir are hla class i molecules almost ubiquitously present on the surface of normal cells . although kir coded by individual genes can either inhibit or activate nk cell function , humans have evolved to collect a large number of such genes within a genomic region rich in immune related genes called the leukocyte receptor cluster on chromosome 19 . this collection of genes is in strong linkage disequilibrium that results in several haplotypes incorporating a sequence of inhibitory and stimulatory kir . since most individuals have several inhibitory kir genes it is likely that each person has at least one kir capable of recognizing at least one autologous hla class i allele . besides this duplication of genes with redundant function but different ligand specificity , kir genes evolved by including several polymorphic sites that could affect the function of intra - cellular signaling domains or ligand ( hla ) recognition . although kir / hla mismatches have been reported to condition the outcome of allogeneic transplantation , at present it is unclear whether kir polymorphism per se has any bearing on disease outcome . in addition , since fcr are mediator of antibody - dependent cytotoxicity it is possible that response to antibody - based therapy could be associated to distinct variants . three subclasses of fcr have been shown to be polymorphic and included fcriia , fcriiia and fcriiib . as this receptors have different effects on leukocyte function including antibody - dependent cellular cytotoxicity , phagocytosis , superoxide generation , degranulation and cytokine production , it is possible that several aspects of the immune system might be strongly affected . a recent review on the subject is available that comprehensively describes the relevance of fcr polymorphisms as prognostic markers for inflammatory diseases and antibody - based immunotherapy . a recent study analyzed the degree of polymorphism in a set of genes associated with innate immune response and found abundant variation in several of them . this study introduces a new dimension of immune polymorphism by adding variation in a system originally thought to be highly conserved capable of recognizing pathogen associated molecular patterns in turn shared by a large group of infectious agents [ 60 - 63 ] . cytokine polymorphism(s ) is becoming a major focus of attention for the understanding of several diseases [ 64 - 71 ] . cytokines , are largely secreted molecules that act on the surrounding microenvironment by providing cell to cell signaling . because of the signaling function , their expression is tightly regulated and most of them are not constitutively expressed . interestingly , as later discussed , most of the polymorphic sites so far identified in cytokine genes have been in non - coding regions containing regulatory sequences . the perturbation of the balance among different cytokines could have implications for the clinical course of many immune diseases as well as organ transplantation . it is possible that balanced polymorphism of immune regulatory genes could have been selected evolutionarily for the beneficial effect of conferring selective advantage in the course of infectious outbreaks . while the relevance of the question is increasingly becoming apparent , with few exceptions , no conclusive information is actually available about the significance of cytokine polymorphism and its practical effects on disease treatment . polymorphisms occur in three main forms , single nucleotide polymorphisms ( snps ) , variable number of tandem repeats and micro - satellites . while some polymorphisms may have direct functional significance by altering directly or indirectly the level of genes expression and/or its function , others may only be useful for the determination of genetic linkage to a particular haplotype associated in turn with a given clinical condition . indeed , a relatively small proportion of polymorphisms that lead to amino acid substitutions fall within the exonic regions ( figure 3 ) . the vast majority of polymorphisms found in cytokine genes and their receptors are located in the promoter , intronic and 3 ' untranslated regions . snp occurring in 3 ' untranslated regions can still affect gene expression and function by altering the stability of rna molecules . in addition , promoter polymorphism may disrupt the binding of transcription factors such as nf-b , jak , stat , irf to regulatory regions . interestingly several signaling and transcription factors central to the regulation of cytokine expression are also polymorphic ( figure 3 ) . number of known polymorphism(s ) of cytokines that occur in known regulatory regions , untranslated gene regions and coding regions . in coding regions only those polymorphisms that result in a chance in protein sequence are counted . the information was compiled to searches based on the following web - sites : ; although some polymorphic loci appear to consistently alter cytokine production most studies suggest that the majority of cytokine polymorphism(s ) have little or no influence on cytokine production and expression . yet polymorphisms in some of these loci have been associated with disease . as an increasing number of studies are being conducted to test whether associations exist between cytokine gene polymorphism and susceptibility to immunologically mediated diseases , cytokine genotypes have been increasingly associated with various diseases of immune or autoimmune nature . often these are complex multi - genic disorders that can be affected by the function of more than one cytokine and/or other genes regulating immune function . therefore , it is not always easy to definitively link the effects of individual cytokine polymorphisms to the etiology , natural history or response to treatment of a disease . with few exceptions such as the mutations in the tnf - rii receptor associated with periodic fevers and the il-2 type cytokine - receptor -chain family variants associated with severe combined immunodeficiency diseases , no cytokine or cytokine receptor polymorphisms have been directly linked to causation of illness . well documented disease associations are beginning to emerge although the relationship between possession of the genetic trait and prevalence of disease occurs with variable strength . a good example is the association of the polymorphism of interleukin ( il)-1 with alzheimer 's disease . and of il-10 with lupus or cancer predisposition . indeed , a list of associations between cytokine , cytokine receptors and disease predisposition , outcome or treatment is ever growing and beyond the purpose of this review . we refer the reader to several recent reviews on the topic that span associations with autoimmune and inflammatory disease [ 82 - 84 ] , occupational disease cancer , allergy , degenerative disease and transplant outcome . in particular , we refer the reader to an updated on line database accessible at that contains information about individual cytokine polymorphisms and relevant disease associations . interestingly , several authors advocate tailored immune therapy based on individual genetic variation of cytokine genes . although this concept may be premature in routine clinical practice , it should be publicized among clinicians and encouraged in the context of clinical trials as genome - wide association studies are becoming feasible with the implementation of high - throughput , cost - effective technologies capable of spanning relevant genomic regions for the detection of known and unknown snp . in general , cytokines are not constitutively produced and stored in the intra - cellular compartment ready for release in response to stimulation . most cytokines expression is triggered by stimulation and their secretion depends on new protein synthesis . this has the advantage of providing a fine regulation of their availability in the extra - cellular space . as a consequence , elaboration of cytokines in response to an inflammatory stimulus transcriptional regulation is critical for the production of many cytokines , transcription factors may play key a role in regulating cytokine - mediated inflammation . genomic analysis has shown that several of these factors are polymorphic in regions that might regulate their function . the toll / interleukin-1 receptor ( tir ) family comprises two groups of transmembrane proteins , which share functional and structural properties . the members of the il-1 receptor ( il-1r ) subfamily are characterized by three extra - cellular immunoglobulin ( ig)-like domains . the members of the toll - like receptor ( tlr ) subfamily recognize alarm signals that can be derived either from pathogens or the host itself . tlr-4 is very important among the tlrs because its ligand is lipopolysaccharide ( lps ) which is a common pathogen component believed to be responsible for the initiation of the immune response during infection . subsequently , several central signaling pathways are activated in parallel , the activation of nf-b being the most prominent event of the inflammatory response . nuclear factor-b ( nf-b ) is a transcription factor that modulates the transcription of a variety of genes , including cytokines and growth factors , adhesion molecules , immune receptors , and acute - phase proteins . nf-b is required for maximal transcription of cytokines including tumor necrosis factor - a ( tnf - a ) , interleukin-1 ( il-1 ) , il-6 , and il-8 , which are thought to be important in the generation of acute inflammatory responses . in turn , excessive cytokine - mediated inflammation is likely to play a fundamental role in the pathogenesis of a variety of disease states [ 101 - 103 ] . the tnf - receptor - associated factor ( traf ) family is a phylogenetically conserved group of scaffold proteins that link receptors of the il-1r / toll and tnf receptor family to signaling cascades , leading to the activation of nf-b and mitogen - activated protein kinases . furthermore , traf proteins serve as a docking platform for a variety of regulators of these signaling pathways and are themselves often regulated at the transcriptional and posttranslational level [ 104 - 108 ] . several of these genes , although predominantly conserved across a wide range of species , are characterized by substantial individual variability in the form of snp ( figure 4 ) . this variation may play a role in determining individual susceptibility to disease and further complicating the interpretation of data related to cytokine polymorphism analysis . number of polymorphism(s ) occurring in selected genes associated with control of the innate immune response . the detection of genetic variation in a given population is important for the understanding of its role in physiological or pathological conditions . allelic discrimination has been predominantly conducted by polymerase chain reaction ( pcr ) . however , pcr - based methods can detect only known polymorphisms since the primers for pcr are designed based on known sites of sequence variation . only unknown polymorphisms that accidentally occur in the region spanned by the primer can be discovered . known snp can be also readily detected using oligo - array - based techniques [ 109 - 112 ] or comparable high - throughput systems [ 113 - 115 ] . oligonucleotide arrays are most commonly based on the principle of competitive hybridization of dna to oligos containing the polymorphism at the centermost position . single base mismatches at a central position of the probe reduce the affinity of the hybridization of the test samples compared to the hybridization of reference samples designed to perfectly match the oligo . when two differentially labeled targets , one representing the test sample and the other representing the reference sample are used for hybridization , competition occurs between the two targets for binding to the two oligonucleotides specific either for the wild type ( reference ) sequence or the snp . a snp - specific hybridization of the test sample will be proportionally higher than the poor hybridization of the reference sample containing the wild type sequence resulting in reduced fluorescence of the reference sample ( signal loss ) . equal signal intensity in test and reference channels indicates no differences for that specific oligo . this technology is dependent upon the design of oligos containing known polymorphic sites and , therefore , it is limited in number of snp that can be identified and can not resolve unknown snps unless all possible permutations are empirically added . detection of unknown snp is not as readily achievable . yet , as occurred during the last decade for hla , it is likely that the number of polymorphic sites will rapidly expand as the investigation of new genomic regions will be broadened to individuals of diverse ethnic background . presently , identification of unknown snp relies on high - throughput sequencing which is burdened by high cost and demanding requirements for sample preparation and interpretation . to improve the efficiency of snp detection , high - density oligonucleotide arrays high - density oligonucleotide arrays adopt in situ oligonucleotide synthesis combined with a computerized photolithographic mask system that allows the addition of one nucleotide at the time in a specific region of the array . the extension of individual oligonucleotide chains is directed by a computerized de - protection of the mask in a defined region of the array . sequence - specific oligonucleotides can be built directly on a solid surface according to a pre - programmed order to cover any sequence combination . in this fashion , any known genomic sequence could potentially be represented on a single oligo array slide . other fabrication techniques utilize pre - synthesized oligonucleotides covalently bound to a solid surface such as glass or micro spheres . these arrays are characterized by extreme accuracy not only for detecting but also in providing definitive sequence information about snp . however , for each genomic region a complex array needs to be assembled as for the 4l ( length of nucleotide ) oligomer probes that query sequential positions in the genome with probes overlapping the previous one of one base . for each position a set of four oligos is prepared identical except at a single position systematically substituted with each of the four nucleotides . thus for a given genomic region a number of oligos equal to the number of bp investigated times 4 is spotted to the array . for instance , to query a 16,569-base pairs ( bp ) sequence 66,276 probes were necessary . although this approach could potentially cover the full genome , it might not be justified for genomic areas with no polymorphism . in addition , preparation of these arrays would be disproportionate for genomic areas with very low density of snp . in those cases it would be preferable to obtain more information about the location of highly polymorphic sites prior to the design of high - density arrays . finally , this approach would not be justifiable in situation where snp occur extremely rarely in a given population . overall , the extraordinary cost of this approach does not justify its use in the context of clinical trials where large patient populations and multi - factorial diseases are studied . a simplified screening tool that could discriminate conserved from polymorphic genomic regions or identify rare individuals carrying unusual snp could dramatically restrict the use of high - throughput sequencing or guide the production of high - density arrays . we recently described a simplified strategy for fluorimetric detection of known and unknown snp by proportional hybridization to oligonucleotide arrays based on optimization of the established principle of signal loss or gain that requires a drastically reduced number of matched or mismatched probes . one set includes overlapping oligos representing an arbitrarily selected " consensus " sequence ( consensus - oligos ) , the other includes oligos specific for known snp ( variant - oligos ) . fluorescence - labeled dna amplified from a homozygous source identical to the consensus represents the reference target and is co - hybridized with a differentially - labeled test sample . lack of hybridization of the test sample to consensus- with simultaneous hybridization to variant - oligos designates a known allele . detection of unknown variants in heterozygous samples depends upon fluorimetric analysis of signal intensity based on the principle that homozygous samples generate twice the amount of signal . this method can identify unknown snp in heterozygous conditions with a sensitivity of 82% and specificity of 90% . although the principle was tested using a library of lymphoid cell lines of know hla phenotype , this strategy is most likely to prove useful for the identification of new snp in yet unexplored regions of the human genome . we believe that , for future clinical trials the design of oligo - array chips based on this principle may allow the coverage of relatively large genomic regions of relevance to the disease investigated and its treatment . high throughput snp detection methods have rapidly confirmed most of the already known polymorphisms identified by conventional techniques and recognized a large number of new snps [ 110,122 - 124 ] . however , most of these studies have been applied to the identification of molecular markers in oncogenesis or to otherwise targeted biological entities . to our knowledge , no systematic searches for multi - genic polymorphisms have been applied to the study of immune pathologies or the interpretation of immune responses during immune therapies . with increasing evidence that immune polymorphism may be a key modulator of immune responses clinical trials for instance , the effect of cytokine gene polymorphism on disease susceptibility has been researched at two levels : studies have relied upon in vitro gene expression induction by stimulation with model immune stimulators such as lps or concavalin a. other studies have simply looked for disease association with individual polymorphism sites or extended haplotypes . only a few studies have integrated both approaches . very few studies have gone farther than studying more that a few cytokines . obviously , the major limitation of the study of immune polymorphism is the extent of the genomic areas that need to be investigated that encompass coding and non - coding regions and the complexity of immune pathology involving an extraordinary number of molecular communications within this extremely adaptable system . however , a systematic multi - genic approach might be extremely important for the determination of disease susceptibility and responsiveness to treatment . we propose that stepwise clinical investigations should be considered in the future that may simply start with the collection of dna from individuals accrued in various experimental protocols . with this valuable source of material it will be possible to answer basic question : 1 ) are genes relevant to particular diseases different among different ethnic groups ? 3 ) is there any correlation between polymorphisms and the natural history of a given disease or its responsiveness to treatment ? 4 ) is there an association between toxicity of therapy and genetic make up ? as an example , patients with cancer undergoing immunotherapy with systemic il-2 , experience a broad and unpredictable range of side effects independent from a linear dose - effect relationship [ 125 - 128 ] . at the same time , il-2 has been shown to induce cancer regression in approximately 20% of such patients in a similarly capricious and inexplicable way [ 129 - 131 ] . indeed , the mechanisms responsible for the therapeutic and toxic effects of systemic administration remain largely unexplained . we have noted that in vitro stimulation with il-2 of peripheral monocytes from patients with metastatic melanoma segregates individuals into two subgroups characterized by high or low production of secondary cytokines . it is possible that such different response stems from polymorphisms in the il-2 receptor and/or down - stream signaling molecules that determine the secretion of cytokine from il-2-induced monocytes . this hypothesis could be easily tested by analyzing such polymorphisms in the context of clinical trials through the preparation of custom made oligo array chips enriched for genes related to the clinical question investigated . obviously , immunogenetic profiling will have to face the controversial regulatory issue of have to deal with genetic information . a fine balance between the benefit to individual patients and to the scientific community on one side and the possible psychological , financial and ethical repercussions on the other will need to be established . possibly , well controlled and regulated data accrual and dissemination should be implemented . however , this problem is germane to all forms of genetic testing and is beyond the purposes of this limited review . in summary , polymorphism is widespread throughout the human genome and most likely to increase in prevalence as the analysis of individuals from different ethnic back grounds will expand . because of its evolving relationship with environmental pathogens , it is likely that the immune system includes a relatively larger number of genes characterized by polymorphisms of functional significance . as a consequence , immune polymorphism may play an important role in disease susceptibility and responsiveness to therapy . it is likely , that most polymorphism relevant to immune pathology are still unknown as suggested by the rapidly increasing databases . the analysis of the impact of individual loci diversity on disease and treatment may be too restrictive . this may be particularly true in complex disease resulting from the involvement of multiple genes having variable effects , which moreover , can vary according to ethnic groups . high throughput technologies of moderate cost should be considered in the future as part of the tools utilized for the interpretation of clinical trials especially in experimental settings . " hla " laboratories should , therefore , broaden their scope to the immunogenetic profiling of genomic regions that might influence not only on transplant outcome but also on autoimmune , infections and neoplastic pathology .

the pathology of humans , in contrast to that of inbred laboratory animals faces the challenge of diversity addressed in genetic terms as polymorphism . thus , unsurprisingly , treatment modalities that successfully can be applied to carefully - selected pre - clinical models only sporadically succeed in the clinical arena . indeed , pre - fabricated experimental models purposefully avoid the basic essence of human pathology : the uncontrollable complexity of disease heterogeneity and the intrinsic diversity of human beings . far from pontificating on this obvious point , this review presents emerging evidence that the study of complex system such as the cytokine network is further complicated by inter - individual differences dictated by increasingly recognized polymorphisms . polymorphism appears widespread among genes of the immune system possibly resulting from an evolutionary adaptation of the organism facing an ever evolving environment . we will refer to this high variability of immune - related genes as immune polymorphism . in this review we will briefly highlight the possible clinical relevance of immune polymorphism and suggest a change in the approach to the study of human pathology , from the targeted study of individual systems to a broader view of the organism as a whole through immunogenetic profiling .

Introduction HLA polymorphism Polymorphisms throughout the genome Immune polymorphism: beyond classical HLA Cytokine polymorphism Polymorphisms associated with cytokine signaling Methods for immunogenetic profiling in clinical immunology

scientists who address the pathophysiology of disease are well aware of this and often resort to the simplification of human pathology through the development of animal models that eliminate this confounding dimension through generations of inbreeding . the immune system is clearly most profoundly affected by the genetic variation of the human species . this observation eventually led to the identification of the human leukocyte antigen ( hla ) system , a nomenclature that refers to the human major histocompatibility complex ( mhc ) . it turned out that the mhc complex includes the most polymorphic genes in the human and wild animals ' genomes and the implications of this polymorphism in relation to transplantation , immune response and autoimmune disease had stirred an ongoing debate [ 3 - 6 ] . conservation is generally considered in biology a structural requirement for function : protein domains that are most conserved are also most likely to be those that are most critical to the function of that protein . conversely , polymorphism is regarded as a dispensable component of the human genome where random mutations are not erased by evolutionary pressure . this concept may very well apply to functions that do not require extensive adaptability of the specie to environmental pressure . molecules like insulin which responds to a well defined and invariable stimulus ( blood glucose ) by reducing its circulating levels with almost mathematical predictability do not need much adaptation and are affected by minimal genetic variation across mammalian species . the immune system , on the other hand , has the more complex task of responding to ever evolving environmental components that enter the organism through different routes in the form of pathogens . this adaptation can occur through genetic recombination throughout life as in the case of antibody formation . this strategy included extensive duplication of genes with redundant function but subtle differences in the way such function is implemented . thus hla molecules are generally conserved in domains of the protein responsible for interactions with conserved components of the t cell receptors and their co - receptors ( like cd8 molecules ) while displaying extensive polymorphism in domains responsible for antigen binding and interactions in variable regions of the t cell receptors . therefore , a first lesson that the hla system has taught us is that polymorphism can occur preferentially in functional domains of a given molecule with dramatic effects on epitope selection and presentation . a more practical question learned from hla is that the extent of recognized polymorphisms of a given gene is directly proportional to the efforts spent for their identification . not coincidentally , hla , being for practical reasons one of most intensely screened genes for polymorphisms , has experienced an exponential increase in the number of variant alleles during the last decade . increase in hla - a , -b and -cw alleles in the last few years . the evolutionarily success of the human specie in broadening the hla repertoire might explain why it has been difficult to pinpoint associations between a particular hla phenotype and susceptibility to infectious processes . indeed , only few examples of such associations have been reported and even in such cases antigen presenting efficiency does not seem to be the explanation . in spite of the difficulty to demonstrate a clear association between hla phenotype and disease susceptibility , it has been clearly demonstrated at the molecular levels that even one amino acid change in the sequence of hla genes can cause dramatic alterations in antigen binding affinity and consequently efficiency of induction of t cell reactivity in vitro and in vivo . this can in turn modulate the immune dominance of individual antigens according to the hla haplotype bore by different individuals . interestingly , in such cases the importance of the structural changes caused by the polymorphic site on the etiology and pathogenesis remains uncharacterized [ 22 - 29 ] . thus , we can conclude that hla has taught us that polymorphisms can occur in functional regions of molecules , they have functional significance and they may have clinical relevance . the completion of the human genome project has provided a reference sequence of all human chromosomes . however , the challenge remains of characterizing the frequency of deviations from this reference among individuals of similar or divergent ethnic background . it remains unclear whether the prevalence of common diseases can be truly attributed to genetic variation due in part to the incomplete information available and in part to the likely overlap in function of several genes regulating the organism . indeed , most studies testing putative associations between genetic variation and disease have focused on one are few genes at the time . however , it is likely that the analysis of individual loci is too restrictive in complex diseases resulting from the involvement of multiple genes . information from the human genome project can not provide comprehensive knowledge of sequence variations because sequences are based on data compiled from few randomly chosen individuals and only few examples of systematic searches for genetic variants within a specific genomic region are available . however , in the context of clinical research a large number of individuals may need to be screened when investigating associations between genetic variation and disease susceptibility or responsiveness to treatment . in such an endeavor , a tool capable of efficiently identifying known and flagging unknown snp could dramatically increase the efficiency of the study of human pathology through direct application of genome - derived information . like for classical hla genes , besides the ubiquitously expressed highly polymorphic " classical " hla class i molecules , humans encode three relatively conserved " non - classical " , selectively expressed ( hla - e , f and g ) mhc class i genes ( also known as mhc - ib ) that evolved at different rates in primates reflecting differential involvement in the modulation of immune responses . their polymorphic variants are not concentrated around the peptide binding groove , yet they seem to have functional significance since most of the mutations are non - synonymous suggesting selective pressure as driving force . mic has also been implicated in transplant rejection as allo - antibodies against them are often found in transplant recipients that may exert complement mediated cytotoxicity against endothelial cells from the graft . other " unusual " mhc - like molecules are present in the genome and have disparate functions including presentation of lipid antigens ( cd1 ) , transport of immunoglobulins ( fc receptor ) and regulation of iron metabolism ( hemochromatosis gene product ) . because of the minimal polymorphism the repertoire of peptides presented is likely to be limited suggesting that peptide binding is necessary to stabilize the molecule rather than being involved in antigen presentation . the peptide binding is highly specific and stabilizes the hla - e protein allowing its migration to the cell surface . thus , surface density of hla - e is an indirect reflection of the number of hla class i alleles expressed by a cell . although kir coded by individual genes can either inhibit or activate nk cell function , humans have evolved to collect a large number of such genes within a genomic region rich in immune related genes called the leukocyte receptor cluster on chromosome 19 . as this receptors have different effects on leukocyte function including antibody - dependent cellular cytotoxicity , phagocytosis , superoxide generation , degranulation and cytokine production , it is possible that several aspects of the immune system might be strongly affected . a recent review on the subject is available that comprehensively describes the relevance of fcr polymorphisms as prognostic markers for inflammatory diseases and antibody - based immunotherapy . a recent study analyzed the degree of polymorphism in a set of genes associated with innate immune response and found abundant variation in several of them . this study introduces a new dimension of immune polymorphism by adding variation in a system originally thought to be highly conserved capable of recognizing pathogen associated molecular patterns in turn shared by a large group of infectious agents [ 60 - 63 ] . because of the signaling function , their expression is tightly regulated and most of them are not constitutively expressed . interestingly , as later discussed , most of the polymorphic sites so far identified in cytokine genes have been in non - coding regions containing regulatory sequences . the perturbation of the balance among different cytokines could have implications for the clinical course of many immune diseases as well as organ transplantation . it is possible that balanced polymorphism of immune regulatory genes could have been selected evolutionarily for the beneficial effect of conferring selective advantage in the course of infectious outbreaks . while the relevance of the question is increasingly becoming apparent , with few exceptions , no conclusive information is actually available about the significance of cytokine polymorphism and its practical effects on disease treatment . while some polymorphisms may have direct functional significance by altering directly or indirectly the level of genes expression and/or its function , others may only be useful for the determination of genetic linkage to a particular haplotype associated in turn with a given clinical condition . indeed , a relatively small proportion of polymorphisms that lead to amino acid substitutions fall within the exonic regions ( figure 3 ) . the vast majority of polymorphisms found in cytokine genes and their receptors are located in the promoter , intronic and 3 ' untranslated regions . in addition , promoter polymorphism may disrupt the binding of transcription factors such as nf-b , jak , stat , irf to regulatory regions . interestingly several signaling and transcription factors central to the regulation of cytokine expression are also polymorphic ( figure 3 ) . the information was compiled to searches based on the following web - sites : ; although some polymorphic loci appear to consistently alter cytokine production most studies suggest that the majority of cytokine polymorphism(s ) have little or no influence on cytokine production and expression . as an increasing number of studies are being conducted to test whether associations exist between cytokine gene polymorphism and susceptibility to immunologically mediated diseases , cytokine genotypes have been increasingly associated with various diseases of immune or autoimmune nature . often these are complex multi - genic disorders that can be affected by the function of more than one cytokine and/or other genes regulating immune function . therefore , it is not always easy to definitively link the effects of individual cytokine polymorphisms to the etiology , natural history or response to treatment of a disease . with few exceptions such as the mutations in the tnf - rii receptor associated with periodic fevers and the il-2 type cytokine - receptor -chain family variants associated with severe combined immunodeficiency diseases , no cytokine or cytokine receptor polymorphisms have been directly linked to causation of illness . well documented disease associations are beginning to emerge although the relationship between possession of the genetic trait and prevalence of disease occurs with variable strength . a good example is the association of the polymorphism of interleukin ( il)-1 with alzheimer 's disease . indeed , a list of associations between cytokine , cytokine receptors and disease predisposition , outcome or treatment is ever growing and beyond the purpose of this review . although this concept may be premature in routine clinical practice , it should be publicized among clinicians and encouraged in the context of clinical trials as genome - wide association studies are becoming feasible with the implementation of high - throughput , cost - effective technologies capable of spanning relevant genomic regions for the detection of known and unknown snp . in general , cytokines are not constitutively produced and stored in the intra - cellular compartment ready for release in response to stimulation . this has the advantage of providing a fine regulation of their availability in the extra - cellular space . as a consequence , elaboration of cytokines in response to an inflammatory stimulus transcriptional regulation is critical for the production of many cytokines , transcription factors may play key a role in regulating cytokine - mediated inflammation . the members of the il-1 receptor ( il-1r ) subfamily are characterized by three extra - cellular immunoglobulin ( ig)-like domains . the members of the toll - like receptor ( tlr ) subfamily recognize alarm signals that can be derived either from pathogens or the host itself . tlr-4 is very important among the tlrs because its ligand is lipopolysaccharide ( lps ) which is a common pathogen component believed to be responsible for the initiation of the immune response during infection . subsequently , several central signaling pathways are activated in parallel , the activation of nf-b being the most prominent event of the inflammatory response . nf-b is required for maximal transcription of cytokines including tumor necrosis factor - a ( tnf - a ) , interleukin-1 ( il-1 ) , il-6 , and il-8 , which are thought to be important in the generation of acute inflammatory responses . in turn , excessive cytokine - mediated inflammation is likely to play a fundamental role in the pathogenesis of a variety of disease states [ 101 - 103 ] . the tnf - receptor - associated factor ( traf ) family is a phylogenetically conserved group of scaffold proteins that link receptors of the il-1r / toll and tnf receptor family to signaling cascades , leading to the activation of nf-b and mitogen - activated protein kinases . several of these genes , although predominantly conserved across a wide range of species , are characterized by substantial individual variability in the form of snp ( figure 4 ) . number of polymorphism(s ) occurring in selected genes associated with control of the innate immune response . only unknown polymorphisms that accidentally occur in the region spanned by the primer can be discovered . known snp can be also readily detected using oligo - array - based techniques [ 109 - 112 ] or comparable high - throughput systems [ 113 - 115 ] . single base mismatches at a central position of the probe reduce the affinity of the hybridization of the test samples compared to the hybridization of reference samples designed to perfectly match the oligo . when two differentially labeled targets , one representing the test sample and the other representing the reference sample are used for hybridization , competition occurs between the two targets for binding to the two oligonucleotides specific either for the wild type ( reference ) sequence or the snp . a snp - specific hybridization of the test sample will be proportionally higher than the poor hybridization of the reference sample containing the wild type sequence resulting in reduced fluorescence of the reference sample ( signal loss ) . yet , as occurred during the last decade for hla , it is likely that the number of polymorphic sites will rapidly expand as the investigation of new genomic regions will be broadened to individuals of diverse ethnic background . to improve the efficiency of snp detection , high - density oligonucleotide arrays high - density oligonucleotide arrays adopt in situ oligonucleotide synthesis combined with a computerized photolithographic mask system that allows the addition of one nucleotide at the time in a specific region of the array . the extension of individual oligonucleotide chains is directed by a computerized de - protection of the mask in a defined region of the array . sequence - specific oligonucleotides can be built directly on a solid surface according to a pre - programmed order to cover any sequence combination . in this fashion , any known genomic sequence could potentially be represented on a single oligo array slide . other fabrication techniques utilize pre - synthesized oligonucleotides covalently bound to a solid surface such as glass or micro spheres . however , for each genomic region a complex array needs to be assembled as for the 4l ( length of nucleotide ) oligomer probes that query sequential positions in the genome with probes overlapping the previous one of one base . for each position a set of four oligos is prepared identical except at a single position systematically substituted with each of the four nucleotides . thus for a given genomic region a number of oligos equal to the number of bp investigated times 4 is spotted to the array . finally , this approach would not be justifiable in situation where snp occur extremely rarely in a given population . overall , the extraordinary cost of this approach does not justify its use in the context of clinical trials where large patient populations and multi - factorial diseases are studied . we recently described a simplified strategy for fluorimetric detection of known and unknown snp by proportional hybridization to oligonucleotide arrays based on optimization of the established principle of signal loss or gain that requires a drastically reduced number of matched or mismatched probes . fluorescence - labeled dna amplified from a homozygous source identical to the consensus represents the reference target and is co - hybridized with a differentially - labeled test sample . although the principle was tested using a library of lymphoid cell lines of know hla phenotype , this strategy is most likely to prove useful for the identification of new snp in yet unexplored regions of the human genome . we believe that , for future clinical trials the design of oligo - array chips based on this principle may allow the coverage of relatively large genomic regions of relevance to the disease investigated and its treatment . high throughput snp detection methods have rapidly confirmed most of the already known polymorphisms identified by conventional techniques and recognized a large number of new snps [ 110,122 - 124 ] . however , most of these studies have been applied to the identification of molecular markers in oncogenesis or to otherwise targeted biological entities . to our knowledge , no systematic searches for multi - genic polymorphisms have been applied to the study of immune pathologies or the interpretation of immune responses during immune therapies . with increasing evidence that immune polymorphism may be a key modulator of immune responses clinical trials for instance , the effect of cytokine gene polymorphism on disease susceptibility has been researched at two levels : studies have relied upon in vitro gene expression induction by stimulation with model immune stimulators such as lps or concavalin a. other studies have simply looked for disease association with individual polymorphism sites or extended haplotypes . obviously , the major limitation of the study of immune polymorphism is the extent of the genomic areas that need to be investigated that encompass coding and non - coding regions and the complexity of immune pathology involving an extraordinary number of molecular communications within this extremely adaptable system . however , a systematic multi - genic approach might be extremely important for the determination of disease susceptibility and responsiveness to treatment . we propose that stepwise clinical investigations should be considered in the future that may simply start with the collection of dna from individuals accrued in various experimental protocols . 3 ) is there any correlation between polymorphisms and the natural history of a given disease or its responsiveness to treatment ? indeed , the mechanisms responsible for the therapeutic and toxic effects of systemic administration remain largely unexplained . it is possible that such different response stems from polymorphisms in the il-2 receptor and/or down - stream signaling molecules that determine the secretion of cytokine from il-2-induced monocytes . this hypothesis could be easily tested by analyzing such polymorphisms in the context of clinical trials through the preparation of custom made oligo array chips enriched for genes related to the clinical question investigated . obviously , immunogenetic profiling will have to face the controversial regulatory issue of have to deal with genetic information . a fine balance between the benefit to individual patients and to the scientific community on one side and the possible psychological , financial and ethical repercussions on the other will need to be established . however , this problem is germane to all forms of genetic testing and is beyond the purposes of this limited review . in summary , polymorphism is widespread throughout the human genome and most likely to increase in prevalence as the analysis of individuals from different ethnic back grounds will expand . because of its evolving relationship with environmental pathogens , it is likely that the immune system includes a relatively larger number of genes characterized by polymorphisms of functional significance . as a consequence , immune polymorphism may play an important role in disease susceptibility and responsiveness to therapy . the analysis of the impact of individual loci diversity on disease and treatment may be too restrictive . this may be particularly true in complex disease resulting from the involvement of multiple genes having variable effects , which moreover , can vary according to ethnic groups . high throughput technologies of moderate cost should be considered in the future as part of the tools utilized for the interpretation of clinical trials especially in experimental settings . " hla " laboratories should , therefore , broaden their scope to the immunogenetic profiling of genomic regions that might influence not only on transplant outcome but also on autoimmune , infections and neoplastic pathology .

parkinson 's disease ( pd ) is neurodegenerative disorder characterized by progressive loss of substantia nigra pars compacta ( snpc ) neurons1,4,5,20 ) . the progressive loss of cells leads to motor dysfunctions including tremor , rigidity , and bradykinesia6,10,28 ) . neural tissue transplantation has been a promising strategy for pd because the transplanted tissues can produce dopamine in the striatum , which has a therapeutic effect in pd patients2,15 ) . however , tissue grafts have the disadvantages of low rates of cell survival and/or development of dyskinesia9 ) . stem - cell transplantation was recently tried , but the effects were controversial due to possible tumorigenesis8 ) , poor differentiation into dopaminergic cells , or low survival rates19 ) . the transplantation of spheroids , or cell aggregations , has been proposed to overcome these problems , because the cell aggregates can extend their axons into the host brain and establish synaptic connections with host neurons following transplantation into specific brain areas13 ) . in our previous study , human brain - derived neural stem cells ( hb - nsc ) which were injected into the striatum of a pd rat model as single - cell suspensions showed partial differentiation to dopaminergic cells , but without therapeutic effect30 ) . we hypothesized that the injected single hb - nsc cell suspensions could partially differentiate into dopaminergic cells , which produced dopamine within the cell , but were unable to secrete it . therefore , in the present study , we transplanted hb - nsc as cell aggregates form into the striatum of pd rats to determine their efficacy compared to single - cell transplantations . we expected that the cell aggregates have the advantage of a pre - established cell network which reported in transplantation of spheroid form13 ) that might promote high rates of survival and differentiation . after the transplantation , we evaluated the results by behavioral tests , pet study and histological study . all animals were housed individually in cages , with free access to food and water in a climate - controlled room . this study was approved by the institutional animal care and use committee of the asan institute for life science . at the beginning of the study , each animal was anesthetized with zoletil 50 ( virbac s.a , carros , france ) and rompun ( bayer , leverkusen , germany ) at doses of 37.5 mg / kg and 6 mg / kg , respectively , using an intraperitoneal ( i.p . ) injection . a total of 22 rats were administered unilateral injections of 14 g 6-ohda hydrochloride ( sigma , st . louis , mo , usa ) in 4 l of 0.9% saline with 0.1% ascorbic acid delivered into the right medial forebrain bundle at the following coordinates : anteroposterior -4.4 mm , lateral + 1.2 mm relative to the bregma and ventral -7.8 mm from the dura by paxinos 's rat atlas , with the tooth bar set at -2.4 mm . the injections were delivered at a rate of 1 l / min using a hamilton syringe ( 33-gauge ) and an automated microsyringe pump ( harvard apparatus , holliston , ma , usa ) . once the injection was finished , the needle was left in place for 5 minutes to prevent backflow of the solution . among 22 rats , 18 rats which showed apomorphine - induced rotation exceeded 6 rotations per min after 4 weeks of 6-ohda injection ( success rate 81.8% ) were selected for this study . then , hb - nsc cells were injected in 9 rats ( hb - nsc group ) and pbs in 9 rats ( control group ) . however , 2 rats were dead during pet study , so the final numbers of experimental animals became 9 in hb - nsc group and 7 in control group . hb - nsc ( san pedro , ca , usa ) were expanded in brain stem - cell growth medium according to the manufacturer 's instructions . for formation of homogenous aggregates , polyethylene glycol ( peg ) hydrogel microwell arrays with a diameter of 500 m were produced using micro - fabrication procedures , as reported previously13 ) . the expanded hb - nsc were allowed to form aggregations within the peg hydrogel microwell arrays . briefly , hb - nsc at an approximate cell density of 110 cells/200 l were deposited into each microwell array , and then placed in a 5% co2 humidified incubator for 30 min to allow the cells to settle . non - adherent cells were carefully removed by washing with pbs , and the adherent cells were allowed to form aggregations within each microwell . the cell aggregations were retrieved from the peg hydrogel after 5 days of culture in the brain stem - cell growth medium ( celprogen , san pedro , ca , usa ) . the cell aggregates are visible with the naked eyes and the diameter of each aggregate is approximately 200 m . and then , the cell aggregation were administered in sterilized pbs at a concentration of 810 cells/8 l . we could evaluate the concentration of cell aggregation by cell counting with trypan blue viability staining which was partially harvested from cell aggregation solution and transformed into cell suspension . the cell aggregations transplanted into the right striatum of the pd rat model ( hb - nsc group , n=9 ) using a 22 g hamilton syringe and a stereotaxic microinjection device ( coordinates : ap + 0.6 mm from the bregma , ml + 3.5 mm from the midline , dv -4.5 mm , nose bar + 2.3 mm ) 4 weeks after the 6-ohda injection . the control group ( n=7 ) received 8 l of pbs ( ph 7.4 ) injected at the same coordinates . all injections used a rate of 1 l / min , and the needle was held in place for 5 min after the injection , followed by withdrawal at a rate of 1 mm / min . postoperatively , all rats received daily cyclosporine a ( chong kun dang pharm , seoul , korea ) at 10 mg / kg ( i.p . ) until the animals were sacrificed . the stepping tests were performed six times : prior to any treatment , 1 week after 6-ohda injection , and 2 , 4 , 6 , and 8 weeks after cell transplantation , as previously described24 ) , with slight modifications . briefly , both hind limbs were firmly held in one hand of the experimenter , while the other hand was used to hold one of the forelimbs . the anterior end of the animal was lowered onto a treadmill ( jeung do bio & plant co. , seoul , korea ) , which was set at a rate of 0.9 m/5 s. the rat 's body remained stationary while the unilateral forelimb was allowed to touch the moving treadmill track for > 5 seconds while the treadmill was operating . all of the experiments were video recorded to count the number of adjusted steps taken in the backward direction . every rat performed the stepping test twice during every session , and the data were averaged . the data are expressed as the percentage of contralateral forelimb steps versus ipsilateral forelimb steps . the rotation tests were conducted 1 week after 6-ohda injection , and 4 and 8 weeks after cell transplantation , as previously described25 ) , with slight modifications . briefly , as soon as 0.5 mg / kg apomorphine ( sigma ; in sterile water , injected subcutaneously ) was administered , the rat was harnessed to an automated rotometer ( panlab , barcelona , spain ) and its rotational behavior was assessed over 45 minutes . data are expressed as the net average rotations ( contralateral - ipsilateral turns ) per minute . 6-ohda - lesioned rats that demonstrated > 6 rotations per minute were selected for inclusion in the present study . all animals were scanned using the micropet focus 120 system ( siemens medical solutions , inc . , erlagen , germany ) , which includes a 1212 array of lutetium oxyorthosilicate and 96 detector blocks . glucose metabolic activity was assessed in the control ( n=3 ) and hb - nsc groups ( n=5 ) using [ f]-fdg at 10 weeks posttransplantation . after fasting for at least 12 h , the rats were injected with 1 mci [ f]-fdg in 0.2 ml of sterile saline via the tail vein . one hour after the injection , the animals were scanned for 30 min under isoflurane anesthesia . dopamine transporter function was assessed with the same animals in the control ( n=3 ) and hb - nsc group ( n=5 ) using [ f]-fp - cit , which binds to the dopamine transporter with high affinity , at 1 or 2 weeks after 6-ohda injection ( pd status before cell transplantation ) and at 5 and 10 weeks after cell transplantation . rats were injected with 1 mci [ f]-fp - cit in 0.4 ml sterile saline via the tail vein , immediately followed by dynamic scanning for 90 minutes . the [ f]-fdg pet data were reconstructed using a 3-dimensional ( 3d ) maximum a posteriori ( map ) algorithm , and the [ f]-fp - cit data were reconstructed using a 2d ordered - subset expectation maximization16 ) . pet images were analyzed using the asipro software ( siemens preclinical solutions , knoxville , tn , usa ) to generate a 3d volume of interest that consisted of three regions of interest ( roi ) . to produce 3d volume of interest , we selected a coronal image which showed highest uptake of striatum , and then , three consecutive rois in axial images which show striatum were taken on the selected coronal image base . data are expressed as the percentage of ipsilateral standardized uptake values based on the mean uptake in the 3d volume of interest against the contralateral standardized uptake values . the equation of standardized uptake values is [ tissue concentration ( mbq / cc)]/[injected dose ( mbq)/body weight ( g ) ] . rats were transcardially and sequentially perfused with 0.9% saline containing 10000 iu heparin ( hanlim pharm , seoul , korea ) and 4% paraformaldehyde in pbs . extracted brain tissues were postfixed overnight in the same fixative , followed by incubation and dehydration in 30% sucrose until they sank . serial sections from frozen tissue , including the striatum ( anteroposterior + 2.5 to 0.0 mm ) , were produced and the brain tissue was cut into 40 m sections on a sliding microtome ( hm450 ; sliding electric microtome , thermo scientific ) . for ihc , striatal sections were incubated overnight with primary antibodies ( anti - th monoclonal 1 : 5000 , sigma aldrich , st . louis , usa ) diluted in pbs ( ph 7.4 ) containing 0.5% bsa ( bioworld , dublin , usa ) . these sections were subsequently incubated for 2 h at room temperature with biotinylated secondary antibodies produced in mouse ( vector laboratories , burlingame , ca , usa ) and diluted 1 : 200 in pbs containing 0.5% bsa . th staining was detected by adding diaminobenzidine ( r&d system , minneapolis , mn , usa ) . diaminobenzidine - treated tissues were dehydrated in a series of alcohol and clearing agents , and a coverslip was applied . th - positive cells were observed in the coronal sections of the striatum using a nikon 80i microscope ( nikon , tokyo , japan ) at 100 magnification using the nis - elements f3.0 software ( nikon , tokyo , japan ) . for if , tissue sections were incubated with blocking solution containing 1% bsa , 0.2% triton x-100 ( sigma aldrich , st . louis , usa ) and 0.05% sodium azide , rinsed in 0.5% bsa in pbs twice and then incubated with anti - th monoclonal ( 1 : 5000 , sigma aldrich , st . louis , usa ) and anti - human 2 microglobulin polyclonal ( 1 : 800 , dako , seoul , korea ) antibodies for 1 h at room temperature . the sections were washed three times and incubated for 90 min with alexa 546-labeled anti - mouse igg ( 1 : 200 , invitrogen , grand island , ny , usa ) and alexa 488-labeled anti - rabbit igg ( 1 : 200 , invitrogen grand island , ny , usa ) . after washing twice , the sections were mounted onto gelatin - coated slides , dried at 50 for 30 min , and cover - slipped with vector mounting ( dako , seoul , korea ) medium for fluorescence . fluorescent images were obtained using a confocal microscope ( tcs - st2 ; leica , wetzlar , germany ) . behavioral parameters were analyzed using mann - whitney u test , and pet data were analyzed by the paired sample t - test for working out the relative importance of each data which was mat - ched one - on - one . all animals were housed individually in cages , with free access to food and water in a climate - controlled room . this study was approved by the institutional animal care and use committee of the asan institute for life science . at the beginning of the study , each animal was anesthetized with zoletil 50 ( virbac s.a , carros , france ) and rompun ( bayer , leverkusen , germany ) at doses of 37.5 mg / kg and 6 mg / kg , respectively , using an intraperitoneal ( i.p . ) injection . a total of 22 rats were administered unilateral injections of 14 g 6-ohda hydrochloride ( sigma , st . louis , mo , usa ) in 4 l of 0.9% saline with 0.1% ascorbic acid delivered into the right medial forebrain bundle at the following coordinates : anteroposterior -4.4 mm , lateral + 1.2 mm relative to the bregma and ventral -7.8 mm from the dura by paxinos 's rat atlas , with the tooth bar set at -2.4 mm . the injections were delivered at a rate of 1 l / min using a hamilton syringe ( 33-gauge ) and an automated microsyringe pump ( harvard apparatus , holliston , ma , usa ) . once the injection was finished , the needle was left in place for 5 minutes to prevent backflow of the solution . among 22 rats , 18 rats which showed apomorphine - induced rotation exceeded 6 rotations per min after 4 weeks of 6-ohda injection ( success rate 81.8% ) were selected for this study . then , hb - nsc cells were injected in 9 rats ( hb - nsc group ) and pbs in 9 rats ( control group ) . however , 2 rats were dead during pet study , so the final numbers of experimental animals became 9 in hb - nsc group and 7 in control group . hb - nsc ( san pedro , ca , usa ) were expanded in brain stem - cell growth medium according to the manufacturer 's instructions . for formation of homogenous aggregates , polyethylene glycol ( peg ) hydrogel microwell arrays with a diameter of 500 m were produced using micro - fabrication procedures , as reported previously13 ) . the expanded hb - nsc were allowed to form aggregations within the peg hydrogel microwell arrays . briefly , hb - nsc at an approximate cell density of 110 cells/200 l were deposited into each microwell array , and then placed in a 5% co2 humidified incubator for 30 min to allow the cells to settle . non - adherent cells were carefully removed by washing with pbs , and the adherent cells were allowed to form aggregations within each microwell . the cell aggregations were retrieved from the peg hydrogel after 5 days of culture in the brain stem - cell growth medium ( celprogen , san pedro , ca , usa ) . the cell aggregates are visible with the naked eyes and the diameter of each aggregate is approximately 200 m . and then , the cell aggregation were administered in sterilized pbs at a concentration of 810 cells/8 l . we could evaluate the concentration of cell aggregation by cell counting with trypan blue viability staining which was partially harvested from cell aggregation solution and transformed into cell suspension . the cell aggregations transplanted into the right striatum of the pd rat model ( hb - nsc group , n=9 ) using a 22 g hamilton syringe and a stereotaxic microinjection device ( coordinates : ap + 0.6 mm from the bregma , ml + 3.5 mm from the midline , dv -4.5 mm , nose bar + 2.3 mm ) 4 weeks after the 6-ohda injection . the control group ( n=7 ) received 8 l of pbs ( ph 7.4 ) injected at the same coordinates . all injections used a rate of 1 l / min , and the needle was held in place for 5 min after the injection , followed by withdrawal at a rate of 1 mm / min . postoperatively , all rats received daily cyclosporine a ( chong kun dang pharm , seoul , korea ) at 10 mg / kg ( i.p . ) until the animals were sacrificed . the stepping tests were performed six times : prior to any treatment , 1 week after 6-ohda injection , and 2 , 4 , 6 , and 8 weeks after cell transplantation , as previously described24 ) , with slight modifications . briefly , both hind limbs were firmly held in one hand of the experimenter , while the other hand was used to hold one of the forelimbs . the anterior end of the animal was lowered onto a treadmill ( jeung do bio & plant co. , seoul , korea ) , which was set at a rate of 0.9 m/5 s. the rat 's body remained stationary while the unilateral forelimb was allowed to touch the moving treadmill track for > 5 seconds while the treadmill was operating . all of the experiments were video recorded to count the number of adjusted steps taken in the backward direction . every rat performed the stepping test twice during every session , and the data were averaged . the data are expressed as the percentage of contralateral forelimb steps versus ipsilateral forelimb steps . the rotation tests were conducted 1 week after 6-ohda injection , and 4 and 8 weeks after cell transplantation , as previously described25 ) , with slight modifications . briefly , as soon as 0.5 mg / kg apomorphine ( sigma ; in sterile water , injected subcutaneously ) was administered , the rat was harnessed to an automated rotometer ( panlab , barcelona , spain ) and its rotational behavior was assessed over 45 minutes . data are expressed as the net average rotations ( contralateral - ipsilateral turns ) per minute . 6-ohda - lesioned rats that demonstrated > 6 rotations per minute were selected for inclusion in the present study . all animals were scanned using the micropet focus 120 system ( siemens medical solutions , inc . , erlagen , germany ) , which includes a 1212 array of lutetium oxyorthosilicate and 96 detector blocks . glucose metabolic activity was assessed in the control ( n=3 ) and hb - nsc groups ( n=5 ) using [ f]-fdg at 10 weeks posttransplantation . after fasting for at least 12 h , the rats were injected with 1 mci [ f]-fdg in 0.2 ml of sterile saline via the tail vein . one hour after the injection , the animals were scanned for 30 min under isoflurane anesthesia . dopamine transporter function was assessed with the same animals in the control ( n=3 ) and hb - nsc group ( n=5 ) using [ f]-fp - cit , which binds to the dopamine transporter with high affinity , at 1 or 2 weeks after 6-ohda injection ( pd status before cell transplantation ) and at 5 and 10 weeks after cell transplantation . rats were injected with 1 mci [ f]-fp - cit in 0.4 ml sterile saline via the tail vein , immediately followed by dynamic scanning for 90 minutes . the [ f]-fdg pet data were reconstructed using a 3-dimensional ( 3d ) maximum a posteriori ( map ) algorithm , and the [ f]-fp - cit data were reconstructed using a 2d ordered - subset expectation maximization16 ) . pet images were analyzed using the asipro software ( siemens preclinical solutions , knoxville , tn , usa ) to generate a 3d volume of interest that consisted of three regions of interest ( roi ) . to produce 3d volume of interest , we selected a coronal image which showed highest uptake of striatum , and then , three consecutive rois in axial images which show striatum were taken on the selected coronal image base . data are expressed as the percentage of ipsilateral standardized uptake values based on the mean uptake in the 3d volume of interest against the contralateral standardized uptake values . the equation of standardized uptake values is [ tissue concentration ( mbq / cc)]/[injected dose ( mbq)/body weight ( g ) ] . rats were transcardially and sequentially perfused with 0.9% saline containing 10000 iu heparin ( hanlim pharm , seoul , korea ) and 4% paraformaldehyde in pbs . extracted brain tissues were postfixed overnight in the same fixative , followed by incubation and dehydration in 30% sucrose until they sank . serial sections from frozen tissue , including the striatum ( anteroposterior + 2.5 to 0.0 mm ) , were produced and the brain tissue was cut into 40 m sections on a sliding microtome ( hm450 ; sliding electric microtome , thermo scientific ) . , striatal sections were incubated overnight with primary antibodies ( anti - th monoclonal 1 : 5000 , sigma aldrich , st . louis , usa ) diluted in pbs ( ph 7.4 ) containing 0.5% bsa ( bioworld , dublin , usa ) . these sections were subsequently incubated for 2 h at room temperature with biotinylated secondary antibodies produced in mouse ( vector laboratories , burlingame , ca , usa ) and diluted 1 : 200 in pbs containing 0.5% bsa . th staining was detected by adding diaminobenzidine ( r&d system , minneapolis , mn , usa ) . diaminobenzidine - treated tissues were dehydrated in a series of alcohol and clearing agents , and a coverslip was applied . th - positive cells were observed in the coronal sections of the striatum using a nikon 80i microscope ( nikon , tokyo , japan ) at 100 magnification using the nis - elements f3.0 software ( nikon , tokyo , japan ) . for if , tissue sections were incubated with blocking solution containing 1% bsa , 0.2% triton x-100 ( sigma aldrich , st . louis , usa ) and 0.05% sodium azide , rinsed in 0.5% bsa in pbs twice and then incubated with anti - th monoclonal ( 1 : 5000 , sigma aldrich , st . louis , usa ) and anti - human 2 microglobulin polyclonal ( 1 : 800 , dako , seoul , korea ) antibodies for 1 h at room temperature . the sections were washed three times and incubated for 90 min with alexa 546-labeled anti - mouse igg ( 1 : 200 , invitrogen , grand island , ny , usa ) and alexa 488-labeled anti - rabbit igg ( 1 : 200 , invitrogen grand island , ny , usa ) . after washing twice , the sections were mounted onto gelatin - coated slides , dried at 50 for 30 min , and cover - slipped with vector mounting ( dako , seoul , korea ) medium for fluorescence . fluorescent images were obtained using a confocal microscope ( tcs - st2 ; leica , wetzlar , germany ) . behavioral parameters were analyzed using mann - whitney u test , and pet data were analyzed by the paired sample t - test for working out the relative importance of each data which was mat - ched one - on - one . the stepping test showed no significant increase in contralateral paw touches up to 4 weeks posttransplantation ; however , at 6 weeks posttransplantation , a significant improvement of 52.9% was measured . at 8 weeks posttransplantation , the increase was 48.9% and this improvement was maintained through to 10 weeks , at which time the increase was 64.2% in the hb - nsc group compared to the control group ( fig . 1a ) . in apomorphine - induced rotation tests , the hb - nsc group showed a slight decrease in contralateral rotations at week 8 , compared to week 4 ; however , the difference was not statistically significant ( fig . there was no significant decrease in uptake of [ f]-fdg in either the control or hb - nsc transplantation group , indicating that glucose metabolism was normal , as is characteristic of pd ( fig . 2a ) . using [ f]-fp - cit to quantify dopamine transporter activity , a gradual increase in radioactivity in the lesion - side striatum was detected in the hb - nsc group and , in particular , there was a statistically significant increase in dopamine trans - porter activity by 10 weeks posttransplantation compared to the pretransplantation state ( 48.53.9% vs. 41.02.6% , respectively ) ( fig . these micropet data are consistent with the results of the stepping test in which significant improvement was detected from 6 weeks posttransplantation onward . ihc and if analysis revealed the presence of tyrosine hydroxylase ( th)-positive cells in the ipsilateral striatum of the hb - nsc transplantation group ( fig . high - magnification images showed that the th - positive cells corresponded to the cells positive for the human 2 microglobulin marker ( fig . the th - positive cells in the striatum were surrounded by a round margin that indicated the area of the transplantation ( fig . these results indicate that the implanted hb - nsc aggregates successfully differentiated into dopaminergic cells within the host striatal tissue . this finding differs from our previous study using single - cell suspensions for injection which demonstrated abnormal parallel staining of dendrites ( fig . dendrites of the stained cells extended in various directions , suggesting network formation with the neighboring cells ( fig . the stepping test showed no significant increase in contralateral paw touches up to 4 weeks posttransplantation ; however , at 6 weeks posttransplantation , a significant improvement of 52.9% was measured . at 8 weeks posttransplantation , the increase was 48.9% and this improvement was maintained through to 10 weeks , at which time the increase was 64.2% in the hb - nsc group compared to the control group ( fig . 1a ) . in apomorphine - induced rotation tests , the hb - nsc group showed a slight decrease in contralateral rotations at week 8 , compared to week 4 ; however , the difference was not statistically significant ( fig . there was no significant decrease in uptake of [ f]-fdg in either the control or hb - nsc transplantation group , indicating that glucose metabolism was normal , as is characteristic of pd ( fig . 2a ) . using [ f]-fp - cit to quantify dopamine transporter activity , a gradual increase in radioactivity in the lesion - side striatum was detected in the hb - nsc group and , in particular , there was a statistically significant increase in dopamine trans - porter activity by 10 weeks posttransplantation compared to the pretransplantation state ( 48.53.9% vs. 41.02.6% , respectively ) ( fig . these micropet data are consistent with the results of the stepping test in which significant improvement was detected from 6 weeks posttransplantation onward . ihc and if analysis revealed the presence of tyrosine hydroxylase ( th)-positive cells in the ipsilateral striatum of the hb - nsc transplantation group ( fig . high - magnification images showed that the th - positive cells corresponded to the cells positive for the human 2 microglobulin marker ( fig . the th - positive cells in the striatum were surrounded by a round margin that indicated the area of the transplantation ( fig . these results indicate that the implanted hb - nsc aggregates successfully differentiated into dopaminergic cells within the host striatal tissue . this finding differs from our previous study using single - cell suspensions for injection which demonstrated abnormal parallel staining of dendrites ( fig . dendrites of the stained cells extended in various directions , suggesting network formation with the neighboring cells ( fig . in previous studies , neural stem cell transplants in pd had difficulty for surviving and differentiating in the host striatum19 ) . over the past decade , clinical trials of neural stem cell transplantation in pd patients as well as animal experiments with pd model used intrastriatal grafts because intrastriatal dopaminergic cell grafts not only are capable of reinnervate with the host striatal tissue but also replace the functions of dopaminergic cells of substantia nigra21,23,26,27 ) . on the contrary , fetal dopaminergic neurons transplanted into the substantia nigra have been reported to regrow axons into the striatum in one report22 ) , but another literature showed failure of extending axons into the striatum12 ) . in this regard , we think the transplantation into substantia nigra does not seem to be effective and appropriate compared to striatal injection and also the injection of enough volume of cells might be difficult in substantia nigra compared to the striatal injection . our previous study on transplantation of hb - nsc as a suspension of single cells showed that the injected cells survived and were demonstrated as positive in th staining , but behavioral tests and [ f]-fp - cit scans showed no improvement30 ) . thus , in this study , aggregation form of hb - nsc were transplanted into the striatum of 6-ohda rat models and the results were evaluated using behavioral tests , multitracer - micropet scans , ihc , and if tissue staining . in the behavioral stepping test , the hb - nsc group showed insignificant increases in contralateral forelimb touches up to 4 weeks after transplantation , but then drastically improved from 6 to 10 weeks posttransplantation . in apomorphine - induced rotation tests , the hb - nsc group exhibited a slight decrease in contralateral rotations by week 8 compared to week 4 posttransplantation , although the difference was not statistically significant . in recent cell transplantation studies , apomorphine - induced rotation tests did not fully reflect the pd status because of apomorphine sensitization18 ) . the significant improvement in the stepping test in the hb - nsc group was not found in the previous single - cell suspension transplant study30 ) . in the stepping test , the drama - tic improvement at 6 weeks posttransplantation suggests an increase in transplanted cell function , previously described as small - world network development by 4 weeks posttransplantation7 ) . the network formed among transplanted hb - nsc or between hb - nsc and host striatal cells could affect cell survival as well as provide therapeutic effects . imaging of dopaminergic neurons by techniques such as [ f]-fp - cit pet scans has been used to diagnose pd3,14 ) . striatal uptake of [ f]-fp - cit , a ligand of the dopamine transporter , decreases in pd . [ f]-fp - cit micropet images were useful and consistent indicators of movement disorders in a rat model in our previous reports29,30 ) . in this study , there was a significant increase in [ f]-fp - cit uptake by 10 weeks posttransplantation compared to the pretransplantation state . these results suggest that the improvements in the behavioral stepping test are the result of dopamine secretion from the transplanted hb - nsc . however , as we described in the methods section , 2 rats in control group were dead during micropet study and comparison between hb - nsc group and control group could not be performed which is a limitation of this study . [ f]-fdg pet scan was performed to evaluate glucose metabolism in the striatum . in typical pd model made by 6-ohda injection into medial forebrain bundle , glucose metabolism of striatum does not decrease . therefore , we studied [ f]-fdg pet at 10 week posttransplantation , and the uptake of bilateral striatum did not show significant differences in control group and hb - nsc group , which suggest successful pd models were made . transplantation of both single - cell suspensions , reported in our previous study30 ) , and aggregates of hb - nsc results in the appearance of th - positive cells in striatum . however , improvements in the stepping tests and increasing uptake of [ f]-fp - cit were demonstrated only after transplantation of hb - nsc aggregates , indicating that dopamine secretion at therapeutic levels only resulted from the trans - plantation of cell aggregates . although single - cell suspensions transplantation resulted in a wide range of th - positive cells in the striatum , the transplanted cells showed abnormal parallel staining which considered to be dendrites of partially differentiated and poorly connected dopaminergic cells ( fig . , no change in [ f]-fp - cit scans in single - cell suspensions transplanted into the striatum suggests that dopamine secretion from the transplanted cells is absent as a result of incomplete differentiation30 ) . by contrast , cell ag - gregate transplantations showed th - positive cells only in a small area in the striatum ( fig . 3b , d ) , but dendrites of the stained cells extended in various directions ( fig . 4d ) suggesting the formation of a network with the neighboring cells . from these data , we hypothesize that the cell aggregates adapt well and efficiently secrete neurotransmitter within the host tissue . the number of cells per transplant in this study was less than 10% of the single - cell suspensions used in the previous study ( 810 vs. 1 10 ) , and this difference in injected cell numbers may explain the small area of th - positive cells compared to previous study . this study has the limitation of showing the advantage of pre - established cell network in cell transplantation because we used cell aggregates not spheroid form which is currently difficult to make and transplant deep into the brain . in addition , the phenomenon of marginal th positive staining in the injection site of cell aggregates could not be explained and quantitative comparison between cell aggregation form and single - cell suspensions was not carried out since the cell numbers of two studies were not similar . in conclusion , this study showed that cell aggregates of hb - nsc transplanted into the striatum not only survive well , but also exert a therapeutic effect in a rat pd model by secreting dopamine . such success was not achieved in the transplantation of single - cell suspensions of hb - nsc . thus , this result suggests that the form of cell transplantation is critical for successful therapy .

objectiveneural tissue transplantation has been a promising strategy for the treatment of parkinson 's disease ( pd ) . however , transplantation has the disadvantages of low - cell survival and/or development of dyskinesia . transplantation of cell aggregates has the potential to overcome these problems , because the cells can extend their axons into the host brain and establish synaptic connections with host neurons . in this present study , aggregates of human brain - derived neural stem cells ( hb - nsc ) were transplanted into a pd animal model and compared to previous report on transplantation of single - cell suspensions.methodsrats received an injection of 6-ohda into the right medial forebrain bundle to generate the pd model and followed by injections of pbs only , or hb - nsc aggregates in pbs into the ipsilateral striatum . behavioral tests , multitracer ( 2-deoxy-2-[18f]-fluoro - d - glucose ( [ 18f]-fdg ) and [ 18f]-n-(3-fluoropropyl)-2-carbomethoxy-3-(4-iodophenyl)nortropane ( [ 18f]-fp - cit ) micropet scans , as well as immunohistochemical ( ihc ) and immunofluorescent ( if ) staining were conducted to evaluate the results.resultsthe stepping test showed significant improvement of contralateral forelimb control in the hb - nsc group from 6 - 10 weeks compared to the control group ( p<0.05 ) . [ 18f]-fp - cit micropet at 10 weeks posttransplantation demonstrated a significant increase in uptake in the hb - nsc group compared to pretransplantation ( p<0.05 ) . in ihc and if staining , tyrosine hydroxylase and human 2 microglobulin ( a human cell marker ) positive cells were visualized at the transplant site.conclusionthese results suggest that the hb - nsc aggregates can survive in the striatum and exert therapeutic effects in a pd model by secreting dopamine .

INTRODUCTION MATERIALS AND METHODS Animal and 6-OHDA lesion procedures Transplantation of aggregates of human brain-derived neural stem cells Behavioral tests Multitracer [ Immunohistochemical and immunofluorescent tissue staining Statistical analysis RESULTS Behavioral tests Multitracer [ Immunohistochemistry and immunofluorescence DISCUSSION CONCLUSION

parkinson 's disease ( pd ) is neurodegenerative disorder characterized by progressive loss of substantia nigra pars compacta ( snpc ) neurons1,4,5,20 ) . neural tissue transplantation has been a promising strategy for pd because the transplanted tissues can produce dopamine in the striatum , which has a therapeutic effect in pd patients2,15 ) . however , tissue grafts have the disadvantages of low rates of cell survival and/or development of dyskinesia9 ) . the transplantation of spheroids , or cell aggregations , has been proposed to overcome these problems , because the cell aggregates can extend their axons into the host brain and establish synaptic connections with host neurons following transplantation into specific brain areas13 ) . in our previous study , human brain - derived neural stem cells ( hb - nsc ) which were injected into the striatum of a pd rat model as single - cell suspensions showed partial differentiation to dopaminergic cells , but without therapeutic effect30 ) . we hypothesized that the injected single hb - nsc cell suspensions could partially differentiate into dopaminergic cells , which produced dopamine within the cell , but were unable to secrete it . therefore , in the present study , we transplanted hb - nsc as cell aggregates form into the striatum of pd rats to determine their efficacy compared to single - cell transplantations . we expected that the cell aggregates have the advantage of a pre - established cell network which reported in transplantation of spheroid form13 ) that might promote high rates of survival and differentiation . after the transplantation , we evaluated the results by behavioral tests , pet study and histological study . at the beginning of the study , each animal was anesthetized with zoletil 50 ( virbac s.a , carros , france ) and rompun ( bayer , leverkusen , germany ) at doses of 37.5 mg / kg and 6 mg / kg , respectively , using an intraperitoneal ( i.p . ) louis , mo , usa ) in 4 l of 0.9% saline with 0.1% ascorbic acid delivered into the right medial forebrain bundle at the following coordinates : anteroposterior -4.4 mm , lateral + 1.2 mm relative to the bregma and ventral -7.8 mm from the dura by paxinos 's rat atlas , with the tooth bar set at -2.4 mm . then , hb - nsc cells were injected in 9 rats ( hb - nsc group ) and pbs in 9 rats ( control group ) . however , 2 rats were dead during pet study , so the final numbers of experimental animals became 9 in hb - nsc group and 7 in control group . hb - nsc ( san pedro , ca , usa ) were expanded in brain stem - cell growth medium according to the manufacturer 's instructions . briefly , hb - nsc at an approximate cell density of 110 cells/200 l were deposited into each microwell array , and then placed in a 5% co2 humidified incubator for 30 min to allow the cells to settle . the cell aggregations transplanted into the right striatum of the pd rat model ( hb - nsc group , n=9 ) using a 22 g hamilton syringe and a stereotaxic microinjection device ( coordinates : ap + 0.6 mm from the bregma , ml + 3.5 mm from the midline , dv -4.5 mm , nose bar + 2.3 mm ) 4 weeks after the 6-ohda injection . the control group ( n=7 ) received 8 l of pbs ( ph 7.4 ) injected at the same coordinates . briefly , as soon as 0.5 mg / kg apomorphine ( sigma ; in sterile water , injected subcutaneously ) was administered , the rat was harnessed to an automated rotometer ( panlab , barcelona , spain ) and its rotational behavior was assessed over 45 minutes . glucose metabolic activity was assessed in the control ( n=3 ) and hb - nsc groups ( n=5 ) using [ f]-fdg at 10 weeks posttransplantation . dopamine transporter function was assessed with the same animals in the control ( n=3 ) and hb - nsc group ( n=5 ) using [ f]-fp - cit , which binds to the dopamine transporter with high affinity , at 1 or 2 weeks after 6-ohda injection ( pd status before cell transplantation ) and at 5 and 10 weeks after cell transplantation . rats were injected with 1 mci [ f]-fp - cit in 0.4 ml sterile saline via the tail vein , immediately followed by dynamic scanning for 90 minutes . data are expressed as the percentage of ipsilateral standardized uptake values based on the mean uptake in the 3d volume of interest against the contralateral standardized uptake values . these sections were subsequently incubated for 2 h at room temperature with biotinylated secondary antibodies produced in mouse ( vector laboratories , burlingame , ca , usa ) and diluted 1 : 200 in pbs containing 0.5% bsa . th - positive cells were observed in the coronal sections of the striatum using a nikon 80i microscope ( nikon , tokyo , japan ) at 100 magnification using the nis - elements f3.0 software ( nikon , tokyo , japan ) . louis , usa ) and anti - human 2 microglobulin polyclonal ( 1 : 800 , dako , seoul , korea ) antibodies for 1 h at room temperature . at the beginning of the study , each animal was anesthetized with zoletil 50 ( virbac s.a , carros , france ) and rompun ( bayer , leverkusen , germany ) at doses of 37.5 mg / kg and 6 mg / kg , respectively , using an intraperitoneal ( i.p . ) louis , mo , usa ) in 4 l of 0.9% saline with 0.1% ascorbic acid delivered into the right medial forebrain bundle at the following coordinates : anteroposterior -4.4 mm , lateral + 1.2 mm relative to the bregma and ventral -7.8 mm from the dura by paxinos 's rat atlas , with the tooth bar set at -2.4 mm . then , hb - nsc cells were injected in 9 rats ( hb - nsc group ) and pbs in 9 rats ( control group ) . however , 2 rats were dead during pet study , so the final numbers of experimental animals became 9 in hb - nsc group and 7 in control group . hb - nsc ( san pedro , ca , usa ) were expanded in brain stem - cell growth medium according to the manufacturer 's instructions . briefly , hb - nsc at an approximate cell density of 110 cells/200 l were deposited into each microwell array , and then placed in a 5% co2 humidified incubator for 30 min to allow the cells to settle . we could evaluate the concentration of cell aggregation by cell counting with trypan blue viability staining which was partially harvested from cell aggregation solution and transformed into cell suspension . the cell aggregations transplanted into the right striatum of the pd rat model ( hb - nsc group , n=9 ) using a 22 g hamilton syringe and a stereotaxic microinjection device ( coordinates : ap + 0.6 mm from the bregma , ml + 3.5 mm from the midline , dv -4.5 mm , nose bar + 2.3 mm ) 4 weeks after the 6-ohda injection . the control group ( n=7 ) received 8 l of pbs ( ph 7.4 ) injected at the same coordinates . the rotation tests were conducted 1 week after 6-ohda injection , and 4 and 8 weeks after cell transplantation , as previously described25 ) , with slight modifications . glucose metabolic activity was assessed in the control ( n=3 ) and hb - nsc groups ( n=5 ) using [ f]-fdg at 10 weeks posttransplantation . dopamine transporter function was assessed with the same animals in the control ( n=3 ) and hb - nsc group ( n=5 ) using [ f]-fp - cit , which binds to the dopamine transporter with high affinity , at 1 or 2 weeks after 6-ohda injection ( pd status before cell transplantation ) and at 5 and 10 weeks after cell transplantation . rats were transcardially and sequentially perfused with 0.9% saline containing 10000 iu heparin ( hanlim pharm , seoul , korea ) and 4% paraformaldehyde in pbs . th - positive cells were observed in the coronal sections of the striatum using a nikon 80i microscope ( nikon , tokyo , japan ) at 100 magnification using the nis - elements f3.0 software ( nikon , tokyo , japan ) . louis , usa ) and anti - human 2 microglobulin polyclonal ( 1 : 800 , dako , seoul , korea ) antibodies for 1 h at room temperature . the stepping test showed no significant increase in contralateral paw touches up to 4 weeks posttransplantation ; however , at 6 weeks posttransplantation , a significant improvement of 52.9% was measured . at 8 weeks posttransplantation , the increase was 48.9% and this improvement was maintained through to 10 weeks , at which time the increase was 64.2% in the hb - nsc group compared to the control group ( fig . in apomorphine - induced rotation tests , the hb - nsc group showed a slight decrease in contralateral rotations at week 8 , compared to week 4 ; however , the difference was not statistically significant ( fig . there was no significant decrease in uptake of [ f]-fdg in either the control or hb - nsc transplantation group , indicating that glucose metabolism was normal , as is characteristic of pd ( fig . using [ f]-fp - cit to quantify dopamine transporter activity , a gradual increase in radioactivity in the lesion - side striatum was detected in the hb - nsc group and , in particular , there was a statistically significant increase in dopamine trans - porter activity by 10 weeks posttransplantation compared to the pretransplantation state ( 48.53.9% vs. 41.02.6% , respectively ) ( fig . these micropet data are consistent with the results of the stepping test in which significant improvement was detected from 6 weeks posttransplantation onward . ihc and if analysis revealed the presence of tyrosine hydroxylase ( th)-positive cells in the ipsilateral striatum of the hb - nsc transplantation group ( fig . high - magnification images showed that the th - positive cells corresponded to the cells positive for the human 2 microglobulin marker ( fig . the th - positive cells in the striatum were surrounded by a round margin that indicated the area of the transplantation ( fig . these results indicate that the implanted hb - nsc aggregates successfully differentiated into dopaminergic cells within the host striatal tissue . this finding differs from our previous study using single - cell suspensions for injection which demonstrated abnormal parallel staining of dendrites ( fig . the stepping test showed no significant increase in contralateral paw touches up to 4 weeks posttransplantation ; however , at 6 weeks posttransplantation , a significant improvement of 52.9% was measured . at 8 weeks posttransplantation , the increase was 48.9% and this improvement was maintained through to 10 weeks , at which time the increase was 64.2% in the hb - nsc group compared to the control group ( fig . in apomorphine - induced rotation tests , the hb - nsc group showed a slight decrease in contralateral rotations at week 8 , compared to week 4 ; however , the difference was not statistically significant ( fig . there was no significant decrease in uptake of [ f]-fdg in either the control or hb - nsc transplantation group , indicating that glucose metabolism was normal , as is characteristic of pd ( fig . using [ f]-fp - cit to quantify dopamine transporter activity , a gradual increase in radioactivity in the lesion - side striatum was detected in the hb - nsc group and , in particular , there was a statistically significant increase in dopamine trans - porter activity by 10 weeks posttransplantation compared to the pretransplantation state ( 48.53.9% vs. 41.02.6% , respectively ) ( fig . these micropet data are consistent with the results of the stepping test in which significant improvement was detected from 6 weeks posttransplantation onward . ihc and if analysis revealed the presence of tyrosine hydroxylase ( th)-positive cells in the ipsilateral striatum of the hb - nsc transplantation group ( fig . high - magnification images showed that the th - positive cells corresponded to the cells positive for the human 2 microglobulin marker ( fig . the th - positive cells in the striatum were surrounded by a round margin that indicated the area of the transplantation ( fig . these results indicate that the implanted hb - nsc aggregates successfully differentiated into dopaminergic cells within the host striatal tissue . this finding differs from our previous study using single - cell suspensions for injection which demonstrated abnormal parallel staining of dendrites ( fig . in previous studies , neural stem cell transplants in pd had difficulty for surviving and differentiating in the host striatum19 ) . over the past decade , clinical trials of neural stem cell transplantation in pd patients as well as animal experiments with pd model used intrastriatal grafts because intrastriatal dopaminergic cell grafts not only are capable of reinnervate with the host striatal tissue but also replace the functions of dopaminergic cells of substantia nigra21,23,26,27 ) . on the contrary , fetal dopaminergic neurons transplanted into the substantia nigra have been reported to regrow axons into the striatum in one report22 ) , but another literature showed failure of extending axons into the striatum12 ) . in this regard , we think the transplantation into substantia nigra does not seem to be effective and appropriate compared to striatal injection and also the injection of enough volume of cells might be difficult in substantia nigra compared to the striatal injection . our previous study on transplantation of hb - nsc as a suspension of single cells showed that the injected cells survived and were demonstrated as positive in th staining , but behavioral tests and [ f]-fp - cit scans showed no improvement30 ) . thus , in this study , aggregation form of hb - nsc were transplanted into the striatum of 6-ohda rat models and the results were evaluated using behavioral tests , multitracer - micropet scans , ihc , and if tissue staining . in the behavioral stepping test , the hb - nsc group showed insignificant increases in contralateral forelimb touches up to 4 weeks after transplantation , but then drastically improved from 6 to 10 weeks posttransplantation . in apomorphine - induced rotation tests , the hb - nsc group exhibited a slight decrease in contralateral rotations by week 8 compared to week 4 posttransplantation , although the difference was not statistically significant . the significant improvement in the stepping test in the hb - nsc group was not found in the previous single - cell suspension transplant study30 ) . in the stepping test , the drama - tic improvement at 6 weeks posttransplantation suggests an increase in transplanted cell function , previously described as small - world network development by 4 weeks posttransplantation7 ) . the network formed among transplanted hb - nsc or between hb - nsc and host striatal cells could affect cell survival as well as provide therapeutic effects . [ f]-fp - cit micropet images were useful and consistent indicators of movement disorders in a rat model in our previous reports29,30 ) . in this study , there was a significant increase in [ f]-fp - cit uptake by 10 weeks posttransplantation compared to the pretransplantation state . these results suggest that the improvements in the behavioral stepping test are the result of dopamine secretion from the transplanted hb - nsc . however , as we described in the methods section , 2 rats in control group were dead during micropet study and comparison between hb - nsc group and control group could not be performed which is a limitation of this study . [ f]-fdg pet scan was performed to evaluate glucose metabolism in the striatum . in typical pd model made by 6-ohda injection into medial forebrain bundle , glucose metabolism of striatum does not decrease . therefore , we studied [ f]-fdg pet at 10 week posttransplantation , and the uptake of bilateral striatum did not show significant differences in control group and hb - nsc group , which suggest successful pd models were made . transplantation of both single - cell suspensions , reported in our previous study30 ) , and aggregates of hb - nsc results in the appearance of th - positive cells in striatum . however , improvements in the stepping tests and increasing uptake of [ f]-fp - cit were demonstrated only after transplantation of hb - nsc aggregates , indicating that dopamine secretion at therapeutic levels only resulted from the trans - plantation of cell aggregates . although single - cell suspensions transplantation resulted in a wide range of th - positive cells in the striatum , the transplanted cells showed abnormal parallel staining which considered to be dendrites of partially differentiated and poorly connected dopaminergic cells ( fig . , no change in [ f]-fp - cit scans in single - cell suspensions transplanted into the striatum suggests that dopamine secretion from the transplanted cells is absent as a result of incomplete differentiation30 ) . by contrast , cell ag - gregate transplantations showed th - positive cells only in a small area in the striatum ( fig . from these data , we hypothesize that the cell aggregates adapt well and efficiently secrete neurotransmitter within the host tissue . the number of cells per transplant in this study was less than 10% of the single - cell suspensions used in the previous study ( 810 vs. 1 10 ) , and this difference in injected cell numbers may explain the small area of th - positive cells compared to previous study . this study has the limitation of showing the advantage of pre - established cell network in cell transplantation because we used cell aggregates not spheroid form which is currently difficult to make and transplant deep into the brain . in addition , the phenomenon of marginal th positive staining in the injection site of cell aggregates could not be explained and quantitative comparison between cell aggregation form and single - cell suspensions was not carried out since the cell numbers of two studies were not similar . in conclusion , this study showed that cell aggregates of hb - nsc transplanted into the striatum not only survive well , but also exert a therapeutic effect in a rat pd model by secreting dopamine . such success was not achieved in the transplantation of single - cell suspensions of hb - nsc .

the ii - vi semiconductor zno is one of the most important multifunctional materials due to its various exotic properties such as direct wide band gap ( 3.37 ev ) and high optical gain of 300 cm ( 100 cm for gan ) at room temperature , large saturation velocity ( 3.2 10 cm / s ) , high breakdown voltage , large exciton binding energy ( 60 mev ) , piezoelectric , biocompatibility , and so on [ 1 - 12 ] . zno can be used in variety of high - technological practical applications such as ultraviolet ( uv ) lasers , light - emitting diodes , photodetectors , piezoelectric transducers and actuators , hydrogen storage , chemical and biosensors , surface acoustic wave guides , solar cells , photocatalysts , etc . the use of zno nanomaterials as photoelectrodes for the fabrication of dye - sensitized solar cells ( dsscs ) has received a great attention due to its compatibility and higher electronic mobility with tio2 nanomaterials and similar electron affinity and band gap ( 3.37 ev at 298 k ) . therefore , some zno nanostructures have been used as photoelectrode materials for the fabrication of dsscs and reported in the literature [ 15 - 21 ] . hsu et al . reported the zno nanorods - based dssc with the electricity conversion efficiency ( ece ) of 0.22% . branched zno nanowires based dsscs , grown by thermal evaporation process at 8001,000 c , with an ece of ~0.46% have been reported by suh et al . . in another report , by using branched zno nanowires grown by mocvd process , the fabricated dsscs exhibited an ece of ~0.5% . cheng et al . also demonstrated the thermally grown zno nanorods - based dssc with the ece of 0.6% . in this paper , we report the direct synthesis of well - crystallized zno nanocombs on fto substrates and their dsscs application . to fabricate the dsscs , the thin films of as - grown zno nanocombs on fto substrates were used as photoanode materials , which exhibited an overall light to electricity conversion efficiency of 0.68% . to the best of our knowledge , the use of zno nanocombs for the fabrication of dsscs is not reported yet in the literature . the high purity metallic zinc powder ( 99.999% ) and oxygen gas were used as source materials . in a typical reaction process , about 1.5 g of metallic zinc powder was put into a ceramic boat and placed at the center of the quartz tube . the furnace temperature was raised up to the desired temperature , and oxygen and nitrogen were fed continuously into the quartz tube furnace with the flow rates of 60 and 240 sccm , respectively . the temperature of the substrate , placed 8-cm away from the source boat , was 570 c . the metallic zinc was vaporized and oxidized with o2 , and finally deposited onto the fto substrate . for dssc fabrication , the prepared zno nanocomb thin - film electrodes was immersed in the ethanolic solution of 0.3 mm cis - bis ( isothiocyanato ) bis(2,2-bipyridyl-4,4-dicarboxylato)-ruthenium ( ii ) bis - tetrabutylammonium ( n719 , solaronix ) at room temperature for 6 h. the dye - adsorbed zno nanocombs thin - film electrodes were then rinsed with acetonitrile and dried under a nitrogen stream . pt counter electrode was prepared by electron beam deposition of a thin layer of pt ( ~ 60 nm ) on the top of ito glass . the pt electrode was placed over the dye - adsorbed zno nanocombs electrode , and the edges of the cell were sealed with 60-m thick sealing sheet ( sx 1170 - 60 , solaronix ) . sealing was accomplished by pressing the two electrodes together on a double hot - plate at a temperature of about 70 c . the electrolyte , consisting of 0.5 m lii , 0.05 mm i2 , and 0.2 m tert - butyl pyridine in acetonitrile , was introduced into the cell through one of two small holes drilled in the counter - electrode . the holes were then covered and sealed with a small square of sealing sheet and microscope objective glass . the light source was 1000-w metal halide lamp , and its radiant power was adjusted with respect to si reference solar cell to about one - sun - light intensity ( 100 mw / cm ) . figure 1a shows the low - magnification fesem image of the zno nanocombs and reveals that the nanocombs are densely grown and uniformly distributed over the large area of the substrate surface . from the high - magnification images , it is seen that the nanocombs are made by two components , i.e. nanorodlike branches and wide ribbonlike stems . the branches ( teeth ) of the nanocombs are uniform and nicely attached along one side of the ribbonlike stem . the width of the stem is ~1.2 0.3 m , and the stem is several micrometers long . the diameter and length of each tooth is ~300 100 nm and ~3 0.5 m respectively . these teeth are arranged in a proper manner with a distance of ~200 50 nm between each other [ figure ( b ) and inset ( b ) ] . the x - ray diffraction ( xrd ) pattern exhibits that the as - grown nanocombs are single - crystalline with the wurtzite hexagonal - phase pure zno ( jcpds # 361451 ) ( fig . except zno , no characteristic peaks for other impurities such as zinc and substrate were observed in the spectrum , which confirms that the obtained products are single - crystalline wurtzite hexagonal - phase zno grown in highdensity on the fto substrate . in addition to this , the energy dispersive spectroscopy ( eds ) confirmed that the as - grown nanocombs are made with almost 1:1 stoichiometry of zinc and oxygen ( fig . . further structural characterization of the grown products was made using the transmission electron microscope ( tem ) and high - resolution tem combined with the selected area electron diffraction ( saed ) pattern . figure 2a shows the low - magnification tem image of the nanocombs , which reveals the full consistency with the fesem observation in terms of morphology and dimensionality . clearly , it is seen in the tem image that the branches of the nanocombs are attached along one side of the ribbonlike stem . the hrtem image of one tooth of comblike structure circled in figure(a ) demonstrated a well - defined lattice fringes with the lattice spacing of 0.52 nm , corresponds to the d - spacing of the crystal plane of the wurtzite hexagonal zno , confirmed that the branches of the comb structures are grown along the direction ( fig . the corresponding saed pattern of a branch of comb projected to the [ 20 ] zone axis is also consistent with hrtem observation ( fig . figure 2c shows the room - temperature photoluminescence ( pl ) spectrum measured using a he cd laser line with an exciton wavelength of 325 nm . the obtained pl spectrum exhibited a narrow peak at ~385 nm in the uv region , also called near band edge emission , and a broad emission peak at ~570 nm in the visible region , also known as deep - level emission . it is well known that the uv emission has been realized to the exciton emission , while the deep - level emission is generally explained as the radial recombination of photo - generated hole with a singly ionized charged state of the oxygen vacancy . typical ( a ) low- and ( b ) high - magnification fesem images ; ( c ) xrd pattern and ( d ) eds spectrum of high density - grown zno nanocombs on fto substrate typical ( a ) low- and ( b ) high - magnification tem image and their corresponding saed pattern [ inset(b ) ] ; and ( c ) room - temperature pl spectrum of as - grown zno nanocombs used for the fabrication of dssc as a wurtzite hexagonal - phase zno possesses a positively charged zn-(0001 ) surfaces that are catalytically active , the negatively charged o-(0001 ) surfaces are chemically inert . the comb stem grows along the [ 2 0 ] direction , while the top and bottom surfaces are zinc and oxygen terminated ( 0001 ) respectively . it is reported that the catalytically active zn - terminated ( 0001 ) surfaces tend to have tiny zn clusters and other zn particles at the growth front , which could provide an active site for the further growth process , and hence comb teeth can grow in front of zinc - terminated ( 0001 ) surfaces . due to higher growth velocity in direction of zno crystals , voltage ( i v ) characteristics for dsscs fabricated with zno nanocombs thin - film electrodes and measured under a simulated illumination with a light intensity of 100 mw / cm ( am = 1.5 ) . a maximum electricity conversion efficiency of 0.68% was achieved by highly branched zno nanocombs thin - film dsscs . the fabricated dsscs also obtained a maximum short - circuit current density ( jsc ) of 3.14 ma / cm with low voc of 0.671 v and low ff of 34% . the low jsc and conversion efficiency reveal that low dye absorption on the surface of zno thin film and result in the low - light harvesting and fast interface recombination rate of electron and holes . it is reported that the interface recombination loss in zno - based dsscs is mostly due to the uncovered oxide surface ( with no dye molecule anchored on ) , where oxide contacts with electrolyte closely and thus increases the probability of charge recombination between the electrons in oxide and the holes in the electrolyte . the low voc can be explained by the gapping between the spikes of zno nanocombs , which also cause direct contact of electrolyte to the fto glass , results the low ff . the low ff and photocurrent may be explained by the fast recombination rate between the photoexcited carriers at the nanocombs and the electrolyte interfaces , which is related to series resistance rs = ( dv / di)i=0 . generally , rs is ascribed to the bulk resistance of semiconductor oxide films , tco electrode , metallic contacts , and electrolyte . from the i v curve , rs of zno nanocombs - based dssc is relatively high ( ~213 cm ) , which increased the charge recombination between the photoexcited carriers at the nanocombs and the redox electrolyte . the high rs results in the low ff and photocurrent . figure 3b shows the uv vis absorption spectrum of desorbed dye obtained from the zno electrodes by dipping the zno nanocombs electrode in 0.1 mm naoh solution for 10 min . it was observed that low dye absorption ( ~3.23 10 mol / cm ) by zno nanocombs film surface electrode was probably due to the nonporous morphologies of the nanocombs . it is well known that the high dye absorption by porous thin film leads to high light - harvesting efficiency . therefore , low jsc and are related to less absorption of dye molecules and insufficient light harvesting from the zno nanocombs thin - film electrodes . 3b demonstrates the general morphologies of zno nanocombs after the dye absorption and , interestingly , there was no distinct change observed in the general morphologies of the nanocombs after dye absorption , hence the nanocombs retain their morphologies after dye absorption . v ) characteristics of zno nanocombs - based dssc and ( b ) typical uv vis absorption spectra of the desorbed dye ( n719 ) from the zno nanocombs electrode thin films . inset of ( b ) exhibits the surface morphology of the comblike structures after the desorption of dye in order to elucidate the charge transfer properties of as - grown zno nanocombs substrates , an electrochemical impedance spectroscopy ( eis ) measurement was used . eis measurements were taken out under the illumination of 100 mw / cm ( am = 1.5 ) by applying a 10 mv ac signal over the frequency range of 10 hz100 khz using a potentiostat with lock in amplifier , as shown in fig . equivalent circuit is composed of the resistance of redox electrolyte solution ( rs ) , the charge transfer resistance at the interface of electrolyte and zno nanocombs ( rct ) , the charge transfer resistance at the interface of zno nanocombs and tco ( rzno / tco ) , the capacitance of accumulation ( of e- ) layer of the zno nanocombs ( cacc ) and space charge capacitance ( csc ) . the value of real impedance ( zre ) at high and medium frequencies represents the rzno / tco and rct . figure 4 exhibits the ac impedance curve of dssc fabricated with thermally grown zno nanocombs electrode . a very high rzno / tco ( 90 ) and rct ( 29.6 ) were obtained for zno nanocombs thin - film electrodes , which are lesser than that of tio2 thin - film electrodes . it is reported that a small rct suggests fast electron transfer , whereas a large rct indicates slow electron transfer . the high rct ( 29.6 ) of zno nanocombs thin - film electrode explains the slow electron transfer , which results in the low photocurrent density and conversion efficiency . therefore , the high charge transfer resistance at zno / electrolyte interface reveals a slow electron transfer through the zno nanocombs thin - film electrode , which results in the low isc , ff , and conversion efficiency of the fabricated dssc . inset shows the equivalent circuit model of the dsscs , wherersis the resistance of redox electrolyte solution , rctthe charge transfer resistance at the interface of electrolyte and zno nanocombs , [ rct ] is the charge transfer resistance at the interface of zno nanocombs and tco [ rzno / tco ] , is the capacitance of accumulation ( of e- ) layer of the zno nanocombs [ cacc ] andcscspace charge capacitance figure 1a shows the low - magnification fesem image of the zno nanocombs and reveals that the nanocombs are densely grown and uniformly distributed over the large area of the substrate surface . from the high - magnification images , it is seen that the nanocombs are made by two components , i.e. nanorodlike branches and wide ribbonlike stems . the branches ( teeth ) of the nanocombs are uniform and nicely attached along one side of the ribbonlike stem . the width of the stem is ~1.2 0.3 m , and the stem is several micrometers long . the diameter and length of each tooth is ~300 100 nm and ~3 0.5 m respectively . these teeth are arranged in a proper manner with a distance of ~200 50 nm between each other [ figure ( b ) and inset ( b ) ] . the x - ray diffraction ( xrd ) pattern exhibits that the as - grown nanocombs are single - crystalline with the wurtzite hexagonal - phase pure zno ( jcpds # 361451 ) ( fig . except zno , no characteristic peaks for other impurities such as zinc and substrate were observed in the spectrum , which confirms that the obtained products are single - crystalline wurtzite hexagonal - phase zno grown in highdensity on the fto substrate . in addition to this , the energy dispersive spectroscopy ( eds ) confirmed that the as - grown nanocombs are made with almost 1:1 stoichiometry of zinc and oxygen ( fig . . further structural characterization of the grown products was made using the transmission electron microscope ( tem ) and high - resolution tem combined with the selected area electron diffraction ( saed ) pattern . figure 2a shows the low - magnification tem image of the nanocombs , which reveals the full consistency with the fesem observation in terms of morphology and dimensionality . clearly , it is seen in the tem image that the branches of the nanocombs are attached along one side of the ribbonlike stem . the hrtem image of one tooth of comblike structure circled in figure(a ) demonstrated a well - defined lattice fringes with the lattice spacing of 0.52 nm , corresponds to the d - spacing of the crystal plane of the wurtzite hexagonal zno , confirmed that the branches of the comb structures are grown along the direction ( fig . the corresponding saed pattern of a branch of comb projected to the [ 20 ] zone axis is also consistent with hrtem observation ( fig . figure 2c shows the room - temperature photoluminescence ( pl ) spectrum measured using a he cd laser line with an exciton wavelength of 325 nm . the obtained pl spectrum exhibited a narrow peak at ~385 nm in the uv region , also called near band edge emission , and a broad emission peak at ~570 nm in the visible region , also known as deep - level emission . it is well known that the uv emission has been realized to the exciton emission , while the deep - level emission is generally explained as the radial recombination of photo - generated hole with a singly ionized charged state of the oxygen vacancy . typical ( a ) low- and ( b ) high - magnification fesem images ; ( c ) xrd pattern and ( d ) eds spectrum of high density - grown zno nanocombs on fto substrate typical ( a ) low- and ( b ) high - magnification tem image and their corresponding saed pattern [ inset(b ) ] ; and ( c ) room - temperature pl spectrum of as - grown zno nanocombs used for the fabrication of dssc as a wurtzite hexagonal - phase zno possesses a positively charged zn-(0001 ) surfaces that are catalytically active , the negatively charged o-(0001 ) surfaces are chemically inert . the comb stem grows along the [ 2 0 ] direction , while the top and bottom surfaces are zinc and oxygen terminated ( 0001 ) respectively . it is reported that the catalytically active zn - terminated ( 0001 ) surfaces tend to have tiny zn clusters and other zn particles at the growth front , which could provide an active site for the further growth process , and hence comb teeth can grow in front of zinc - terminated ( 0001 ) surfaces . due to higher growth velocity in direction of zno crystals , figure 3a shows the current density voltage ( i v ) characteristics for dsscs fabricated with zno nanocombs thin - film electrodes and measured under a simulated illumination with a light intensity of 100 mw / cm ( am = 1.5 ) . a maximum electricity conversion efficiency of 0.68% was achieved by highly branched zno nanocombs thin - film dsscs . the fabricated dsscs also obtained a maximum short - circuit current density ( jsc ) of 3.14 ma / cm with low voc of 0.671 v and low ff of 34% . the low jsc and conversion efficiency reveal that low dye absorption on the surface of zno thin film and result in the low - light harvesting and fast interface recombination rate of electron and holes . it is reported that the interface recombination loss in zno - based dsscs is mostly due to the uncovered oxide surface ( with no dye molecule anchored on ) , where oxide contacts with electrolyte closely and thus increases the probability of charge recombination between the electrons in oxide and the holes in the electrolyte . the low voc can be explained by the gapping between the spikes of zno nanocombs , which also cause direct contact of electrolyte to the fto glass , results the low ff . the low ff and photocurrent may be explained by the fast recombination rate between the photoexcited carriers at the nanocombs and the electrolyte interfaces , which is related to series resistance rs = ( dv / di)i=0 . generally , rs is ascribed to the bulk resistance of semiconductor oxide films , tco electrode , metallic contacts , and electrolyte . from the i v curve , rs of zno nanocombs - based dssc is relatively high ( ~213 cm ) , which increased the charge recombination between the photoexcited carriers at the nanocombs and the redox electrolyte . the high rs results in the low ff and photocurrent . figure 3b shows the uv vis absorption spectrum of desorbed dye obtained from the zno electrodes by dipping the zno nanocombs electrode in 0.1 mm naoh solution for 10 min . it was observed that low dye absorption ( ~3.23 10 mol / cm ) by zno nanocombs film surface electrode was probably due to the nonporous morphologies of the nanocombs . it is well known that the high dye absorption by porous thin film leads to high light - harvesting efficiency . therefore , low jsc and are related to less absorption of dye molecules and insufficient light harvesting from the zno nanocombs thin - film electrodes . 3b demonstrates the general morphologies of zno nanocombs after the dye absorption and , interestingly , there was no distinct change observed in the general morphologies of the nanocombs after dye absorption , hence the nanocombs retain their morphologies after dye absorption . v ) characteristics of zno nanocombs - based dssc and ( b ) typical uv vis absorption spectra of the desorbed dye ( n719 ) from the zno nanocombs electrode thin films . inset of ( b ) exhibits the surface morphology of the comblike structures after the desorption of dye in order to elucidate the charge transfer properties of as - grown zno nanocombs substrates , an electrochemical impedance spectroscopy ( eis ) measurement was used . eis measurements were taken out under the illumination of 100 mw / cm ( am = 1.5 ) by applying a 10 mv ac signal over the frequency range of 10 hz100 khz using a potentiostat with lock in amplifier , as shown in fig . 4 . according to the diffusion recombination model proposed by bisquert et al . , an equivalent circuit representing dsscs was illustrated ( inset of fig . equivalent circuit is composed of the resistance of redox electrolyte solution ( rs ) , the charge transfer resistance at the interface of electrolyte and zno nanocombs ( rct ) , the charge transfer resistance at the interface of zno nanocombs and tco ( rzno / tco ) , the capacitance of accumulation ( of e- ) layer of the zno nanocombs ( cacc ) and space charge capacitance ( csc ) . the value of real impedance ( zre ) at high and medium frequencies represents the rzno / tco and rct . figure 4 exhibits the ac impedance curve of dssc fabricated with thermally grown zno nanocombs electrode . a very high rzno / tco ( 90 ) and rct ( 29.6 ) were obtained for zno nanocombs thin - film electrodes , which are lesser than that of tio2 thin - film electrodes . it is reported that a small rct suggests fast electron transfer , whereas a large rct indicates slow electron transfer . the high rct ( 29.6 ) of zno nanocombs thin - film electrode explains the slow electron transfer , which results in the low photocurrent density and conversion efficiency . therefore , the high charge transfer resistance at zno / electrolyte interface reveals a slow electron transfer through the zno nanocombs thin - film electrode , which results in the low isc , ff , and conversion efficiency of the fabricated dssc . inset shows the equivalent circuit model of the dsscs , wherersis the resistance of redox electrolyte solution , rctthe charge transfer resistance at the interface of electrolyte and zno nanocombs , [ rct ] is the charge transfer resistance at the interface of zno nanocombs and tco [ rzno / tco ] , is the capacitance of accumulation ( of e- ) layer of the zno nanocombs [ cacc ] andcscspace charge capacitance in summary , well - crystallized zno nanocombs were directly grown onto the fto substrate via noncatalytic simple thermal evaporation process and utilized as photoanode materials to fabricate the dsscs . the fabricated dsscs demonstrated an overall light to electricity conversion efficiency of ~0.68% with a fill factor of 34% , short - circuit current of 3.14 ma / cmand open - circuit voltage of 0.671 v. this research opens a new way to utilize various kinds of zno nanostructures as photoanode material for the fabrication of efficient dsscs . this work has been done through the service contract between najran university , saudi arabia and chonbuk national university , south korea . author would like to thank professor yoon - bong hahn , school of semiconductor and chemical engineering , chonbuk national university and dr . d. h. kim , hanyang university , south korea for useful discussions and helps to carry out the experiments . this work was partially supported by the research project funded by najran university , najran , saudi arabia .

dye - sensitized solar cells ( dsscs ) were fabricated by using well - crystallized zno nanocombs directly grown onto the fluorine - doped tin oxide ( fto ) via noncatalytic thermal evaporation process . the thin films of as - grown zno nanocombs were used as photoanode materials to fabricate the dsscs , which exhibited an overall light to electricity conversion efficiency of 0.68% with a fill factor of 34% , short - circuit current of 3.14 ma / cm2 , and open - circuit voltage of 0.671 v. to the best of our knowledge , this is first report in which thin film of zno nanocombs was used as photoanode materials to fabricate the dsscs .

Introduction Experimental Details Results and Discussion Structural and Optical Properties of As-grown ZnO Nanocombs Photovoltaic Properties of As-grown ZnO Nanocombs Conclusion Acknowledgements

the ii - vi semiconductor zno is one of the most important multifunctional materials due to its various exotic properties such as direct wide band gap ( 3.37 ev ) and high optical gain of 300 cm ( 100 cm for gan ) at room temperature , large saturation velocity ( 3.2 10 cm / s ) , high breakdown voltage , large exciton binding energy ( 60 mev ) , piezoelectric , biocompatibility , and so on [ 1 - 12 ] . the use of zno nanomaterials as photoelectrodes for the fabrication of dye - sensitized solar cells ( dsscs ) has received a great attention due to its compatibility and higher electronic mobility with tio2 nanomaterials and similar electron affinity and band gap ( 3.37 ev at 298 k ) . therefore , some zno nanostructures have been used as photoelectrode materials for the fabrication of dsscs and reported in the literature [ 15 - 21 ] . reported the zno nanorods - based dssc with the electricity conversion efficiency ( ece ) of 0.22% . branched zno nanowires based dsscs , grown by thermal evaporation process at 8001,000 c , with an ece of ~0.46% have been reported by suh et al . in another report , by using branched zno nanowires grown by mocvd process , the fabricated dsscs exhibited an ece of ~0.5% . also demonstrated the thermally grown zno nanorods - based dssc with the ece of 0.6% . in this paper , we report the direct synthesis of well - crystallized zno nanocombs on fto substrates and their dsscs application . to fabricate the dsscs , the thin films of as - grown zno nanocombs on fto substrates were used as photoanode materials , which exhibited an overall light to electricity conversion efficiency of 0.68% . to the best of our knowledge , the use of zno nanocombs for the fabrication of dsscs is not reported yet in the literature . the high purity metallic zinc powder ( 99.999% ) and oxygen gas were used as source materials . the furnace temperature was raised up to the desired temperature , and oxygen and nitrogen were fed continuously into the quartz tube furnace with the flow rates of 60 and 240 sccm , respectively . the metallic zinc was vaporized and oxidized with o2 , and finally deposited onto the fto substrate . for dssc fabrication , the prepared zno nanocomb thin - film electrodes was immersed in the ethanolic solution of 0.3 mm cis - bis ( isothiocyanato ) bis(2,2-bipyridyl-4,4-dicarboxylato)-ruthenium ( ii ) bis - tetrabutylammonium ( n719 , solaronix ) at room temperature for 6 h. the dye - adsorbed zno nanocombs thin - film electrodes were then rinsed with acetonitrile and dried under a nitrogen stream . the pt electrode was placed over the dye - adsorbed zno nanocombs electrode , and the edges of the cell were sealed with 60-m thick sealing sheet ( sx 1170 - 60 , solaronix ) . the electrolyte , consisting of 0.5 m lii , 0.05 mm i2 , and 0.2 m tert - butyl pyridine in acetonitrile , was introduced into the cell through one of two small holes drilled in the counter - electrode . the holes were then covered and sealed with a small square of sealing sheet and microscope objective glass . the width of the stem is ~1.2 0.3 m , and the stem is several micrometers long . these teeth are arranged in a proper manner with a distance of ~200 50 nm between each other [ figure ( b ) and inset ( b ) ] . the x - ray diffraction ( xrd ) pattern exhibits that the as - grown nanocombs are single - crystalline with the wurtzite hexagonal - phase pure zno ( jcpds # 361451 ) ( fig . except zno , no characteristic peaks for other impurities such as zinc and substrate were observed in the spectrum , which confirms that the obtained products are single - crystalline wurtzite hexagonal - phase zno grown in highdensity on the fto substrate . in addition to this , the energy dispersive spectroscopy ( eds ) confirmed that the as - grown nanocombs are made with almost 1:1 stoichiometry of zinc and oxygen ( fig . figure 2a shows the low - magnification tem image of the nanocombs , which reveals the full consistency with the fesem observation in terms of morphology and dimensionality . the hrtem image of one tooth of comblike structure circled in figure(a ) demonstrated a well - defined lattice fringes with the lattice spacing of 0.52 nm , corresponds to the d - spacing of the crystal plane of the wurtzite hexagonal zno , confirmed that the branches of the comb structures are grown along the direction ( fig . the obtained pl spectrum exhibited a narrow peak at ~385 nm in the uv region , also called near band edge emission , and a broad emission peak at ~570 nm in the visible region , also known as deep - level emission . it is well known that the uv emission has been realized to the exciton emission , while the deep - level emission is generally explained as the radial recombination of photo - generated hole with a singly ionized charged state of the oxygen vacancy . typical ( a ) low- and ( b ) high - magnification fesem images ; ( c ) xrd pattern and ( d ) eds spectrum of high density - grown zno nanocombs on fto substrate typical ( a ) low- and ( b ) high - magnification tem image and their corresponding saed pattern [ inset(b ) ] ; and ( c ) room - temperature pl spectrum of as - grown zno nanocombs used for the fabrication of dssc as a wurtzite hexagonal - phase zno possesses a positively charged zn-(0001 ) surfaces that are catalytically active , the negatively charged o-(0001 ) surfaces are chemically inert . it is reported that the catalytically active zn - terminated ( 0001 ) surfaces tend to have tiny zn clusters and other zn particles at the growth front , which could provide an active site for the further growth process , and hence comb teeth can grow in front of zinc - terminated ( 0001 ) surfaces . due to higher growth velocity in direction of zno crystals , voltage ( i v ) characteristics for dsscs fabricated with zno nanocombs thin - film electrodes and measured under a simulated illumination with a light intensity of 100 mw / cm ( am = 1.5 ) . a maximum electricity conversion efficiency of 0.68% was achieved by highly branched zno nanocombs thin - film dsscs . the fabricated dsscs also obtained a maximum short - circuit current density ( jsc ) of 3.14 ma / cm with low voc of 0.671 v and low ff of 34% . the low jsc and conversion efficiency reveal that low dye absorption on the surface of zno thin film and result in the low - light harvesting and fast interface recombination rate of electron and holes . it is reported that the interface recombination loss in zno - based dsscs is mostly due to the uncovered oxide surface ( with no dye molecule anchored on ) , where oxide contacts with electrolyte closely and thus increases the probability of charge recombination between the electrons in oxide and the holes in the electrolyte . the low voc can be explained by the gapping between the spikes of zno nanocombs , which also cause direct contact of electrolyte to the fto glass , results the low ff . generally , rs is ascribed to the bulk resistance of semiconductor oxide films , tco electrode , metallic contacts , and electrolyte . from the i v curve , rs of zno nanocombs - based dssc is relatively high ( ~213 cm ) , which increased the charge recombination between the photoexcited carriers at the nanocombs and the redox electrolyte . figure 3b shows the uv vis absorption spectrum of desorbed dye obtained from the zno electrodes by dipping the zno nanocombs electrode in 0.1 mm naoh solution for 10 min . it was observed that low dye absorption ( ~3.23 10 mol / cm ) by zno nanocombs film surface electrode was probably due to the nonporous morphologies of the nanocombs . it is well known that the high dye absorption by porous thin film leads to high light - harvesting efficiency . 3b demonstrates the general morphologies of zno nanocombs after the dye absorption and , interestingly , there was no distinct change observed in the general morphologies of the nanocombs after dye absorption , hence the nanocombs retain their morphologies after dye absorption . v ) characteristics of zno nanocombs - based dssc and ( b ) typical uv vis absorption spectra of the desorbed dye ( n719 ) from the zno nanocombs electrode thin films . inset of ( b ) exhibits the surface morphology of the comblike structures after the desorption of dye in order to elucidate the charge transfer properties of as - grown zno nanocombs substrates , an electrochemical impedance spectroscopy ( eis ) measurement was used . equivalent circuit is composed of the resistance of redox electrolyte solution ( rs ) , the charge transfer resistance at the interface of electrolyte and zno nanocombs ( rct ) , the charge transfer resistance at the interface of zno nanocombs and tco ( rzno / tco ) , the capacitance of accumulation ( of e- ) layer of the zno nanocombs ( cacc ) and space charge capacitance ( csc ) . figure 4 exhibits the ac impedance curve of dssc fabricated with thermally grown zno nanocombs electrode . a very high rzno / tco ( 90 ) and rct ( 29.6 ) were obtained for zno nanocombs thin - film electrodes , which are lesser than that of tio2 thin - film electrodes . the high rct ( 29.6 ) of zno nanocombs thin - film electrode explains the slow electron transfer , which results in the low photocurrent density and conversion efficiency . therefore , the high charge transfer resistance at zno / electrolyte interface reveals a slow electron transfer through the zno nanocombs thin - film electrode , which results in the low isc , ff , and conversion efficiency of the fabricated dssc . inset shows the equivalent circuit model of the dsscs , wherersis the resistance of redox electrolyte solution , rctthe charge transfer resistance at the interface of electrolyte and zno nanocombs , [ rct ] is the charge transfer resistance at the interface of zno nanocombs and tco [ rzno / tco ] , is the capacitance of accumulation ( of e- ) layer of the zno nanocombs [ cacc ] andcscspace charge capacitance figure 1a shows the low - magnification fesem image of the zno nanocombs and reveals that the nanocombs are densely grown and uniformly distributed over the large area of the substrate surface . the width of the stem is ~1.2 0.3 m , and the stem is several micrometers long . the x - ray diffraction ( xrd ) pattern exhibits that the as - grown nanocombs are single - crystalline with the wurtzite hexagonal - phase pure zno ( jcpds # 361451 ) ( fig . except zno , no characteristic peaks for other impurities such as zinc and substrate were observed in the spectrum , which confirms that the obtained products are single - crystalline wurtzite hexagonal - phase zno grown in highdensity on the fto substrate . in addition to this , the energy dispersive spectroscopy ( eds ) confirmed that the as - grown nanocombs are made with almost 1:1 stoichiometry of zinc and oxygen ( fig . the hrtem image of one tooth of comblike structure circled in figure(a ) demonstrated a well - defined lattice fringes with the lattice spacing of 0.52 nm , corresponds to the d - spacing of the crystal plane of the wurtzite hexagonal zno , confirmed that the branches of the comb structures are grown along the direction ( fig . the corresponding saed pattern of a branch of comb projected to the [ 20 ] zone axis is also consistent with hrtem observation ( fig . it is well known that the uv emission has been realized to the exciton emission , while the deep - level emission is generally explained as the radial recombination of photo - generated hole with a singly ionized charged state of the oxygen vacancy . typical ( a ) low- and ( b ) high - magnification fesem images ; ( c ) xrd pattern and ( d ) eds spectrum of high density - grown zno nanocombs on fto substrate typical ( a ) low- and ( b ) high - magnification tem image and their corresponding saed pattern [ inset(b ) ] ; and ( c ) room - temperature pl spectrum of as - grown zno nanocombs used for the fabrication of dssc as a wurtzite hexagonal - phase zno possesses a positively charged zn-(0001 ) surfaces that are catalytically active , the negatively charged o-(0001 ) surfaces are chemically inert . it is reported that the catalytically active zn - terminated ( 0001 ) surfaces tend to have tiny zn clusters and other zn particles at the growth front , which could provide an active site for the further growth process , and hence comb teeth can grow in front of zinc - terminated ( 0001 ) surfaces . due to higher growth velocity in direction of zno crystals , figure 3a shows the current density voltage ( i v ) characteristics for dsscs fabricated with zno nanocombs thin - film electrodes and measured under a simulated illumination with a light intensity of 100 mw / cm ( am = 1.5 ) . a maximum electricity conversion efficiency of 0.68% was achieved by highly branched zno nanocombs thin - film dsscs . the fabricated dsscs also obtained a maximum short - circuit current density ( jsc ) of 3.14 ma / cm with low voc of 0.671 v and low ff of 34% . the low jsc and conversion efficiency reveal that low dye absorption on the surface of zno thin film and result in the low - light harvesting and fast interface recombination rate of electron and holes . it is reported that the interface recombination loss in zno - based dsscs is mostly due to the uncovered oxide surface ( with no dye molecule anchored on ) , where oxide contacts with electrolyte closely and thus increases the probability of charge recombination between the electrons in oxide and the holes in the electrolyte . the low voc can be explained by the gapping between the spikes of zno nanocombs , which also cause direct contact of electrolyte to the fto glass , results the low ff . the low ff and photocurrent may be explained by the fast recombination rate between the photoexcited carriers at the nanocombs and the electrolyte interfaces , which is related to series resistance rs = ( dv / di)i=0 . generally , rs is ascribed to the bulk resistance of semiconductor oxide films , tco electrode , metallic contacts , and electrolyte . from the i v curve , rs of zno nanocombs - based dssc is relatively high ( ~213 cm ) , which increased the charge recombination between the photoexcited carriers at the nanocombs and the redox electrolyte . it was observed that low dye absorption ( ~3.23 10 mol / cm ) by zno nanocombs film surface electrode was probably due to the nonporous morphologies of the nanocombs . it is well known that the high dye absorption by porous thin film leads to high light - harvesting efficiency . 3b demonstrates the general morphologies of zno nanocombs after the dye absorption and , interestingly , there was no distinct change observed in the general morphologies of the nanocombs after dye absorption , hence the nanocombs retain their morphologies after dye absorption . v ) characteristics of zno nanocombs - based dssc and ( b ) typical uv vis absorption spectra of the desorbed dye ( n719 ) from the zno nanocombs electrode thin films . inset of ( b ) exhibits the surface morphology of the comblike structures after the desorption of dye in order to elucidate the charge transfer properties of as - grown zno nanocombs substrates , an electrochemical impedance spectroscopy ( eis ) measurement was used . according to the diffusion recombination model proposed by bisquert et al . equivalent circuit is composed of the resistance of redox electrolyte solution ( rs ) , the charge transfer resistance at the interface of electrolyte and zno nanocombs ( rct ) , the charge transfer resistance at the interface of zno nanocombs and tco ( rzno / tco ) , the capacitance of accumulation ( of e- ) layer of the zno nanocombs ( cacc ) and space charge capacitance ( csc ) . figure 4 exhibits the ac impedance curve of dssc fabricated with thermally grown zno nanocombs electrode . a very high rzno / tco ( 90 ) and rct ( 29.6 ) were obtained for zno nanocombs thin - film electrodes , which are lesser than that of tio2 thin - film electrodes . the high rct ( 29.6 ) of zno nanocombs thin - film electrode explains the slow electron transfer , which results in the low photocurrent density and conversion efficiency . therefore , the high charge transfer resistance at zno / electrolyte interface reveals a slow electron transfer through the zno nanocombs thin - film electrode , which results in the low isc , ff , and conversion efficiency of the fabricated dssc . inset shows the equivalent circuit model of the dsscs , wherersis the resistance of redox electrolyte solution , rctthe charge transfer resistance at the interface of electrolyte and zno nanocombs , [ rct ] is the charge transfer resistance at the interface of zno nanocombs and tco [ rzno / tco ] , is the capacitance of accumulation ( of e- ) layer of the zno nanocombs [ cacc ] andcscspace charge capacitance in summary , well - crystallized zno nanocombs were directly grown onto the fto substrate via noncatalytic simple thermal evaporation process and utilized as photoanode materials to fabricate the dsscs . the fabricated dsscs demonstrated an overall light to electricity conversion efficiency of ~0.68% with a fill factor of 34% , short - circuit current of 3.14 ma / cmand open - circuit voltage of 0.671 v. this research opens a new way to utilize various kinds of zno nanostructures as photoanode material for the fabrication of efficient dsscs .

host genome variants affect the complex biology involved in pharmacokinetics / pharmacodynamics and need to be addressed to identify the critical factors determining treatment outcomes . childhood acute lymphoblastic leukemia ( all ) has for several reasons been a model disease for such research owing to its frequency , well - described epidemiology , clinical characteristics and biologic profiles within cytogenetically defined subsets . in general , childhood all is very chemosensitive , and patients are almost uniformly treated within collaborative groups that stratify treatment according to known molecular aberrations in the leukemic clone , clinical characteristics and treatment response . however , even with risk group - adapted treatment there is a wide diversity in cure rates , partly explainable by both host and cancer genomes . several candidate gene studies have associated inherited polymorphisms with treatment response and cure rates in childhood all . still , the biologic relevance and interpretation of multiple single - nucleotide polymorphisms ( snps ) identified in recent genome - wide association studies ( gwas ) remain unclear , as the functions of several of the most significant genes are unknown , and effect sizes of single snp associations are in any case extremely limited . to address some of these limitations and complement the gwas approach , we applied a multiplexed targeted sequencing method allowing screening of 25 00034 000 preselected snps within biologic domains potentially relevant to childhood all , allowing both single variants and multiple snps acting in the same pathways to be explored for association with relapse , and finally combining it with known clinical risk factors in a predictive profile . danish patients were 115 years of age and diagnosed 19922008 with b - cell precursor all ( bcp - all ) or t - lineage all and treated according to the nordic society for pediatric hematology and oncology ( nopho ) all92 ( n=227 ) or nopho all2000 protocols ( n=268 ) ( figure 1 , supplementary online material and supplementary tables 1 ) . these two treatment protocols are very similar and included a 4-week , 3-drug induction phase ( prednisolone , vincristine and doxorubicin ( with intrathecal methotrexate ) ) with no glucocorticosteroid prephase , a risk group - adapted consolidation phase and methotrexate/6-mercaptopurine maintenance therapy up until 2 to 2.5 years from diagnosis ( detailed in supplementary online material ) . end of induction minimal residual disease ( mrd ) measurements were available for 73% of nopho all2000 patients , but not included in risk stratification . for cross - protocol validation of relapse - predictive host genomic variants , 500 german childhood all patients 118 years at diagnosis were included , all treated according to the bfm all2000 protocol . the bfm all2000 protocol included a 1-week prednisolone prephase , a 4-week , 4-drug induction phase ( prednisolone or dexamethasone , vincristine , doxorubicin and l - asparaginase ( with intrathecal methotrexate ) ) , a risk group - adapted consolidation phase and methotrexate/6-mercaptopurine maintenance therapy until 2 years from diagnosis ( supplementary online material ) . a total of 352 danish and 426 german patients were eligible for the final relapse - risk analysis ( figure 1 and supplementary tables s1 ) . patients were enrolled between 1992 and 2008 with a median follow - up of 7.6 years ( 50% range : 5.39.2 years ) for patients in first remission . in this study , owing to the complexity of the bioinformatic analysis and the application of nonlinear machine learning , and as it is uncertain how to weight competing events ( induction failures , deaths in remission and second cancers ) , we chose , in this exploratory , hypothesis - generating study , to exclude patients with such events already at the time of diagnosis of all ( figure 1 ) . the study was conducted in accordance with the declaration of helsinki principles of 1975 , and approved by the danish data protection agency , the committee on biomedical research ethics and by the ethics committee of the hannover medical school , hannover , germany . snp selection and bait design have been previously described in detail ( wesolowska et al . and supplementary online material ) . snps were selected to cover all known and putative clinically relevant genetic variation with regard to childhood all treatment ( 13 drugs ) , drug pharmacokinetics and pharmacodynamics , relapse risk and several toxicities ( supplementary figure s1 ) . in short , clinically important genes and snps for the 13 most commonly administered antileukemic drugs were evaluated with regard to genes encoding proteins involved in metabolism , transport , target proteins , regulation of drug - target response and to some extent drug - related toxicity ( e.g. coagulation , immune function ) ( supplementary online material ) . initially , this was carried out by literature curation , and then expanded to cover additional aspects of response to chemotherapy such as genes encoding proteins involved in apoptosis pathways and dna repair . this also included consultations with experts within such areas and through various online resources ( supplementary online material ) . finally , known drug protein associations and first - order protein protein interactions were evaluated and added ( supplementary online material and supplementary figure s1 ) . the selected genes were then screened for known polymorphisms with putative functional consequence on their transcript ( supplementary table s3 ) . baits for the sureselect target enrichment system ( protocol version 1.2 april 2009 ; agilent technologies , santa clara , ca , usa ) were designed for all identified snps . the first design included baits targeting 25 086 clinically relevant snps in 1540 genes ( supplementary figure s2 ) . as baits covered more genomic material than just the targeted snps , readouts on the targeted regions covered 116 646 known human variants ( supplementary table s4 ) . this panel was subsequently updated after sequencing the first group of patients ( from nopho all2000 ) ( supplementary online material and supplementary tables s3 and s4 ) . thus , the nopho all92 and bfm all2000 cohorts were screened using an expanded bait design , covering 33 683 snps in 2254 genes . the patients were genotyped for either of the two snp panels by multiplex targeted sequencing as described previously . briefly , the agilent sureselect target enrichment system protocol ( protocol version 1.2 april 2009 ; agilent technologies ) was modified , allowing nucleotide barcoding , and then pooling of samples before target enrichment and sequencing . genomic dna from leukemia remission samples was sheared , purified , end - repaired and 3 adenylated . custom - made adaptors containing unique four - base barcodes were ligated to the dna fragments before size selection and amplification . the dna libraries were subsequently mixed in groups of up to eight samples , and the pooled libraries were hybridized to the custom - designed baits . posthybridization amplification pcr was performed , and the pooled libraries were sequenced ( supplementary online material ) . the sequencing reads were mapped to the reference human genome ( grch37 ) , and snp calling was performed ( supplementary figure s3 ) . the threshold set for snp calling was minimum 10 sequencing depth . patient samples not clustering together with the hapmap central european ancestry samples in the principal component analysis ( supplementary figures s4 and s5 ) were removed . to make sure that the data did not contain any bias arising either from sequencing or data processing , the observed minor allele frequencies ( mafs ) of the genotyped snps were plotted against the hapmap ceu - reported mafs for the 5962 snps , where data were available ( supplementary figure s7 ) . furthermore , snps for which < 50% of the patients could be genotyped at the minimum depth of 10 and snps with maf < 1% were excluded from the analyses . to validate readouts , genotyping from this experiment was compared with genotype calls obtained from the illumina human 1m - duo snp chip performed on a subset of patients ( n=275 , overlap of 2394 snps on both platforms ) . the mean genotyping concordance of those two methods was 98.2% ( supplementary online material ) . single snps were explored for associations with risk of relapse using a fisher 's exact test separately for the nopho and bfm cohorts . p - values obtained were corrected for multiple testing by adaptive permutation , and subsequently only snps with adjusted p - values below 0.05 in both cohorts were considered significant . besides investigating such contribution of individual variations , the combined effects of multiple snps acting in the same biologic pathways were investigated using nonlinear machine learning . all pathways from the reactome database and 12-drug metabolism pathways from the pharmgkb database ( ref . 27 in supplementary online material ) , with up to 193 snps in a pathway , were interrogated . briefly , ann models including all combinations of up to three snps together with white blood cell counts ( wbc ) and age at diagnosis were tested within each pathway and stepwise expanded with up to 15 snps in a pathway , if threefold cross - validated performance improved ( supplementary online material ) ( figure 2 ) . the best model for each pathway was then chosen , and the most informative pathway models were combined with clinical information in classification and regression tree ( cart ) analysis assigning for each pathway a score from 1 to 10 for every patient based on the relapse prediction from the neural network . as induction therapy differed between the danish and german cohorts , each of these was dichotomously subdivided based on the approximate median end of induction mrd levels in each cohort being 10 and 10 in nopho and bfm cohorts , respectively ( supplementary online material ) . single snps were explored for associations with risk of relapse using a fisher 's exact test separately for the nopho and bfm cohorts . p - values obtained were corrected for multiple testing by adaptive permutation , and subsequently only snps with adjusted p - values below 0.05 in both cohorts were considered significant . besides investigating such contribution of individual variations , the combined effects of multiple snps acting in the same biologic pathways were investigated using nonlinear machine learning . all pathways from the reactome database and 12-drug metabolism pathways from the pharmgkb database ( ref . 27 in supplementary online material ) , with up to 193 snps in a pathway , were interrogated . briefly , ann models including all combinations of up to three snps together with white blood cell counts ( wbc ) and age at diagnosis were tested within each pathway and stepwise expanded with up to 15 snps in a pathway , if threefold cross - validated performance improved ( supplementary online material ) ( figure 2 ) . the best model for each pathway was then chosen , and the most informative pathway models were combined with clinical information in classification and regression tree ( cart ) analysis assigning for each pathway a score from 1 to 10 for every patient based on the relapse prediction from the neural network . as induction therapy differed between the danish and german cohorts , each of these was dichotomously subdivided based on the approximate median end of induction mrd levels in each cohort being 10 and 10 in nopho and bfm cohorts , respectively ( supplementary online material ) . of the genotyped patients ( n=869 ) , 808 fulfilled the quality control and the european ancestry criteria ( figure 1 ) , of which 778 patients could be included in the final relapse - risk analysis . the majority of genotyped variants had very low heterogeneity ( maf < 1% ) between patients and thus did not contribute with sufficient power to the single snp analysis . thus , a total of 4260 and 3865 snps in the nopho and the bfm cohorts were included in the relapse - risk analysis , respectively . the qq plots showed good agreement with the null distribution and absence of genomic inflation ( supplementary figure s8 ) , and several loci were significantly associated with risk of relapse ( supplementary figure s9 ) . a total of 188 and 152 snps were associated with relapse risk in the nopho and bfm cohorts , respectively ( supplementary tables s5 and s6 ) . as some of these will reflect chance findings , we included in the subsequent analyses only the 11 snps that were associated with relapse risk in both cohorts . importantly , these 11 snps were related to risk of relapse independent of other known risk factors ( table 1 , supplementary table s7 and supplementary online material ) . as further support of their biologic significance , kaplan meier analyses for these snps showed a general tendency of gene dose effects ( 0 versus 1 versus 2 alleles associated with increased risk of relapse ) with log - rank trend p - values ranging from 4.8 10 for rs3216144 ( matrix metalloproteinase 7 , mmp7 ) to 0.03 for rs35721373 ( dysferlin , dysf ) , respectively ( supplementary figure s9 ) . next , functional snps were grouped by biologic pathways , and the relevance of the pathways to risk of relapse was assessed by training anns on different combinations of snps from each pathway , allowing nonlinear correlations between snps . the top - associated reactome pathway ranked by matthew 's correlation coefficient ( mcc , ranging from 0 to 1 ) was atp - binding cassette ( abc ) family protein - mediated transport ' ( mcc=0.33 , area under the receiver - operator curve ( auc)=0.69 ) ( supplementary table s8 ) . similarly , 12-drug metabolism pathways from the pharmgkb database relevant for the administered drugs were investigated , and the top pathways were vinka alkaloid pathway , pharmacokinetics ' ( mcc=0.321 , auc=0.72 ) , glucocorticoid pathway ( peripheral tissue ) , pharmacodynamics ' ( mcc=0.320 , auc=0.75 ) and methotrexate pathway ( brain cell ) , pharmacokinetics ' ( mcc=0.320 , auc=0.70 ) ( supplementary table s9 ) . to explore and illustrate the combined significance of clinical data and host genomic findings , cart analysis was applied to subclassify sequentially patients in a multivariate model , including the 426 patients from both cohorts with complete information on sex , age , immunophenotype , wbc , leukemia karyotype , end of induction mrd , risk group and genotypes of the 11 cross - cohort - associated snps ( figure 3a ) . end of induction mrd levels divided the patients into two major groups : one group with high mrd levels ( i.e. above median for that cohort ) and a high risk of relapse , which could be further stratified by snps in the myeloperoxidase ( mpo ) , estrogen receptor 1 ( esr1 ) , lamin b1 ( lmnb1 ) and mmp7 genes , and another large group with low mrd levels and low cumulative relapse risk . as the latter group , because of its size , accounts for 25% of all relapses , a subsequent cart analysis with pathway profiles for the top 10 reactome and the pharmgkb pathways was added ( all having aucs of approximately 0.70 ) , which indicated a role of abc transporters and glucocorticosteroid transcription regulation pathways ( figure 3b ) . finally , based on mrd , wbc , the aforementioned host genomic data and the resulting observed incidence of relapse ( figures 3a and b ) , we could define three large subsets of patients with significant differences in the risk of relapse ( p<0.001 ) . with 92% of projected relapses by kaplan meier analysis having been observed in the total cohort , the two extreme subgroups of patients had 6-year cumulative risks of relapse of 4% ( 95% confidence interval : 1.66.3% ) for the best outcome group ( 71.5% of all patients ) and 76% ( 95% confidence interval : 4190% ) for the worst outcome group ( 5% of all patients ) ( figure 3c and table 2 ) , leaving age , immunophenotype ( bcp versus t - all ) , cytogenetics ( table 2 ; high risk , that is , t(9;22 ) , hypodiploid , t(4;11 ) ; low risk , that is , high hyperdiploid , t(12;21 ) ; other ) and risk group nonsignificant with this approach . as mrd was not available for all patients , an additional cart analysis was performed not including mrd ( supplementary figure s10 ) . survival analysis taking into account the time to event was also performed ( supplementary figure s11 ) , with results largely agreeing with the model in figure 3 . multivariate regression analysis to predict risk of relapse found all the features selected by cart analysis to be statistically significant , with mrd being the most significant clinical factor ( p=7.9 10 ) , mpo rs28730837 being the most significant snp region ( p=0.002 ) and glucocorticosteroid pathway ( transcription regulation , pharmacodynamics ) ( p=2.0 10 ) being the most significant pathway . today , many high - risk patients do not respond to intensified treatment , but most relapses occur among non - high - risk patients . the present study , using genomic candidate gene genotyping and front - line bioinformatics analyses , provides a novel biology / pharmacology - driven approach for outcome prediction and goes beyond conventional genome - wide association studies ( gwas ) approaches , while still being more cost - effective than whole - genome sequencing ( for cost details see supplementary online material ) . the rapidly growing understanding of the complex human genome and its derived functional biology allows selection of candidate snps based on the current knowledge of pharmacogenomics , disease mechanisms , signaling pathways and protein interactions . large - scale , genomic candidate gene setup facilitates not only single snp investigations but also associations of multiple variations grouped by their putative function . further associations of combinations of snps grouped by biologic pathways tested with neural network models enable detection of meaningful nonlinear snp interactions . the results obtained through these strategies can provide conclusions at new levels of genomic complexity , collectively emphasizing the importance of specific biologic mechanisms for the phenotype . the 11 cross - cohort relapse - associated snps resided in genes previously suggested as markers for leukemia aggressiveness , involved in steroid response , implicated in resistance mechanisms or toxicities of certain drugs ( supplementary online material ) . the pathway analysis strongly indicated an importance of the abc family protein - mediated transport , activation of matrix metalloproteinases , toll - like receptor cascade signaling and various signal - transduction pathways , as well as involvement of the cell cycle . the top - associated pathway abc family protein - mediated transport ' confirms the role of pharmacogenomics in drug response by pointing to the importance of drug transport . the atp - dependent drug efflux pumps have broad substrate specificities ; they influence drug accumulation and are associated with the development of resistance to anticancer drugs . matrix metalloproteinases are involved in tumor progression , whereas the toll - like receptor cascades and nucleotide - binding oligomerization domain - containing protein 1 and 2 signaling pathways support a role for the innate immune system function for proper drug response or all biology . it is noteworthy that the three top drug metabolism pathways for relapse risk corresponded to pharmacodynamics of glucocorticosteroids and pharmacokinetics of vinka alkaloids and methotrexate . this emphasizes the clinical significance of glucocorticosteroid therapy and cell cycle - arresting agents such as vincristine and methotrexate , and also highlights the potential profound consequence of adverse drug disposition on the effect of these agents . as risk factors linked to host genomics , leukemia biology , treatment response markers and drug pharmacokinetics accumulate , cart analysis facilitates integration of the different layers of molecular complexity with patients ' clinical characteristics to identify groups of patients with distinct treatment outcomes , and accordingly candidates for such treatment adaption . thus , combining patients ' genotypes with their clinical features is likely to explain treatment outcome better than single snps . even though the binary recursive partitioning of the cart methodology offers an approach to patient classifications that is easier to perceive from a biologic point of view and to apply in the clinical setting , cart does not necessarily outperform conventional , stepwise , multivariate regression analyses . interactions between variables ( and combinations hereof ) may be missed , and the downstream decision branching may be unstable if one variable is removed . although the cart analysis presented here classified patients into highly different prognostic subsets , and conventional regression analysis confirmed the most significant clinical feature , snp and pathway , larger independent data sets will be needed to determine which of these two biostatistical approaches is superior for relapse prediction . in future trials , the small group of patients who have the highest risk of relapse can be offered intensified treatments , or be candidates for phase 2 trials , whereas treatment reduction to avoid specific toxicities may be relevant for the large subset of patients who have a projected risk of relapse of < 5% . in this context , the least relapse - predictive drug metabolism pathways may point to which drug doses potentially could be safely reduced in complex combination chemotherapy , and/or to which patients a specific drug is important owing to the patient 's genomic variants . although further verification is needed to strengthen the findings , the present approach has , when compared with other large - scale studies , the clear advantage of specifically targeting potential causative variants , which reduces the need to investigate genomic patterns of linkage disequilibrium , or conducting follow - up fine - mapping studies . functional prioritization of the potential snps to be genotyped eliminates the difficulties linked to gwas when mapping significant variants to genes and genotypes , and can be more directly combined in functional analyses . as an example , only one of the 134 snps associated to relapse risk in the recent childhood all gwas study of yang et al . was also genotyped in the present study , as the remaining snps resided in noncoding or functionally unannotated regions , and it was not found significant . moreover , as the associated snps in most cases were difficult to map to the gene on which they exert their effects , it is difficult to evaluate the involved biologic mechanisms . the present study combines large - scale genetic investigations of a gwas approach with the targeted focus of a candidate gene approach . thus , the major strength of our approach is that a wide panel of functional snps was selected with various prior assumptions of their potential relevance for childhood all treatment outcome . owing to annotation to specific pathways , the results obtained are easier to interpret , and eventually to implement in the clinic based on existing knowledge of all disease mechanisms and pharmacokinetics and pharmacodynamics of administered drugs . thus , the snp / pathway - based approach can also be applied for prediction of specific side effects of antileukemic therapy . host genome profiles may then indicate drugs with little importance for cure but associated with a high risk for side effects for specific patients , and this information could be applied for tailored therapy . accordingly , the strategies and findings of this paper will be validated in the ongoing nordic / baltic nopho all2008 protocol addressing risk grouping , occurrence of toxicities ( including infections ) and risk of relapse before they are included into future treatment allocation . we acknowledge that not covering the whole genome will certainly miss unknown , important genetic components of treatment response , which may benefit from alternative genome - wide screening approaches such as gwas or whole - exome / transcriptome / genome sequencing , although the latter is still burdened by high costs owing to required patient numbers for statistical significance , and requires extensive follow - up for unannotated variants to be credibly accepted . in conclusion , this large - scale integration of the knowledge of disease mechanisms and drug pharmacokinetics / pharmacodynamics in host genome studies in childhood all can offer both significant improvements to the current relapse prediction algorithms , and , importantly , indicate specific directions for leukemia response and toxicity risk - based treatments adaptation for the individual patient .

childhood acute lymphoblastic leukemia survival approaches 90% . new strategies are needed to identify the 1015% who evade cure . we applied targeted , sequencing - based genotyping of 25 000 to 34 000 preselected potentially clinically relevant single - nucleotide polymorphisms ( snps ) to identify host genome profiles associated with relapse risk in 352 patients from the nordic all92/2000 protocols and 426 patients from the german berlin frankfurt munster ( bfm ) all2000 protocol . patients were enrolled between 1992 and 2008 ( median follow - up : 7.6 years ) . eleven cross - validated snps were significantly associated with risk of relapse across protocols . snp and biologic pathway level analyses associated relapse risk with leukemia aggressiveness , glucocorticosteroid pharmacology / response and drug transport / metabolism pathways . classification and regression tree analysis identified three distinct risk groups defined by end of induction residual leukemia , white blood cell count and variants in myeloperoxidase ( mpo ) , estrogen receptor 1 ( esr1 ) , lamin b1 ( lmnb1 ) and matrix metalloproteinase-7 ( mmp7 ) genes , atp - binding cassette transporters and glucocorticosteroid transcription regulation pathways . relapse rates ranged from 4% ( 95% confidence interval ( ci ) : 1.66.3% ) for the best group ( 72% of patients ) to 76% ( 95% ci : 4190% ) for the worst group ( 5% of patients , p<0.001 ) . validation of these findings and similar approaches to identify snps associated with toxicities may allow future individualized relapse and toxicity risk - based treatments adaptation .

Introduction Materials and methods SNP associations Results Discussion Supplementary Material

host genome variants affect the complex biology involved in pharmacokinetics / pharmacodynamics and need to be addressed to identify the critical factors determining treatment outcomes . childhood acute lymphoblastic leukemia ( all ) has for several reasons been a model disease for such research owing to its frequency , well - described epidemiology , clinical characteristics and biologic profiles within cytogenetically defined subsets . however , even with risk group - adapted treatment there is a wide diversity in cure rates , partly explainable by both host and cancer genomes . several candidate gene studies have associated inherited polymorphisms with treatment response and cure rates in childhood all . still , the biologic relevance and interpretation of multiple single - nucleotide polymorphisms ( snps ) identified in recent genome - wide association studies ( gwas ) remain unclear , as the functions of several of the most significant genes are unknown , and effect sizes of single snp associations are in any case extremely limited . to address some of these limitations and complement the gwas approach , we applied a multiplexed targeted sequencing method allowing screening of 25 00034 000 preselected snps within biologic domains potentially relevant to childhood all , allowing both single variants and multiple snps acting in the same pathways to be explored for association with relapse , and finally combining it with known clinical risk factors in a predictive profile . danish patients were 115 years of age and diagnosed 19922008 with b - cell precursor all ( bcp - all ) or t - lineage all and treated according to the nordic society for pediatric hematology and oncology ( nopho ) all92 ( n=227 ) or nopho all2000 protocols ( n=268 ) ( figure 1 , supplementary online material and supplementary tables 1 ) . end of induction minimal residual disease ( mrd ) measurements were available for 73% of nopho all2000 patients , but not included in risk stratification . for cross - protocol validation of relapse - predictive host genomic variants , 500 german childhood all patients 118 years at diagnosis were included , all treated according to the bfm all2000 protocol . the bfm all2000 protocol included a 1-week prednisolone prephase , a 4-week , 4-drug induction phase ( prednisolone or dexamethasone , vincristine , doxorubicin and l - asparaginase ( with intrathecal methotrexate ) ) , a risk group - adapted consolidation phase and methotrexate/6-mercaptopurine maintenance therapy until 2 years from diagnosis ( supplementary online material ) . a total of 352 danish and 426 german patients were eligible for the final relapse - risk analysis ( figure 1 and supplementary tables s1 ) . patients were enrolled between 1992 and 2008 with a median follow - up of 7.6 years ( 50% range : 5.39.2 years ) for patients in first remission . in this study , owing to the complexity of the bioinformatic analysis and the application of nonlinear machine learning , and as it is uncertain how to weight competing events ( induction failures , deaths in remission and second cancers ) , we chose , in this exploratory , hypothesis - generating study , to exclude patients with such events already at the time of diagnosis of all ( figure 1 ) . snp selection and bait design have been previously described in detail ( wesolowska et al . snps were selected to cover all known and putative clinically relevant genetic variation with regard to childhood all treatment ( 13 drugs ) , drug pharmacokinetics and pharmacodynamics , relapse risk and several toxicities ( supplementary figure s1 ) . in short , clinically important genes and snps for the 13 most commonly administered antileukemic drugs were evaluated with regard to genes encoding proteins involved in metabolism , transport , target proteins , regulation of drug - target response and to some extent drug - related toxicity ( e.g. this also included consultations with experts within such areas and through various online resources ( supplementary online material ) . finally , known drug protein associations and first - order protein protein interactions were evaluated and added ( supplementary online material and supplementary figure s1 ) . baits for the sureselect target enrichment system ( protocol version 1.2 april 2009 ; agilent technologies , santa clara , ca , usa ) were designed for all identified snps . the first design included baits targeting 25 086 clinically relevant snps in 1540 genes ( supplementary figure s2 ) . this panel was subsequently updated after sequencing the first group of patients ( from nopho all2000 ) ( supplementary online material and supplementary tables s3 and s4 ) . thus , the nopho all92 and bfm all2000 cohorts were screened using an expanded bait design , covering 33 683 snps in 2254 genes . the patients were genotyped for either of the two snp panels by multiplex targeted sequencing as described previously . briefly , the agilent sureselect target enrichment system protocol ( protocol version 1.2 april 2009 ; agilent technologies ) was modified , allowing nucleotide barcoding , and then pooling of samples before target enrichment and sequencing . the dna libraries were subsequently mixed in groups of up to eight samples , and the pooled libraries were hybridized to the custom - designed baits . the sequencing reads were mapped to the reference human genome ( grch37 ) , and snp calling was performed ( supplementary figure s3 ) . to make sure that the data did not contain any bias arising either from sequencing or data processing , the observed minor allele frequencies ( mafs ) of the genotyped snps were plotted against the hapmap ceu - reported mafs for the 5962 snps , where data were available ( supplementary figure s7 ) . furthermore , snps for which < 50% of the patients could be genotyped at the minimum depth of 10 and snps with maf < 1% were excluded from the analyses . to validate readouts , genotyping from this experiment was compared with genotype calls obtained from the illumina human 1m - duo snp chip performed on a subset of patients ( n=275 , overlap of 2394 snps on both platforms ) . single snps were explored for associations with risk of relapse using a fisher 's exact test separately for the nopho and bfm cohorts . p - values obtained were corrected for multiple testing by adaptive permutation , and subsequently only snps with adjusted p - values below 0.05 in both cohorts were considered significant . all pathways from the reactome database and 12-drug metabolism pathways from the pharmgkb database ( ref . 27 in supplementary online material ) , with up to 193 snps in a pathway , were interrogated . briefly , ann models including all combinations of up to three snps together with white blood cell counts ( wbc ) and age at diagnosis were tested within each pathway and stepwise expanded with up to 15 snps in a pathway , if threefold cross - validated performance improved ( supplementary online material ) ( figure 2 ) . the best model for each pathway was then chosen , and the most informative pathway models were combined with clinical information in classification and regression tree ( cart ) analysis assigning for each pathway a score from 1 to 10 for every patient based on the relapse prediction from the neural network . as induction therapy differed between the danish and german cohorts , each of these was dichotomously subdivided based on the approximate median end of induction mrd levels in each cohort being 10 and 10 in nopho and bfm cohorts , respectively ( supplementary online material ) . single snps were explored for associations with risk of relapse using a fisher 's exact test separately for the nopho and bfm cohorts . p - values obtained were corrected for multiple testing by adaptive permutation , and subsequently only snps with adjusted p - values below 0.05 in both cohorts were considered significant . all pathways from the reactome database and 12-drug metabolism pathways from the pharmgkb database ( ref . 27 in supplementary online material ) , with up to 193 snps in a pathway , were interrogated . briefly , ann models including all combinations of up to three snps together with white blood cell counts ( wbc ) and age at diagnosis were tested within each pathway and stepwise expanded with up to 15 snps in a pathway , if threefold cross - validated performance improved ( supplementary online material ) ( figure 2 ) . the best model for each pathway was then chosen , and the most informative pathway models were combined with clinical information in classification and regression tree ( cart ) analysis assigning for each pathway a score from 1 to 10 for every patient based on the relapse prediction from the neural network . as induction therapy differed between the danish and german cohorts , each of these was dichotomously subdivided based on the approximate median end of induction mrd levels in each cohort being 10 and 10 in nopho and bfm cohorts , respectively ( supplementary online material ) . of the genotyped patients ( n=869 ) , 808 fulfilled the quality control and the european ancestry criteria ( figure 1 ) , of which 778 patients could be included in the final relapse - risk analysis . the qq plots showed good agreement with the null distribution and absence of genomic inflation ( supplementary figure s8 ) , and several loci were significantly associated with risk of relapse ( supplementary figure s9 ) . a total of 188 and 152 snps were associated with relapse risk in the nopho and bfm cohorts , respectively ( supplementary tables s5 and s6 ) . as some of these will reflect chance findings , we included in the subsequent analyses only the 11 snps that were associated with relapse risk in both cohorts . importantly , these 11 snps were related to risk of relapse independent of other known risk factors ( table 1 , supplementary table s7 and supplementary online material ) . as further support of their biologic significance , kaplan meier analyses for these snps showed a general tendency of gene dose effects ( 0 versus 1 versus 2 alleles associated with increased risk of relapse ) with log - rank trend p - values ranging from 4.8 10 for rs3216144 ( matrix metalloproteinase 7 , mmp7 ) to 0.03 for rs35721373 ( dysferlin , dysf ) , respectively ( supplementary figure s9 ) . next , functional snps were grouped by biologic pathways , and the relevance of the pathways to risk of relapse was assessed by training anns on different combinations of snps from each pathway , allowing nonlinear correlations between snps . the top - associated reactome pathway ranked by matthew 's correlation coefficient ( mcc , ranging from 0 to 1 ) was atp - binding cassette ( abc ) family protein - mediated transport ' ( mcc=0.33 , area under the receiver - operator curve ( auc)=0.69 ) ( supplementary table s8 ) . similarly , 12-drug metabolism pathways from the pharmgkb database relevant for the administered drugs were investigated , and the top pathways were vinka alkaloid pathway , pharmacokinetics ' ( mcc=0.321 , auc=0.72 ) , glucocorticoid pathway ( peripheral tissue ) , pharmacodynamics ' ( mcc=0.320 , auc=0.75 ) and methotrexate pathway ( brain cell ) , pharmacokinetics ' ( mcc=0.320 , auc=0.70 ) ( supplementary table s9 ) . to explore and illustrate the combined significance of clinical data and host genomic findings , cart analysis was applied to subclassify sequentially patients in a multivariate model , including the 426 patients from both cohorts with complete information on sex , age , immunophenotype , wbc , leukemia karyotype , end of induction mrd , risk group and genotypes of the 11 cross - cohort - associated snps ( figure 3a ) . end of induction mrd levels divided the patients into two major groups : one group with high mrd levels ( i.e. above median for that cohort ) and a high risk of relapse , which could be further stratified by snps in the myeloperoxidase ( mpo ) , estrogen receptor 1 ( esr1 ) , lamin b1 ( lmnb1 ) and mmp7 genes , and another large group with low mrd levels and low cumulative relapse risk . as the latter group , because of its size , accounts for 25% of all relapses , a subsequent cart analysis with pathway profiles for the top 10 reactome and the pharmgkb pathways was added ( all having aucs of approximately 0.70 ) , which indicated a role of abc transporters and glucocorticosteroid transcription regulation pathways ( figure 3b ) . finally , based on mrd , wbc , the aforementioned host genomic data and the resulting observed incidence of relapse ( figures 3a and b ) , we could define three large subsets of patients with significant differences in the risk of relapse ( p<0.001 ) . with 92% of projected relapses by kaplan meier analysis having been observed in the total cohort , the two extreme subgroups of patients had 6-year cumulative risks of relapse of 4% ( 95% confidence interval : 1.66.3% ) for the best outcome group ( 71.5% of all patients ) and 76% ( 95% confidence interval : 4190% ) for the worst outcome group ( 5% of all patients ) ( figure 3c and table 2 ) , leaving age , immunophenotype ( bcp versus t - all ) , cytogenetics ( table 2 ; high risk , that is , t(9;22 ) , hypodiploid , t(4;11 ) ; low risk , that is , high hyperdiploid , t(12;21 ) ; other ) and risk group nonsignificant with this approach . as mrd was not available for all patients , an additional cart analysis was performed not including mrd ( supplementary figure s10 ) . survival analysis taking into account the time to event was also performed ( supplementary figure s11 ) , with results largely agreeing with the model in figure 3 . multivariate regression analysis to predict risk of relapse found all the features selected by cart analysis to be statistically significant , with mrd being the most significant clinical factor ( p=7.9 10 ) , mpo rs28730837 being the most significant snp region ( p=0.002 ) and glucocorticosteroid pathway ( transcription regulation , pharmacodynamics ) ( p=2.0 10 ) being the most significant pathway . today , many high - risk patients do not respond to intensified treatment , but most relapses occur among non - high - risk patients . the present study , using genomic candidate gene genotyping and front - line bioinformatics analyses , provides a novel biology / pharmacology - driven approach for outcome prediction and goes beyond conventional genome - wide association studies ( gwas ) approaches , while still being more cost - effective than whole - genome sequencing ( for cost details see supplementary online material ) . large - scale , genomic candidate gene setup facilitates not only single snp investigations but also associations of multiple variations grouped by their putative function . further associations of combinations of snps grouped by biologic pathways tested with neural network models enable detection of meaningful nonlinear snp interactions . the results obtained through these strategies can provide conclusions at new levels of genomic complexity , collectively emphasizing the importance of specific biologic mechanisms for the phenotype . the 11 cross - cohort relapse - associated snps resided in genes previously suggested as markers for leukemia aggressiveness , involved in steroid response , implicated in resistance mechanisms or toxicities of certain drugs ( supplementary online material ) . the pathway analysis strongly indicated an importance of the abc family protein - mediated transport , activation of matrix metalloproteinases , toll - like receptor cascade signaling and various signal - transduction pathways , as well as involvement of the cell cycle . the top - associated pathway abc family protein - mediated transport ' confirms the role of pharmacogenomics in drug response by pointing to the importance of drug transport . the atp - dependent drug efflux pumps have broad substrate specificities ; they influence drug accumulation and are associated with the development of resistance to anticancer drugs . matrix metalloproteinases are involved in tumor progression , whereas the toll - like receptor cascades and nucleotide - binding oligomerization domain - containing protein 1 and 2 signaling pathways support a role for the innate immune system function for proper drug response or all biology . it is noteworthy that the three top drug metabolism pathways for relapse risk corresponded to pharmacodynamics of glucocorticosteroids and pharmacokinetics of vinka alkaloids and methotrexate . this emphasizes the clinical significance of glucocorticosteroid therapy and cell cycle - arresting agents such as vincristine and methotrexate , and also highlights the potential profound consequence of adverse drug disposition on the effect of these agents . as risk factors linked to host genomics , leukemia biology , treatment response markers and drug pharmacokinetics accumulate , cart analysis facilitates integration of the different layers of molecular complexity with patients ' clinical characteristics to identify groups of patients with distinct treatment outcomes , and accordingly candidates for such treatment adaption . although the cart analysis presented here classified patients into highly different prognostic subsets , and conventional regression analysis confirmed the most significant clinical feature , snp and pathway , larger independent data sets will be needed to determine which of these two biostatistical approaches is superior for relapse prediction . in future trials , the small group of patients who have the highest risk of relapse can be offered intensified treatments , or be candidates for phase 2 trials , whereas treatment reduction to avoid specific toxicities may be relevant for the large subset of patients who have a projected risk of relapse of < 5% . in this context , the least relapse - predictive drug metabolism pathways may point to which drug doses potentially could be safely reduced in complex combination chemotherapy , and/or to which patients a specific drug is important owing to the patient 's genomic variants . although further verification is needed to strengthen the findings , the present approach has , when compared with other large - scale studies , the clear advantage of specifically targeting potential causative variants , which reduces the need to investigate genomic patterns of linkage disequilibrium , or conducting follow - up fine - mapping studies . functional prioritization of the potential snps to be genotyped eliminates the difficulties linked to gwas when mapping significant variants to genes and genotypes , and can be more directly combined in functional analyses . as an example , only one of the 134 snps associated to relapse risk in the recent childhood all gwas study of yang et al . thus , the snp / pathway - based approach can also be applied for prediction of specific side effects of antileukemic therapy . host genome profiles may then indicate drugs with little importance for cure but associated with a high risk for side effects for specific patients , and this information could be applied for tailored therapy . accordingly , the strategies and findings of this paper will be validated in the ongoing nordic / baltic nopho all2008 protocol addressing risk grouping , occurrence of toxicities ( including infections ) and risk of relapse before they are included into future treatment allocation . we acknowledge that not covering the whole genome will certainly miss unknown , important genetic components of treatment response , which may benefit from alternative genome - wide screening approaches such as gwas or whole - exome / transcriptome / genome sequencing , although the latter is still burdened by high costs owing to required patient numbers for statistical significance , and requires extensive follow - up for unannotated variants to be credibly accepted . in conclusion , this large - scale integration of the knowledge of disease mechanisms and drug pharmacokinetics / pharmacodynamics in host genome studies in childhood all can offer both significant improvements to the current relapse prediction algorithms , and , importantly , indicate specific directions for leukemia response and toxicity risk - based treatments adaptation for the individual patient .

the igf system plays crucial roles in biological processes such as proliferation , differentiation , invasion , tumor expansion , migration , and survival . it can be used to predict clinical outcome , , diagnosis , endocrine responsiveness , cancer progression or inhibition of cancer growth , and apoptosis . the ligands of the igf system , igf - i and igf - ii , mediate the growth and development of organisms . they bind to igfir and initiate multiple cellular phenotypes , induce cell proliferation , suppress apoptosis , and promote differentiation . igfbp5s regulate the functions of igfs by binding to conserved amino terminal domains of igfs , , and this action can extend the half - life of igfs or restrict their function . igfbp5 , expressed in both normal and cancer tissues , regulates the growth and development of tissues and cells , such as myoblasts , thyroid carcinoma cells , uterine leiomyomata cells , neural cells , and muscle - derived tumor rhabdomyosarcoma cells . igfbp5 has both igf - dependent and igf - independent effects. igfs are deemed as cell survival factors that are up - regulated in tumorigenesis and their inhibition by igfbps induce cell death . however , igfbp5 acts as a survival factor during myogenesis via an igf - independent mechanism . overexpression of wild - type igfbp5 ( wtigfbp5 ) decreased muscle igfir phosphorylation , probably via binding to and restricting the function of igf - i . igfbp5 has also been found to increase akt phosphorylation through an igfir - independent mechanism . both transient overexpression and administration of exogenous igfbp5 in a breast cancer cell line caused cell cycle arrest and apoptosis . examined igf - independent effects of igfbp5 during development using mice that express mutant igfbp5 ( mutigfbp5 ) , which does not bind igfs . they found that overexpression of wtigfbp5 resulted in increased total and free serum igf - i , but overexpression of mutigfbp5 did not change serum igf - i concentrations . furthermore , de0spite being highly expressed in the murine model , mutigfbp5 had an undetectable effect on the concentration of members of the igf axis in the circulation relative to wtigfbp5 . in addition , overexpression of wtigfbp5 rescued the lethal phenotype of mice carrying the maternal igf2r - null allele , but mutlgfbp5 did not . notably , overexpression of wtigfbp5 activated the p38 mapk pathway in an igf - independent manner . this is the first study to report igf - independent actions of igfbp5 in a mouse model . igf - i affects cells in the growth plate through endocrine , paracrine , and autocrine mechanisms. igf - i also enhances igf - i - driven chondrocyte proliferation . evaluated the expression profile of igf system components in proliferating and differentiating growth plate chondrocytes using two cell culture models : rcj3.1c5.18 ( rcj ) mesenchymal chondrogenic cells that , without biochemical or oncogenic transformation , do not express igf - i , and rat chondrocytes of the growth cartilage in primary culture . they found that igf - i and especially igfbp5 gene expression was increased during chondrocyte differentiation . to evaluate a possible functional role of igfbp5 on chondrocytes , rcj cells were transfected with a vector containing human igfbp5 cdna , cultured in serum - deprived media , and then treated with or without igf - i . igfbp5-transfected cells showed higher levels of expression of igfbp5 in response to igf - i treatment than did control cells . igfbp5 promotes the igf - i - enhanced differentiation of rcj cells but does not promote chondrocyte differentiation on its own . igfbp5-overexpressing cells showed enhanced igf - i - stimulated phosphorylation of akt , a member of pi3 kinase ( pi3k ) pathway , but mapk/ erk1/2 cascade was down - regulated . this result suggests that igfbp5 promotes the igf - i - enhanced differentiation of rcj cells , especially increases the activity of the pi3k pathway in a specific manner . for example , overexpression of igfbp5 may inhibit igf - i activity in malignant pleural mesothelioma ( mpm ) . consistent with this , igfbp5 was found to block the migration of oecm-1 , a head and neck squamous cell carcinoma ( hnscc ) cell line , which is stimulated by igf - i . in contrast , igfbp5 activity is also associated with elevated igf activity in several tumors . igfbp5 can be up - regulated by either physiologic ( cell differentiation ) or pathologic ( cancer ) processes . it can also be down - regulated in some cases . given the nature of its variable expression pattern and effects in both normal and cancer tissues , igfbp5 is considered neither a tumor suppressor nor an oncogene . mpm is an aggressive neoplasm of the serosal lining of the pleural cavity arising from mesothelial cells . igfbp5 was weakly expressed in mpm cell lines and tissue samples , though igf - i was overexpressed in all mpm specimens . evaluated igfbp5 rna expression among icc , normal liver ( nl ) , chronic liver disease ( cld ) , hepatocellular carcinoma ( hcc ) , and extrahepatic adenocarcinomas ( breast , colon , stomach , ovary , and lung ) and found that igfbp5 expression was higher in icc than in hcc , nl , cld , or adenocarcinomas originating from other organs . on the other hand , umemura et al . found that expression of igfbp5 was affected by gankyrin in hcc samples and that both igfbp5 and gankyrin may play oncogenic roles in early stages of human hepatocarcinogenesis . furthermore , human osteosarcoma cell line ( u2os ) and a well differentiated hepatocyte - derived cellular carcinoma cell line ( huh-7 ) overexpressing gankyrin had a respective 5.2-fold and 1.7-fold increase in igfbp5 mrna levels compared to noncancerous tissues . . showed that igfbp5 expression was strongly up - regulated during hepatic stallete cell transdifferen - tiation in vitro and during the development of liver fibrosis in vivo . in their current study , they determined that igfbp5 extended the survival of human hepatic stellate cell line , lx2 , a model for partially activated hepatic stellate cells , but did not affect cell proliferation . igfbp5 induced collagen type i , alpha 1 ( col1a1 ) by 70% , tissue inhibitor of metalloproteinase ( timp ) vmetallopeptidase inhibitor 1 ( timp1 ) by 70% , and matrix metallopeptidase 1 by 100% in lx2 cells , suggesting that igfbp5 may be a fibrotic marker . microarray studies have revealed that igfbp5 is overexpressed in pancreatic adenocarcinoma . when transfected into panc-1 pancreatic cancer cells , igfbp5 inhibited cell growth in serum - free culture and acted as a tumor suppressor . in contrast , igfbp5 did not affect the growth of bxpc-3 cells when it was transfected into the cells . additionally , pancreatic cancer pac cells can overexpress autocrine growth factors and overexpression of these growth factors and their receptors is associated with increased tumorigenicity in pancreatic cancer . bxpc-3 cells overexpressing igfbp5 exhibited high dna replication and cell numbers in the absence of serum , but the opposite phenotype was observed in panc-1 cells . in addition , igfbp5 promoted cell cycle in bxpc-3 cells but led to g2/m arrest in panc-1 cells . these findings support the notion that igfbp5 has cell - specific and environment - specific effects . thus , igfbp5 has cell- and environment - specific effects and its individualized effects must be considered for all cells and cancer types igfbp5 is also expressed in ovarian cancers and may play a role in the development of high - grade serous carcinoma of the ovaries . wang et al . have revealed that undifferentiated carcinoma , serous carcinoma , and transitional cell carcinomas have significantly higher expression levels of igfbp5 when compared to clear cell or mucinous carcinomas . mice bearing tumors generated by injection with skov3 ovarian cancer cells experienced decreased tumor growth after treatment with recombinant igfbp5 . development of cervical intraepithelial neoplasia ( cin ) and cc from normal cervical tissue is a gradual process , and the occurrence and development of these diseases is related with persistent human papilloma virus infection . hou et al . demonstrated that igfbp5 levels were highest in cin samples ( 91.9% ) , followed by in normal cervical tissues ( 71.4% ) , and lowest in cc tissues ( 45% ) . in cc samples , igfbp5 is negatively correlated with lymph node metastasis , cc progression , and high differentiation . the samples in early stage show increased igfbp5 expression , whereas those in late stage show decreased igfbp5 expression . in contrast , igfbp5 is detected in squamous cell cervical cancer and associated with progression of squamous cell carcinoma at a preneoplastic stage . igfbp5 is expressed in the mammary and breast tissues of mammals , , and it is thought to be necessary for normal mammary gland involution and to possibly regulate mammary gland morphogenesis in response to hormone stimulation . in addition , igfbp5 has also been found to have diverse effects in breast cancer cells . igfbp5 inhibits cell growth when transiently expressed in the mda - mb-231 and hs578 t breast cancer cell lines . both stable and adenovirus - mediated expression of igfbp5 in these cell lines result in a significant decrease in dna synthesis , but only adenovirus - mediated transfection of igfbp5 cause g2/m arrest compared with vector controls . as suggested by an in vivo model , plasma levels of igfbp5 plasma levels of igfbp5 are 1.5-fold higher in tumor - bearing mice than in non - tumor - bearing mice . nevertheless , li et al . found no significant correlation between the mrna level of igfbp5 and tumor size , clinical stage , or nuclear grade . they determined that igfbp5 mrna levels were up - regulated in breast cancers relative to normal breast tissues . they also found a positive correlation between igfbp5 mrna levels and the status of hormone receptors , including estrogen receptor ( er ) and progesteron receptor ( pr ) , and a negative link between igfbp5 mrna levels and distant metastasis or lymph node status . in contrast , hao et al . revealed that igfbp5 protein expression was elevated in lymph node metastasis samples compared with primary breast tumor samples . igfbp5 could be a useful marker of cancerous tissue and metastasis , but it could have diverse effects on growth of cancer cells depending on cell type and expression method . in addition to being up - regulated , igfbp5 is down - regulated in several cancers , including mpm , and in some immortal cell lines . also , igfbp5 is overexpressed in invasive non - functioning pituitary adenomas ( nfpas ) . igfbp5 expression may also vary between subclasses of the same cancer , as observed in glioblastomas . igfbp5 levels in colorectal cell lines(dld-1 , hct116 , sw837 , ht-29 , and sw48 ) were 10-fold lower than those in normal human fibroblasts . similarly , igfbp5 expression was elevated in glial cells co - cultured with retinoblastoma cells . a hypoxic microenvironment contributes to tumor development , and hypoxia - inducible factor-1 ( hif-1 ) plays a role in this process . to understand the molecular effects of hypoxia and hif-1 , the human genome u133a array was used to determine the gene expression profile of nci - h446 small cell lung cancer cells cultured in a hypoxic environment after transfection with ad5-hif-1 or ad5-sihif-1. in addition to other genes , igfbp5 is up - regulated at both the mrna and protein levels in the cells transfected with hif-1. igfbp5 expression can also be regulated by other proteins . in a study comparing colonic mucosa specimens from 12 colorectal cancer patients and 10 healthy controls , fos , the v - fos fbj murine osteosarcoma viral oncogene homolog , was found to activate igfbp5 in colorectal cancer but down - regulate it in normal colon tissue . the effects of igfbp5 on the biological activity of cells can change depending on several factors , . the protein interacts with many molecules that impact its effects on cellular characteristics . among its numerous effects in cells , in some cell lines or tissues , it can enhance migration , , differentiation , and cell attachment , whereas it can block cell motility , induce retention of cell morphology , and reinforce adhesion in others . cell migration , motility , and attachment to the extracellular matrix ( ecm ) are important features for cancer progression and metastasis process . ecm components like vitronectin ( vn ) , are important for cell migration and attachment to ecm , . the stimulatory effect of igf - i : igfbp5:vn complexes on cell migration has been shown in skin keratinocytes and mcf-7 breast cancer cells , . furthermore , elevated levels of igfbp5 have been associated with breast cancer metastasis . igfbp5 expression is higher in metastatic breast carcinomas with axillary lymph node involvement than in primary breast carcinoma . igfbp5 was also found to be elevated in lymph node metastasis samples than in primary tumor samples . found that expression of igfbp5 was higher in t1 invasive breast carcinoma than in benign breast epithelium . moreover , hs578 t breast cancer epithelial cells treated with igfbp5 and igf - i show reduced cell attachment . treatment with recombinant igfbp5 protein or transfection with igfbp5 plasmid decreases growth , stimulates migration , and reduces adhesion of hnscc cells , and this effect is igf - independent . thus , igfbp5 is an effective regulator of cell migration and adhesion and may control these processes through an igf - independent pathway . we have mentioned positive effects of igfbp5 on cell migration , but igfbp5 also has opposite effects on cell motility . igfbp5 specifically inhibits vascular endothelial growth factor - induced endothelial cell proliferation and , thus , effectively suppresses the formation of blood vessels in vitro and in vivo . protein kinase c was reported to promote igfbp5-mediated cell adhesion in hs578 t breast cancer cells . in a recent study , wtigfbp5 and mutigfbp5 were transfected to mda - mb-435 breast cancer cells and this study revealed a negative role of igfbp5 on cell motility . also , a nuclear localization signal ( nls ) mutant form of igfbp5 is generated by site - directed mutagenesis . mutigfbp5 and wtigfbp5 were transfected into mda - mb-435 breast cancer cells to generate stable clones overexpressing either mutigfbp5 or wtigfbp5 . cells overexpressing mutigfbp5 has significantly higher proliferation and migration rates than do cells overexpressing wtigfbp5 . cellular localization analyses have revealed that nls mutations cause an accumulation of the protein in cytoplasm . these results indicate that subcellular localization of igfbp5 affects the growth and migration of breast cancer cells . thus , cytoplasmic accumulation of igfbp5 could induce cell growth and motility , which could impact cancer development and progression . igfbp5 has also been found to inhibit igf - i - induced proliferation and migration of smooth muscle cells . igfbp5 has been reported to promote activation and migration of peripheral blood mononuclear cells ( pbmcs ) and these effects on migration were induced via the mapk pathway and independent of igf - i . found that igfbp5 stimulated the migration of rat mesanglial cells in an igf - independent and rgd - independent manner , and mccaig et al . revealed that igfbp5 alone reduced cell adhesion that increased after co - administration of igfbp5 and igf - i these results indicate that igfbp5 could induce migration of cells in an igf - i - dependent or -independent manner and igf - i treatment could reverse the activity of igfbp5 . since migration of cancer cells is an important point for metastasis , a great attention should be paid to interaction of igfbp5 and igf - i in metastasis and invasion of cancer . lan-5 neuroblastoma cells differentiate towards a neuronal phenotype and have elevated expression of igfbp5 when treated with all - trans retinoic acid ( ra ) at micromolar concentrations . rna interference ( rnai)-mediated knockdown of igfbp5 prevents neuroblastoma cells from differentiating towards a neuronal phenotype as evidenced by the absence of detectable neuronal markers and neurofilaments after treatment with ra for different durations . compared to controls , co - treatment with recombinant igfbp5 and ra rescues differentiation of cells in which igfbp5 expression was knocked down . igfbp5 is the major protein secreted by skeletal muscles , and its expression is induced during muscle differentiation . igfbp5 inhibits skeletal muscle cell differentiation via binding igf receptors and thereby blocking igf action . in contrast , the addition of purified bone - derived human igfbp5 to muscle cells enhances differentiation . ren et al . revealed that the expression of igf - ii and igfbp5 was elevated during myogenic differentiation and found that knockdown of igfbp5 impaired myogenic differentiation of c2c12 mouse myoblast cells . in these studies , igfbp5 was found to be overexpressed in activated hepatic stallete cells , so it may be a marker for hepatic stallete cell activation . igfbp5-overexpressing cells have high levels of osteocalcin , which is an indicator of advanced , differentiated osteoblast cells . thus , schneider et al . suggested that igfbp5 may promote osteoblast cell differentiation despite not finding a direct effect of igfbp5 on osteocalcin expression . there are numerous studies on the role of igfbp5 in survival and apoptosis of both normal and cancer cells . . showed that igfbp5 activated caspase-8 and caspase-9 , causing apoptosis through bcl-2 in the intrinsic apoptotic pathway in mda - mb-231 breast cancer cells . igfbp5-expressing mda - mb-231 cells had elevated levels of jun n - terminal kinase ( jnk ) , which sensitized the cells to tumor necrosis factor- ( tnf ) . similar to its effects in mda - mb-231 cells , endogenous igfbp5 inhibits the proliferation of mda - mb-435 breast cancer cells , and igfbp5 has been reported to inhibit cell growth and cause g2/m arrest in human breast cancer and panc-1 pancreatic cancer cells , , . notably , igfbp5 do not induce apoptosis but decreased the number of cells in human hepatocellular carcinoma . other studies have focused on the antiapoptoic role of igfbp5 . in normal cells like myoblasts , igfbp5 has been reported to inhibit apoptosis during cell differentiation . a study of rnai - mediated igfbp5 knockdown in neuroblastoma cells revealed that loss of igfbp5 resulted in inhibition of cell growth . in these cells , igfbp5 shows both igf - dependent and igf - independent effects that are related with cell proliferation . suppression of igfbp5 expression provoked apoptotic morphology , such as hypodiploid dna content and activation of caspase-3 and caspase-7 . the role of igfbp5 in cellular senesence was first described by kim et al .. the knockdown of igfbp5 in old human primary endothelial cells triggered anti - aging effects . notably , excess igf - i has been found in old cells compared to young cells , but this trend is not observed for igf - ii . the expression pattern of igfbp5 in different types of cancers reflects its role in cell proliferation . igfbp5 could contribute to survival effects in breast cancer cells via repressing the mitochondria - independent apoptosis pathway , and induce the anti - apoptotic effects of igf - i in prostate cancer cells . retinoic acid inhibits the growth of hpv - negative cc cells by inducing the igfbp5 expression . thus igfbp5 is an important player of cc cell growth , but definite mechanism is unknown . as described previously , igfbp5 induces the response to igf - i stimulation in prostate cancer cells whereas reduces the response in osteosarcoma cells . in huvec cells , igfbp5 inhibits proliferation , but this effect could be reversed by silencing igfbp5 gene expression . igfbp5 was found to enhance dna replication and cell proliferation in bxpc-3 cells after serum starvation . as indicated above , there are numerous studies on the effects of igfbp5 on cell proliferation , some of which show a negative effect of igfbp5 on proliferation , but some show positive effect ; however , the exact molecular mechanisms that cause these effects are not determined yet . igfbp5 can be up - regulated by either physiologic ( cell differentiation ) or pathologic ( cancer ) processes . it can also be down - regulated in some cases . given the nature of its variable expression pattern and effects in both normal and cancer tissues , igfbp5 is considered neither a tumor suppressor nor an oncogene . mpm is an aggressive neoplasm of the serosal lining of the pleural cavity arising from mesothelial cells . igfbp5 was weakly expressed in mpm cell lines and tissue samples , though igf - i was overexpressed in all mpm specimens . evaluated igfbp5 rna expression among icc , normal liver ( nl ) , chronic liver disease ( cld ) , hepatocellular carcinoma ( hcc ) , and extrahepatic adenocarcinomas ( breast , colon , stomach , ovary , and lung ) and found that igfbp5 expression was higher in icc than in hcc , nl , cld , or adenocarcinomas originating from other organs . on the other hand , umemura et al . found that expression of igfbp5 was affected by gankyrin in hcc samples and that both igfbp5 and gankyrin may play oncogenic roles in early stages of human hepatocarcinogenesis . furthermore , human osteosarcoma cell line ( u2os ) and a well differentiated hepatocyte - derived cellular carcinoma cell line ( huh-7 ) overexpressing gankyrin had a respective 5.2-fold and 1.7-fold increase in igfbp5 mrna levels compared to noncancerous tissues . . showed that igfbp5 expression was strongly up - regulated during hepatic stallete cell transdifferen - tiation in vitro and during the development of liver fibrosis in vivo . in their current study , they determined that igfbp5 extended the survival of human hepatic stellate cell line , lx2 , a model for partially activated hepatic stellate cells , but did not affect cell proliferation . igfbp5 induced collagen type i , alpha 1 ( col1a1 ) by 70% , tissue inhibitor of metalloproteinase ( timp ) vmetallopeptidase inhibitor 1 ( timp1 ) by 70% , and matrix metallopeptidase 1 by 100% in lx2 cells , suggesting that igfbp5 may be a fibrotic marker . microarray studies have revealed that igfbp5 is overexpressed in pancreatic adenocarcinoma . when transfected into panc-1 pancreatic cancer cells , igfbp5 inhibited cell growth in serum - free culture and acted as a tumor suppressor . in contrast , igfbp5 did not affect the growth of bxpc-3 cells when it was transfected into the cells . additionally , pancreatic cancer pac cells can overexpress autocrine growth factors and overexpression of these growth factors and their receptors is associated with increased tumorigenicity in pancreatic cancer . bxpc-3 cells overexpressing igfbp5 exhibited high dna replication and cell numbers in the absence of serum , but the opposite phenotype was observed in panc-1 cells . in addition , igfbp5 promoted cell cycle in bxpc-3 cells but led to g2/m arrest in panc-1 cells . these findings support the notion that igfbp5 has cell - specific and environment - specific effects . thus , igfbp5 has cell- and environment - specific effects and its individualized effects must be considered for all cells and cancer types igfbp5 is also expressed in ovarian cancers and may play a role in the development of high - grade serous carcinoma of the ovaries . wang et al . have revealed that undifferentiated carcinoma , serous carcinoma , and transitional cell carcinomas have significantly higher expression levels of igfbp5 when compared to clear cell or mucinous carcinomas . mice bearing tumors generated by injection with skov3 ovarian cancer cells experienced decreased tumor growth after treatment with recombinant igfbp5 . development of cervical intraepithelial neoplasia ( cin ) and cc from normal cervical tissue is a gradual process , and the occurrence and development of these diseases is related with persistent human papilloma virus infection . hou et al . demonstrated that igfbp5 levels were highest in cin samples ( 91.9% ) , followed by in normal cervical tissues ( 71.4% ) , and lowest in cc tissues ( 45% ) . in cc samples , igfbp5 is negatively correlated with lymph node metastasis , cc progression , and high differentiation . the samples in early stage show increased igfbp5 expression , whereas those in late stage show decreased igfbp5 expression . in contrast , igfbp5 is detected in squamous cell cervical cancer and associated with progression of squamous cell carcinoma at a preneoplastic stage . igfbp5 is expressed in the mammary and breast tissues of mammals , , and it is thought to be necessary for normal mammary gland involution and to possibly regulate mammary gland morphogenesis in response to hormone stimulation . in addition , igfbp5 has also been found to have diverse effects in breast cancer cells . igfbp5 inhibits cell growth when transiently expressed in the mda - mb-231 and hs578 t breast cancer cell lines . both stable and adenovirus - mediated expression of igfbp5 in these cell lines result in a significant decrease in dna synthesis , but only adenovirus - mediated transfection of igfbp5 cause g2/m arrest compared with vector controls . as suggested by an in vivo model , plasma levels of igfbp5 plasma levels of igfbp5 are 1.5-fold higher in tumor - bearing mice than in non - tumor - bearing mice . nevertheless , li et al . found no significant correlation between the mrna level of igfbp5 and tumor size , clinical stage , or nuclear grade . they determined that igfbp5 mrna levels were up - regulated in breast cancers relative to normal breast tissues . they also found a positive correlation between igfbp5 mrna levels and the status of hormone receptors , including estrogen receptor ( er ) and progesteron receptor ( pr ) , and a negative link between igfbp5 mrna levels and distant metastasis or lymph node status . in contrast , hao et al . revealed that igfbp5 protein expression was elevated in lymph node metastasis samples compared with primary breast tumor samples . igfbp5 could be a useful marker of cancerous tissue and metastasis , but it could have diverse effects on growth of cancer cells depending on cell type and expression method . in addition to being up - regulated , igfbp5 is down - regulated in several cancers , including mpm , and in some immortal cell lines . also , igfbp5 is overexpressed in invasive non - functioning pituitary adenomas ( nfpas ) . igfbp5 expression may also vary between subclasses of the same cancer , as observed in glioblastomas . igfbp5 levels in colorectal cell lines(dld-1 , hct116 , sw837 , ht-29 , and sw48 ) were 10-fold lower than those in normal human fibroblasts . similarly , igfbp5 expression was elevated in glial cells co - cultured with retinoblastoma cells . a hypoxic microenvironment contributes to tumor development , and hypoxia - inducible factor-1 ( hif-1 ) plays a role in this process . to understand the molecular effects of hypoxia and hif-1 , the human genome u133a array was used to determine the gene expression profile of nci - h446 small cell lung cancer cells cultured in a hypoxic environment after transfection with ad5-hif-1 or ad5-sihif-1. in addition to other genes , igfbp5 is up - regulated at both the mrna and protein levels in the cells transfected with hif-1. igfbp5 expression can also be regulated by other proteins . in a study comparing colonic mucosa specimens from 12 colorectal cancer patients and 10 healthy controls , fos , the v - fos fbj murine osteosarcoma viral oncogene homolog , was found to activate igfbp5 in colorectal cancer but down - regulate it in normal colon tissue . the effects of igfbp5 on the biological activity of cells can change depending on several factors , . the protein interacts with many molecules that impact its effects on cellular characteristics . among its numerous effects in cells , in some cell lines or tissues , it can enhance migration , , differentiation , and cell attachment , whereas it can block cell motility , induce retention of cell morphology , and reinforce adhesion in others . cell migration , motility , and attachment to the extracellular matrix ( ecm ) are important features for cancer progression and metastasis process . ecm components like vitronectin ( vn ) , are important for cell migration and attachment to ecm , . the stimulatory effect of igf - i : igfbp5:vn complexes on cell migration has been shown in skin keratinocytes and mcf-7 breast cancer cells , . furthermore , elevated levels of igfbp5 have been associated with breast cancer metastasis . igfbp5 expression is higher in metastatic breast carcinomas with axillary lymph node involvement than in primary breast carcinoma . igfbp5 was also found to be elevated in lymph node metastasis samples than in primary tumor samples . consistent with this , wang et al . found that expression of igfbp5 was higher in t1 invasive breast carcinoma than in benign breast epithelium . moreover , hs578 t breast cancer epithelial cells treated with igfbp5 and igf - i show reduced cell attachment . treatment with recombinant igfbp5 protein or transfection with igfbp5 plasmid decreases growth , stimulates migration , and reduces adhesion of hnscc cells , and this effect is igf - independent . thus , igfbp5 is an effective regulator of cell migration and adhesion and may control these processes through an igf - independent pathway . we have mentioned positive effects of igfbp5 on cell migration , but igfbp5 also has opposite effects on cell motility . igfbp5 specifically inhibits vascular endothelial growth factor - induced endothelial cell proliferation and , thus , effectively suppresses the formation of blood vessels in vitro and in vivo . protein kinase c was reported to promote igfbp5-mediated cell adhesion in hs578 t breast cancer cells . in a recent study , wtigfbp5 and mutigfbp5 were transfected to mda - mb-435 breast cancer cells and this study revealed a negative role of igfbp5 on cell motility . also , a nuclear localization signal ( nls ) mutant form of igfbp5 is generated by site - directed mutagenesis . mutigfbp5 and wtigfbp5 were transfected into mda - mb-435 breast cancer cells to generate stable clones overexpressing either mutigfbp5 or wtigfbp5 . cells overexpressing mutigfbp5 has significantly higher proliferation and migration rates than do cells overexpressing wtigfbp5 . cellular localization analyses have revealed that nls mutations cause an accumulation of the protein in cytoplasm . these results indicate that subcellular localization of igfbp5 affects the growth and migration of breast cancer cells . thus , cytoplasmic accumulation of igfbp5 could induce cell growth and motility , which could impact cancer development and progression . igfbp5 has also been found to inhibit igf - i - induced proliferation and migration of smooth muscle cells . igfbp5 has been reported to promote activation and migration of peripheral blood mononuclear cells ( pbmcs ) and these effects on migration were induced via the mapk pathway and independent of igf - i . found that igfbp5 stimulated the migration of rat mesanglial cells in an igf - independent and rgd - independent manner , and mccaig et al . revealed that igfbp5 alone reduced cell adhesion that increased after co - administration of igfbp5 and igf - i . these results indicate that igfbp5 could induce migration of cells in an igf - i - dependent or -independent manner and igf - i treatment could reverse the activity of igfbp5 . since migration of cancer cells is an important point for metastasis , a great attention should be paid to interaction of igfbp5 and igf - i in metastasis and invasion of cancer . lan-5 neuroblastoma cells differentiate towards a neuronal phenotype and have elevated expression of igfbp5 when treated with all - trans retinoic acid ( ra ) at micromolar concentrations . rna interference ( rnai)-mediated knockdown of igfbp5 prevents neuroblastoma cells from differentiating towards a neuronal phenotype as evidenced by the absence of detectable neuronal markers and neurofilaments after treatment with ra for different durations . compared to controls , co - treatment with recombinant igfbp5 and ra rescues differentiation of cells in which igfbp5 expression was knocked down . igfbp5 is the major protein secreted by skeletal muscles , and its expression is induced during muscle differentiation . igfbp5 inhibits skeletal muscle cell differentiation via binding igf receptors and thereby blocking igf action . in contrast , the addition of purified bone - derived human igfbp5 to muscle cells enhances differentiation . ren et al . revealed that the expression of igf - ii and igfbp5 was elevated during myogenic differentiation and found that knockdown of igfbp5 impaired myogenic differentiation of c2c12 mouse myoblast cells . in these studies , igfbp5 was found to be overexpressed in activated hepatic stallete cells , so it may be a marker for hepatic stallete cell activation . igfbp5-overexpressing cells have high levels of osteocalcin , which is an indicator of advanced , differentiated osteoblast cells . thus , schneider et al . suggested that igfbp5 may promote osteoblast cell differentiation despite not finding a direct effect of igfbp5 on osteocalcin expression . there are numerous studies on the role of igfbp5 in survival and apoptosis of both normal and cancer cells . . showed that igfbp5 activated caspase-8 and caspase-9 , causing apoptosis through bcl-2 in the intrinsic apoptotic pathway in mda - mb-231 breast cancer cells . igfbp5-expressing mda - mb-231 cells had elevated levels of jun n - terminal kinase ( jnk ) , which sensitized the cells to tumor necrosis factor- ( tnf ) . similar to its effects in mda - mb-231 cells , endogenous igfbp5 inhibits the proliferation of mda - mb-435 breast cancer cells , and igfbp5 has been reported to inhibit cell growth and cause g2/m arrest in human breast cancer and panc-1 pancreatic cancer cells , , . notably , igfbp5 do not induce apoptosis but decreased the number of cells in human hepatocellular carcinoma . other studies have focused on the antiapoptoic role of igfbp5 . in normal cells like myoblasts , a study of rnai - mediated igfbp5 knockdown in neuroblastoma cells revealed that loss of igfbp5 resulted in inhibition of cell growth . in these cells , igfbp5 shows both igf - dependent and igf - independent effects that are related with cell proliferation . suppression of igfbp5 expression provoked apoptotic morphology , such as hypodiploid dna content and activation of caspase-3 and caspase-7 . the role of igfbp5 in cellular senesence was first described by kim et al .. the knockdown of igfbp5 in old human primary endothelial cells triggered anti - aging effects . on the other hand , overexpression of igfbp5 in young cells results in aging . notably , excess igf - i has been found in old cells compared to young cells , but this trend is not observed for igf - ii . the expression pattern of igfbp5 in different types of cancers reflects its role in cell proliferation . igfbp5 could contribute to survival effects in breast cancer cells via repressing the mitochondria - independent apoptosis pathway , and induce the anti - apoptotic effects of igf - i in prostate cancer cells . retinoic acid inhibits the growth of hpv - negative cc cells by inducing the igfbp5 expression . thus igfbp5 is an important player of cc cell growth , but definite mechanism is unknown . as described previously , igfbp5 induces the response to igf - i stimulation in prostate cancer cells whereas reduces the response in osteosarcoma cells . in huvec cells , igfbp5 inhibits proliferation , but this effect could be reversed by silencing igfbp5 gene expression . igfbp5 was found to enhance dna replication and cell proliferation in bxpc-3 cells after serum starvation . as indicated above , there are numerous studies on the effects of igfbp5 on cell proliferation , some of which show a negative effect of igfbp5 on proliferation , but some show positive effect ; however , the exact molecular mechanisms that cause these effects are not determined yet . igfbp5 is involved in many signaling pathways that regulate the biological functions of cells . using transgenic mice overexpressing wtigfbp5 , kuemmerle et al . , showed that up - regulation of igfbp5 reduced igfir phosphorylation , but this did not affect the p - akt level in myogenic cells . overexpression of igfbp5 may activate the p38 mapk pathway , and igf - i may use the same pathway to stimulate igfbp5 expression . igfbp5 binding to its receptor induced the p38 mapk and erk1/2 pathway activation , thereby stimulating growth and igf - i secretion in human intestinal muscle cells . moreover , igf - i stimulated igfbp5 expression via the pi3k pathway during schwann cell differentiation . used two specific pharmacologic inhibitors of the p42/44 mapk pathway , u0126 and pd098059 , to evaluate igf - i - induced igfbp5 mrna expression in rat growth plate chondrocytes . both inhibitors were capable of suppressing phosphorylation of erk1/2 but did not alter total erk1/2 concentration in chondrocyte cell lysates . similarly , co - incubation of igf - i with u0126 did not affect igf - i - induced igfbp5 mrna expression . however , co - incubation of igf - i with the pi3k inhibitor ly294002 abolished the stimulatory effect of igf - i on igfbp5 mrna expression . thus , igf - i stimulates igfbp5 mrna expression via the pi3k pathway in rat growth plate chondrocytes . along the same line , other studies revealed that igfbp5 mrna expression induced by igf - i is mediated via the pi3k pathway in vascular smooth muscle cells and primary schwann cells . in contrast , xin et al . found that igf - i - induced igfbp5 expression occurs via the p42/44 mapk pathway in rat intestinal smooth muscle cells , and kuemmerle revealed that igfbp5 mrna expression was induced by igf - i via the p42/44 mapk and pi3k pathways . moreover , igf - i - induced igfbp5 expression was reported to occur via the mapk pathway in rat intestinal smooth muscle cells and via the pi3k and mapk pathways in mammary fibroblats and human intestinal smooth muscle cells , . kuemmerle suggested that igfbp5 stimulates cell growth and igf - i secretion via the mkk3/6-p38 and ras - erk1/2 pathways , and found that a positive feedback mechanism links igf - i and igfbp5 in human intestinal muscle cells . igf - i , via interacting with its cognate receptor that was facilitated by igfbp5 , activated the pi3k and erk1/2 pathways that mediate enhanced proliferation and secretion of igfbp5 , . in turn , igfbp5 , via interacting with its cognate receptor , activates the p38 mapk and erk1/2 pathways . johnson et al . transfected full - length human igfbp5 into two pancreatic cancer cell lines , bxcpc-3 and panc-1 . they found that p - akt levels were elevated after serum deprivation in bxcpc-3 cells expressing igfbp5 , but akt phosphorylation was reduced in panc-1 cells expressing igfbp5 after treatment with mek1/2 inhibitor , they found that erk1/2 phosphorylation was 2.6-fold higher in bxpc-3 cells expressing igfbp5 than in primary bxpc-3 cells and that igfbp5-mediated growth was inhibited when the mapk pathway was blocked with mek1/2 inhibitor . these results suggested that the mapk pathway is necessary for igfbp5-enhanced cell growth after serum deprivation . in addition , pathway analysis revealed that erk1/2-independent pathways contribute to the increase in akt phosphorylation associated with igfbp5 and that inhibition of the mapk or pi3k pathway in igfbp5-expressing panc-1 cells may shift signaling to the other pathway . showed that parathyroid hormone ( pth ) induced igfbp5 protein expression in umr106 - 01 osteosarcoma cells and found that activation of cyclic amp , protein lipase c , and protein kinase c increased igfbp5 mrna level . they also found that pth mediates the activation of igfbp5 gene transcription in osteoblast cells . also , akt was phosphorylated through igfir phosphorylation , which was induced by igfbp5 . taken together , these studies suggest that igf - i and igfbp5 have complex molecular mechanisms that require further investigation . any dysregulation in these pathways could cause deterioration of cellular functions such as proliferation , growth , and migration , leading to aggressiveness and metastatic properties in cancers . together with g - proteins , igfbp5 can induce phosphorylation of erk1/2 and p38 mapk . igfbp5 can act independently of igf - i to stimulate proliferation and up - regulate igf - i production through ras - dependent activation of grb - sos - mek - erk1/2 and p38 mapk . igfbp5 and igf - i secretion are linked by a positive feedback mechanism that reinforces their individual effects on cell growth . expression of igfir is stimulated by steroid and other hormones , resulting in an increase in erk1/2 pathway activation , which in turn stimulates igfbp5 production and proliferation . this suggests a positive feedback loop between igfbp5 and erk1/2 . in some cancer cell lines , the small gtpases rac , rho , and cdc42 are requisite co - factors in ras - dependent raf activation and subsequent activation of erk1/2 . igfbp5-induced growth mediated by the erk1/2 or p38 mapk pathways has been shown to involve these small gtpases , as well as raf1 , mkk1/2 , and shh , but the molecular interactions of these proteins remains unclear . igfbp5 was observed to have a clinical importance in various cancers such as breast cancer , , , ovarian cancer , glioblastoma , and nfpa , and it was also found to be a useful marker for diagnosis and differentiation of cc and cin . although igbfp5 mrna was found to be overexpressed in invasive nfpas and not in non - invasive nfpas , this differential expression was not confirmed at the protein level . igfbp5 expression has been found to correlate with prognosis , clinical outcome , survival , response to therapy , and drug resistance , either negatively or positively . igfbp5 is one of a group of genes that has been shown to predict the prognosis of primary breast tumors in a dynamic manner and reverse tamoxifen resistance . in addition , lower expression levels of igfbp5 are reportedly associated with shorter overall survival after tamoxifen ( tam ) therapy . igfbp5 expression was also found to predict response to exemestane therapy in er - positive ( er ) breast cancer and to be associated with poor clinical outcome and development of metastatic or recurrent disease in patients with malignant fibrous histiocytomas(mfh ) , pleomorphic sarcomas , and not otherwise specified ( nos ) sarcomas . because the expression levels of estrogen - regulated genes have been considered potential predictive markers for endocrine therapy for breast cancer , the expression of igfbp4 , an estrogen - induced gene , and igfbp5 , an estrogen - repressed gene , was analyzed in human breast cancer tissues with quantitative real - time reverse transcription - polymerase chain reaction ( rt - pcr ) . igfbp4 and igfbp5 mrna levels were found to be positively correlated with er and pr status and negatively correlated with her2 overexpression . igfbp4 expression was also found to be an independent prognostic factor for disease - free survival in er breast cancers . analysis of 116 patients with er breast cancer revealed that higher levels of igfbp4 mrna or lower levels of igfbp5 mrna in tumors were associated with better prognosis and disease - free survival . to evaluate the clinical significance of igfbp5 in breast cancer , mrna expression levels have been analyzed in normal breast tissues , primary tumors , and lymph node metastases using rt - pcr . igfbp5 mrna expression was found to be positively correlated with invasion of axillary lymph nodes and the status of hormonal receptor , and it was also found to be associated with poor outcome of breast cancer in patients with positive lymph nodes and negative er status . tissue microarray and immunohistochemical analysis of 164 cases of t1 breast carcinoma , either with or without axillary lymph node involvement , revealed that igfbp5 and igfbp2 might serve as useful markers of lymph node metastasis in small invasive breast carcinomas . assessed the gene expression changes after tam treatment in an er estrogen - responsive breast cancer xenograft model to predict endocrine sensitivity . zr-75 - 1 breast cancer cells were implanted into female nu / nu mice , which were separated into two treatment groups : tam ( the experimental group ) or estradiol ( e2 ) ( the control group ) . microarray analysis was used to assess gene expression changes at days 0 , 1 , 2 , 4 , 7 , and 14 , and hierarchical clustering identified 6 time - related gene expression patterns ( set 1 - 6 ) . these 6 sets of genes were separated into three groups by early / transient responses , continuous / late responses , and variable responses . the continuous / late response group contained set 5 and set 6 genes , which have been down - regulated and up - regulated , respectively , in response to therapy . igfbp5 and trefoil factor 3 ( tff3 ) , set 6 and set 5 genes , respectively , exhibit a continuous / late response to treatment . the study of pretreatment and post - treatment samples of 28 patients revealed that the protein expression change in igfbp5 , tff3 or both was significantly associated with change in tumor volume . higher igfbp5 gene expression was observed in patients with poor prognosis , but there was no significant difference at day 1 . four data sets representing a total of 404 patients have revealed that the genes most differentially expressed on days 2 , 4 , and 7 after treatment with tam were able to predict prognosis . the authors speculated that early / transient expression changes are more likely to be causative and primary events for tumor volume decrease , whereas continuous / late expression changes may be consequential and secondary to the volume changes . these results also suggest that igfbp5 may be a good biomarker for outcome after tam treatment . because mouse mammary gland involution resembles a wound healing response with suppressed inflammation and because inflammation is also associated with tumor development , stein et al . day 3 of mouse mammary gland involution ( inv3 ) and cluster 5 ( c5 ) genes showed the strongest predictive power for metastasis and survival . when hierarchical ordered partitioning and collapsing hybrid ( hopach ) clustering method was applied , the mouse mammary gland involution inv3/c5 signature could not cluster the data set into good or poor survival . however , when hierarchical clustering was used , the inv3/c5 signature was a good predictor of overall and metastasis - free survival . the authors concluded that the gene signature was possibly a combination of weaker markers making its predictive power dependent on the clustering method used in different data sets . based on this finding , we conclude that results of the previous study , which identified 6 time - related gene expression patterns in response to tam treatment using hierarchical clustering , depend on the clustering method . it was observed that igfbp5 warrants reversing tam resistance both in vivo and in vitro . igfbp5 knockdown in mcf7 human breast cancer cells has been shown to induce tam resistance in vitro due to concomitant loss of er expression and signaling . tam - resistant mcf7 cells , which were selected from cultures , have also been found to have a reduced igfbp5 expression . both tam - resistant cells and igfbp5-knockdown cells could be resensitized to tam by treatment with exogenous recombinant igfbp5 protein . in vivo , tam resistance of a mouse tumor xenograft model generated from igfbp5-knockdown mcf7 cells these findings have been verified by igfbp5 immunohistochemical staining in a cohort of tumor samples from 153 patients with breast cancer , indicating that low igfbp5 expression was associated with shorter overall survival after tam therapy . igfbp5 expression is not only associated with tam ; it is also associated with exemestane therapy response in er breast cancers . fifteen postmenopausal patients over 70 years who were diagnosed with primary er breast cancer were treated daily with exemestane for 6 months . before and after treatment patients who responded to therapy showed higher expression of her2 or igfbp5 in both the cytoplasm and nuclei . however , cytoplasmic expression of igfbp5 was increased in post - treatment sections , regardless of treatment response . these results suggest that the expression of igfbp5 can be influenced by exemestane treatment and that nuclear igfbp5 expression level is likely a predictor of response . cdna microarray and immunohistochemistry analysis of squamous cell carcinomas ( sccs ) and their adjacent normal squamous epithelia revealed that igfbp5 mrna and protein levels were significantly decreased in sccs . premalignant cin lesions and advanced cin3 lesions showed significantly weaker or negative staining for igfbp5 compared to normal squamous epithelia , suggesting a role for igfbp5 in cancer progression in cervical epithelia . the expression levels of igfbp5 and cellular fas - associated death domain - like interleukin-1-converting enzyme ( flice)-like inhibitory protein ( cflip ) were measured in cc , cin , and normal cervical tissue samples by rt - pcr and immunohistochemistry . stages ii and iii cin tissues had the highest igfbp5 protein expression , and cc samples had decreased protein levels relative to controls . the igfbp5 mrna levels in cc and cin samples , relative to the controls , were higher and lower , respectively . however , a positive correlation was found between the degree of pathologic change and expression levels of cflip protein and mrna . these results indicate that igfbp5 expression is up - regulated during cin progression and down - regulated in invasive cc . therefore , igfbp5 and cflip may be useful markers for diagnosing and differentiating cin and cc . serial analysis of gene expression ( sage ) of hcc and icc cdna libraries revealed distinct gene expression patterns for hcc and icc . gene expression was validated with real - time rt - pcr prior to comparing the icc library with the gastric , colon , prostate , and breast cancer libraries . the biglycan ( bgn ) , igfbp5 , and claudin-4 ( cldn4 ) genes were identified as icc - specific markers . discrimination analysis was done with randomly selected 53 samples from the total 74 samples and showed an efficient receiver operating characteristic ( roc ) curve with an area under curve ( auc ) value of 0.987 . confirmation of the discrimination analysis was completed with the remaining 21 samples and the z - score was found to be positive for all icc samples . these results indicate that a combination of the bgn , igfbp5 , and cldn4 genes could be used to distinguish icc from hcc or metastatic adenocarcinoma , but a validation using a larger cohort is needed to confirm this result . tissue microarray and immunohistochemistry analysis of normal ovarian surface epithelia and high - grade ovarian carcinomas of different histological types revealed that igfbp2 and igfbp5 are overexpressed in high - grade carcinomas , suggesting a role of igfbp5 in the development of high - grade ovarian carcinomas . down - regulation of igfbp5 and igfbp3 and up - regulation of igfbp4 has been reported in the er , estrogen - responsive ovarian cancer cell line pe04 after exposure to e2 . use of er-specific and er-specific agonists revealed that changes produced by er-specific agonist were very similar to those produced by e2 , suggesting that er is the main modulator of igfbp expression . semi - quantitative immunohistochemistry analysis of paraffin - fixed sections obtained from ovarian cancer patients treated with letrozole revealed that the expression of igfbp5 and igfbp3 was reduced but igfbp4 expression was increased in nonprogressive ( ca125 reduction or a minimal increase of < 50% ) tumors . multivariate logistic regression analysis reveals that igfbp3 is the most powerful predictor of lack of stable disease and er status was found to add power to the prediction . combined expressions of er plus igfbp4 and er plus igfbp5 also have predictive power , but they are inferior to igfbp3 alone . the combinations of igfbp4 plus er and igfbp5 plus er are superior to igfbp3 alone but are still inferior to the combination of igfbp3 with er. igfbp5 shows clinical importance in hnscc in addition , a functional polymorphism in the igfbp5 promoter was recently found to be associated with risk of late - stage hnscc . in this study , which included 1082 patients with hnscc and cancer - free controls , differential binding of transcription factor activator protein 1 ( ap-1 ) to igfbp5 promoter with the 1195c variant was associated with risk of late - stage hnscc when compared to the t variant genotype . thus , this polymorphism could be a marker for susceptibility to late - stage hnscc . however , there are limited studies on the polymorphisms of igfbp5 , and further study is needed to evaluate polymorphic differences of igfbp5 in different cancers . recently , shersher et al . used immunobead assays to measure serum levels of igfbp5 in non - small cell lung cancer patients with different nodal status and metastatic progression . they found that low serum igfbp5 levels correlated strongly with a positive nodal status and disease recurrence and also predicted poor recurrence - free survival . these results , along with previous findings , suggest that igfbp5 is an important player of carcinogenesis in various types of cancers and could be a strong biomarker for identifying non - small cell lung cancer progression and patient outcome . igfbp5 is involved in carcinogenesis , treatment , and diagnosis processes either negatively or positively depending on the type of tissue or cell and its micro - environment . some inconsistent findings summarized above could be due to the complexity of the igf pathway and process of carcinogenesis . it is not clear that if igfbp5 is a tumor suppressor or an oncogene , a predictor of good prognosis or bad prognosis , biomarker of survival or disease progression . it binds to igfs , interacts with cell surface and matrix components , and becomes modified post - translationally , leading to altered cell proliferation , apoptosis , survival , and migration . despite a great deal in secondary structural analyses , igfbp5 localizes in both the cytoplasm and nucleus , and its localization affects cell growth , migration , and proliferation properties directly and indirectly by inducing the nuclear uptake of other proteins . because igfbp5 has tissue - specific and cell - specific effects , further studies should take this into consideration . furthermore , we speculate that igfbp5 could have individual effects depending on the activities and expression patterns of its interacting proteins . because cytoplasmic expression increases in post - treatment sections of breast cancer samples and overexpression of the nls mutant form in breast cancer cells causes higher proliferation and migration rates , we hypothesize that proteins that interact with igfbp5 can cause the protein to accumulate in the cytoplasm . for these reasons , igfbp5 needs to be investigated in terms of its function as well as its direct and indirect binding partners . this will reveal its potentially extensive and significant use in cancer prognosis , differential diagnosis , and treatment . a hypothetical comparative model for igfbp5 in breast cancer and normal tissue a , igfbp5 enters the cell , probably by binding to a cell surface receptor through its receptor - binding domain . the nls sequence directs the protein to the nucleus , where it is transported across the nuclear membrane by importins . b , igfbp5 enters the cell through its potential cell surface receptor ( 1 ) . the protein is imported into the nucleus by importins ( 2 ) or by free diffusion ( 3 ) , thereby inducing apoptosis and suppressing cell growth and proliferation . igfbp5 can also interact with cytoplasmic proteins ( 4 ) , which blocks the nls sequence or changes the three - dimentional structure of the protein ( 5 and 6 ) , thereby preventing nuclear uptake and causing igfbp5 to accumulate in the cytoplasm . cytoplasmic accumulation of igfbp5 decreases or blocks apoptosis and may cause metastasis in some cancers . the inhibitory and stimulatory affects of igfbp5 are thought to proceed via the mapk , pi3k , ras - erk1/2 , and mkk3 pathways . to determine exact molecular mechanism and properties of igfbp5 , molecular interactions with proteins in these pathways is expected to increase the prognostic and clinical significance of igfbp5 , thereby serving to overcome obstacles concerning the prediction of clinical outcome , drug resistance , and response to therapy . because igfbp5 has different effects on normal and cancer tissues , drug development related to igfbp5 may result in cancer cell - specific treatment strategies .

insulin - like growth factor - binding proteins ( igfbps ) are critical regulators of the mitogenic activity of insulin - like growth factors ( igfs ) . igfbp5 , one of these igfbps , has special structural features , including a nuclear transport domain , heparin - binding motif , and igf / extracellular matrix / acid - labile subunit - binding sites . furthermore , igfbp5 has several functional effects on carcinogenesis and even normal cell processes , such as cell growth , death , motility , and tissue remodeling . these biological effects are sometimes related with igf ( igf - dependent effects ) and sometimes not ( igf - independent effects ) . the functional role of igfbp5 is most likely determined in a cell - type and tissue - type specific manner but also depends on cell context , especially in terms of the diversity of interacting proteins and the potential for nuclear localization . clinical findings show that igfbp5 has the potential to be a useful clinical biomarker for predicting response to therapy and clinical outcome of cancer patients . in this review , we summarize the functional diversity and clinical importance of igfbp5 in different types of cancers .

IGF-dependent or IGF-independent Functions of IGFBP5 Importance of differentially expressed IGFBP5 in cancer tissues Migration, differentiation, and metastasis effects of IGFBP5 in cancer Cell survival and apoptotic effects of IGFBP5 in cancer Association of IGFBP5 with Signaling Pathways Prognostic and Clinical Importance of IGFBP5 in Cancer Conclusions

it can be used to predict clinical outcome , , diagnosis , endocrine responsiveness , cancer progression or inhibition of cancer growth , and apoptosis . the ligands of the igf system , igf - i and igf - ii , mediate the growth and development of organisms . igfbp5 , expressed in both normal and cancer tissues , regulates the growth and development of tissues and cells , such as myoblasts , thyroid carcinoma cells , uterine leiomyomata cells , neural cells , and muscle - derived tumor rhabdomyosarcoma cells . igfbp5 has both igf - dependent and igf - independent effects. examined igf - independent effects of igfbp5 during development using mice that express mutant igfbp5 ( mutigfbp5 ) , which does not bind igfs . this is the first study to report igf - independent actions of igfbp5 in a mouse model . evaluated the expression profile of igf system components in proliferating and differentiating growth plate chondrocytes using two cell culture models : rcj3.1c5.18 ( rcj ) mesenchymal chondrogenic cells that , without biochemical or oncogenic transformation , do not express igf - i , and rat chondrocytes of the growth cartilage in primary culture . to evaluate a possible functional role of igfbp5 on chondrocytes , rcj cells were transfected with a vector containing human igfbp5 cdna , cultured in serum - deprived media , and then treated with or without igf - i . igfbp5-transfected cells showed higher levels of expression of igfbp5 in response to igf - i treatment than did control cells . this result suggests that igfbp5 promotes the igf - i - enhanced differentiation of rcj cells , especially increases the activity of the pi3k pathway in a specific manner . evaluated igfbp5 rna expression among icc , normal liver ( nl ) , chronic liver disease ( cld ) , hepatocellular carcinoma ( hcc ) , and extrahepatic adenocarcinomas ( breast , colon , stomach , ovary , and lung ) and found that igfbp5 expression was higher in icc than in hcc , nl , cld , or adenocarcinomas originating from other organs . thus , igfbp5 has cell- and environment - specific effects and its individualized effects must be considered for all cells and cancer types igfbp5 is also expressed in ovarian cancers and may play a role in the development of high - grade serous carcinoma of the ovaries . development of cervical intraepithelial neoplasia ( cin ) and cc from normal cervical tissue is a gradual process , and the occurrence and development of these diseases is related with persistent human papilloma virus infection . in cc samples , igfbp5 is negatively correlated with lymph node metastasis , cc progression , and high differentiation . igfbp5 is expressed in the mammary and breast tissues of mammals , , and it is thought to be necessary for normal mammary gland involution and to possibly regulate mammary gland morphogenesis in response to hormone stimulation . both stable and adenovirus - mediated expression of igfbp5 in these cell lines result in a significant decrease in dna synthesis , but only adenovirus - mediated transfection of igfbp5 cause g2/m arrest compared with vector controls . they also found a positive correlation between igfbp5 mrna levels and the status of hormone receptors , including estrogen receptor ( er ) and progesteron receptor ( pr ) , and a negative link between igfbp5 mrna levels and distant metastasis or lymph node status . igfbp5 could be a useful marker of cancerous tissue and metastasis , but it could have diverse effects on growth of cancer cells depending on cell type and expression method . in addition to being up - regulated , igfbp5 is down - regulated in several cancers , including mpm , and in some immortal cell lines . in a study comparing colonic mucosa specimens from 12 colorectal cancer patients and 10 healthy controls , fos , the v - fos fbj murine osteosarcoma viral oncogene homolog , was found to activate igfbp5 in colorectal cancer but down - regulate it in normal colon tissue . cell migration , motility , and attachment to the extracellular matrix ( ecm ) are important features for cancer progression and metastasis process . treatment with recombinant igfbp5 protein or transfection with igfbp5 plasmid decreases growth , stimulates migration , and reduces adhesion of hnscc cells , and this effect is igf - independent . thus , igfbp5 is an effective regulator of cell migration and adhesion and may control these processes through an igf - independent pathway . we have mentioned positive effects of igfbp5 on cell migration , but igfbp5 also has opposite effects on cell motility . in a recent study , wtigfbp5 and mutigfbp5 were transfected to mda - mb-435 breast cancer cells and this study revealed a negative role of igfbp5 on cell motility . also , a nuclear localization signal ( nls ) mutant form of igfbp5 is generated by site - directed mutagenesis . igfbp5 has been reported to promote activation and migration of peripheral blood mononuclear cells ( pbmcs ) and these effects on migration were induced via the mapk pathway and independent of igf - i . found that igfbp5 stimulated the migration of rat mesanglial cells in an igf - independent and rgd - independent manner , and mccaig et al . revealed that igfbp5 alone reduced cell adhesion that increased after co - administration of igfbp5 and igf - i these results indicate that igfbp5 could induce migration of cells in an igf - i - dependent or -independent manner and igf - i treatment could reverse the activity of igfbp5 . since migration of cancer cells is an important point for metastasis , a great attention should be paid to interaction of igfbp5 and igf - i in metastasis and invasion of cancer . in these studies , igfbp5 was found to be overexpressed in activated hepatic stallete cells , so it may be a marker for hepatic stallete cell activation . in these cells , igfbp5 shows both igf - dependent and igf - independent effects that are related with cell proliferation . suppression of igfbp5 expression provoked apoptotic morphology , such as hypodiploid dna content and activation of caspase-3 and caspase-7 . the role of igfbp5 in cellular senesence was first described by kim et al .. the knockdown of igfbp5 in old human primary endothelial cells triggered anti - aging effects . the expression pattern of igfbp5 in different types of cancers reflects its role in cell proliferation . igfbp5 could contribute to survival effects in breast cancer cells via repressing the mitochondria - independent apoptosis pathway , and induce the anti - apoptotic effects of igf - i in prostate cancer cells . as described previously , igfbp5 induces the response to igf - i stimulation in prostate cancer cells whereas reduces the response in osteosarcoma cells . evaluated igfbp5 rna expression among icc , normal liver ( nl ) , chronic liver disease ( cld ) , hepatocellular carcinoma ( hcc ) , and extrahepatic adenocarcinomas ( breast , colon , stomach , ovary , and lung ) and found that igfbp5 expression was higher in icc than in hcc , nl , cld , or adenocarcinomas originating from other organs . igfbp5 induced collagen type i , alpha 1 ( col1a1 ) by 70% , tissue inhibitor of metalloproteinase ( timp ) vmetallopeptidase inhibitor 1 ( timp1 ) by 70% , and matrix metallopeptidase 1 by 100% in lx2 cells , suggesting that igfbp5 may be a fibrotic marker . these findings support the notion that igfbp5 has cell - specific and environment - specific effects . thus , igfbp5 has cell- and environment - specific effects and its individualized effects must be considered for all cells and cancer types igfbp5 is also expressed in ovarian cancers and may play a role in the development of high - grade serous carcinoma of the ovaries . development of cervical intraepithelial neoplasia ( cin ) and cc from normal cervical tissue is a gradual process , and the occurrence and development of these diseases is related with persistent human papilloma virus infection . in cc samples , igfbp5 is negatively correlated with lymph node metastasis , cc progression , and high differentiation . igfbp5 is expressed in the mammary and breast tissues of mammals , , and it is thought to be necessary for normal mammary gland involution and to possibly regulate mammary gland morphogenesis in response to hormone stimulation . both stable and adenovirus - mediated expression of igfbp5 in these cell lines result in a significant decrease in dna synthesis , but only adenovirus - mediated transfection of igfbp5 cause g2/m arrest compared with vector controls . they also found a positive correlation between igfbp5 mrna levels and the status of hormone receptors , including estrogen receptor ( er ) and progesteron receptor ( pr ) , and a negative link between igfbp5 mrna levels and distant metastasis or lymph node status . igfbp5 could be a useful marker of cancerous tissue and metastasis , but it could have diverse effects on growth of cancer cells depending on cell type and expression method . in addition to being up - regulated , igfbp5 is down - regulated in several cancers , including mpm , and in some immortal cell lines . in a study comparing colonic mucosa specimens from 12 colorectal cancer patients and 10 healthy controls , fos , the v - fos fbj murine osteosarcoma viral oncogene homolog , was found to activate igfbp5 in colorectal cancer but down - regulate it in normal colon tissue . cell migration , motility , and attachment to the extracellular matrix ( ecm ) are important features for cancer progression and metastasis process . treatment with recombinant igfbp5 protein or transfection with igfbp5 plasmid decreases growth , stimulates migration , and reduces adhesion of hnscc cells , and this effect is igf - independent . thus , igfbp5 is an effective regulator of cell migration and adhesion and may control these processes through an igf - independent pathway . we have mentioned positive effects of igfbp5 on cell migration , but igfbp5 also has opposite effects on cell motility . in a recent study , wtigfbp5 and mutigfbp5 were transfected to mda - mb-435 breast cancer cells and this study revealed a negative role of igfbp5 on cell motility . also , a nuclear localization signal ( nls ) mutant form of igfbp5 is generated by site - directed mutagenesis . thus , cytoplasmic accumulation of igfbp5 could induce cell growth and motility , which could impact cancer development and progression . igfbp5 has been reported to promote activation and migration of peripheral blood mononuclear cells ( pbmcs ) and these effects on migration were induced via the mapk pathway and independent of igf - i . found that igfbp5 stimulated the migration of rat mesanglial cells in an igf - independent and rgd - independent manner , and mccaig et al . revealed that igfbp5 alone reduced cell adhesion that increased after co - administration of igfbp5 and igf - i . these results indicate that igfbp5 could induce migration of cells in an igf - i - dependent or -independent manner and igf - i treatment could reverse the activity of igfbp5 . since migration of cancer cells is an important point for metastasis , a great attention should be paid to interaction of igfbp5 and igf - i in metastasis and invasion of cancer . in these studies , igfbp5 was found to be overexpressed in activated hepatic stallete cells , so it may be a marker for hepatic stallete cell activation . there are numerous studies on the role of igfbp5 in survival and apoptosis of both normal and cancer cells . similar to its effects in mda - mb-231 cells , endogenous igfbp5 inhibits the proliferation of mda - mb-435 breast cancer cells , and igfbp5 has been reported to inhibit cell growth and cause g2/m arrest in human breast cancer and panc-1 pancreatic cancer cells , , . in these cells , igfbp5 shows both igf - dependent and igf - independent effects that are related with cell proliferation . suppression of igfbp5 expression provoked apoptotic morphology , such as hypodiploid dna content and activation of caspase-3 and caspase-7 . the role of igfbp5 in cellular senesence was first described by kim et al .. the knockdown of igfbp5 in old human primary endothelial cells triggered anti - aging effects . the expression pattern of igfbp5 in different types of cancers reflects its role in cell proliferation . as described previously , igfbp5 induces the response to igf - i stimulation in prostate cancer cells whereas reduces the response in osteosarcoma cells . as indicated above , there are numerous studies on the effects of igfbp5 on cell proliferation , some of which show a negative effect of igfbp5 on proliferation , but some show positive effect ; however , the exact molecular mechanisms that cause these effects are not determined yet . overexpression of igfbp5 may activate the p38 mapk pathway , and igf - i may use the same pathway to stimulate igfbp5 expression . found that igf - i - induced igfbp5 expression occurs via the p42/44 mapk pathway in rat intestinal smooth muscle cells , and kuemmerle revealed that igfbp5 mrna expression was induced by igf - i via the p42/44 mapk and pi3k pathways . kuemmerle suggested that igfbp5 stimulates cell growth and igf - i secretion via the mkk3/6-p38 and ras - erk1/2 pathways , and found that a positive feedback mechanism links igf - i and igfbp5 in human intestinal muscle cells . igf - i , via interacting with its cognate receptor that was facilitated by igfbp5 , activated the pi3k and erk1/2 pathways that mediate enhanced proliferation and secretion of igfbp5 , . any dysregulation in these pathways could cause deterioration of cellular functions such as proliferation , growth , and migration , leading to aggressiveness and metastatic properties in cancers . igfbp5 and igf - i secretion are linked by a positive feedback mechanism that reinforces their individual effects on cell growth . igfbp5 was observed to have a clinical importance in various cancers such as breast cancer , , , ovarian cancer , glioblastoma , and nfpa , and it was also found to be a useful marker for diagnosis and differentiation of cc and cin . igfbp5 expression has been found to correlate with prognosis , clinical outcome , survival , response to therapy , and drug resistance , either negatively or positively . igfbp5 expression was also found to predict response to exemestane therapy in er - positive ( er ) breast cancer and to be associated with poor clinical outcome and development of metastatic or recurrent disease in patients with malignant fibrous histiocytomas(mfh ) , pleomorphic sarcomas , and not otherwise specified ( nos ) sarcomas . igfbp5 mrna expression was found to be positively correlated with invasion of axillary lymph nodes and the status of hormonal receptor , and it was also found to be associated with poor outcome of breast cancer in patients with positive lymph nodes and negative er status . these results also suggest that igfbp5 may be a good biomarker for outcome after tam treatment . based on this finding , we conclude that results of the previous study , which identified 6 time - related gene expression patterns in response to tam treatment using hierarchical clustering , depend on the clustering method . the expression levels of igfbp5 and cellular fas - associated death domain - like interleukin-1-converting enzyme ( flice)-like inhibitory protein ( cflip ) were measured in cc , cin , and normal cervical tissue samples by rt - pcr and immunohistochemistry . the biglycan ( bgn ) , igfbp5 , and claudin-4 ( cldn4 ) genes were identified as icc - specific markers . confirmation of the discrimination analysis was completed with the remaining 21 samples and the z - score was found to be positive for all icc samples . these results indicate that a combination of the bgn , igfbp5 , and cldn4 genes could be used to distinguish icc from hcc or metastatic adenocarcinoma , but a validation using a larger cohort is needed to confirm this result . tissue microarray and immunohistochemistry analysis of normal ovarian surface epithelia and high - grade ovarian carcinomas of different histological types revealed that igfbp2 and igfbp5 are overexpressed in high - grade carcinomas , suggesting a role of igfbp5 in the development of high - grade ovarian carcinomas . however , there are limited studies on the polymorphisms of igfbp5 , and further study is needed to evaluate polymorphic differences of igfbp5 in different cancers . these results , along with previous findings , suggest that igfbp5 is an important player of carcinogenesis in various types of cancers and could be a strong biomarker for identifying non - small cell lung cancer progression and patient outcome . despite a great deal in secondary structural analyses , igfbp5 localizes in both the cytoplasm and nucleus , and its localization affects cell growth , migration , and proliferation properties directly and indirectly by inducing the nuclear uptake of other proteins . because igfbp5 has tissue - specific and cell - specific effects , further studies should take this into consideration . furthermore , we speculate that igfbp5 could have individual effects depending on the activities and expression patterns of its interacting proteins . for these reasons , igfbp5 needs to be investigated in terms of its function as well as its direct and indirect binding partners . a hypothetical comparative model for igfbp5 in breast cancer and normal tissue a , igfbp5 enters the cell , probably by binding to a cell surface receptor through its receptor - binding domain . to determine exact molecular mechanism and properties of igfbp5 , molecular interactions with proteins in these pathways is expected to increase the prognostic and clinical significance of igfbp5 , thereby serving to overcome obstacles concerning the prediction of clinical outcome , drug resistance , and response to therapy . because igfbp5 has different effects on normal and cancer tissues , drug development related to igfbp5 may result in cancer cell - specific treatment strategies .

all echocardiographic and pc mri scans were carried out for research purposes only , with research ethics committee approval and signed parental consent . infants were recruited from stable infants admitted to the neonatal intensive care unit or postnatal ward at queen charlotte 's and chelsea hospital , london , at any time during admission . echocardiographic images were acquired using a vivid 7 ultrasound machine ( ge healthcare , milwaukee , wi ) with a 10-mhz sector probe . before the study of this cohort all examinations were either performed by or directly supervised by a neonatologist with > 10 years ' experience in functional echocardiography ( a.m.g . ) . all subjects were screened for congenital heart disease , including the exclusion of bilateral superior venae cavae . in all cases , an additional cohort of infants was subsequently examined by echocardiography to investigate the repeatability of quantification of svc flow volume . examinations in these infants were all performed by a single investigator ( b.f . ) after standardization of approach between operators in 10 infants . aortic dimension was assessed from the parasternal long - axis view , with high - definition zoom to the aortic valve , and diameter was assessed at the valve hinge points at end - systole ( figure 1a , dashed line ) . aortic flow velocity was assessed by pulsed - wave doppler from an optimized apical five - chamber view , with the pulsed - wave doppler gate placed at the level of the aortic valve ( figure 1b ) . care was taken to minimize angulation between the doppler beam and flow direction in the left ventricular outflow tract . first , diameter was assessed from a modified parasternal long - axis view as initial described by kluckow and evans , hereafter described as the sagittal approach . high - definition zoom was used to focus on the superior vena cava as it begins to open up into the right atrium ( figure 2a , dashed line ) , with maximum and minimum diameters through the cardiac cycle taken from b - mode images . because of concerns about the irregular shape of the superior vena cava , we also measured vessel area directly from an axial view , again using high - definition zoom and tracing maximum and minimum cross - sectional area from the b - mode images ( figure 2b , dashed line ) . svc flow velocity was assessed using pulsed - wave doppler from a low subcostal view as described by kluckow and evans , with the ultrasound probe moved caudally until a clear length of the superior vena cava could be seen entering the right atrium , where the pulsed - wave doppler gate was placed ( figure 2c ) . in all cases , three to five consecutive cycles were analyzed , except in the case of svc flow velocity , for which eight to 10 cycles were used to reduce the impact of respiratory variability . all flow quantification was performed offline using echopac software ( ge healthcare , milwaukee , wi ) by investigators blind to the pc mri results . pc mri was performed using a 3-t scanner ( philips medical systems , best , the netherlands ) using a specialized eight - channel pediatric body receive coil for infants weighing > 2 kg or a small - extremity receive coil for infants weighing < 2 kg . the methodology has previously been described , but in summary , infants were allowed to fall into a natural sleep after a feeding , without the use of sedation or anesthesia . they received nasal continuous positive airway pressure or low - flow oxygen support as clinically indicated , and a specially trained pediatrician was in attendance at all times . single - slice pc mri acquisition sequences with in - plane spatial resolution of 0.6 mm , slice thickness of 4 mm , repetition time of 5.9 ms , echo time of 3.1 ms , and 20 phases per cardiac cycle were used . field of view and matrix were altered to maintain spatial resolution at 0.6 mm while minimizing scan duration . the velocity encoding was calibrated for the range of 120 cm / sec for lvo sequences and 60 cm / sec for svc sequences . depending on heart rate and heart rate stability , the acquisition time for each two - dimensional pc mri scan ranged between 2 and 4 min . pilot scans were acquired to view the vessels of interest to ascertain the straightest section of the vessel adequate for the slice thickness of the pc mri sequences and to position the imaging plane perpendicular to the centerline of the vessel to minimize partial volume effects . lvo was quantified immediately distal to the level of the aortic valve ( figure 3a ) . volume of flow in the superior vena cava was quantified at the level of the pulmonary trunk as the superior vena cava begins to open into the right atrium ( figure 3b ) . sequence analysis and flow volume quantification for pc data sets were performed using a commercial workstation ( viewforum ; philips medical systems ) . automated vessel edge detection was used for all vessels of interest , with manual correction as necessary . once defined in the first cardiac phase , the software tracks the vessel of interest over the cardiac cycle using edge detection algorithms . flow is then calculated at each time point of the cardiac cycle , generating a flow curve and volume of flow value for each vessel of interest . measures of flow obtained by echocardiography and pc mri were compared using simple linear regression and also as described by bland and altman : the mean bias and limits of agreement ( loa , or repeatability coefficient ; 1.96 sd of differences ) were calculated . repeatability index ( ri ; loa / mean of measures ) was also calculated to allow comparison of repeatability between different measures . echocardiographic images were acquired using a vivid 7 ultrasound machine ( ge healthcare , milwaukee , wi ) with a 10-mhz sector probe . before the study of this cohort all examinations were either performed by or directly supervised by a neonatologist with > 10 years ' experience in functional echocardiography ( a.m.g . ) . all subjects were screened for congenital heart disease , including the exclusion of bilateral superior venae cavae . in all cases , an additional cohort of infants was subsequently examined by echocardiography to investigate the repeatability of quantification of svc flow volume . examinations in these infants were all performed by a single investigator ( b.f . ) after standardization of approach between operators in 10 infants . aortic dimension was assessed from the parasternal long - axis view , with high - definition zoom to the aortic valve , and diameter was assessed at the valve hinge points at end - systole ( figure 1a , dashed line ) . aortic flow velocity was assessed by pulsed - wave doppler from an optimized apical five - chamber view , with the pulsed - wave doppler gate placed at the level of the aortic valve ( figure 1b ) . care was taken to minimize angulation between the doppler beam and flow direction in the left ventricular outflow tract . first , diameter was assessed from a modified parasternal long - axis view as initial described by kluckow and evans , hereafter described as the sagittal approach . high - definition zoom was used to focus on the superior vena cava as it begins to open up into the right atrium ( figure 2a , dashed line ) , with maximum and minimum diameters through the cardiac cycle taken from b - mode images . because of concerns about the irregular shape of the superior vena cava , we also measured vessel area directly from an axial view , again using high - definition zoom and tracing maximum and minimum cross - sectional area from the b - mode images ( figure 2b , dashed line ) . svc flow velocity was assessed using pulsed - wave doppler from a low subcostal view as described by kluckow and evans , with the ultrasound probe moved caudally until a clear length of the superior vena cava could be seen entering the right atrium , where the pulsed - wave doppler gate was placed ( figure 2c ) . in all cases , three to five consecutive cycles were analyzed , except in the case of svc flow velocity , for which eight to 10 cycles were used to reduce the impact of respiratory variability . all flow quantification was performed offline using echopac software ( ge healthcare , milwaukee , wi ) by investigators blind to the pc mri results . pc mri was performed using a 3-t scanner ( philips medical systems , best , the netherlands ) using a specialized eight - channel pediatric body receive coil for infants weighing > 2 kg or a small - extremity receive coil for infants weighing < 2 kg . the methodology has previously been described , but in summary , infants were allowed to fall into a natural sleep after a feeding , without the use of sedation or anesthesia . they received nasal continuous positive airway pressure or low - flow oxygen support as clinically indicated , and a specially trained pediatrician was in attendance at all times . single - slice pc mri acquisition sequences with in - plane spatial resolution of 0.6 mm , slice thickness of 4 mm , repetition time of 5.9 ms , echo time of 3.1 ms , and 20 phases per cardiac cycle were used . field of view and matrix were altered to maintain spatial resolution at 0.6 mm while minimizing scan duration . the velocity encoding was calibrated for the range of 120 cm / sec for lvo sequences and 60 cm / sec for svc sequences . depending on heart rate and heart rate stability , the acquisition time for each two - dimensional pc mri scan ranged between 2 and 4 min . pilot scans were acquired to view the vessels of interest to ascertain the straightest section of the vessel adequate for the slice thickness of the pc mri sequences and to position the imaging plane perpendicular to the centerline of the vessel to minimize partial volume effects . lvo was quantified immediately distal to the level of the aortic valve ( figure 3a ) . volume of flow in the superior vena cava was quantified at the level of the pulmonary trunk as the superior vena cava begins to open into the right atrium ( figure 3b ) . sequence analysis and flow volume quantification for pc data sets were performed using a commercial workstation ( viewforum ; philips medical systems ) . automated vessel edge detection was used for all vessels of interest , with manual correction as necessary . once defined in the first cardiac phase , the software tracks the vessel of interest over the cardiac cycle using edge detection algorithms . flow is then calculated at each time point of the cardiac cycle , generating a flow curve and volume of flow value for each vessel of interest . measures of flow obtained by echocardiography and pc mri were compared using simple linear regression and also as described by bland and altman : the mean bias and limits of agreement ( loa , or repeatability coefficient ; 1.96 sd of differences ) were calculated . repeatability index ( ri ; loa / mean of measures ) was also calculated to allow comparison of repeatability between different measures . paired pc mri and echocardiographic examinations were performed in 49 infants with a median gestational age of 32.57 weeks ( range , 24.4339.14 ) weeks and a median weight at birth of 1,750 g ( range , 5253,760 g ) . the median postnatal age at scan was 11 days ( range , 184 days ) , with a median corrected gestational age of 34.43 weeks ( range , 27.4340 weeks ) and a median weight at scan of 1,880 g ( range , 6603,760 g ) . the median interval between pc mri and echocardiographic scanning was 3.67 hours ( range , 0.2533.0 hours ) , with pc mri being performed before echocardiography in 46 of 49 infants . no infant had a significant change in clinical condition between pc mri and echocardiography , and no infant received intervention for a blood transfusion or change in vasoactive medications or treatment for patent ductus arteriosus between the two scans . one infant was found to have a small ( 3-mm ) muscular ventricular septal defect , and eight infants were found to have patent ductus arteriosus . the means and standard deviations of aortic valve diameter , left ventricular outflow velocity - time integral ( vti ; equivalent to stroke distance ) , heart rate , and lvo assessed using echocardiography and pc mri are shown in table 1 . significant disparities are seen in the echocardiographic and pc mri measurements of vessel diameter and vti because the two techniques quantify flow at slightly different sites in the left ventricular outflow tract . however , despite the disparities in the assessment of diameter and vti , echocardiographic assessment of flow volume ( lvo ) showed a strong correlation with that by pc mri ( r = 0.91 , r = 0.83 ; figure 4 ) . bland - altman analysis demonstrated a small mean bias between pc mri and echocardiography of 9.6 ml / kg / min , with loa of 79.2 to + 60.0 ml / kg / min , corresponding to an ri of 28.2% ( figure 4 ) . the means and standard deviations of svc diameter ( measured sagittally using echocardiography and axially using pc mri ) , svc vti ( stroke distance ) , heart rate , and svc flow volume assessed using echocardiography and pc mri are shown in table 2 . again , significant disparities were seen in the echocardiographic and pc mri measurements of vessel diameter and vti . however , echocardiographic assessment of volume of svc flow showed a poor correlation with that by pc mri ( r = 0.47 , r = 0.22 ; figure 5 ) . bland - altman analysis demonstrated a mean bias between pc mri and echocardiography of 13.7 ml / kg / min ( loa , 89.1 to + 61.7 ml / kg / min ; ri , 68.0% ; figure 5 ) . echocardiographic assessments of svc dimensions ( from a sagittal diameter measurement ) systematically underestimated true svc area : the mean pc mri estimate of area was 18.9 mm , the mean echocardiographic estimate of area 9.97 mm , and the mean bias 8.9 mm ( loa , 19.8 to + 2.6 mm ; ri , 77.4% ) . the mean pc mri estimate of stroke distance was 7.28 cm , the mean echocardiographic estimate 14.5 cm , and the mean bias 7.2 cm ( loa , 2.0 to + 12.4 cm ; ri , 48.0% ) . echocardiographic assessments of svc area directly from the axial view ( mean , 16.7 mm ) more closely reflected svc area as assessed by pc mri ( mean , 18.4 mm ) . calculating svc flow volume from axial area measurement and applying a 50% reduction to stroke distance to compensate for systematic overestimation of vti by echocardiography gave a slightly improved correlation with pc mri ( r = 0.54 , r = 0.29 ) . bland - altman analysis demonstrated a mean bias between pc mri and echocardiography of 2.6 ml / kg / min ( loa , 53.4 to + 58.6 ml / kg / min ; ri , 54.5% ) . infants ' heart rates tended to be slightly higher during echocardiography than pc mri ( 147 vs 141 beats / min , p = .054 ) and showed significant variability ( r = 0.69 ; loa , 34.7 to + 22.5 beats / min ; ri , 19.9% ) , potentially reflecting differing levels of arousal . scan - rescan repeatability of echocardiographic quantification of volume of svc flow was assessed in an additional cohort of 20 infants with a median corrected gestational age of 31.84 weeks ( range , 26.7136.57 weeks ) and a median weight at scan of 1,518 g ( range , 6002,800 g ) . the mean vti was 11.8 3.6 cm , with mean bias on repeat scanning of 0.19 cm ( loa , 3.9 to + 4.3 cm ; ri , 34% ) . the mean svc diameter measured sagittally was 3.9 1.2 mm , with mean bias on repeat scanning of 0.01 mm ( loa , 0.49 to + 0.47 mm ; ri , 12% ) . squaring of diameter to estimate area from sagittal svc measurements showed mean bias of 0.0 mm ( loa , 4.0 to + 4.0 mm ; ri , 29% ) . the mean svc area measured axially was 16.1 6.7 mm , with mean bias on repeat scanning of 0.3 mm ( loa , 2.7 to + 2.0 mm ; ri , 14% ) . the means and standard deviations of aortic valve diameter , left ventricular outflow velocity - time integral ( vti ; equivalent to stroke distance ) , heart rate , and lvo assessed using echocardiography and pc mri are shown in table 1 . significant disparities are seen in the echocardiographic and pc mri measurements of vessel diameter and vti because the two techniques quantify flow at slightly different sites in the left ventricular outflow tract . however , despite the disparities in the assessment of diameter and vti , echocardiographic assessment of flow volume ( lvo ) showed a strong correlation with that by pc mri ( r = 0.91 , r = 0.83 ; figure 4 ) . bland - altman analysis demonstrated a small mean bias between pc mri and echocardiography of 9.6 ml / kg / min , with loa of 79.2 to + 60.0 ml / kg / min , corresponding to an ri of 28.2% ( figure 4 ) . the means and standard deviations of svc diameter ( measured sagittally using echocardiography and axially using pc mri ) , svc vti ( stroke distance ) , heart rate , and svc flow volume assessed using echocardiography and pc mri are shown in table 2 . again , significant disparities were seen in the echocardiographic and pc mri measurements of vessel diameter and vti . however , echocardiographic assessment of volume of svc flow showed a poor correlation with that by pc mri ( r = 0.47 , r = 0.22 ; figure 5 ) . bland - altman analysis demonstrated a mean bias between pc mri and echocardiography of 13.7 ml / kg / min ( loa , 89.1 to + 61.7 ml / kg / min ; ri , 68.0% ; figure 5 ) . echocardiographic assessments of svc dimensions ( from a sagittal diameter measurement ) systematically underestimated true svc area : the mean pc mri estimate of area was 18.9 mm , the mean echocardiographic estimate of area 9.97 mm , and the mean bias 8.9 mm ( loa , 19.8 to + 2.6 mm ; ri , 77.4% ) . the mean pc mri estimate of stroke distance was 7.28 cm , the mean echocardiographic estimate 14.5 cm , and the mean bias 7.2 cm ( loa , 2.0 to + 12.4 cm ; ri , 48.0% ) . echocardiographic assessments of svc area directly from the axial view ( mean , 16.7 mm ) more closely reflected svc area as assessed by pc mri ( mean , 18.4 mm ) . calculating svc flow volume from axial area measurement and applying a 50% reduction to stroke distance to compensate for systematic overestimation of vti by echocardiography gave a slightly improved correlation with pc mri ( r = 0.54 , r = 0.29 ) . bland - altman analysis demonstrated a mean bias between pc mri and echocardiography of 2.6 ml / kg / min ( loa , 53.4 to + 58.6 ml / kg / min ; ri , 54.5% ) . infants ' heart rates tended to be slightly higher during echocardiography than pc mri ( 147 vs 141 beats / min , p = .054 ) and showed significant variability ( r = 0.69 ; loa , 34.7 to + 22.5 beats / min ; ri , 19.9% ) , potentially reflecting differing levels of arousal . scan - rescan repeatability of echocardiographic quantification of volume of svc flow was assessed in an additional cohort of 20 infants with a median corrected gestational age of 31.84 weeks ( range , 26.7136.57 weeks ) and a median weight at scan of 1,518 g ( range , 6002,800 g ) . the mean vti was 11.8 3.6 cm , with mean bias on repeat scanning of 0.19 cm ( loa , 3.9 to + 4.3 cm ; ri , 34% ) . the mean svc diameter measured sagittally was 3.9 1.2 mm , with mean bias on repeat scanning of 0.01 mm ( loa , 0.49 to + 0.47 mm ; ri , 12% ) . squaring of diameter to estimate area from sagittal svc measurements showed mean bias of 0.0 mm ( loa , 4.0 to + 4.0 mm ; ri , 29% ) . the mean svc area measured axially was 16.1 6.7 mm , with mean bias on repeat scanning of 0.3 mm ( loa , 2.7 to + 2.0 mm ; ri , 14% ) . improvements in circulatory care in preterm newborns are urgently required , but progress is limited by inaccuracy in regularly used circulatory biomarkers . there is increasing acceptance that arterial blood pressure is a poor surrogate of circulatory health during the circulatory transition of preterm infants . arterial blood pressure shows little if any association with the volume of systemic blood flow and has an uncertain relationship with the volume of cerebral blood flow , and trials of randomized interventions on the basis of blood pressure thresholds have shown no apparent benefit on long - term outcomes . echocardiographic techniques in newborns can be performed in real time at the bedside , by a range of appropriately trained personnel , and if performed carefully need not significantly disturb infants ' cardiorespiratory status . although there is significant variation in the use of targeted neonatal echocardiography at different centers , there is an increasing appreciation that the techniques may be able to advance care . we have presented the first pc mri validation of multiple echocardiographic measures of blood flow volume in preterm infants . although pc mri is not a flawless gold standard , it produces accurate measures of flow volume that can be validated ex vivo and that show repeatability in the neonatal population far superior to that seen with echocardiography . lvo and svc flow volume as assessed by pc mri had scan - rescan ris ( equivalent to the 95% confidence interval ) of 11.5% and 12.8% , respectively . in the absence of any true gold standard , and acknowledging that all other gold standards such as the fick method and thermodilution , also have intrinsic variability , we feel that pc mri is the best comparator currently available for the validation of echocardiographic findings in this population . our findings broadly support the use of echocardiographic assessments of lvo , which appear to be performed with a relatively high degree of accuracy . although there was a significant disparity between echocardiography and pc mri measures of aortic diameter and vti , it should be noted that echocardiography quantifies flow at a single point at the aortic valve annulus , the narrowest part of the outflow tract , whereas pc mri quantifies flow distal to this point at which the aortic sinuses increase vessel diameter ( figure 1 ) and for which stroke distance averaged over the entire vessel will necessarily be lower . when absolute volume of lvo was calculated from the respective echocardiographic and pc mri parameters the statistical correlation between echocardiographic and pc mri measures of lvo might be somewhat enhanced by the wide range of lvo values seen in the newborn population , in which a patent ductus arteriosus frequently leads to supraphysiologic levels of lvo . assessment of repeatability by the method suggested by bland and altman is not affected by the range of values within a population and provides a more robust assessment of a test 's performance . echocardiographic assessment of lvo in the neonatal population showed a tendency to underestimate flow ( mean bias , 9.6 ml / kg / min ) , but this was a disparity of < 4% of the flow volume . although loa of about 70 ml / kg / min ( approximately 28% of flow ) are higher than desirable , it should be appreciated that echocardiography and mri were performed up to 33 hours apart , and a degree of variability may be due to true biologic fluctuation rather than inconsistencies in measurement . to highlight this , we note that the loa for heart rate , a variable presumably measured with complete accuracy , were 29 beats / min ( an ri of 19.9% ) in this cohort . the small size of infants in this cohort ( mean weight at scan , 1,890 g ) will also mean that minor alterations in transducer position are likely to produce significant errors . however the validation against pc mri for lvo seen in this cohort ( r = 0.91 ) actually compares well with pc mri validation of two - dimensional doppler assessment of left ventricular stroke volume in adults ( r = 0.80 ) , three - dimensional doppler assessment of mitral valve flow in children ( r = 0.92 ) , and even three - dimensional doppler validation of left ventricular stroke volume in anaesthetized animals ( r = 0.91 ) . echocardiographic assessments of svc flow volume appear to be less robust and consequently should be used with caution in the clinical environment . echocardiography systematically underestimated the area of the superior vena cava , with area measures being about half those demonstrated by pc mri . because the true cross - sectional area of the superior vena cava would not be expected to change significantly in the area visualized , we feel that errors in svc dimension quantification are partly related to the asymmetric nature of the vessel . for much of its course , the superior vena cava is closely adherent to the ascending aorta , and it molds to the shape of this higher pressure vessel . cross - sectional images of the vessel visualized by echocardiography and pc mri are shown in figures 2b and 3b . imaging in a sagittal plane and using an imaging window as close as possible to the midline as originally described will lead to estimation of svc diameter toward the narrower tail end of its often crescent - shaped cross - section ( figure 3b ) . although it is possible that others may be able to perform the image acquisitions from the sagittal view with improved repeatability compared with our group , it should be noted that there is significant variation in normative values produced from different centers and that even assessment of svc flow volume from a single prerecorded image acquisition has been shown to have significant variability . echocardiography also systematically overestimated svc stroke distance , with stroke distance measures being about double those demonstrated by pc mri . this can best be demonstrated in the pulsed - wave doppler images , in which rather than seeing an open envelope of flow ( as seen in the aorta ; figure 1b ) , there is a filled envelope of flow ( figure 2c ) in the superior vena cava , showing that not all flow in the vessel is accelerating to near the maximal velocity . because stroke distance is calculated from the area under the velocity - time trace , all flow will be assumed to be at the maximal velocity . by combining measures of dimension ( which is underestimated by echocardiography ) and vti ( which is overestimated by echocardiography ) to produce estimates of svc flow , the resultant flow measures are in a biologically plausible range but with a high degree of variability . direct assessment of svc area from the axial view was more accurate and showed improved scan - rescan repeatability compared with measures of svc area produced by squaring diameter measures taken from the sagittal view ( ri , 14% vs 29% ) . however , even using the improved axial measurement of svc flow and a corrected measure of stroke distance , the 95% confidence limits for the echocardiographic estimation of svc flow volume were still 54.5% of the pc mri measure . this may be related to errors in the assessment of vti from the subcostal view ( scan - rescan ri , 34% ) . although echocardiographic assessment of lvo appears to be relatively robust , it is of limited clinical value in the neonatal unit setting , in which the majority of sick preterm infants will have patent ductus arteriosus , meaning that lvo does not represent systemic blood flow and is in fact a better marker of pulmonary flow volume . although the volume of right ventricular output may be considered a marker of systemic blood flow , it also includes the volume of intra - atrial shunting , which may be as much as 50% of lvo in a premature newborn . assessment of svc flow is predictive of adverse outcomes in populations of preterm infants and has significant potential to be used as outcome measures in clinical trials , but in our opinion , its limited accuracy gives it limited clinical utility in identifying circulatory failure in individual infants . however , estimating the vessel area directly from the axial view offers some improvement in performance , and there is scope for the technique to be further improved in the future , such as by estimation of vti from the suprasternal instead of the subcostal view .

backgroundthe echocardiographic assessment of circulatory function in sick newborn infants has the potential to improve patient care . however , measurements are prone to error and have not been sufficiently validated . phase - contrast magnetic resonance imaging ( mri ) provides highly validated measures of blood flow and has recently been applied to the newborn population . the aim of this study was to validate measures of left ventricular output and superior vena caval flow volume in newborn infants.methodsechocardiographic and mri assessments were performed within 1 working day of each other in a cohort of newborn infants.resultsexaminations were performed in 49 infants with a median corrected gestational age at scan of 34.43 weeks ( range , 27.4340 weeks ) and a median weight at scan of 1,880 g ( range , 6603,760 g ) . echocardiographic assessment of left ventricular output showed a strong correlation with mri assessment ( r2 = 0.83 ; mean bias , 9.6 ml / kg / min ; limits of agreement , 79.6 to + 60.0 ml / kg / min ; repeatability index , 28.2% ) . echocardiographic assessment of superior vena caval flow showed a poor correlation with mri assessment ( r2 = 0.22 ; mean bias , 13.7 ml / kg / min ; limits of agreement , 89.1 to + 61.7 ml / kg / min ; repeatability index , 68.0% ) . calculating superior vena caval flow volume from an axial area measurement and applying a 50% reduction to stroke distance to compensate for overestimation gave a slightly improved correlation with mri ( r2 = 0.29 ; mean bias , 2.6 ml / kg / min ; limits of agreement , 53.4 to + 58.6 ml / kg / min ; repeatability index , 54.5%).conclusionsechocardiographic assessment of left ventricular output appears relatively robust in newborn infant . echocardiographic assessment of superior vena caval flow is of limited accuracy in this population , casting doubt on the utility of the measurement for diagnostic decision making .

Methods Echocardiographic Measures LVO SVC Flow Volume PC MRI Acquisition Statistical Analysis Results Echocardiographic Assessment of LVO Assessment of SVC Flow with Diameter Measured in the Sagittal Plane Assessment of SVC Area from an Axial View Variability in Heart Rate Scan-Rescan Repeatability of Quantification of SVC Flow by Echocardiography Discussion Conclusions

before the study of this cohort all examinations were either performed by or directly supervised by a neonatologist with > 10 years ' experience in functional echocardiography ( a.m.g . ) in all cases , an additional cohort of infants was subsequently examined by echocardiography to investigate the repeatability of quantification of svc flow volume . high - definition zoom was used to focus on the superior vena cava as it begins to open up into the right atrium ( figure 2a , dashed line ) , with maximum and minimum diameters through the cardiac cycle taken from b - mode images . because of concerns about the irregular shape of the superior vena cava , we also measured vessel area directly from an axial view , again using high - definition zoom and tracing maximum and minimum cross - sectional area from the b - mode images ( figure 2b , dashed line ) . pilot scans were acquired to view the vessels of interest to ascertain the straightest section of the vessel adequate for the slice thickness of the pc mri sequences and to position the imaging plane perpendicular to the centerline of the vessel to minimize partial volume effects . lvo was quantified immediately distal to the level of the aortic valve ( figure 3a ) . volume of flow in the superior vena cava was quantified at the level of the pulmonary trunk as the superior vena cava begins to open into the right atrium ( figure 3b ) . sequence analysis and flow volume quantification for pc data sets were performed using a commercial workstation ( viewforum ; philips medical systems ) . flow is then calculated at each time point of the cardiac cycle , generating a flow curve and volume of flow value for each vessel of interest . measures of flow obtained by echocardiography and pc mri were compared using simple linear regression and also as described by bland and altman : the mean bias and limits of agreement ( loa , or repeatability coefficient ; 1.96 sd of differences ) were calculated . before the study of this cohort all examinations were either performed by or directly supervised by a neonatologist with > 10 years ' experience in functional echocardiography ( a.m.g . ) in all cases , an additional cohort of infants was subsequently examined by echocardiography to investigate the repeatability of quantification of svc flow volume . high - definition zoom was used to focus on the superior vena cava as it begins to open up into the right atrium ( figure 2a , dashed line ) , with maximum and minimum diameters through the cardiac cycle taken from b - mode images . because of concerns about the irregular shape of the superior vena cava , we also measured vessel area directly from an axial view , again using high - definition zoom and tracing maximum and minimum cross - sectional area from the b - mode images ( figure 2b , dashed line ) . pilot scans were acquired to view the vessels of interest to ascertain the straightest section of the vessel adequate for the slice thickness of the pc mri sequences and to position the imaging plane perpendicular to the centerline of the vessel to minimize partial volume effects . lvo was quantified immediately distal to the level of the aortic valve ( figure 3a ) . sequence analysis and flow volume quantification for pc data sets were performed using a commercial workstation ( viewforum ; philips medical systems ) . flow is then calculated at each time point of the cardiac cycle , generating a flow curve and volume of flow value for each vessel of interest . measures of flow obtained by echocardiography and pc mri were compared using simple linear regression and also as described by bland and altman : the mean bias and limits of agreement ( loa , or repeatability coefficient ; 1.96 sd of differences ) were calculated . paired pc mri and echocardiographic examinations were performed in 49 infants with a median gestational age of 32.57 weeks ( range , 24.4339.14 ) weeks and a median weight at birth of 1,750 g ( range , 5253,760 g ) . the median postnatal age at scan was 11 days ( range , 184 days ) , with a median corrected gestational age of 34.43 weeks ( range , 27.4340 weeks ) and a median weight at scan of 1,880 g ( range , 6603,760 g ) . the median interval between pc mri and echocardiographic scanning was 3.67 hours ( range , 0.2533.0 hours ) , with pc mri being performed before echocardiography in 46 of 49 infants . the means and standard deviations of aortic valve diameter , left ventricular outflow velocity - time integral ( vti ; equivalent to stroke distance ) , heart rate , and lvo assessed using echocardiography and pc mri are shown in table 1 . however , despite the disparities in the assessment of diameter and vti , echocardiographic assessment of flow volume ( lvo ) showed a strong correlation with that by pc mri ( r = 0.91 , r = 0.83 ; figure 4 ) . bland - altman analysis demonstrated a small mean bias between pc mri and echocardiography of 9.6 ml / kg / min , with loa of 79.2 to + 60.0 ml / kg / min , corresponding to an ri of 28.2% ( figure 4 ) . the means and standard deviations of svc diameter ( measured sagittally using echocardiography and axially using pc mri ) , svc vti ( stroke distance ) , heart rate , and svc flow volume assessed using echocardiography and pc mri are shown in table 2 . however , echocardiographic assessment of volume of svc flow showed a poor correlation with that by pc mri ( r = 0.47 , r = 0.22 ; figure 5 ) . bland - altman analysis demonstrated a mean bias between pc mri and echocardiography of 13.7 ml / kg / min ( loa , 89.1 to + 61.7 ml / kg / min ; ri , 68.0% ; figure 5 ) . echocardiographic assessments of svc dimensions ( from a sagittal diameter measurement ) systematically underestimated true svc area : the mean pc mri estimate of area was 18.9 mm , the mean echocardiographic estimate of area 9.97 mm , and the mean bias 8.9 mm ( loa , 19.8 to + 2.6 mm ; ri , 77.4% ) . the mean pc mri estimate of stroke distance was 7.28 cm , the mean echocardiographic estimate 14.5 cm , and the mean bias 7.2 cm ( loa , 2.0 to + 12.4 cm ; ri , 48.0% ) . calculating svc flow volume from axial area measurement and applying a 50% reduction to stroke distance to compensate for systematic overestimation of vti by echocardiography gave a slightly improved correlation with pc mri ( r = 0.54 , r = 0.29 ) . bland - altman analysis demonstrated a mean bias between pc mri and echocardiography of 2.6 ml / kg / min ( loa , 53.4 to + 58.6 ml / kg / min ; ri , 54.5% ) . infants ' heart rates tended to be slightly higher during echocardiography than pc mri ( 147 vs 141 beats / min , p = .054 ) and showed significant variability ( r = 0.69 ; loa , 34.7 to + 22.5 beats / min ; ri , 19.9% ) , potentially reflecting differing levels of arousal . scan - rescan repeatability of echocardiographic quantification of volume of svc flow was assessed in an additional cohort of 20 infants with a median corrected gestational age of 31.84 weeks ( range , 26.7136.57 weeks ) and a median weight at scan of 1,518 g ( range , 6002,800 g ) . the mean vti was 11.8 3.6 cm , with mean bias on repeat scanning of 0.19 cm ( loa , 3.9 to + 4.3 cm ; ri , 34% ) . the mean svc diameter measured sagittally was 3.9 1.2 mm , with mean bias on repeat scanning of 0.01 mm ( loa , 0.49 to + 0.47 mm ; ri , 12% ) . squaring of diameter to estimate area from sagittal svc measurements showed mean bias of 0.0 mm ( loa , 4.0 to + 4.0 mm ; ri , 29% ) . the mean svc area measured axially was 16.1 6.7 mm , with mean bias on repeat scanning of 0.3 mm ( loa , 2.7 to + 2.0 mm ; ri , 14% ) . the means and standard deviations of aortic valve diameter , left ventricular outflow velocity - time integral ( vti ; equivalent to stroke distance ) , heart rate , and lvo assessed using echocardiography and pc mri are shown in table 1 . however , despite the disparities in the assessment of diameter and vti , echocardiographic assessment of flow volume ( lvo ) showed a strong correlation with that by pc mri ( r = 0.91 , r = 0.83 ; figure 4 ) . bland - altman analysis demonstrated a small mean bias between pc mri and echocardiography of 9.6 ml / kg / min , with loa of 79.2 to + 60.0 ml / kg / min , corresponding to an ri of 28.2% ( figure 4 ) . the means and standard deviations of svc diameter ( measured sagittally using echocardiography and axially using pc mri ) , svc vti ( stroke distance ) , heart rate , and svc flow volume assessed using echocardiography and pc mri are shown in table 2 . however , echocardiographic assessment of volume of svc flow showed a poor correlation with that by pc mri ( r = 0.47 , r = 0.22 ; figure 5 ) . bland - altman analysis demonstrated a mean bias between pc mri and echocardiography of 13.7 ml / kg / min ( loa , 89.1 to + 61.7 ml / kg / min ; ri , 68.0% ; figure 5 ) . echocardiographic assessments of svc dimensions ( from a sagittal diameter measurement ) systematically underestimated true svc area : the mean pc mri estimate of area was 18.9 mm , the mean echocardiographic estimate of area 9.97 mm , and the mean bias 8.9 mm ( loa , 19.8 to + 2.6 mm ; ri , 77.4% ) . the mean pc mri estimate of stroke distance was 7.28 cm , the mean echocardiographic estimate 14.5 cm , and the mean bias 7.2 cm ( loa , 2.0 to + 12.4 cm ; ri , 48.0% ) . calculating svc flow volume from axial area measurement and applying a 50% reduction to stroke distance to compensate for systematic overestimation of vti by echocardiography gave a slightly improved correlation with pc mri ( r = 0.54 , r = 0.29 ) . bland - altman analysis demonstrated a mean bias between pc mri and echocardiography of 2.6 ml / kg / min ( loa , 53.4 to + 58.6 ml / kg / min ; ri , 54.5% ) . infants ' heart rates tended to be slightly higher during echocardiography than pc mri ( 147 vs 141 beats / min , p = .054 ) and showed significant variability ( r = 0.69 ; loa , 34.7 to + 22.5 beats / min ; ri , 19.9% ) , potentially reflecting differing levels of arousal . scan - rescan repeatability of echocardiographic quantification of volume of svc flow was assessed in an additional cohort of 20 infants with a median corrected gestational age of 31.84 weeks ( range , 26.7136.57 weeks ) and a median weight at scan of 1,518 g ( range , 6002,800 g ) . the mean vti was 11.8 3.6 cm , with mean bias on repeat scanning of 0.19 cm ( loa , 3.9 to + 4.3 cm ; ri , 34% ) . the mean svc diameter measured sagittally was 3.9 1.2 mm , with mean bias on repeat scanning of 0.01 mm ( loa , 0.49 to + 0.47 mm ; ri , 12% ) . squaring of diameter to estimate area from sagittal svc measurements showed mean bias of 0.0 mm ( loa , 4.0 to + 4.0 mm ; ri , 29% ) . the mean svc area measured axially was 16.1 6.7 mm , with mean bias on repeat scanning of 0.3 mm ( loa , 2.7 to + 2.0 mm ; ri , 14% ) . arterial blood pressure shows little if any association with the volume of systemic blood flow and has an uncertain relationship with the volume of cerebral blood flow , and trials of randomized interventions on the basis of blood pressure thresholds have shown no apparent benefit on long - term outcomes . we have presented the first pc mri validation of multiple echocardiographic measures of blood flow volume in preterm infants . in the absence of any true gold standard , and acknowledging that all other gold standards such as the fick method and thermodilution , also have intrinsic variability , we feel that pc mri is the best comparator currently available for the validation of echocardiographic findings in this population . although there was a significant disparity between echocardiography and pc mri measures of aortic diameter and vti , it should be noted that echocardiography quantifies flow at a single point at the aortic valve annulus , the narrowest part of the outflow tract , whereas pc mri quantifies flow distal to this point at which the aortic sinuses increase vessel diameter ( figure 1 ) and for which stroke distance averaged over the entire vessel will necessarily be lower . when absolute volume of lvo was calculated from the respective echocardiographic and pc mri parameters the statistical correlation between echocardiographic and pc mri measures of lvo might be somewhat enhanced by the wide range of lvo values seen in the newborn population , in which a patent ductus arteriosus frequently leads to supraphysiologic levels of lvo . echocardiographic assessment of lvo in the neonatal population showed a tendency to underestimate flow ( mean bias , 9.6 ml / kg / min ) , but this was a disparity of < 4% of the flow volume . although loa of about 70 ml / kg / min ( approximately 28% of flow ) are higher than desirable , it should be appreciated that echocardiography and mri were performed up to 33 hours apart , and a degree of variability may be due to true biologic fluctuation rather than inconsistencies in measurement . to highlight this , we note that the loa for heart rate , a variable presumably measured with complete accuracy , were 29 beats / min ( an ri of 19.9% ) in this cohort . the small size of infants in this cohort ( mean weight at scan , 1,890 g ) will also mean that minor alterations in transducer position are likely to produce significant errors . however the validation against pc mri for lvo seen in this cohort ( r = 0.91 ) actually compares well with pc mri validation of two - dimensional doppler assessment of left ventricular stroke volume in adults ( r = 0.80 ) , three - dimensional doppler assessment of mitral valve flow in children ( r = 0.92 ) , and even three - dimensional doppler validation of left ventricular stroke volume in anaesthetized animals ( r = 0.91 ) . echocardiography systematically underestimated the area of the superior vena cava , with area measures being about half those demonstrated by pc mri . because the true cross - sectional area of the superior vena cava would not be expected to change significantly in the area visualized , we feel that errors in svc dimension quantification are partly related to the asymmetric nature of the vessel . for much of its course , the superior vena cava is closely adherent to the ascending aorta , and it molds to the shape of this higher pressure vessel . imaging in a sagittal plane and using an imaging window as close as possible to the midline as originally described will lead to estimation of svc diameter toward the narrower tail end of its often crescent - shaped cross - section ( figure 3b ) . although it is possible that others may be able to perform the image acquisitions from the sagittal view with improved repeatability compared with our group , it should be noted that there is significant variation in normative values produced from different centers and that even assessment of svc flow volume from a single prerecorded image acquisition has been shown to have significant variability . by combining measures of dimension ( which is underestimated by echocardiography ) and vti ( which is overestimated by echocardiography ) to produce estimates of svc flow , the resultant flow measures are in a biologically plausible range but with a high degree of variability . direct assessment of svc area from the axial view was more accurate and showed improved scan - rescan repeatability compared with measures of svc area produced by squaring diameter measures taken from the sagittal view ( ri , 14% vs 29% ) . however , even using the improved axial measurement of svc flow and a corrected measure of stroke distance , the 95% confidence limits for the echocardiographic estimation of svc flow volume were still 54.5% of the pc mri measure . although echocardiographic assessment of lvo appears to be relatively robust , it is of limited clinical value in the neonatal unit setting , in which the majority of sick preterm infants will have patent ductus arteriosus , meaning that lvo does not represent systemic blood flow and is in fact a better marker of pulmonary flow volume . although the volume of right ventricular output may be considered a marker of systemic blood flow , it also includes the volume of intra - atrial shunting , which may be as much as 50% of lvo in a premature newborn . assessment of svc flow is predictive of adverse outcomes in populations of preterm infants and has significant potential to be used as outcome measures in clinical trials , but in our opinion , its limited accuracy gives it limited clinical utility in identifying circulatory failure in individual infants . however , estimating the vessel area directly from the axial view offers some improvement in performance , and there is scope for the technique to be further improved in the future , such as by estimation of vti from the suprasternal instead of the subcostal view .

stool consistency , measured by the bristol stool scale ( bss ) , reflects differences in water content and activity in the colon ecosystem and is correlated with colon transit time . ( low scores : firm stool and slow transit , high scores : loose stool and fast transit ) . species richness is proposed to be a major marker for gut health because of the idea that high bacterial richness and diversity often reflect ecosystem stability and resilience together with the consistent association between disease and a reduction in the number of species in a faecal sample . faecal metagenomes have been shown to stratify into enterotypes , splitting the human population around three possible constellations in terms of their gut microbial ecosystem ; likewise firmicutes : bacteroidetes and prevotellaceae : bacteroidetes ratios have been put forward as important stratifiers for gut microbiomes . observed species richness declines with higher bss scores reaching its minimum in individuals with loose stool . enterotypes are distinctly distributed over the bss scores : the prevotella ( p ) enterotype is more abundant in subjects with loose stool while the ruminococcaceae - bacteroides ( rb ) enterotype completely dominates the harder stool samples . within the rb enterotype , methanobrevibacter and akkermansia a similar observation can be made for oxalobacter and butyricimonas , while bacteroides is more abundant in loose stool . microbiota growth potential is positively correlated with bss scores in the rb enterotype , hinting to transit time as a selective force on microbial life - strategies . here we show that major alterations in species richness or community composition could partially be reflecting variation in stool consistency , hampering the identification , but especially robustness and reproducibility of disease markers . this study therefore stresses the urgent necessity of these measurements in gut microbiota research and clinical studies . despite recent efforts undertaken to characterise the healthy colon microbiota,14 many parameters potentially affecting microbial composition and metabolic capacity remain underexplored . the identification of such confounding factors , including diet,5 6 history of antibiotics intake,7 and inflammation status,8 9 is essential to define the boundaries of a healthy gut ecosystem and , hence , to identify robust disease markers in clinical microbiome association studies . transit time is a key determinant of the gut microbial habitat , affecting nutrient and water absorption along the intestine as well as setting the pace of luminal microbial population clearance during egestion.10 as direct measurement of transit time is often impractical or mildly invasive , it has generally been neglected in microbiome studies so far . however , stool consistency as categorised by the bristol stool scale ( bss ) scores has been put forward as a proxy for colonic transit rate,1012 allowing assessment of its impact even when applying home - based sampling protocols . the use of the bss is widespread in clinical studies and has been advised for the assessment of constipation and diarrhoea in functional bowel disorders.1315 the bss classifies human faeces into seven consistency categories , with highest scores corresponding to loose stools and fast transit , while lower scores stand for hard stools and longer colon transit times.1012 16 each consistency category reflects differences in moisture content of faecal material , with decreased water activity associated with prolonged intestinal transit limiting microbial growth through reduced nutrient mobility and hampered enzymatic activity.17 hence , bss categorisation summarises the impact of two major and obviously related selective forces shaping the gut ecosystem : rate of intestinal transit and water activity . here , we used self - reported bss scores combined with 16s rdna illumina amplicon profiles of faecal samples of 53 healthy volunteers to assess potential associations between microbiota composition and stool consistency . in a healthy western population , more than half of the stools passed are predicted to belong to the central bss categories 3 and 4.11 however , increased occurrence of harder stools has been reported to be more common among women.11 12 18 hence , in order to increase chances of sampling a more uniform distribution of stools over all bss categories , we limited inclusion to female volunteers . fifty - three healthy women , aged 2055 years ( median 42.5 ) , were recruited as part of the flemish gut flora project . none were diagnosed with cancer or ibd , or had taken diarrhoea inhibitors , laxatives or prebiotics in the week before sampling , nor antibiotics within 3 months before sampling ( see online supplementary table s1 ) . the aims of the project and the commitments required were explained by means of an information brochure and all participants signed a statement of informed consent . the participants general practitioner recorded their medical history , together with height , weight , and waist and hip circumferences . volunteers recorded time of defaecation and bss and reported this information together with information about general diet and health status in a questionnaire . after frozen transport to a collection point in their neighbourhood and subsequent transport on dry ice , samples were stored at 80c within 72 h after delivery at the collection point until dna extraction . dna extraction was performed according to godon et al.19 to amplify the variable region 4 of the 16s rrna gene , we used the 515f and 806r primers ( gtgccagcmgccgcggtaa and ggactachvgggtwtctaat , respectively ) modified to contain illumina adapters and barcode sequences to allow for directional sequencing . amplifications were performed in triplicate as 25 l reactions containing 2 l of diluted template ( 1:10 ) , 2.5 l of 10x accuprime pcr buffer i , 0.1 l of accuprime taq high fidelity ( 5 u/l ) , and 0.5 l of 515f and 1.0 l of 806r primer ( 10 m of each primer ) . thermal cycling consisted of an initial denaturation step ( 3 min at 94c ) , followed by 30 cycles of denaturation ( 45 s at 94c ) , annealing ( 60 s at 50c ) and 90 s extension at 72c . amplicons were quantified on the agilent 2100 bioanalyzer ( agilent technologies , santa clara , california , usa ) and pooled in equimolar concentrations . fragment size was selected ( 400 bp ) by cutting the main band from the agarose gel to reduce non - specific products of amplification . a final library size and quantification sequencing was performed on the illumina miseq platform ( miseq reagent kit v.2 , 500 cycles ) according to the manufacturer 's specifications to generate paired - end reads of 250 bases in length in each direction . the overlapping paired - end reads were merged using fastq - join20 and processed with macqiime v.1.8 . only illumina reads with a length > 250 bp and an average quality score above 30 , were retained for further analysis . reads were assigned to operational taxonomic units ( otus ) by de novo otu picking through the qiime pipeline . this way 99.6% , 87.6% , 53.4% and 12.5% of the reads were assigned to order , family , genus and species levels , respectively . closed otu picking with qiime against the greengenes 2013 database was performed in addition for a more stringent taxonomic assignment . from otu abundance and their respective taxonomic classifications , feature abundance matrices were calculated at different taxonomic levels , representing otu and taxa abundance per sample . to compare the different samples , sample counts were rarefied to 26 260 reads for the de novo otu picking data set and 26 024 reads for closed otu picking and trimmed for the consequently absent otus with the phyloseq package based on the minimum of the sum of taxa abundances in rv.3.0.1 . clustering of the samples into enterotypes was done using the cluster package21 according to instructions available on http://enterotyping.embl.de . optimal number of clusters was determined by the calinski - harabasz index . in addition , enterotyping was performed with dirichlet multinomial mixtures ( dmm ) using the dirichletmultinomial 1.6.0 package in r.22 laplace was used to determine the optimal number of clustering by penalising model complexity . clusters were assigned the enterotype ruminoccoccaceae - bacteroides or prevotella based on the taxa dominating the enterotype as in the original article of arumugam et al23 ( see online supplementary table s2 ) . species richness ( observed , chao1 ) and diversity measurements ( shannon ) were calculated using the phyloseq package.24 each sample s microbiota growth potential was estimated as the average of the maximum growth rates ( maxgrs ) of the genera , weighted by their abundance in the sample . the maxgrs of gut - reference species ( img v4ref ) were estimated from genomic traits25 and the median was assigned to the genus . correlations between species richness , microbiota growth potential , and relative bacterial taxa abundance with bss were assessed by spearman 's rank order correlation , as implemented in r. p values were corrected for multiple testing with benjamini - hochberg false discovery rate correction ( q value ) . spearman 's rank order correlation was used to check the correlation between enterotype and transit time ( bss ) , based on the percentage of each enterotype in each bss category . significance of differences in microbiota growth potential between enterotypes was assessed by wilcoxon signed rank test . fifty - three healthy women , aged 2055 years ( median 42.5 ) , were recruited as part of the flemish gut flora project . none were diagnosed with cancer or ibd , or had taken diarrhoea inhibitors , laxatives or prebiotics in the week before sampling , nor antibiotics within 3 months before sampling ( see online supplementary table s1 ) . the aims of the project and the commitments required were explained by means of an information brochure and all participants signed a statement of informed consent . the participants general practitioner recorded their medical history , together with height , weight , and waist and hip circumferences . volunteers recorded time of defaecation and bss and reported this information together with information about general diet and health status in a questionnaire . faecal samples were frozen at 20c immediately after collection by the participants . after frozen transport to a collection point in their neighbourhood and subsequent transport on dry ice , samples were stored at 80c within 72 h after delivery at the collection point until dna extraction . dna extraction was performed according to godon et al.19 to amplify the variable region 4 of the 16s rrna gene , we used the 515f and 806r primers ( gtgccagcmgccgcggtaa and ggactachvgggtwtctaat , respectively ) modified to contain illumina adapters and barcode sequences to allow for directional sequencing . amplifications were performed in triplicate as 25 l reactions containing 2 l of diluted template ( 1:10 ) , 2.5 l of 10x accuprime pcr buffer i , 0.1 l of accuprime taq high fidelity ( 5 u/l ) , and 0.5 l of 515f and 1.0 l of 806r primer ( 10 m of each primer ) . thermal cycling consisted of an initial denaturation step ( 3 min at 94c ) , followed by 30 cycles of denaturation ( 45 s at 94c ) , annealing ( 60 s at 50c ) and 90 s extension at 72c . amplicons were quantified on the agilent 2100 bioanalyzer ( agilent technologies , santa clara , california , usa ) and pooled in equimolar concentrations . fragment size was selected ( 400 bp ) by cutting the main band from the agarose gel to reduce non - specific products of amplification . a final library size and quantification sequencing was performed on the illumina miseq platform ( miseq reagent kit v.2 , 500 cycles ) according to the manufacturer 's specifications to generate paired - end reads of 250 bases in length in each direction . the overlapping paired - end reads were merged using fastq - join20 and processed with macqiime v.1.8 . only illumina reads with a length > 250 bp and an average quality score above 30 , were retained for further analysis . reads were assigned to operational taxonomic units ( otus ) by de novo otu picking through the qiime pipeline . this way 99.6% , 87.6% , 53.4% and 12.5% of the reads closed otu picking with qiime against the greengenes 2013 database was performed in addition for a more stringent taxonomic assignment . from otu abundance and their respective taxonomic classifications , feature abundance matrices were calculated at different taxonomic levels , representing otu and taxa abundance per sample . to compare the different samples , sample counts were rarefied to 26 260 reads for the de novo otu picking data set and 26 024 reads for closed otu picking and trimmed for the consequently absent otus with the phyloseq package based on the minimum of the sum of taxa abundances in rv.3.0.1 . clustering of the samples into enterotypes was done using the cluster package21 according to instructions available on http://enterotyping.embl.de . in addition , enterotyping was performed with dirichlet multinomial mixtures ( dmm ) using the dirichletmultinomial 1.6.0 package in r.22 laplace was used to determine the optimal number of clustering by penalising model complexity . clusters were assigned the enterotype ruminoccoccaceae - bacteroides or prevotella based on the taxa dominating the enterotype as in the original article of arumugam et al23 ( see online supplementary table s2 ) . species richness ( observed , chao1 ) and diversity measurements ( shannon ) were calculated using the phyloseq package.24 each sample s microbiota growth potential was estimated as the average of the maximum growth rates ( maxgrs ) of the genera , weighted by their abundance in the sample . the maxgrs of gut - reference species ( img v4ref ) were estimated from genomic traits25 and the median was assigned to the genus . correlations between species richness , microbiota growth potential , and relative bacterial taxa abundance with bss were assessed by spearman 's rank order correlation , as implemented in r. p values were corrected for multiple testing with benjamini - hochberg false discovery rate correction ( q value ) . spearman 's rank order correlation was used to check the correlation between enterotype and transit time ( bss ) , based on the percentage of each enterotype in each bss category . significance of differences in microbiota growth potential between enterotypes was assessed by wilcoxon signed rank test . a healthy gut microbial ecosystem is generally thought to be characterised by high bacterial richness and diversity , presumed to reflect ecosystem stability and resilience.26 27 faecal microbiome analyses have revealed a seemingly consistent association between disease and a reduction in the observed or estimated number of species in a sample , suggesting bacterial richness to be a major marker for gut health.26 27 here , we identify stool consistency to be strongly associated to faecal microbial richness . indeed , observed species richness significantly declines with stool firmness ( spearman 's r=0.45 , p=0.0007 ) , reaching its minimum in diarrhoea - afflicted individuals ( figure 1a ) . estimation of total species richness ( chao1 ) confirms this negative correlation ( r=0.41 , p=0.003 , data not shown ) and the signal remains significant with the application of a more stringent taxonomical assignment ( closed reference otu picking , see methods ; trend also confirmed in a data set excluding patients with ibs , online supplementary figure s7 , and in a data set of 24 men , online supplementary figure s8 ) . these results are in line with anecdotal reports of decreased microbial richness associated with osmotic diarrhoea.28 stool consistency variation drives species richness and human enterotypes . correlation between ( a ) observed species richness and stool consistency , defined by bristol stool scale ( bss ) ( spearman 's correlation , r=0.45 , p=0.0007 ) and ( b ) enterotype distribution and stool consistency ( bss ) ; blue : ruminococcaceae - bacteroides ( rb ) enterotype ( r=0.88 , p=0.019 ) , green : p enterotype ( r=0.88 , p=0.019 ) . previously , faecal metagenomes have been shown to stratify into enterotypes,23 splitting the human population around three possible constellations in terms of their gut microbial community structure . as enterotypes have also been linked to richness gradients,8 9 we analysed the distribution of enterotypes over stool consistency scores . applying multiple clustering approaches , we find that our current data set optimally separates into two distinct clusters ( see online supplementary table s2 ) . named after the dominating taxa as in the reference publication,23 we refer to the clusters observed as the ruminococcaceae - bacteroides ( rb ) and the prevotella ( p ) enterotype ( 77% and 23% of total samples , respectively ; partitioning around medoids ( pam ) clustering with jensen - shannon distance ) . the enterotypes observed are distinctly distributed over bss scores : while the p enterotype is more abundant in individuals with loose stools ( r : 0.88 , p=0.019 ) , the rb enterotype completely dominates firmer samples ( r : 0.88 , p=0.019 ; figure 1b ; trend also confirmed in men , online supplementary figure s8 ) . these observations are substantiated by the analysis of the firmicutes : bacteroidetes and prevotellaceae : bacteroidetes abundance ratios ( both put forward as alternatives to enterotyping29 ) over stool score ( respectively r=0.42 , p=0.001 and r=0.04 , p=0.77 ; see online supplementary figure s1a , b ) . of note , within the rb enterotype , ruminococcaceae abundance positively correlates with bss scores ( r=0.37 , p=0.016 ) , while bacteroides populations increase in looser stools ( r=0.43 , p=0.004 ) ( see online supplementary figure s2 ) . hence , the enhanced prevalence of the rb enterotype in the harder stool categories is driven by the samples with a high ruminococcaceae : bacteroides ratio , which would classify them as ruminococcaceae enterotype according to the terminology originally suggested by arumugam et al.23 as even within a single enterotype low richness samples are more abundant in looser stool ( see online supplementary figure s3 ) , the richness signal is thus not a mere consequence of enterotype distribution across consistency scores . although the identification of environmental factors shaping or contributing to enterotype stratification is still ongoing , the potentially diverging impact of diet has been emphasised previously.5 30 more specifically , the occurrence of the p enterotype has been linked to a rural , fibre - rich diet.5 31 as non - fermentable fibre consumption is thought to increase water content and plasticity of stool,32 this observation fits with the increased prevalence of the p enterotype in the loose stool categories . however , the question whether dietary fibre consumption or the resulting changes in transit rate or water activity are driving prevotella blooms , remains currently unanswered . stool consistency variation is associated with shifts in faecal microbiota composition at the level of community structure and diversity , and it correlates with abundance gradients of individual genera ( see online supplementary table s3 ) . within the rb enterotype , methanobrevibacter and akkermansia populations increase with stool firmness ( and are thus more prevalent in slow transit individuals ) . a similar observation can be made for oxalobacter and butyricimonas , while bacteroides is more abundant in loose stools ( see online supplementary figure s4 ) . most of these associations remain significant in the total data set ( see table 1 and online supplementary figure s5 ) or when applying a more stringent taxonomic assignment ( see online supplementary table s4 ) . genera abundances significantly correlated with stool consistency genera abundances significantly correlated with stool consistency ( bss ) ( q<0.1 ) in the total data set , the rb enterotype , or the p enterotype . bss , bristol stool scale ; na , not assigned ; rb , ruminococcaceae - bacteroides . the increased abundance of methanogens such as methanobrevibacter in harder stools confirms previous reports of elevated methane production in constipated individuals.33 it has been suggested that methane plays an active role in the delay of transit by slowing down intestinal motility.34 moreover , hydrogen removal through methane production alters the whole of gut fermentation processes , which could potentially affect colon peristalsis.33 alternatively , increased abundance could reflect the fitness of a genus to grow in conditions of slowed - down intestinal transit . as firmer stool consistencies correspond with reduced ecosystem water activity , associated fluctuations in microbial abundances could also result from species - specific resistance to water stress . the human colon ecosystem is an open , nutrient - rich and continuous - flow environment . in order to avoid washout , the residing bacteria can either reproduce at a sufficiently high growth rate or attach to or colonise host tissues.35 hence , transit time may act as a selective force on gut bacterial growth rates . indeed , higher fluctuations in nutrient availability and microbiota population size induced by decreased colon transit time would be the text - book selection pressure for fast growing bacteria , outgrowing their slow growing counterparts whenever resources are available ( r - selection).36 also enhanced water activity and the associated increase of nutrient mobility in loose stools will contribute to the selective force imposed by accelerated transit . indications for the suggested correlation between bacterial growth rates and rates of passage come from in vitro work with continuous flow fermenters3739where dilution rates determine pace of growth of the bacteria cultured and in vivo observations of higher faecal bacterial biomass associated with shorter transit times.40 41 to assess this hypothesis , we estimated each sample s microbiota growth potential as the average of the maxgr25 ( see methods ) of the genera , weighted by their abundance in the sample ( based on de novo otu as well as closed reference otu assignment ; online supplementary table s5 ) . we find that microbiota growth potential is indeed positively correlated with stool score , and hence colon transit time , in the rb enterotype ( r=0.34 , p=0,028 ; figure 2 ) . strikingly , the average growth rate of the p enterotype samples is lower than the one of the rb samples ( median microbiota growth potential 0.38/h vs 0.52/h , respectively , wilcoxon p<10 ; online supplementary figure s6 ) . this is largely driven by the higher abundance of relatively slow - growing prevotella spp in these samples ( median relative abundance of prevotella of 0.48671 vs 0.00026 in the p - enterotype and rb - enterotype , respectively ) . on one hand , the absence of correlation between growth rate and colon transit time in the p enterotype could suggest that species in this enterotype resort to an alternative strategy to avoid washout , namely a higher degree of adherence to host tissues . the fact that prevotella is indeed able to bind collagen and degrade mucin oligosaccharides support this hypothesis.42 43 alternatively , our results could indicate that stool consistency and hence water activity in prevotella individuals are independent of accelerated transit , and mainly reflect increased faecal water - binding capacity , for example , related to fibre consumption . overall , as faeces and library dilution series have been shown not to affect compositional readouts in 16s amplicon analyses44and the results obtained are thus not due to technical issues arising from the variation in water content our results hint to transit time as one of the determining selective forces on microbial life - strategies . microbiota growth potential correlates to faster intestinal transit in the ruminococcaceae - bacteroides ( rb ) enterotype . microbiota growth potential over stool consistency ( bristol stool scale ( bss ) ) , which is proposed as a proxy for transit time , in the rb - enterotype ( r=0.34 , p=0.028 ) . the results of the present study indicate that major alterations in species richness or community composition could partially be reflecting variation in stool consistency , hampering the identification , and affecting robustness and reproducibility of disease markers . while constipation and or diarrhoea are often seen as indicators of a dysbiotic colon microbiota potentially contributing to disease symptoms , onset or evolvement , stool consistency or transit time are not always taken into account as confounders . we illustrate this concern using a recent study by scheperjans et al45 evaluating the role of the microbiota in parkinson 's disease . the authors detected a lower abundance of prevotellaceae combined with an increased ruminococcaceae population in patients compared with healthy controls . however , as constipation has been reported as an early symptom of parkinson s disease46 and given the overlap between the microbiome signal observed and our findings of bss - associated fluctuations in microbial abundances , there might be a risk that the microbiota signature proposed is a mere consequence of stool consistency differences between healthy and diseased individuals . to their credit , the authors did include the wexner constipation score in the study yet this score is primarily focused on long - term assessment of severe constipation ( ie , extreme bss 1 ) and would not compensate for day - to - day variation in bss in patients as well as controls.47 inclusion of stool consistency records or colon transit time measurements as a confounder would have allowed better disentangling of signals exclusively associated to the disease and those associated to stool consistency . together , our results demonstrate a profound association between stool consistency and all major readouts of gut microbiota composition . as increased colon transit time has been linked to enhanced proteolytic fermentation4850 and associated production of potentially deleterious metabolites , the observed negative correlation between bss - assessed passage rate and microbiome richness as reported here challenges the currently dominating view of high richness being directly associated to host health . furthermore , we here identify gut microbial compositional differences with stool consistency on community scale and genus level and evaluate a hypothesised biological mechanism of how transit time might shape the gut microbiota through selective pressure on microbial life - strategies . although longitudinal studies combining stool score records and direct transit time measurements are necessary to consolidate the observed correlations and proposed hypotheses , the strength of the associations between bss and species richness , enterotypes and community composition emphasise the crucial importance of stool consistency assessment and confounder analysis in gut microbiota research and clinical studies .

objectivethe assessment of potentially confounding factors affecting colon microbiota composition is essential to the identification of robust microbiome based disease markers . here , we investigate the link between gut microbiota variation and stool consistency using bristol stool scale classification , which reflects faecal water content and activity , and is considered a proxy for intestinal colon transit time.designthrough 16s rdna illumina profiling of faecal samples of 53 healthy women , we evaluated associations between microbiome richness , bacteroidetes : firmicutes ratio , enterotypes , and genus abundance with self - reported , bristol stool scale - based stool consistency . each sample s microbiota growth potential was calculated to test whether transit time acts as a selective force on gut bacterial growth rates.resultsstool consistency strongly correlates with all known major microbiome markers . it is negatively correlated with species richness , positively associated to the bacteroidetes : firmicutes ratio , and linked to akkermansia and methanobrevibacter abundance . enterotypes are distinctly distributed over the bss - scores . based on the correlations between microbiota growth potential and stool consistency scores within both enterotypes , we hypothesise that accelerated transit contributes to colon ecosystem differentiation . while shorter transit times can be linked to increased abundance of fast growing species in ruminococcaceae - bacteroides samples , hinting to a washout avoidance strategy of faster replication , this trend is absent in prevotella - enterotyped individuals . within this enterotype adherence to host tissue therefore appears to be a more likely bacterial strategy to cope with washout.conclusionsthe strength of the associations between stool consistency and species richness , enterotypes and community composition emphasises the crucial importance of stool consistency assessment in gut metagenome - wide association studies .

What is already known on this subject? What are the new findings? How might it impact on clinical practice in the foreseeable future? Introduction Methods Sample data Characterisation of the bacterial component of the gut microbiota by variable region 4 rDNA sequencing Statistical analysis Results Conclusion Supplementary Material

stool consistency , measured by the bristol stool scale ( bss ) , reflects differences in water content and activity in the colon ecosystem and is correlated with colon transit time . species richness is proposed to be a major marker for gut health because of the idea that high bacterial richness and diversity often reflect ecosystem stability and resilience together with the consistent association between disease and a reduction in the number of species in a faecal sample . faecal metagenomes have been shown to stratify into enterotypes , splitting the human population around three possible constellations in terms of their gut microbial ecosystem ; likewise firmicutes : bacteroidetes and prevotellaceae : bacteroidetes ratios have been put forward as important stratifiers for gut microbiomes . enterotypes are distinctly distributed over the bss scores : the prevotella ( p ) enterotype is more abundant in subjects with loose stool while the ruminococcaceae - bacteroides ( rb ) enterotype completely dominates the harder stool samples . microbiota growth potential is positively correlated with bss scores in the rb enterotype , hinting to transit time as a selective force on microbial life - strategies . here we show that major alterations in species richness or community composition could partially be reflecting variation in stool consistency , hampering the identification , but especially robustness and reproducibility of disease markers . this study therefore stresses the urgent necessity of these measurements in gut microbiota research and clinical studies . the identification of such confounding factors , including diet,5 6 history of antibiotics intake,7 and inflammation status,8 9 is essential to define the boundaries of a healthy gut ecosystem and , hence , to identify robust disease markers in clinical microbiome association studies . transit time is a key determinant of the gut microbial habitat , affecting nutrient and water absorption along the intestine as well as setting the pace of luminal microbial population clearance during egestion.10 as direct measurement of transit time is often impractical or mildly invasive , it has generally been neglected in microbiome studies so far . however , stool consistency as categorised by the bristol stool scale ( bss ) scores has been put forward as a proxy for colonic transit rate,1012 allowing assessment of its impact even when applying home - based sampling protocols . the use of the bss is widespread in clinical studies and has been advised for the assessment of constipation and diarrhoea in functional bowel disorders.1315 the bss classifies human faeces into seven consistency categories , with highest scores corresponding to loose stools and fast transit , while lower scores stand for hard stools and longer colon transit times.1012 16 each consistency category reflects differences in moisture content of faecal material , with decreased water activity associated with prolonged intestinal transit limiting microbial growth through reduced nutrient mobility and hampered enzymatic activity.17 hence , bss categorisation summarises the impact of two major and obviously related selective forces shaping the gut ecosystem : rate of intestinal transit and water activity . here , we used self - reported bss scores combined with 16s rdna illumina amplicon profiles of faecal samples of 53 healthy volunteers to assess potential associations between microbiota composition and stool consistency . in a healthy western population , more than half of the stools passed are predicted to belong to the central bss categories 3 and 4.11 however , increased occurrence of harder stools has been reported to be more common among women.11 12 18 hence , in order to increase chances of sampling a more uniform distribution of stools over all bss categories , we limited inclusion to female volunteers . fifty - three healthy women , aged 2055 years ( median 42.5 ) , were recruited as part of the flemish gut flora project . the participants general practitioner recorded their medical history , together with height , weight , and waist and hip circumferences . dna extraction was performed according to godon et al.19 to amplify the variable region 4 of the 16s rrna gene , we used the 515f and 806r primers ( gtgccagcmgccgcggtaa and ggactachvgggtwtctaat , respectively ) modified to contain illumina adapters and barcode sequences to allow for directional sequencing . amplifications were performed in triplicate as 25 l reactions containing 2 l of diluted template ( 1:10 ) , 2.5 l of 10x accuprime pcr buffer i , 0.1 l of accuprime taq high fidelity ( 5 u/l ) , and 0.5 l of 515f and 1.0 l of 806r primer ( 10 m of each primer ) . a final library size and quantification sequencing was performed on the illumina miseq platform ( miseq reagent kit v.2 , 500 cycles ) according to the manufacturer 's specifications to generate paired - end reads of 250 bases in length in each direction . this way 99.6% , 87.6% , 53.4% and 12.5% of the reads were assigned to order , family , genus and species levels , respectively . to compare the different samples , sample counts were rarefied to 26 260 reads for the de novo otu picking data set and 26 024 reads for closed otu picking and trimmed for the consequently absent otus with the phyloseq package based on the minimum of the sum of taxa abundances in rv.3.0.1 . clusters were assigned the enterotype ruminoccoccaceae - bacteroides or prevotella based on the taxa dominating the enterotype as in the original article of arumugam et al23 ( see online supplementary table s2 ) . species richness ( observed , chao1 ) and diversity measurements ( shannon ) were calculated using the phyloseq package.24 each sample s microbiota growth potential was estimated as the average of the maximum growth rates ( maxgrs ) of the genera , weighted by their abundance in the sample . correlations between species richness , microbiota growth potential , and relative bacterial taxa abundance with bss were assessed by spearman 's rank order correlation , as implemented in r. p values were corrected for multiple testing with benjamini - hochberg false discovery rate correction ( q value ) . spearman 's rank order correlation was used to check the correlation between enterotype and transit time ( bss ) , based on the percentage of each enterotype in each bss category . significance of differences in microbiota growth potential between enterotypes was assessed by wilcoxon signed rank test . fifty - three healthy women , aged 2055 years ( median 42.5 ) , were recruited as part of the flemish gut flora project . faecal samples were frozen at 20c immediately after collection by the participants . after frozen transport to a collection point in their neighbourhood and subsequent transport on dry ice , samples were stored at 80c within 72 h after delivery at the collection point until dna extraction . dna extraction was performed according to godon et al.19 to amplify the variable region 4 of the 16s rrna gene , we used the 515f and 806r primers ( gtgccagcmgccgcggtaa and ggactachvgggtwtctaat , respectively ) modified to contain illumina adapters and barcode sequences to allow for directional sequencing . a final library size and quantification sequencing was performed on the illumina miseq platform ( miseq reagent kit v.2 , 500 cycles ) according to the manufacturer 's specifications to generate paired - end reads of 250 bases in length in each direction . this way 99.6% , 87.6% , 53.4% and 12.5% of the reads closed otu picking with qiime against the greengenes 2013 database was performed in addition for a more stringent taxonomic assignment . to compare the different samples , sample counts were rarefied to 26 260 reads for the de novo otu picking data set and 26 024 reads for closed otu picking and trimmed for the consequently absent otus with the phyloseq package based on the minimum of the sum of taxa abundances in rv.3.0.1 . clusters were assigned the enterotype ruminoccoccaceae - bacteroides or prevotella based on the taxa dominating the enterotype as in the original article of arumugam et al23 ( see online supplementary table s2 ) . species richness ( observed , chao1 ) and diversity measurements ( shannon ) were calculated using the phyloseq package.24 each sample s microbiota growth potential was estimated as the average of the maximum growth rates ( maxgrs ) of the genera , weighted by their abundance in the sample . the maxgrs of gut - reference species ( img v4ref ) were estimated from genomic traits25 and the median was assigned to the genus . correlations between species richness , microbiota growth potential , and relative bacterial taxa abundance with bss were assessed by spearman 's rank order correlation , as implemented in r. p values were corrected for multiple testing with benjamini - hochberg false discovery rate correction ( q value ) . spearman 's rank order correlation was used to check the correlation between enterotype and transit time ( bss ) , based on the percentage of each enterotype in each bss category . significance of differences in microbiota growth potential between enterotypes was assessed by wilcoxon signed rank test . a healthy gut microbial ecosystem is generally thought to be characterised by high bacterial richness and diversity , presumed to reflect ecosystem stability and resilience.26 27 faecal microbiome analyses have revealed a seemingly consistent association between disease and a reduction in the observed or estimated number of species in a sample , suggesting bacterial richness to be a major marker for gut health.26 27 here , we identify stool consistency to be strongly associated to faecal microbial richness . indeed , observed species richness significantly declines with stool firmness ( spearman 's r=0.45 , p=0.0007 ) , reaching its minimum in diarrhoea - afflicted individuals ( figure 1a ) . estimation of total species richness ( chao1 ) confirms this negative correlation ( r=0.41 , p=0.003 , data not shown ) and the signal remains significant with the application of a more stringent taxonomical assignment ( closed reference otu picking , see methods ; trend also confirmed in a data set excluding patients with ibs , online supplementary figure s7 , and in a data set of 24 men , online supplementary figure s8 ) . these results are in line with anecdotal reports of decreased microbial richness associated with osmotic diarrhoea.28 stool consistency variation drives species richness and human enterotypes . correlation between ( a ) observed species richness and stool consistency , defined by bristol stool scale ( bss ) ( spearman 's correlation , r=0.45 , p=0.0007 ) and ( b ) enterotype distribution and stool consistency ( bss ) ; blue : ruminococcaceae - bacteroides ( rb ) enterotype ( r=0.88 , p=0.019 ) , green : p enterotype ( r=0.88 , p=0.019 ) . as enterotypes have also been linked to richness gradients,8 9 we analysed the distribution of enterotypes over stool consistency scores . applying multiple clustering approaches , we find that our current data set optimally separates into two distinct clusters ( see online supplementary table s2 ) . named after the dominating taxa as in the reference publication,23 we refer to the clusters observed as the ruminococcaceae - bacteroides ( rb ) and the prevotella ( p ) enterotype ( 77% and 23% of total samples , respectively ; partitioning around medoids ( pam ) clustering with jensen - shannon distance ) . the enterotypes observed are distinctly distributed over bss scores : while the p enterotype is more abundant in individuals with loose stools ( r : 0.88 , p=0.019 ) , the rb enterotype completely dominates firmer samples ( r : 0.88 , p=0.019 ; figure 1b ; trend also confirmed in men , online supplementary figure s8 ) . these observations are substantiated by the analysis of the firmicutes : bacteroidetes and prevotellaceae : bacteroidetes abundance ratios ( both put forward as alternatives to enterotyping29 ) over stool score ( respectively r=0.42 , p=0.001 and r=0.04 , p=0.77 ; see online supplementary figure s1a , b ) . hence , the enhanced prevalence of the rb enterotype in the harder stool categories is driven by the samples with a high ruminococcaceae : bacteroides ratio , which would classify them as ruminococcaceae enterotype according to the terminology originally suggested by arumugam et al.23 as even within a single enterotype low richness samples are more abundant in looser stool ( see online supplementary figure s3 ) , the richness signal is thus not a mere consequence of enterotype distribution across consistency scores . although the identification of environmental factors shaping or contributing to enterotype stratification is still ongoing , the potentially diverging impact of diet has been emphasised previously.5 30 more specifically , the occurrence of the p enterotype has been linked to a rural , fibre - rich diet.5 31 as non - fermentable fibre consumption is thought to increase water content and plasticity of stool,32 this observation fits with the increased prevalence of the p enterotype in the loose stool categories . stool consistency variation is associated with shifts in faecal microbiota composition at the level of community structure and diversity , and it correlates with abundance gradients of individual genera ( see online supplementary table s3 ) . genera abundances significantly correlated with stool consistency genera abundances significantly correlated with stool consistency ( bss ) ( q<0.1 ) in the total data set , the rb enterotype , or the p enterotype . bss , bristol stool scale ; na , not assigned ; rb , ruminococcaceae - bacteroides . the increased abundance of methanogens such as methanobrevibacter in harder stools confirms previous reports of elevated methane production in constipated individuals.33 it has been suggested that methane plays an active role in the delay of transit by slowing down intestinal motility.34 moreover , hydrogen removal through methane production alters the whole of gut fermentation processes , which could potentially affect colon peristalsis.33 alternatively , increased abundance could reflect the fitness of a genus to grow in conditions of slowed - down intestinal transit . in order to avoid washout , the residing bacteria can either reproduce at a sufficiently high growth rate or attach to or colonise host tissues.35 hence , transit time may act as a selective force on gut bacterial growth rates . indeed , higher fluctuations in nutrient availability and microbiota population size induced by decreased colon transit time would be the text - book selection pressure for fast growing bacteria , outgrowing their slow growing counterparts whenever resources are available ( r - selection).36 also enhanced water activity and the associated increase of nutrient mobility in loose stools will contribute to the selective force imposed by accelerated transit . indications for the suggested correlation between bacterial growth rates and rates of passage come from in vitro work with continuous flow fermenters3739where dilution rates determine pace of growth of the bacteria cultured and in vivo observations of higher faecal bacterial biomass associated with shorter transit times.40 41 to assess this hypothesis , we estimated each sample s microbiota growth potential as the average of the maxgr25 ( see methods ) of the genera , weighted by their abundance in the sample ( based on de novo otu as well as closed reference otu assignment ; online supplementary table s5 ) . we find that microbiota growth potential is indeed positively correlated with stool score , and hence colon transit time , in the rb enterotype ( r=0.34 , p=0,028 ; figure 2 ) . strikingly , the average growth rate of the p enterotype samples is lower than the one of the rb samples ( median microbiota growth potential 0.38/h vs 0.52/h , respectively , wilcoxon p<10 ; online supplementary figure s6 ) . on one hand , the absence of correlation between growth rate and colon transit time in the p enterotype could suggest that species in this enterotype resort to an alternative strategy to avoid washout , namely a higher degree of adherence to host tissues . the fact that prevotella is indeed able to bind collagen and degrade mucin oligosaccharides support this hypothesis.42 43 alternatively , our results could indicate that stool consistency and hence water activity in prevotella individuals are independent of accelerated transit , and mainly reflect increased faecal water - binding capacity , for example , related to fibre consumption . overall , as faeces and library dilution series have been shown not to affect compositional readouts in 16s amplicon analyses44and the results obtained are thus not due to technical issues arising from the variation in water content our results hint to transit time as one of the determining selective forces on microbial life - strategies . microbiota growth potential correlates to faster intestinal transit in the ruminococcaceae - bacteroides ( rb ) enterotype . microbiota growth potential over stool consistency ( bristol stool scale ( bss ) ) , which is proposed as a proxy for transit time , in the rb - enterotype ( r=0.34 , p=0.028 ) . the results of the present study indicate that major alterations in species richness or community composition could partially be reflecting variation in stool consistency , hampering the identification , and affecting robustness and reproducibility of disease markers . while constipation and or diarrhoea are often seen as indicators of a dysbiotic colon microbiota potentially contributing to disease symptoms , onset or evolvement , stool consistency or transit time are not always taken into account as confounders . the authors detected a lower abundance of prevotellaceae combined with an increased ruminococcaceae population in patients compared with healthy controls . however , as constipation has been reported as an early symptom of parkinson s disease46 and given the overlap between the microbiome signal observed and our findings of bss - associated fluctuations in microbial abundances , there might be a risk that the microbiota signature proposed is a mere consequence of stool consistency differences between healthy and diseased individuals . to their credit , the authors did include the wexner constipation score in the study yet this score is primarily focused on long - term assessment of severe constipation ( ie , extreme bss 1 ) and would not compensate for day - to - day variation in bss in patients as well as controls.47 inclusion of stool consistency records or colon transit time measurements as a confounder would have allowed better disentangling of signals exclusively associated to the disease and those associated to stool consistency . together , our results demonstrate a profound association between stool consistency and all major readouts of gut microbiota composition . as increased colon transit time has been linked to enhanced proteolytic fermentation4850 and associated production of potentially deleterious metabolites , the observed negative correlation between bss - assessed passage rate and microbiome richness as reported here challenges the currently dominating view of high richness being directly associated to host health . furthermore , we here identify gut microbial compositional differences with stool consistency on community scale and genus level and evaluate a hypothesised biological mechanism of how transit time might shape the gut microbiota through selective pressure on microbial life - strategies . although longitudinal studies combining stool score records and direct transit time measurements are necessary to consolidate the observed correlations and proposed hypotheses , the strength of the associations between bss and species richness , enterotypes and community composition emphasise the crucial importance of stool consistency assessment and confounder analysis in gut microbiota research and clinical studies .

there are several important reasons for such a focus , but a key issue is no doubt the relationship between poverty and ill - health and premature death , shown not least by several classical and historical investigations [ 1 , 2 ] . the finding of the social gradient is also of interest when going from these historical studies to present discussions about poverty , inequality , and population health , as it indicates that not only the very poorest sections were hit but that relative poverty was also of importance . it is well known that countries with high absolute poverty rates today ( e.g. , world bank indicators of 1 or 2 us dollars a day ) also tend to be those with low life expectancy and high mortality risks . but what is the relationship between relative poverty rates and mortality risks among the richer countries of the world ? assuming that the poorest people in rich countries do not live under absolute poverty , the relationship between variations in relative poverty rates and variations in mortality rates may seem less self - evident . however , this relationship has been at the centre of one of the most debated topics within the field of public health research and social epidemiology in recent decades , namely , the health impact of income inequality . it is actually one foundation of the so - called wilkinson hypothesis , which basically states that it is not the level of affluence as such that matters among rich countries but rather how the pie of total economic resources is distributed . this hypothesis is articulated in relation to the whole social structure , thus stating that it is income inequality as such , not only poverty , that kills . however , most evidence , on both the macrolevel of countries and the microlevel of individuals , suggests a curvilinear association between income and health , which implies that health gains can be made by transferring money from the richer to the poorer . if this is so , it means that not only income inequality but also and even more evident variations in poverty rates should be associated with population health . but can we evidence that cross - national variations in relative poverty rates are related to cross - national variations in survival possibilities in relatively rich countries ? in this study , we conduct a comparative analysis of the relationship between poverty and mortality across 26 developed countries over time . we utilize data from the luxembourg income study to construct age - related poverty rates across countries and time covering the period from around 1980 to 2005 , merged with data on age- and gender - specific mortality data from the human mortality database . it is reasonable to assume that the consequences of poverty differ between gender and age groups . it is also a well - known fact that the causes of death vary by gender and age . for example , it has been shown that there were larger socioeconomic differences in men 's mortality patterns than women 's . it follows that it seems to be a reasonable hypothesis that the poverty and mortality relationship could also be different between women and men . therefore , it is important that we conduct sex- and age - disaggregated analyses . in the next section , we briefly present some of the arguments and empirical evidence of relevance to our study . we then present our results , and the paper ends with a concluding discussion about our findings . as mentioned , the idea that income inequality could influence population health was noted already in the typical curvilinear association of the so - called rodgers curve . partly based on empirical data , rodgers presented a model of how smaller income disparities and relative poverty at societal level are linked to better public health through differential impacts on individual health status among both low- and high - income earners . he argued that the health returns of income diminish at higher income levels , implying that this relationship is curvilinear [ 7 , 8 ] . in the rodgers example ( figure 1 ) , the health of the low - income person x1 is much poorer than that of the high - income person x2 at t1 . redistributing income from x2 to x1 at t2 will result in an unchanged average income ( x- ) , while average health ( y - t2 ) improves . this is simply the result of the health gain among the poor ( yx1 ) being larger than the health loss among the rich ( yx2 ) as a consequence of this income redistribution . rodgers also presented results from cross - national , cross - sectional analysis supporting the specification that countries with lower inequality had higher life expectancy . although rodgers , and later wilkinson , articulated how the whole income distribution could make a difference , it is evident from the hypothesis that what should particularly make a difference is how the relatively poor fare and how large a fraction of the population is at risk of poverty . the topic of income inequality and health has become a small research industry within social epidemiology , with some influences from economics and sociology , and numerous studies have been published , especially on the relationship between income inequality across american states and various health outcomes . one major review was largely in favour of the hypothesis , whereas another was sceptical . a meta - analysis of multilevel studies linking income inequality to mortality and self - rated health lent support to the idea . a recent global study investigating 140 countries also lends support to the hypothesis , but only in low- and middle - income countries . in cross - national analyses , not least with regard to population health and poverty , it has become common procedure to group countries according to their specific mix of welfare production , that is , welfare regime [ 15 , 16 ] . as noted by several authors [ 17 , 18 ] , the welfare modelling business has become a central part of welfare state research , starting with esping - andersen 's famous trichotomy that he labelled on the basis of main political ideologies : the liberal , social - democratic , and conservative / corporatist regimes . the idea behind the regime approach goes beyond the welfare state in the stricter sense by looking at the nexus of the state , markets , and family . while esping - andersen identified three welfare state regimes among the countries he analysed , it has subsequently been common procedure to also include and identify additional clusters of southern and eastern european countries . although our overall aim is to study the link between poverty and mortality , it is of obvious interest to note variations in this relationship by welfare state regime and to adjust our analyses by regime . not least within the news project , initiated in collaboration with the who commission on social determinants of health , a number of studies were produced linking the specific design , generosity and coverage of social policy programmes to overall and age - specific mortality on the one hand , and to morbidity on the other [ 2024 ] . these studies focused on the cash side of the welfare state and supported the idea that cash programmes of the welfare state have been of importance to public health during the second half of the 20th century . these studies did not investigate the role of welfare services , nor did they study any specific mechanisms behind the associations found . however , the ability of these programmes to alleviate poverty was often referred to as a key factor in cross - national variations in mortality rates . of course , the programmes of the welfare state are likely to also influence other more proximal health - related factors that could influence mortality risks . in this study we will explore the relative poverty argument directly , by making use of the best sources for comparative studies on poverty and mortality over a 25-year period . we partly overcome the small - n problem that occurs in most cross - national studies by using multiple waves of data for each country included . although we will not examine the mechanisms , it is still necessary to briefly mention some of the possible multiple pathways linking relative poverty and mortality . overall mortality has decreased in recent decades in developed countries ( with russia as the only exception ) . the question is whether the incidence of relative poverty has delayed or prevented a fall in mortality in the countries included in our analysis . the experience of living in relative poverty may be connected to unhealthy habits and continuous stress , as well as negative consequences more or less directly stemming from a lack of resources , for example , not being able to consume healthy food or live in adequate housing , or moving to a neighbourhood with more safety , better primary health care or better schools and other services . we believe many of these factors may work in a causal chain rather than as contradictory mechanisms . in so far as psychosocial processes are at work , it seems more reasonable to assume that they have a material base than to regard the material and psychosocial as representing two opposite and mutually exclusive poles . our two main data sources are the luxembourg income study ( lis ) and the human mortality database ( hmd ) . the lis is a cross - national harmonized database that includes multiple waves of microdata for a number of countries . it has a focus on income inequality and poverty , but also includes a great deal of information on aspects such as family situation , and employment status . wave 1 started around 1980 with approximate five - year intervals , so that wave 6 of the data is from around 2005 ( for a thorough presentation of the database see ) . the lis is commonly regarded as the best source of cross - national comparisons of poverty and income inequality . the hmd , maintained by the university of california , berkeley , and the max planck institute of demographic research , provides detailed open access mortality and population data for a number of countries for years reaching from the 1800s to around 2010 . currently , the hmd includes information for 37 countries , which are partly the same and partly different to those in the lis database . in our study , we include all countries from the lis that have at least two waves of data from the same original survey source , and for these countries , all lis waves for which mortality data were also available in the hmd for corresponding years . this led to a country sample of 26 rich countries with two to six waves , a total of 122 data points ( see table 1 ) . countries included are australia , austria , belgium , canada , the czech republic , denmark , finland , france , germany , hungary , ireland , israel , italy , luxembourg , the netherlands , norway , poland , russia , the slovak republic , slovenia , spain , sweden , switzerland , taiwan , the united kingdom , and the united states . we investigate four nested models in which the dependent variable is the logged mortality rate and the exposure variable is the poverty rate . mortality rates were assessed for three gender - specific age classes : infants ( aged < 1 year ) , children ( aged 117 years ) , and adults ( aged 2564 years ) . data on deaths and populations at risk were collected for one - year age bands for each country from the hmd for all lis waves , and for three following years of each wave . while infant mortality rates were used as such , age - standardized mortality rates for the age groups 117 and 2564 were calculated to adjust for the different age structures of the countries . in these calculations the age - standardized rates thus represent what the crude rates would be if the populations of the countries had the same age distribution as the european standard population . the age - standardized mortality rates were assessed as deaths per 1,000 person years , over four - year periods ( from each wave until three years later ) , to allow for exposure time . however , for infant mortality , we only took into account the immediate year . in the multivariate regressions , poverty rates were calculated using a standard income poverty head - count measurement in which individuals living in households with equivalent disposable income lower than a certain percentage of median income are regarded as poor . accordingly , we measure income after taking into account welfare state transfers and taxes . in order to be able to compare households of different sizes , each household 's disposable income is divided by the square root of the number of persons in the household . the proportion of poor households will of course be partly determined by where we set the threshold . evidently , the nature of poverty in terms of both the size of income and , for the countries analysed here , its consequences will become more severe the further we move from the national median . in our analyses we have employed a more severe definition than the usual 60% threshold , setting the poverty threshold at 40% of the national median . poverty rates in each country and each wave were calculated separately for children ( aged < 18 ) and working - age adults ( aged 2565 ) . with the data at hand , we can not have a perfect age match between the poverty rates and the mortality rates . thus , the total child poverty rates are used as the exposure for both our child mortality analyses , and there is also a one - year mismatch for the adults . the latter mismatch is highly unlikely to have any effect on our results . as confounders we consider the following variables . the lis wave number is included to allow for time - related changes in poverty and mortality rates . the wave number also is an indicator variable pertaining to the more or less automatic decline in mortality that takes place in all countries . gdp per capita/1,000 us dollars was derived from penn 's world tables that contain information on the gdp per capita levels for all the countries included in our analyses . the gdp levels are adjusted to changes in cost of living across time and space and are given in 2005 us dollars . administrative costs are also included , but the inclusion of the costs for running the schemes is not a major problem as these costs comprise only 24% of all expenditure . a more nuanced way of studying the impact of welfare spending would have been to use disaggregate spending data , that is , to separate cash and in kind benefits used for children , elderly , health care , various income maintenance programs , and so forth . however , this kind of analysis falls outside the scope of this particular paper and is a task for future studies . here we simply assume that the overall social spending level reflects the state 's commitment to citizens ' welfare . and russia and taiwan are not included in this database ; therefore , data for these countries are derived from other sources [ 29 , 30 ] . because data for russia and taiwan are adapted from nonstandard oecd sources , they are not totally comparable . therefore , we have run sensitivity tests with and without these countries . the omission of taiwan did not change the results , while the exclusion of russia had a strong impact . our data set is unbalanced ; that is , data are not available for all countries and all years ; therefore , we also run control analyses for the balanced data . whereas the omission of russia had the strongest impact , the omission of other outliers was not highly significant ( see further discussion below ) . we also include dummy variables for the welfare state regime each country belongs to ( see table 1 ) . the latter variables were added in the model one at time to better investigate their associations . our analytical approach is first to inspect bivariate plots to observe the general pattern of the relationship between age - specific mortality rates and the background variables . we start by looking at developmental pattern in mortality over cross - sections and welfare regimes . thereafter , we proceed to multivariate analyses to observe how the bivariate relationships will change when other variables are included in the regression models . for regression analyses , we used pooled cross - sectional time - series methods . these methods take advantage of the panel structure of the data while taking care of the correlations of data points between waves using panel - corrected standard errors [ 3133 ] . in these analyses , we use country as the panel variable and wave as the time variable . although partially solving the small n - problems , the pooled cross - sectional time - series method results in problems of spatial and longitudinal autocorrelation and heterogeneity . there are a number of regression techniques available to deal with the special problems of analysing pooled data , each with its own weaknesses , and results seem to be highly sensitive to the specific method applied [ 3438 ] . pooled regressions were run using the stata 12 cross - sectional time - series package using prais - winsten regressions . here we tested two possible ways to model the autocorrelation : ( 1 ) the psar(1 ) model uses autoregressive ( ar1 ) autocorrelation that is panel specifically calculated . the positive side is that it is tailored for each panel separately , and the negative side is that it may be unstable if there are few cross - sections ; ( 2 ) the ar(1 ) model uses an autocorrelation structure that is common for all panels . in order to further test the robustness of our results the results were robust for the different methods applied , and although the standard errors varied the interpretations of the results did not . figures 2(a)2(f ) show the magnitude and variability of infant , child , and adult standardized mortality rates by sex in different welfare state regimes and over time . we begin by looking more closely at infant mortality rates ( figures 2(a ) and 2(b ) ) . in the regression models to be shown later we will use logged mortality for girls and boys together , but in these more descriptive figures we show the raw figures for each sex . the box - plots display medians and distributions of country - based and wave - based observations around the median values . the first observation from the box - plot figure is that infant mortality rates among boys are higher than among girls ( 7.99 per 1,000 boys and 6.43 per 1,000 girls , on average across countries and time points ) . while this gender gap holds for all welfare regimes there is regime- and country - based variation in the width of the gap , widest in the postsocialist countries . as can be seen , the mortality rates are the lowest in the nordic group ( on average 4.56 for girls and 5.92 for boys ) and there is relatively small variation between the four countries included in this cluster . the starting levels are already low , and there is a modest absolute decline in infant mortality . on the other end of the continuum , we find the former socialist countries to have both the highest mortality rates and the highest variation between nations , but also the highest absolute decline in infant mortality . there is substantial variation over time as well , and as evident in figure 2(b ) , there is a downward trend over the waves concerning both levels and cross - national variation . bivariate scatterplots between infant mortality rates and the background variables we will later use in our multivariate models are shown in figure 3(a ) . in pooled data , that is , where all cross - sections are merged into one , the relationships between predictors and infant mortality rates are in the expected direction but not always convincingly high . the overall correlation in the pooled data is the highest between infant mortality rate and gdp , indicating that infant mortality is conditioned by the wealth of the nation and all the factors linked to gdp . however , gdp is not only an indicator of economic prosperity but also represents a more general modernization trend that includes better food , better health care , better sanitation , access to clean water , and so forth factors regarded as important in combatting infant mortality . in line with earlier research , we can also note the curvilinearity in the association between economic prosperity and infant mortality . the second strongest correlation in the pooled data is the one between mortality and social spending , representing the magnitude of the public commitment to the social protection of the populace . here as well the pattern is rather constant over cross - sections ( figure 3(a ) ) : the larger the share of gdp that is made up of social spending , the lower the infant mortality rate . contrary to our initial expectations , the link between infant mortality and child poverty rates is also relatively low in the pooled data . whereas relative poverty is rather strongly correlated with mortality in the first and last waves , the correlations in waves 2 and 3 are rather weak . our interim conclusion is that the level of prosperity of the country and the magnitude of the welfare state matter , and that the impact of the welfare state is mirrored in lower levels of child poverty and inequality , which in turn partially combat new - born deaths . an intriguing question is to what extent , if any , these bivariate relationships are robust when they are analysed simultaneously . in table 2 , we present results from regression analyses in which we step - wise include additional variables such as trend ( wave ) , gdp per capita ( 1,000 us dollars in 2005 values ) , social spending and , finally , the welfare state regimes as dummies . in the last model ( 4 ) the nordic welfare regime is used as a reference and is left out of the models . we ran the models separately for infant girls and boys , because the results turned out to be very similar we show them for both sexes combined . however , detailed results of the gender - specific analyses can be found in tables 5(a ) and 5(b ) . in the first model of table 2 , including only the poverty rate and the wave variable , the coefficient for poverty is significant . the coefficient of the association between poverty and logged mortality rate from this model can be statistically interpreted as follows : a one percentage - point increase in child poverty corresponds to about a 2% increase in infant mortality . the introduction of gdp per capita/1,000 us dollars ( model 2 ) does not change the picture . the inclusion of social spending ( model 3 ) , as expected , leads to an attenuation of the poverty estimate by about 40% . the statistical explanation for the strong attenuation of the poverty estimate when social spending is added is the strong association between social spending and poverty rates . so it seems that the welfare state matters for relative poverty , and relative poverty matters for infant mortality . finally , when welfare regimes are introduced ( model 4 ) , the poverty estimate remains about the same . the welfare regimes obviously capture not only different welfare state characteristics but also different levels of economic prosperity , since the coefficient for gdp totally disappears . in model 4 , controlling for poverty , wave , gdp , social spending and welfare regime , infant mortality rates are significantly higher in central european , liberal , and especially postsocialist regime types compared to the benchmarking nordic regime ( the reference category ) , while the southern european and other regimes do not significantly deviate from the nordic one . these regime differences are notable , especially if one bears in mind that they are not captured by differences in poverty , economic prosperity , or social spending . this evident variation between the regime types highlights that the causes of differences in population health statistics are multifactorial , and we are not able to fully capture this with the variables in our regression models . when we move from the new - borns to older children , the risk of death radically diminishes . this is also reflected in the age - standardized mortality rates in the age group 1 to 17 . figures 2(c ) and 2(d ) give the variability in these rates by welfare state regime type and across waves . again , there is an overrepresentation of boys in the death toll ( the average age - standardized mortality rate over countries and time points is 0.22 per 1,000 girls and 0.33 per 1,000 boys aged 117 ) . in the postsocialist regime , the average death rate ( 0.27 among girls , 0.42 among boys ) is about 1.5-fold compared to the low nordic numbers ( 0.17 among girls , 0.26 among boys ) . in this age group , the country group stands out with relatively high mortality rates but also very large variation . as seen in figure 2(d ) , there is a clear trend here as well towards lower death rates in time ( in average , from 0.31 to 0.15 among girls and from 0.49 to 0.21 among boys ) . the fact that we have an unbalanced panel can of course influence the magnitude of this downward trend , but the overall trend is general within all countries . figure 3(b ) shows the crude relations between the pooled data of the age - standardized death rates and the three main explanatory factors . especially social spending has a relatively strong association with child mortality , whereas the associations of gdp and relative child poverty with mortality are more modest . in order to cross - check the extent to which the results are biased by the former communist countries , we ran controls in which the countries in the postsocialist cluster were excluded . in general , correlations between gdp and mortality became weaker , but the signs were not changed . correlations between social spending and mortality became stronger , however , and correlations between poverty and mortality became stronger or remained almost the same . in table 3 we show the results of our pooled cross - sectional time series analysis for this age group for girls and boys together , because the results of gender - specific analyses proved to be almost identical in terms of regression coefficients ( see tables 6(a ) and 6(b ) ) . the analytical strategy is basically the same as for infants , although the logged age - standardized mortality rates are now calculated as the average of the lis years ' mortality plus the following three years ' mortality to allow for exposure time on mortality after our poverty measurements . the basic story for the age group 1 to 17 is also very much the same as what we showed for the infants ( table 2 ) . the poverty estimates , especially in the two first models , and estimates for most of the other variables have similar magnitude as in the case of infant mortality . an important exception to this is the welfare regime . in terms of mortality among children aged 117 , the liberal and southern european regimes fare significantly better compared to the nordic regime after the other covariates are adjusted for . , we can note that the poverty estimate actually doubles when welfare regime is adjusted for ( compare models 3 and 4 ) . we will return to this finding in our final discussion . when comparing adult and mortality rates with child mortality rates , interesting shifts in the rank order of good and bad regimes can be observed . whereas in both groups of children ( figures 2(a)2(d ) ) the nordic welfare cluster displayed the lowest mortality rates , among adults the southern european cluster outperforms the nordic one ( figure 2(e ) ) . the figure reveals the exceptionally high mortality rates among males in the postsocialist countries , where the average age - standardized mortality rate across time points is as high as 10.06 for men and 3.69 for women ; the corresponding figures for the southern european cluster are 4.43 and 1.93 . as in the case of child mortality , there is a general downward trend over time ( figure 2(f ) ) . figure 3(c ) once again displays the well - known curvilinear relationship between mortality and gdp per capita ; the mirror picture of this is the relationship between gdp and life expectancy . neither social spending nor relative adult poverty rate displays any clear - cut relationship with mortality . although the relationship is somewhat different in different waves , the general message is that the bivariate plots basically show no association . results from pooled cross - sectional regressions are shown separately for women ( table 4(a ) ) and men ( table 4(b ) ) . in general , the association between poverty and mortality is weaker in the models for the adult population than for children . starting with the results for women , we can note that poverty remains significantly and positively associated with mortality across all four models . the general picture that the poverty estimate attenuates when social spending is controlled for is also evident for women in the same manner as we saw earlier for infants and children . somewhat oddly , we find that the poverty estimate actually increases when welfare regime type is also adjusted for ( compare model 4 to model 3 ) . scrutinizing the estimate for regime , we note that the association between regime and adult mortality is different to that between regime and infant mortality ( table 2 ) , but somewhat similar to that between regime and child mortality ( table 3 ) . compared to the nordic regime , the central and especially southern european regime types show statistically significantly lower mortality rates , whereas the postsocialist regime shows higher mortality rates among women when poverty , wave , gdp , and social spending are controlled for . turning to the results for men ( table 4(b ) ) , the picture is somewhat less clear . although the poverty estimates as such are not lower than for women , the variability , as evident in the high p - values , is much higher . and , somewhat strangely , the poverty estimate has its largest value and is clearly significant only in the final model . the postsocialist cluster has an extremely high estimate , especially considering all the other covariates we have adjusted for . apart from this cluster we can note that , in comparison to the nordic regime , the southern european , liberal , and other regimes have lower adjusted male mortality rates . this difference between southern and northern europe has also been corroborated by other cross - national research on mortality differences . but here it seems as if these differences , for both women and men , are accentuated by the fact that we control for the other welfare state - related variables . simultaneously , this also accentuates the effects of poverty for both women and men . we have performed a number of sensitivity analyses with regard to the inclusion / exclusion of countries and setting a higher poverty threshold . we also tested the impact of income inequality ( as expressed by the gini coefficient ) . we argue that the 40% poverty threshold comes closer to the absolute poverty level , not least combined with the national wealth indicator ( gdp ) , than the 60% poverty threshold , which comes closer to income inequality measured using the gini coefficient . however , as gini and poverty measures are strongly correlated they can not be used simultaneously as explanatory variables . the correlation between the whole population - level gini and relative poverty rate with 40% threshold is 0.85 , and gini and relative poverty rate with 60% threshold is 0.89 . when it comes to deviant cases we have one country that stands out : russia . during the time span covered by this study , russia had high poverty rates and extreme death risks , especially visible among adult males therefore , when we reran all regressions omitting russia , the estimates changed substantially . by and large our poverty estimates attenuated by about a third for infants , the degree of attenuation varied across the models for children aged 117 , and among adults the poverty estimate became insignificant . this choice was made mainly for theoretical reasons , as we suggest that it is the long - term and broad difference in poverty that matters rather than any yearly fluctuations [ 36 , 41 ] . we realize that models focusing on change would capture unmeasured heterogeneity but , on the other hand , such models also increase the noise - to - signal ratio . in the end , in line with babones in his comparative analysis of income inequality and health , we note that a major complication for any fixed effects model is the remarkable stability for both variables over time . however , it should be noted that some of the fixed effects are still included through the dummies for the lis wave and the welfare state regimes . another methodological concern in our study is the fact that we have an unbalanced panel structure . in other words , we have different countries in different waves . although statistically speaking our method takes this into account , it may still have an influence on our findings . this is an analogy to the finding from a simulation analysis by pop et al . suggesting that the composition of the sample of high - income countries may be crucial . still , in sensitivity analyses we found that a balanced panel gave largely similar results . in summary , it seems that the relative poverty rates are of importance to child mortality for the sample of countries and the period examined . this is also in line with earlier research showing a stronger association between income inequality and infant and child mortality than between income inequality and adult mortality . found evidence of socioeconomic resources in childhood and later in life having both direct and indirect effects on mortality patterns . primarily two models have been suggested in life course epidemiology : accumulation and critical periods during life . poor socioeconomic resources in childhood are associated with morbidity patterns in adulthood , particularly diseases such as stomach cancer and hemorrhagic stroke . the magnitudes of deprivation among children and the effect of the association vary between countries and have also been shown to be influenced by the design of the welfare state , such as choices of social policy / redistribution . the aim of this study was to analyse the effect of relative poverty upon mortality rates among three age groups infants , children , and working - age adults , also stratified by gender . we used a low threshold ( 40% of median ) to measure relative poverty , which thereby measures more severe poverty prevalence . our time period is 19802005 , and we have an unbalanced time series for 26 countries belonging to the rich world but also including postsocialist countries from eastern europe . we have recently seen a number of studies that go beyond the cross - sectional picture between income inequality and mortality [ 14 , 42 , 46 , 47 ] . to our knowledge , this is one of the first studies to go beyond the cross - sectional picture with a focus on poverty rather than inequality . there is ample evidence of profound differences in poverty across welfare regimes [ 4850 ] , suggesting that poverty , welfare regime , and mortality also may be interrelated . our results are basically the following : we find support for the assumption that the prevalence of poverty is of importance . the strength and level of significance vary depending on which additional variables are included in the model . when social spending is included , the poverty estimate for children attenuates by a third . a statistical explanation for this is the strong and robust association between poverty and social spending . when thinking about the order of impacts , one can , with overwhelming empirical support , argue that social spending is causally related to poverty : the higher the spending level in a country , the lower the poverty levels . we anticipated that if we took into consideration the welfare regimes ' belongingness , the relative role of poverty rates would be eaten up . however , for children the effect on the poverty estimate was negligible , and for adults the inclusion of welfare regime fortified the connection between poverty and mortality . this does not influence the poverty and mortality association for children , but it is important to note that the regime type as such has a clear influence on child mortality , even when controlling for gdp and social spending . in other words , this result suggests that there are other regime - specific factors that are important . for adults , interestingly enough , for both women and men we find that the poverty estimate becomes stronger when welfare state regime type is also controlled for . the reason for this is not self - evident , but from earlier research we know that several of the southern european countries are ranked at the top of life expectancy figures in europe and worldwide . we also know that they are less favourably ranked when it comes to poverty rates . in a sense , the regime variable captures whatever it is that is specifically health - promoting in these countries , and the resulting poverty estimate is thereby adjusted for that regime - specific aspect . another intriguing result , then , is that welfare regimes do not treat all age groups similarly . when it comes to the nordic welfare model , it seems to be good for infants and children but is no longer superior in older age groups , and some central and southern european countries outperform it . the results also show exceptionally high mortality rates among males living in the postsocialist countries . this result , in turn , indicates that welfare state and poverty have an impact on mortality , but there are other factors in play , such as drinking and eating habits and the way healthy and unhealthy behaviour is distributed between socioeconomic groups , according to income and education attainment levels . our study is definitely not the final answer to the question of whether or not the prevalence of poverty in relatively rich countries still has an influence on death risks . our study is somewhat different to most of the cross - country studies linking poverty and mortality . they have used either a more worldwide inclusion of countries ( but the question then becomes somewhat different ) or a much smaller sample of countries and have particularly been totally cross - sectional in their design . moreover , we have used age - specific analysis when it comes to both poverty calculations and mortality rates , thereby further specifying the tests . finally , as our study is based on large - scale macrophenomena we obviously have several mediating factors . however , a policy recommendation from our study is that national governments invest in eliminating child poverty . this is likely to have positive population health effects from both a short- and long - term perspective . from cross - national poverty analyses we know that universal , redistributive social policies are key instruments in reducing poverty ; if such policies are also coupled with social investment policies for young children , such as high - quality day care , this not only reduces poverty here and now but is also likely to be a good investment for the future .

a prime objective of welfare state activities is to take action to enhance population health and to decrease mortality risks . for several centuries , poverty has been seen as a key social risk factor in these respects . consequently , the fight against poverty has historically been at the forefront of public health and social policy . the relationship between relative poverty rates and population health indicators is less self - evident , notwithstanding the obvious similarity to the debated topic of the relationship between population health and income inequality . in this study we undertake a comparative analysis of the relationship between relative poverty and mortality across 26 countries over time , with pooled cross - sectional time series analysis . we utilize data from the luxembourg income study to construct age - specific poverty rates across countries and time covering the period from around 1980 to 2005 , merged with data on age- and gender - specific mortality data from the human mortality database . our results suggest not only an impact of relative poverty but also clear differences by welfare regime that partly goes beyond the well - known differences in poverty rates between welfare regimes .

1. Introduction 2. Material 3. Analytical Approach, Methods, and Variables 4. Results and Discussion 5. Conclusions

there are several important reasons for such a focus , but a key issue is no doubt the relationship between poverty and ill - health and premature death , shown not least by several classical and historical investigations [ 1 , 2 ] . the finding of the social gradient is also of interest when going from these historical studies to present discussions about poverty , inequality , and population health , as it indicates that not only the very poorest sections were hit but that relative poverty was also of importance . but what is the relationship between relative poverty rates and mortality risks among the richer countries of the world ? assuming that the poorest people in rich countries do not live under absolute poverty , the relationship between variations in relative poverty rates and variations in mortality rates may seem less self - evident . however , this relationship has been at the centre of one of the most debated topics within the field of public health research and social epidemiology in recent decades , namely , the health impact of income inequality . this hypothesis is articulated in relation to the whole social structure , thus stating that it is income inequality as such , not only poverty , that kills . however , most evidence , on both the macrolevel of countries and the microlevel of individuals , suggests a curvilinear association between income and health , which implies that health gains can be made by transferring money from the richer to the poorer . if this is so , it means that not only income inequality but also and even more evident variations in poverty rates should be associated with population health . but can we evidence that cross - national variations in relative poverty rates are related to cross - national variations in survival possibilities in relatively rich countries ? in this study , we conduct a comparative analysis of the relationship between poverty and mortality across 26 developed countries over time . we utilize data from the luxembourg income study to construct age - related poverty rates across countries and time covering the period from around 1980 to 2005 , merged with data on age- and gender - specific mortality data from the human mortality database . it is also a well - known fact that the causes of death vary by gender and age . for example , it has been shown that there were larger socioeconomic differences in men 's mortality patterns than women 's . it follows that it seems to be a reasonable hypothesis that the poverty and mortality relationship could also be different between women and men . as mentioned , the idea that income inequality could influence population health was noted already in the typical curvilinear association of the so - called rodgers curve . partly based on empirical data , rodgers presented a model of how smaller income disparities and relative poverty at societal level are linked to better public health through differential impacts on individual health status among both low- and high - income earners . in the rodgers example ( figure 1 ) , the health of the low - income person x1 is much poorer than that of the high - income person x2 at t1 . this is simply the result of the health gain among the poor ( yx1 ) being larger than the health loss among the rich ( yx2 ) as a consequence of this income redistribution . rodgers also presented results from cross - national , cross - sectional analysis supporting the specification that countries with lower inequality had higher life expectancy . although rodgers , and later wilkinson , articulated how the whole income distribution could make a difference , it is evident from the hypothesis that what should particularly make a difference is how the relatively poor fare and how large a fraction of the population is at risk of poverty . the topic of income inequality and health has become a small research industry within social epidemiology , with some influences from economics and sociology , and numerous studies have been published , especially on the relationship between income inequality across american states and various health outcomes . a meta - analysis of multilevel studies linking income inequality to mortality and self - rated health lent support to the idea . in cross - national analyses , not least with regard to population health and poverty , it has become common procedure to group countries according to their specific mix of welfare production , that is , welfare regime [ 15 , 16 ] . as noted by several authors [ 17 , 18 ] , the welfare modelling business has become a central part of welfare state research , starting with esping - andersen 's famous trichotomy that he labelled on the basis of main political ideologies : the liberal , social - democratic , and conservative / corporatist regimes . the idea behind the regime approach goes beyond the welfare state in the stricter sense by looking at the nexus of the state , markets , and family . although our overall aim is to study the link between poverty and mortality , it is of obvious interest to note variations in this relationship by welfare state regime and to adjust our analyses by regime . not least within the news project , initiated in collaboration with the who commission on social determinants of health , a number of studies were produced linking the specific design , generosity and coverage of social policy programmes to overall and age - specific mortality on the one hand , and to morbidity on the other [ 2024 ] . these studies focused on the cash side of the welfare state and supported the idea that cash programmes of the welfare state have been of importance to public health during the second half of the 20th century . however , the ability of these programmes to alleviate poverty was often referred to as a key factor in cross - national variations in mortality rates . of course , the programmes of the welfare state are likely to also influence other more proximal health - related factors that could influence mortality risks . in this study we will explore the relative poverty argument directly , by making use of the best sources for comparative studies on poverty and mortality over a 25-year period . although we will not examine the mechanisms , it is still necessary to briefly mention some of the possible multiple pathways linking relative poverty and mortality . the question is whether the incidence of relative poverty has delayed or prevented a fall in mortality in the countries included in our analysis . our two main data sources are the luxembourg income study ( lis ) and the human mortality database ( hmd ) . it has a focus on income inequality and poverty , but also includes a great deal of information on aspects such as family situation , and employment status . wave 1 started around 1980 with approximate five - year intervals , so that wave 6 of the data is from around 2005 ( for a thorough presentation of the database see ) . the lis is commonly regarded as the best source of cross - national comparisons of poverty and income inequality . in our study , we include all countries from the lis that have at least two waves of data from the same original survey source , and for these countries , all lis waves for which mortality data were also available in the hmd for corresponding years . mortality rates were assessed for three gender - specific age classes : infants ( aged < 1 year ) , children ( aged 117 years ) , and adults ( aged 2564 years ) . data on deaths and populations at risk were collected for one - year age bands for each country from the hmd for all lis waves , and for three following years of each wave . while infant mortality rates were used as such , age - standardized mortality rates for the age groups 117 and 2564 were calculated to adjust for the different age structures of the countries . in these calculations the age - standardized rates thus represent what the crude rates would be if the populations of the countries had the same age distribution as the european standard population . evidently , the nature of poverty in terms of both the size of income and , for the countries analysed here , its consequences will become more severe the further we move from the national median . with the data at hand , we can not have a perfect age match between the poverty rates and the mortality rates . thus , the total child poverty rates are used as the exposure for both our child mortality analyses , and there is also a one - year mismatch for the adults . the lis wave number is included to allow for time - related changes in poverty and mortality rates . a more nuanced way of studying the impact of welfare spending would have been to use disaggregate spending data , that is , to separate cash and in kind benefits used for children , elderly , health care , various income maintenance programs , and so forth . our analytical approach is first to inspect bivariate plots to observe the general pattern of the relationship between age - specific mortality rates and the background variables . we start by looking at developmental pattern in mortality over cross - sections and welfare regimes . for regression analyses , we used pooled cross - sectional time - series methods . although partially solving the small n - problems , the pooled cross - sectional time - series method results in problems of spatial and longitudinal autocorrelation and heterogeneity . pooled regressions were run using the stata 12 cross - sectional time - series package using prais - winsten regressions . in order to further test the robustness of our results the results were robust for the different methods applied , and although the standard errors varied the interpretations of the results did not . figures 2(a)2(f ) show the magnitude and variability of infant , child , and adult standardized mortality rates by sex in different welfare state regimes and over time . the first observation from the box - plot figure is that infant mortality rates among boys are higher than among girls ( 7.99 per 1,000 boys and 6.43 per 1,000 girls , on average across countries and time points ) . as can be seen , the mortality rates are the lowest in the nordic group ( on average 4.56 for girls and 5.92 for boys ) and there is relatively small variation between the four countries included in this cluster . on the other end of the continuum , we find the former socialist countries to have both the highest mortality rates and the highest variation between nations , but also the highest absolute decline in infant mortality . there is substantial variation over time as well , and as evident in figure 2(b ) , there is a downward trend over the waves concerning both levels and cross - national variation . in pooled data , that is , where all cross - sections are merged into one , the relationships between predictors and infant mortality rates are in the expected direction but not always convincingly high . however , gdp is not only an indicator of economic prosperity but also represents a more general modernization trend that includes better food , better health care , better sanitation , access to clean water , and so forth factors regarded as important in combatting infant mortality . the second strongest correlation in the pooled data is the one between mortality and social spending , representing the magnitude of the public commitment to the social protection of the populace . here as well the pattern is rather constant over cross - sections ( figure 3(a ) ) : the larger the share of gdp that is made up of social spending , the lower the infant mortality rate . contrary to our initial expectations , the link between infant mortality and child poverty rates is also relatively low in the pooled data . whereas relative poverty is rather strongly correlated with mortality in the first and last waves , the correlations in waves 2 and 3 are rather weak . our interim conclusion is that the level of prosperity of the country and the magnitude of the welfare state matter , and that the impact of the welfare state is mirrored in lower levels of child poverty and inequality , which in turn partially combat new - born deaths . in the last model ( 4 ) the nordic welfare regime is used as a reference and is left out of the models . however , detailed results of the gender - specific analyses can be found in tables 5(a ) and 5(b ) . the coefficient of the association between poverty and logged mortality rate from this model can be statistically interpreted as follows : a one percentage - point increase in child poverty corresponds to about a 2% increase in infant mortality . the statistical explanation for the strong attenuation of the poverty estimate when social spending is added is the strong association between social spending and poverty rates . so it seems that the welfare state matters for relative poverty , and relative poverty matters for infant mortality . finally , when welfare regimes are introduced ( model 4 ) , the poverty estimate remains about the same . the welfare regimes obviously capture not only different welfare state characteristics but also different levels of economic prosperity , since the coefficient for gdp totally disappears . in model 4 , controlling for poverty , wave , gdp , social spending and welfare regime , infant mortality rates are significantly higher in central european , liberal , and especially postsocialist regime types compared to the benchmarking nordic regime ( the reference category ) , while the southern european and other regimes do not significantly deviate from the nordic one . these regime differences are notable , especially if one bears in mind that they are not captured by differences in poverty , economic prosperity , or social spending . this evident variation between the regime types highlights that the causes of differences in population health statistics are multifactorial , and we are not able to fully capture this with the variables in our regression models . when we move from the new - borns to older children , the risk of death radically diminishes . figures 2(c ) and 2(d ) give the variability in these rates by welfare state regime type and across waves . again , there is an overrepresentation of boys in the death toll ( the average age - standardized mortality rate over countries and time points is 0.22 per 1,000 girls and 0.33 per 1,000 boys aged 117 ) . in the postsocialist regime , the average death rate ( 0.27 among girls , 0.42 among boys ) is about 1.5-fold compared to the low nordic numbers ( 0.17 among girls , 0.26 among boys ) . in this age group , the country group stands out with relatively high mortality rates but also very large variation . figure 3(b ) shows the crude relations between the pooled data of the age - standardized death rates and the three main explanatory factors . in table 3 we show the results of our pooled cross - sectional time series analysis for this age group for girls and boys together , because the results of gender - specific analyses proved to be almost identical in terms of regression coefficients ( see tables 6(a ) and 6(b ) ) . the analytical strategy is basically the same as for infants , although the logged age - standardized mortality rates are now calculated as the average of the lis years ' mortality plus the following three years ' mortality to allow for exposure time on mortality after our poverty measurements . in terms of mortality among children aged 117 , the liberal and southern european regimes fare significantly better compared to the nordic regime after the other covariates are adjusted for . figure 3(c ) once again displays the well - known curvilinear relationship between mortality and gdp per capita ; the mirror picture of this is the relationship between gdp and life expectancy . although the relationship is somewhat different in different waves , the general message is that the bivariate plots basically show no association . results from pooled cross - sectional regressions are shown separately for women ( table 4(a ) ) and men ( table 4(b ) ) . in general , the association between poverty and mortality is weaker in the models for the adult population than for children . compared to the nordic regime , the central and especially southern european regime types show statistically significantly lower mortality rates , whereas the postsocialist regime shows higher mortality rates among women when poverty , wave , gdp , and social spending are controlled for . turning to the results for men ( table 4(b ) ) , the picture is somewhat less clear . apart from this cluster we can note that , in comparison to the nordic regime , the southern european , liberal , and other regimes have lower adjusted male mortality rates . we have performed a number of sensitivity analyses with regard to the inclusion / exclusion of countries and setting a higher poverty threshold . we also tested the impact of income inequality ( as expressed by the gini coefficient ) . we argue that the 40% poverty threshold comes closer to the absolute poverty level , not least combined with the national wealth indicator ( gdp ) , than the 60% poverty threshold , which comes closer to income inequality measured using the gini coefficient . during the time span covered by this study , russia had high poverty rates and extreme death risks , especially visible among adult males therefore , when we reran all regressions omitting russia , the estimates changed substantially . in the end , in line with babones in his comparative analysis of income inequality and health , we note that a major complication for any fixed effects model is the remarkable stability for both variables over time . however , it should be noted that some of the fixed effects are still included through the dummies for the lis wave and the welfare state regimes . in summary , it seems that the relative poverty rates are of importance to child mortality for the sample of countries and the period examined . the magnitudes of deprivation among children and the effect of the association vary between countries and have also been shown to be influenced by the design of the welfare state , such as choices of social policy / redistribution . the aim of this study was to analyse the effect of relative poverty upon mortality rates among three age groups infants , children , and working - age adults , also stratified by gender . our time period is 19802005 , and we have an unbalanced time series for 26 countries belonging to the rich world but also including postsocialist countries from eastern europe . we have recently seen a number of studies that go beyond the cross - sectional picture between income inequality and mortality [ 14 , 42 , 46 , 47 ] . to our knowledge , this is one of the first studies to go beyond the cross - sectional picture with a focus on poverty rather than inequality . there is ample evidence of profound differences in poverty across welfare regimes [ 4850 ] , suggesting that poverty , welfare regime , and mortality also may be interrelated . a statistical explanation for this is the strong and robust association between poverty and social spending . when thinking about the order of impacts , one can , with overwhelming empirical support , argue that social spending is causally related to poverty : the higher the spending level in a country , the lower the poverty levels . we anticipated that if we took into consideration the welfare regimes ' belongingness , the relative role of poverty rates would be eaten up . however , for children the effect on the poverty estimate was negligible , and for adults the inclusion of welfare regime fortified the connection between poverty and mortality . this does not influence the poverty and mortality association for children , but it is important to note that the regime type as such has a clear influence on child mortality , even when controlling for gdp and social spending . the reason for this is not self - evident , but from earlier research we know that several of the southern european countries are ranked at the top of life expectancy figures in europe and worldwide . in a sense , the regime variable captures whatever it is that is specifically health - promoting in these countries , and the resulting poverty estimate is thereby adjusted for that regime - specific aspect . this result , in turn , indicates that welfare state and poverty have an impact on mortality , but there are other factors in play , such as drinking and eating habits and the way healthy and unhealthy behaviour is distributed between socioeconomic groups , according to income and education attainment levels . our study is somewhat different to most of the cross - country studies linking poverty and mortality . they have used either a more worldwide inclusion of countries ( but the question then becomes somewhat different ) or a much smaller sample of countries and have particularly been totally cross - sectional in their design . moreover , we have used age - specific analysis when it comes to both poverty calculations and mortality rates , thereby further specifying the tests . from cross - national poverty analyses we know that universal , redistributive social policies are key instruments in reducing poverty ; if such policies are also coupled with social investment policies for young children , such as high - quality day care , this not only reduces poverty here and now but is also likely to be a good investment for the future .

the theme of infanticide has received increasing attention from historians in recent years . reasons for this growing interest include curiosity into the daily lives of poor women , and also the sharp contrast between a strong ideology of motherhood and a crime that seems to completely contradict the image of maternity . scholars have studied the socio - economic backgrounds of these women , their trials and punishments , as well as the influence of enlightenment humanitarianism , including sympathy for the plight of child murderers , who came to be seen as victims rather than criminals . dutch historians have mostly focused on the socio - economic backgrounds of child murderers in the eighteenth , and to a lesser extent , nineteenth century . these studies all indicate that most of the women accused of newborn child murder were unmarried servants who tried to hide their pregnancy and birth out of shame . fearing the loss of their jobs and livelihood as unmarried mothers , as well as their honour , they resorted to murdering their babies . similar to research in other countries , dutch historians have assumed only a limited number of cases of child murder were actually tried before the courts , pointing to an unknown dark number. besides the socio - economic background and the presumed motivations of these murdering mothers , historians have studied the dutch laws on infanticide , including the sentences in practice , which i will elaborate on below . here , it is relevant to note that dutch scholars have paid scant attention to the role of forensic medicine and psychiatry in court cases of infanticide , even though doctors were indispensable in cases of infanticide . from the early modern period physicians were called in to examine a dead baby s body , to establish whether it was stillborn or born alive and possibly murdered . sometimes the bodies of women who were accused of infanticide were searched for signs of recent pregnancy . physicians vehemently debated the crime of child murder and the weight of their medical findings in these cases , both amongst themselves and with legal scholars . this article explores the influence of forensic doctors and psychiatrists in dutch court cases of infanticide in the period 18111911 . in 1811 the french code pnal was introduced in the netherlands and in 1911 the concept of puerperal insanity ( insanity during or directly after birth ) was used for the first time in court , as part of the question whether jannetje j. , a servant girl , was accountable for her crime . in contrast to england , where reference to puerperal insanity was used as a line of defence since 1822 , in the netherlands this concept did not play a role in nineteenth and early twentieth century court cases of child murder . in order to account for this absence , first i suggest more attention is paid to the connections between body and mind in contemporary formulations of affect , which provided room to discuss forms of insanity without the need to resort to the new science of psychiatry . second , i propose a new approach to the history of forensic medicine , emphasising travelling knowledge. i have adapted the concept of travelling knowledge from insights presented by scholars from different disciplines . historians and sociologists of knowledge have already stressed how knowledge is socially constructed and negotiated . in particular , science and technology studies have demonstrated how knowledge is produced in laboratories and how factual information comes to be seen as such . the idea of travelling knowledge has mostly been applied to the appropriation of scientific knowledge from other continents in the early modern period . historians have traced how scientific knowledge has been passed on between people , countries and continents , showing that the process of travel [ ] involves much more than simply moving something from one place to another so that , in the end , it remains the same even if found elsewhere . there is no displacement , whether it be of people , instruments , objects , or data , without some sort of work , material as well as social , which enables their extraction from a given place and context , and insertion in a new place and new set of relations . the process of travel [ ] involves much more than simply moving something from one place to another so that , in the end , it remains the same even if found elsewhere . there is no displacement , whether it be of people , instruments , objects , or data , without some sort of work , material as well as social , which enables their extraction from a given place and context , and insertion in a new place and new set of relations . not only in the field of history of science has the mobility of knowledge been emphasised . cultural theorist mieke bal , as well , has introduced the notion of travelling concepts in the humanities , showing how certain concepts travel between academic disciplines , in the meantime changing these disciplines , while also changing themselves . in a similar vein , literary critic and theorist stephen greenblatt recently presented a mobility studies manifesto , urging scholars to take seriously the concept of mobility in cultural history . greenblatt advocates studying mobility in a literal sense , pointing to the physical , infrastructural , and institutional conditions of movement , but he also encourages us to pay attention to hidden as well as conspicuous movements of peoples , objects , images , texts , and ideas . importantly , greenblatt underscores that mobility studies should identify and analyse the contact zones where cultural goods are exchanged. applying these ideas on cultural mobility to medical history , i propose to trace the way medical knowledge travelled to the courtroom and , on its way , was adapted to the judicial context . from this perspective , the courtroom might be regarded as a contact zone with its own rules of behaviour , shaping the insights from the medical field as doctors and psychiatrists were called in as expert witnesses . these rules might change when the law is reformulated , when medical expertise changes , or when other cultural images or expectations influence the judicial process . in this article , i aim to show how medical knowledge of mania puerperalis , or puerperal insanity , travelled from england and germany to the netherlands , but , more importantly , which barriers subsequently prevented this knowledge from being applied in court cases of infanticide . the courtroom might be seen as a contact zone between the medical and judicial fields , both fields stimulating the exchange of knowledge , but also dismissing the appropriation of new ideas . the concept of travelling knowledge might help the formulation of theoretical approaches to the history of forensic medicine . although the history of forensic medicine has recently received more attention , the field remains undertheorised . often , it is presented as the unfolding of better technological knowledge , like the introduction of chemical tests , culminating in the application of dna , that ultimate instrument of truth , in court cases in the 1980s . however , progressivist histories of medicine have been under scrutiny for some decades , for instance by cultural historians , who underline the social construction of science , medicine and disease . social - constructionist approaches have , however , hardly been applied to the history of forensic medicine some important works excepted . for instance , in his study of nineteenth - century norwegian practices of forensic psychiatry , sklevg distinguishes between two approaches . the first he designates as the historical sociology of professions , exploring the growing power of medicine and the law as professions , their competition and their pursuit for greater public esteem . the second perspective revolves around a foucauldian discourse analysis and regards medicine and the law as cooperative , not as competing branches of the medico - legal apparatus in the nineteenth century . in the latter approach , an impersonal scientific discourse , disciplining criminals , is seen to have taken precedence over a specific body of lawyers , judges or doctors . sklevg eventually regards both approaches as complementary , but found little evidence for prestige as a strategic professional goal , thereby concluding that the model of professional interest does not carry sufficient analytical potential . rather than describing the history of forensic psychiatry as a medicalization of law , sklevg shows how , in norway , psychiatrists in the nineteenth century also cooperated with the law , and sometimes opposed general medicine . by focusing on broader contemporary cultural conceptions of mankind , he concludes that a separation between body and mind in the course of the nineteenth century opened up a space for psychiatrists to claim expertise on the mind . in addition to his emphasis on the body mind dichotomy and his focus on shifting alliances between medicine , psychiatry and law , i would like to propose another perspective , that of travelling knowledge , to show how knowledge from the field of medicine was transferred to the courts , or , in the case of puerperal insanity , was barred from the legal arena . in order to apply this concept to dutch court cases of infanticide , i have studied the legal records of all ( fifty - seven ) cases of infanticide tried before the courts of north - holland and amsterdam . in addition to these records , my sources include textbooks of forensic medicine as well as legal and medical texts on the role of physicians and psychiatrists in cases of child murder . before i analyse these sources , however , i will sketch the characteristics of dutch laws on infanticide in the nineteenth century . in 1774 the enlightened humanitarian dutch doctor petrus camper advocated leniency for women accused of child murder . camper pointed to the stringent social laws on marriage which made unmarried mothers into victims , rather than perpetrators . however , he was also criticised for his views . in other countries , as well , enlightened ideas on crime and punishment influenced debates on infanticide . thomas laqueur has shown how humanitarian narrative in its attention to the body not only as the locus of pain but also as the common bond between those who suffer and those who would help , fostered compassion and empathy . sheena sommers , applying laqueur s concept of the humanitarian narrative to eighteenth - century court cases of infanticide , argues that medical expertise of physicians , having replaced the traditional knowledge of midwives , left room for empathy towards the murdering mothers , regardless of or even because of its uncertainty . mark jackson critically discusses the contemporary , progressivist cultural discourse on humanitarianism in relation to crime and punishment . according to jackson , a new medical discourse and the increasing influence of scientific testimonies in court were more influential in the changing view on crime and punishment . moreover , the role of forensic medicine and psychiatry in promoting legal reform was small , especially in comparison with england . in the eighteenth century the numbers of women prosecuted for infanticide in the netherlands were relatively low and the verdicts lenient : judges were unwilling to sentence the perpetrators to death . although punishment became less severe from the end of the eighteenth century , resulting from increasing sympathy for the plight of unmarried mothers , the death penalty for child murder was not abolished in the netherlands until 1854 . in 1811 the dutch adopted the french code pnal , which penalised infanticide with the death penalty , leading to a high number of acquittals since the materialist french law hardly allowed for extenuating circumstances and the death penalty was seen as too severe by many people . between 1811 and 1870 about fifty death sentences were proclaimed for child murder in the netherlands , but most of the convicts received pardon from the king and were subsequently given prison sentences of fifteen to twenty years . generally , acquittal on the basis of insanity was possible , but partial non - responsibility did not exist , probably explaining why , until the late nineteenth century , few medical doctors were called on to establish the sanity of the accused in dutch courts of law . the code pnal , used in the netherlands until 1886 , was based on the enlightenment principle of sentencing criminals proportionately to the crime committed , dismissing the personality of the criminal to avoid arbitrariness . this focus on material evidence is also thought to have contributed to the small role of psychiatrists in court cases . liberal and social reformers succeeded in reforming infanticide law only in 1854 , when the death penalty was abolished for unmarried women who committed infanticide for the first time and was replaced by a prison sentence of five to ten years . in 1870 the death penalty was abolished for all other cases of child murder . in 1886 , when a new dutch criminal law was introduced , the death penalty as such was abolished and more attention was paid to the strong fear experienced by the murdering mother . the law from 1854 explicitly mentioned the unmarried mother s fear of discovery of her pregnancy , and her resulting abnormal psychic and physical condition before and during delivery , as a reason to kill her child . in 1886 importantly , this law stated that , unlike in other crimes , a plea for extenuating circumstances was impossible , since these circumstances had already been assumed in the specific description of the crime in the law : the section on child murder mentioned the execution of a decision made under the influence of fear of the discovery of her upcoming delivery. this probably contributed to a dismissal of the overall mental condition of the mother . since the law already assumed these women s state of bewilderment during delivery , further inquiry as to the cause would be unnecessary . the new dutch penal code from 1886 furthermore introduced a distinction between murder and manslaughter of children , the difference depending on the moment ( before or during the delivery ) when the mother had decided to kill her baby . in contrast to england , where infanticide was mostly prosecuted as murder or manslaughter until 1922 , child murder was a separate section in dutch law and a law punishing the hiding of a pregnancy in and of itself did not exist . the age of the newborn child was , moreover , neither specified in the french code pnal nor in the new dutch laws introduced in the second half of the nineteenth century . these new laws took into account that child murder was a last resort for mostly poor , unmarried mothers and therefore showed awareness of the difficult plight of these women . this new cultural awareness dovetailed with sympathy for prostitutes , who were increasingly regarded as victims of male lust as well . in the netherlands , christian politicians and feminists advocated the abolition of the regulation of prostitution , especially in the second half of the nineteenth century and the first decades of the twentieth . nevertheless , prostitution and adultery were much more important themes in this political struggle over female sexuality than infanticide . in the netherlands so far only one case of infanticide has been found during which psychiatrists were called in as expert witnesses to establish whether the accused had been suffering from insanity , a condition that could potentially exonerate her . in general , few dutch doctors were invited into the courtroom to assess the mental condition of criminals . in france , for instance , psychiatrists became involved in court cases to establish the accountability of suspects in the 1820s , coining the concept of monomania . nevertheless , despite many french medical studies on puerperal insanity , it seems that the latter notion was rarely applied in cases of child murder before 1901 . in germany as well , physicians expertise was part of legal proceedings from the eighteenth century . in cases of infanticide , eighteenth - century german doctors sometimes described the mental condition of the mother , but did not use the concept of mania puerperalis , although german physicians had published on that topic since the seventeenth century . subsequently , psychiatrists seem to have played a minor role in dutch , german and french cases of infanticide . their lack of involvement stands in sharp contrast to their colleagues in england , however , probably relating to the different legal systems . the adversarial nature of legal proceedings in england , as contrasted with the inquisitorial system in continental law , could explain this difference . local physicians and professors in medicine were only called in randomly as expert witnesses in court . in contrast to france , there were no court - appointed physicians in the netherlands . a law from 1838 determined that expert statements were not to be considered as evidence by and of themselves . donker and faber have suggested that the late application of the psychiatric concept of mania puerperalis in the netherlands was due to the specific formulations of the dutch laws on infanticide , in combination with the emphasis on material evidence like physical proof and witness statements in the dutch legal system . however , they have neither done extensive research to back up this suggestion , nor examined the much broader and important role of forensic medicine as opposed to forensic psychiatry in cases of infanticide . specifically , their isolated focus on the mind dismisses in advance more complicated and entangled body mind constellations . this article , therefore , further explores the cause of the extremely late reference to potential insanity in cases of infanticide , compared to britain , by analysing textbooks of forensic medicine , medical and legal journals , in addition to court cases . i will describe the relationship between body and mind in court cases of infanticide and argue that the emphasis on material evidence and the particular formulation of dutch laws on infanticide were indeed grounds for a late application of the concept of mania puerperalis , as donker and faber have suggested . in addition i propose to take into account the contemporary cultural and medical discourses on the connections between body and mind , which left enough space to articulate mental confusion without referring to academic psychiatric concepts and thus made the acceptance of new psychiatric knowledge less urgent , thus explaining why this knowledge did not travel to the courtroom . therefore in the following paragraphs i explore legal records , medical textbooks and articles in medical and legal journals to see whether the possible causes of the scant dutch attention to mental aspects in cases of infanticide ( a general focus on material evidence in the code pnal ; no options for partial accountability ; and the impossibility of pleas for extenuating circumstances from 1854 ) , as suggested in the secondary literature , can be corroborated . i will argue that all of these factors played a role , but do not suffice to explain the minor significance of states of mind . equally important are the presence of a vocabulary on emotion , both medical and vernacular , which accorded room to mental aspects . in this particular discourse , body and mind in order to account for the late entrance of psychiatric expertise to dutch cases of infanticide , the first question we need to address is whether the concept of mania puerperalis was known in the netherlands . whereas french , german and english physicians and psychiatrists developed this notion , sometimes in relation to infanticide , this knowledge did not complete its journey to the netherlands , as this section will show . interestingly , the notion was introduced by a dutch doctor who had read foreign texts describing the concept , but for several reasons it was not regarded as important enough to be used on a regular basis by dutch physicians or legal scholars . although medical theories on madness caused by pregnancy and childbirth had circulated since antiquity mostly seen as caused by the sedimentation of the mother s milk in the brain the french psychiatrist jean - tienne dominique esquirol ( 17721840 ) dismissed the paradigm of humours and began the empirical observation of women s psychoses directly after childbirth . he located the origins of mental illness in the passions of the soul , rather than in organic causes . several german doctors had written on post - partum mania and melancholia from the seventeenth century , viewing them until the beginning of the nineteenth century as intellectual disturbances and regarding the affective components of the psychoses merely as epiphenomena . until the middle of the nineteenth century , german psychiatrists generally included all forms of post - partum psychosis under the heading of mania puerperarum , including those with melancholic symptoms , not yet distinguishing between insanity and mood disorder . nancy theriot has described how the term puerperal insanity was constructed by nineteenth - century american doctors and patients . most physicians divided the disease into phases of the reproductive process ( pregnancy , lactation , parturition ) or into maniacal , melancholic and depressive puerperal insanity . insanity of parturition or puerperal mania , characterised by maniacal symptoms like an abnormal state of excitement and a hostility to husband or child , was regarded as the most common type of puerperal insanity . theriot argues the disease was not only a product of male physicians notions of proper femininity , but also reflected contemporary ideas on the connections between mind and body in mental illness . puerperal insanity can be interpreted as a socially constructed disease , reflecting both the gender constraints of the nineteenth century and the professional battles accompanying medical specialization. in these french , german and american medical discussions on the aetiology of puerperal insanity , the crime of infanticide hardly played a role . another french psychiatrist , however , louis - victor marc , elaborated on the connection between puerperal madness and child murder . marc regarded women s reproductive functions as vital causes of mental illness , but also left room for hereditary predispositions and emotional triggers . focusing mostly on physical causes of the mental instabilities associated with pregnancy and the postpartum period , marc suggested that , although more research had to be done into the possible exoneration of the mother accused of infanticide based on her mental conditions , this was certainly an option . in england , the london obstetrician dr robert gooch produced the first detailed account in english of puerperal insanity , described by hilary marland as very much a disorder of the nineteenth century and from 1822 puerperal insanity was used in defence pleas , mediating between the wrath provoked by high levels of child murder and the sympathetic approach towards mothers who committed the crime. as daniel grey has recently shown , the diagnosis remained a frequent feature in englishwomen s infanticide trials until the first two decades of the twentieth century . in ireland , as well , the concept was often put forward in cases of newborn child murder , and the link was made in the medical literature from the second half of the nineteenth century . however , in america considerable resistance was shown toward the application of this psychiatric notion to late nineteenth- and early twentieth - century infanticide cases . janet galley has suggested three reasons why american doctors , lawyers and the general public rejected the connection between infanticide and female insanity . first , the american system of state - based criminal law implied that there was neither a uniform legal response to infanticide or insanity , nor national studies into the phenomenon . second , at the end of the nineteenth century , the number of infanticide cases in america decreased due to the creation of foundling homes and homes for unwed mothers , as well as the declining cost of rubber condoms . the lower number of cases led to fewer demands on state legal communities to change the existing laws in relation to the crime of infanticide . third , many americans regarded the insanity defence in general as a way for criminals to escape their punishment . american beliefs in free will and the republican ideal of individual responsibility worked against the notion of unaccountability on the basis of mental illness . these american arguments against the use of the insanity defence in cases of infanticide differed completely from the dutch ones . in the netherlands , mania puerperalis was described in dutch for the first time by dr a. moll in 1822 in a medical journal , summarising the research by gooch , based on an article in a german journal . moll , who wrote the first dutch textbook of forensic medicine , published in 1825 , briefly mentioned mania puerperalis in his chapter on insanity and accountability , classifying it under melancholia , but did not refer to the psychological condition of parturient women in the chapter on infanticide , which was limited to the material and physical traces on the bodies of baby and mother . therefore , contrary to what other dutch historians have assumed , the notion of mania puerperalis or puerperal insanity was introduced in the netherlands in 1822 . nevertheless , in the medical literature few connections were made with insanity pleas in cases of infanticide . it would be incorrect , however , to state that forensic doctors did not display any interest in the mental state of the mother in these cases . although most of the authors of textbooks of forensic medicine did not explicitly mention mania puerperalis , several devoted some lines to the psychic condition of the mother and its relationship to legal accountability . already at the end of the eighteenth century , the aforementioned enlightened doctor petrus camper referred to the emotions experienced by women during pregnancy and delivery . whereas pregnancy makes even married women melancholic , camper noted , unmarried , solitary women , who are despised by the community , experience fear , pain and desperation during delivery , leading in that terrible moment of deep emotion to the killing of their child . camper s reference to emotions as causing women s disturbance during childbirth was very common in medical and alienist literature and quite easily overlapped with early psychiatric notions . in 1824 , for instance , medical doctor j.h . de he s agreed with camper s plea for a more humane treatment of he claimed that , in the netherlands , the advice given by camper fifty years ago , to give heed to the psychic condition of the mother , seems to have disappeared. de he s located the cause for this disappearance in the decline of dutch forensic medicine , especially in comparison with germany , thus siding with many other doctors who complained about the sorry state of dutch forensic science during the nineteenth century . camper was regarded by de he s as far ahead of his time , applying the teachings of psychic diseases to the practice of law in a period in which this was still very imperfect . referring to moll and van eldik s article from 1822 , de he s underlined the existence of melancholia and mania before , during and after childbirth . he located the cause of puerperal insanity in the genitals , triggered by emotional upheaval . he left open the possibility of these women being prone to the disease by a certain condition of the brain . referring to wigand , de he s saw the sudden surge of blood to the head , increasing the passions , as prompting madness . it was the task of the forensic physician to find out if violence committed to a child had been performed in a state of melancholia or mania . de he s , then , was one of the few forensic doctors who explicitly advocated attention to the mind of the mother . he regarded this focus , however , as in line with older notions of emotional upheaval , indicating how the new psychiatric discourse fused with an already existing discourse on emotions . thus the notion of puerperal mania travelled to the netherlands from france , germany and england . although it was taken up by moll and de he s , several factors might have contributed to halting this notion from further travel to the courtroom . second , it overlapped to an important extent with an older vocabulary of emotion , thus making its application less urgent , as i will explain below . third , a contemporary , omnipresent notion of the inadequacy of dutch forensic medicine might also account for the lack of interest in the application of new research to forensic medicine . moreover , another explanatory factor might have been the disagreement over the mania women possibly suffered in regard to childbirth . legal scholar de feyfer in his dissertation treatise on child murder ( 1866 ) pointed to the lack of agreement between scholars in regard to the temporary insanity of child murderesses during and after birth . still , he admitted that unmarried women , who often had a very quick delivery because of their vehement emotions , could easily attain a state of mental confusion . thus far , forensic doctors had not paid enough attention to the psychic condition of the mother , according to de feyfer , having focused too much on the material facts , whereas there was every reason to regard them as not fully accountable for their actions . heeding the tight connection between soul and body in human beings , these women s physical sufferings caused a limitation of the rational mind . in de feyfer s text , the connection between body and mind was central , emotions being inextricably connected to the psyche moreover , he confirmed the neglect by dutch forensic doctors of the mental condition of criminal mothers , pointing to the material aspects of dutch law . this claim was underlined by dr w. koster , who also concluded in his textbook of forensic medicine from 1877 that , in most cases , the forensic doctor only had to unveil the facts relevant to finding whether the baby was violently killed , without mentioning the psychic state of the mother . whereas during the first half of the nineteenth century , doctors were mostly concerned with classifying puerperal insanity and discussed its existence in general , during the second half of the nineteenth century they debated whether post - partum madness could serve to claim diminished legal responsibility . as i have shown above , the french psychiatrist marc was the first to suggest that puerperal insanity could actually function as such . however , the famous french forensic doctor tardieu disagreed with him in a text written in 1868 . both tardieu and , in 1897 , pathologist and forensic scholar paul brouardel doubted the existence of acute insanity during and directly after childbirth . in their textbook of forensic obstetrics ( 1908 ) , the dutch doctor hector treub and the legal scholar a. tak refuted the opinions of these french scholars . treub and tak , referring to austrian - german psychiatrist richard von krafft - ebing s studies on consciousness and mania , regarded attacks of mania transitoria in parturient and recently delivered women and transitory deliria on an epileptic and hysterical basis as potential grounds for non - accountability . simon thomas had argued in the dutch journal for criminal law that the emphasis on fear in the 1886 dutch law on infanticide , and the description of special affects in the accompanying explanatory memorandum , did not imply a reference to a psychic condition that made women unaccountable for their actions . thus medical and legal opinions on the existence of puerperal insanity , as well as its relationship to unaccountability , continued to differ at the beginning of the twentieth century . to summarise , we have seen that knowledge of the concept of mania puerperalis existed in the netherlands , introduced by moll . fleeting references in dutch medical journals , as well as a limited number of allusions in other texts on forensic medicine , indicate an awareness of the condition . however , several barriers served to keep this knowledge from being generally accepted and used in court cases . some regarded the mentioning of fear in the law itself as a reason not to look for further evidence of psychic illness . last , it seems that the discussion of emotions in relation to infanticide left considerable room to include psychic components . for example , in an article in a dutch medical journal , the anonymous authors vaguely referred to new research into anthropology and psychology to argue that the term this adjective was intended to leave space for a lenient sentence for the mother , since the unmarried mother , directly after birth , and after the recently suffered labour pains , has not yet felt full love for the new - born baby , and the birth itself so powerfully impacts on the body and mind of the woman , to be able to assume , that the murder committed immediately after birth was committed in a sickly and psychically excited condition . since , according to the authors , this phase of physical and psychic confusion could not be pinned down to an exact time frame , the term importantly , the authors use the term abnormal somatic condition , referring to the interconnection of body and mind . i shall explore this relationship between body , mind and affect further by looking at the role of forensic doctors in dutch court cases of infanticide in order to account for the barriers that prevented psychiatric expertise from travelling to the courtroom , in contrast to medical expertise on the body of the child , which did find its way to court . the unmarried mother , directly after birth , and after the recently suffered labour pains , has not yet felt full love for the new - born baby , and the birth itself so powerfully impacts on the body and mind of the woman , to be able to assume , that the murder committed immediately after birth was committed in a sickly and psychically excited condition . physicians played an important role in cases of infanticide . in nearly every instance , doctors were called in to inspect the body of the dead baby , to establish whether it had been born alive . they assessed whether it was full term , and they searched for signs of violence . an autopsy was also performed to show the condition of the inner organs , especially of the lungs , and included the findings of the infamous lung test . this test , invented at the end of the seventeenth century , consisted of placing the lungs in a bucket of water in order to see whether they sank or floated . floating lungs were thought to indicate the presence of oxygen , hence pointing to a live birth . it was considered to be faulty , especially since the lungs could contain gas that was often present in corpses , which could also make them float . throughout the eighteenth and nineteenth centuries , physicians studied more accurate lung tests and other signs to use in addition to the test . nevertheless , it continued to be used , although only as one part of increasingly extensive post - mortem reports . in all the cases consulted for which sufficient documentation has been preserved , the lung test was executed and , in all of these except for one where the results were inconclusive it was concluded that the baby had been born alive . it then depended on the confession of the accused , witness statements and other material evidence whether the judge would convict the mother . it is , however , difficult to reconstruct the exact weight of the forensic evidence . especially in cases dating from the first half of the nineteenth century , judges and lawyers often approached forensic evidence critically . in a case from 1839 , for instance , a judge criticised the results of a lung test , indicating that the child , having respired , had been born alive and strangled by the umbilical cord . he argued that a child could have breathed before and during birth , yet still be born dead . since neither these signs , nor the other evidence , provided enough certainty , the accused was acquitted . similarly , in a case from 1852 the judge deliberated that the lung test was considered an one explanation for the qualms expressed by judges in regard to the lung test might be that they were reluctant to sentence women accused of infanticide to death , a dilemma they no longer faced in the second half of the nineteenth century . in addition , as the visum repertum ( autopsy report ) became more extensive , the lung test decreased in importance , as other bodily evidence was increasingly taken into account . still , until the beginning of the twentieth century lawyers for the defence would invite professors of medicine to court to underscore the uncertainty of the lung test . the different values attached to the lung test by people in different locations with differing interests points to the shape that knowledge might take while travelling to other places . whereas in medicine , as evidenced by textbooks of forensic medicine , the lung test could be promoted wholeheartedly or with some reserve , in the courtroom different criteria applied . for example , in a case from 1845 the defence called in a professor of medicine and a medical doctor as additional witnesses , who claimed the lung test only presumed that breathing took place , and that there were examples of breathing and life before birth . similarly , in a case from 1857 the defence lawyer included professor of medicine van geuns , who was critical of the premises of the medical report . that the child had been mature and breathing at birth could be concluded from the report with some measure of probability , but not as strong evidence. the judge admitted that the medical report had not provided undisputed certainty , but a high degree of probability was enough to give reinou vette , a thirty - four - year - old female worker , the death penalty . thus forensic doctors appointed by the authorities often claimed certainty in regard to the results of the lung test , but when this knowledge travelled to the courtroom the judge or the medical experts hired by the defence transformed this into probability or a lack of certainty . disagreement amongst the several experts involved in the investigation and court case could cause severe problems . in a case from 1822 the defence s request for more experts was rejected by the judge , since that would give the impression that the report had been subjected to the judgement of other experts , which would be little respectful to the experts appointed by the judges , and which will make it impossible to find experts in future , which is already difficult now . to question further experts , professors against professors cannot be advantageous for the interests of justice . the report had been subjected to the judgement of other experts , which would be little respectful to the experts appointed by the judges , and which will make it impossible to find experts in future , which is already difficult now . to question further experts , professors against professors equally , in texts on forensic medicine authors were concerned with the image of forensic medicine if several doctors disagreed with each other in public court cases . ten houte de lange complained that forensic medicine in holland was a disgrace for science , a plaything for the cleverness of jurists. in a case dating from 1884 , the accused was acquitted because the three experts interrogated at the trial contradicted the findings of the post - mortem performed by two other doctors , even though she had made a confession : she had put her fingers in the mouth and throat of the baby , but the experts disagreed over whether that had actually caused the death or whether the child had died because of problems experienced during birth . the court eventually ruled that , because of the great differences in expert opinion , the guilt of the accused had not been sufficiently proved . in a similar case , the doctors disagreed over the cause of death ( bleeding or suffocation ) and therefore it could not be proved that the mother had murdered her baby by pulling a string attached to the baby s neck . paradoxically , although medical expertise was considered a vital element of the evidence sought in cases of infanticide , it was not accepted uncritically by judges , especially in the first half of the nineteenth century , possibly to avoid sentencing the accused women to death . generally , however , in the second half of the nineteenth century , the court agreed with expert opinion when it came to bodily and material evidence . the important role of the forensic doctor in regard to the examination of bodies contrasts with the lack of expertise sought in the realm of the mind . the absence of forensic psychiatrists and their scientific vocabulary , however , does not imply reference to the mind was completely absent . to express the state of mind of the accused during or directly after delivery , as well as her accountability , the vocabulary of emotion was appropriated . confusion or a baffled emotional state . however , the language of affect could probe more deeply into the mind , as evidenced by expressions like bewilderment of affect ( verbijsterde gemoedsaandoening ) , fears in movements of the soul ( angsten in zielsontroering ) , utterly moved , desperate , and sufficiently devoid of senses ( geheel ontroerd , wanhoopend en genoegzaam zinnenloos ) . when , for instance , in 1882 twenty - one - year - old servant elisabeth kelder told the court that , during the delivery , her heart had felt like stone and she herself had felt mad like a crazy person , the records do not evidence any response by the judge and a medical expert was not called in . similarly , in 1885 twenty - two - year - old servant jantje van tuil described her condition during her delivery as nearly bordering on madness , preventing her from thinking . they underlined her rational behaviour , leading the court to hold the defendant accountable for her crime . these could refer to a state of consciousness , and hence to potential responsibility and accountability . in the following case , dating from 1888 , this allusion to affect comes clearly to the fore : twenty - four - year - old servant helena van krenk at first claimed not to remember whether she had pinched her baby in the neck before or after its death . neither could she recall if the umbilical cord was torn before or after the child had suffocated . dr luchtmans stated that women in the circumstances experienced by the accused were not generally in a normal condition . however , in his opinion this also depended on the individual , since one person gets into affect more easily than others. since he had neither observed the suspect before , nor after the birth , he had never seen her in affect. he had the impression she had a calm character . the term in affect was not a conventional medical term and here equated emotion , consciousness and memory . the lower court first exonerated her from child murder , since premeditation had not been proved . she was convicted of manslaughter and sentenced to a prison sentence of one year and five months . the court of appeal , however , ruled that the killing of her child had been premeditated and convicted her of infanticide , with a prison sentence of two years and six months . thus eventually the medical evidence on the accused s mental condition did not contribute to her acquittal . expert opinion could , however , endorse exoneration in regard to the speed of birth , corroborating women s claims . many women testified in court that their babies had escaped them during birth and had fallen on the floor or in the lavatory , causing their deaths . interestingly , forensic doctors connected body and mind in their explanation of fast deliveries , as in the case of maria van dam , a thirty - three - year - old servant accused of infanticide : the inconsistency in her answers [ during interrogation wr ] testifies to the desperation of the prisoner both before , during and after the delivery , causing her to be unable to notice the events and to give a full testimony of them [ ] the emotions of a girl surprised by labour can be deregulated by fear so badly that she is hardly conscious of all that is happening , and can not clearly represent them ; also , these affects influence these parts used during labour this fear especially works on the functioning of the womb which might cause convulsions of the womb , possibly triggering a faster delivery . the inconsistency in her answers [ during interrogation wr ] testifies to the desperation of the prisoner both before , during and after the delivery , causing her to be unable to notice the events and to give a full testimony of them [ ] the emotions of a girl surprised by labour can be deregulated by fear so badly that she is hardly conscious of all that is happening , and can not clearly represent them ; also , these affects influence these parts used during labour this fear especially works on the functioning of the womb which might cause convulsions of the womb , possibly triggering a faster delivery . importantly , this reference to the mind is very much connected to the body and used to explain the fast and sudden delivery , rather than the motive of the accused and her possible unsound mind and lack of responsibility for her actions . the sheer importance of the discourse on affect is shown in the terms used during the trial of jannetje jurgens , the first case in the netherlands where psychiatrists asserted that the female perpetrator probably suffered from a mental condition while committing child murder . dr herman schoo , who was one of the two physicians who conducted the autopsy , but was also asked for his opinion on the mental state of the suspect , stated that in his opinion the suspect had committed the crime in a fear psychosis. the other expert , dr jacob voorhoeve , suggested that the accused might not have known what she was doing , that is , did not commit the crime in full consciousness. physician dr frederik meijers wrote in his report on the mental state of jannetje that she was suffering from acute nervous oedema , indicated by her thick , swollen tongue. meijers described how the fear of her pregnancy being discovered led to a state of sickly excitement of the nerves , which decreases her accountability to a minimum , but can not be thought to be completely absent. dr schoo agreed with him during the appeal and added that , although he had spoken of fear psychosis during the first trial , he had meant a sickly excitement of the nerves , the wording used by meijers in his report . both experts furthermore stated that the fact that jannetje had hardly felt any pain during the delivery pointed to her pathological excitement of the nerves , the lack of feeling being one of the symptoms of this nervous condition . clearly , the physical experience of pain during delivery was directly connected to the woman s mental condition . this should not surprise us , considering that from the early days of psychiatry for instance , in the description of monomania the status of the passions or affections had been unclear and the difference between normal and pathological passions was not specified . thus a considerable overlap between the language of psychiatry and the more common language of affect remained . this language could , but did not always , explain the particular mental and physical condition of the female suspect and exonerate her . the strong focus of the dutch law on material evidence , however , meant that the state of mind of the accused was hardly ever the sole focus of the interrogation and therefore rarely served as the only line of defence . this article aimed to provide new source material to study the absence of psychiatric expertise in dutch cases of infanticide , and to explore the use of the concept of travelling knowledge as an approach to the history of forensic medicine . travelling knowledge might refer literally to medical knowledge and its potential application in the field of law , being received and dispersed in a different country . in the case of mania puerperalis , the dutch doctor moll introduced it into the netherlands by referring to the work of the english doctor gooch , having read about it in a german journal . more interestingly , however , travelling knowledge can point to the fields of medicine and law as contact zones inviting , promoting or barring the transmission of knowledge . in the netherlands , the concept of mania puerperalis was not applied to actual court cases of infanticide for several reasons . first , physicians and legal scholars continued to doubt the existence of this form of mania . also , the specific formulation of the law strongly determined what medical evidence was needed in court cases . not only did the code pnal generally emphasise material evidence , the laws on infanticide specifically mentioned fear and therefore additional reference to a mental condition of the accused was not needed . most importantly , i argue , the existing vocabulary on emotion , both vernacular and medical , allowed for an analysis of psychic components , connecting body and mind in the description of the mental state of the mother during and after birth , as well as of the rapidity of her delivery . to conclude , specific national formulations of the law might have been the biggest barrier to the application of psychiatric knowledge , while the presence of an older , useful vocabulary of affect might have been a more hidden obstacle to the introduction of new mental concepts .

this article aims to explain why the notion of mania puerperalis , or puerperal insanity , was not used in the netherlands to exonerate women accused of infanticide , in contrast to other countries . it applies the concept of travelling knowledge as an approach to the history of forensic medicine , pointing to the fields of medicine and law as contact zones inviting , promoting or barring the transmission of knowledge . although the notion of mania puerperalis was known in the netherlands from 1822 on , psychiatric expertise was not requested in actual court cases of infanticide for several reasons . first , physicians and legal scholars continued to doubt the existence of this form of mania . moreover , it was not always directly connected to infanticide . also , the specific formulation of the law strongly determined what medical evidence was needed in court cases . not only did the code pnal generally emphasise material evidence , the laws on infanticide specifically mentioned fear and therefore an additional reference to the mental condition of the accused was not needed . most importantly , the article argues that the existing vocabulary on emotion , both vernacular and medical , already allowed for an analysis of psychic components .

Introduction Dutch Laws on Infanticide Puerperal Insanity and Travelling Knowledge Court Cases of Infanticide Expert Opinion and Affect Conclusion

similar to research in other countries , dutch historians have assumed only a limited number of cases of child murder were actually tried before the courts , pointing to an unknown dark number. here , it is relevant to note that dutch scholars have paid scant attention to the role of forensic medicine and psychiatry in court cases of infanticide , even though doctors were indispensable in cases of infanticide . this article explores the influence of forensic doctors and psychiatrists in dutch court cases of infanticide in the period 18111911 . in 1811 the french code pnal was introduced in the netherlands and in 1911 the concept of puerperal insanity ( insanity during or directly after birth ) was used for the first time in court , as part of the question whether jannetje j. , a servant girl , was accountable for her crime . in contrast to england , where reference to puerperal insanity was used as a line of defence since 1822 , in the netherlands this concept did not play a role in nineteenth and early twentieth century court cases of child murder . second , i propose a new approach to the history of forensic medicine , emphasising travelling knowledge. i have adapted the concept of travelling knowledge from insights presented by scholars from different disciplines . the idea of travelling knowledge has mostly been applied to the appropriation of scientific knowledge from other continents in the early modern period . historians have traced how scientific knowledge has been passed on between people , countries and continents , showing that the process of travel [ ] involves much more than simply moving something from one place to another so that , in the end , it remains the same even if found elsewhere . not only in the field of history of science has the mobility of knowledge been emphasised . cultural theorist mieke bal , as well , has introduced the notion of travelling concepts in the humanities , showing how certain concepts travel between academic disciplines , in the meantime changing these disciplines , while also changing themselves . in this article , i aim to show how medical knowledge of mania puerperalis , or puerperal insanity , travelled from england and germany to the netherlands , but , more importantly , which barriers subsequently prevented this knowledge from being applied in court cases of infanticide . the concept of travelling knowledge might help the formulation of theoretical approaches to the history of forensic medicine . although the history of forensic medicine has recently received more attention , the field remains undertheorised . often , it is presented as the unfolding of better technological knowledge , like the introduction of chemical tests , culminating in the application of dna , that ultimate instrument of truth , in court cases in the 1980s . social - constructionist approaches have , however , hardly been applied to the history of forensic medicine some important works excepted . the first he designates as the historical sociology of professions , exploring the growing power of medicine and the law as professions , their competition and their pursuit for greater public esteem . the second perspective revolves around a foucauldian discourse analysis and regards medicine and the law as cooperative , not as competing branches of the medico - legal apparatus in the nineteenth century . rather than describing the history of forensic psychiatry as a medicalization of law , sklevg shows how , in norway , psychiatrists in the nineteenth century also cooperated with the law , and sometimes opposed general medicine . in addition to his emphasis on the body mind dichotomy and his focus on shifting alliances between medicine , psychiatry and law , i would like to propose another perspective , that of travelling knowledge , to show how knowledge from the field of medicine was transferred to the courts , or , in the case of puerperal insanity , was barred from the legal arena . in order to apply this concept to dutch court cases of infanticide , i have studied the legal records of all ( fifty - seven ) cases of infanticide tried before the courts of north - holland and amsterdam . in addition to these records , my sources include textbooks of forensic medicine as well as legal and medical texts on the role of physicians and psychiatrists in cases of child murder . before i analyse these sources , however , i will sketch the characteristics of dutch laws on infanticide in the nineteenth century . sheena sommers , applying laqueur s concept of the humanitarian narrative to eighteenth - century court cases of infanticide , argues that medical expertise of physicians , having replaced the traditional knowledge of midwives , left room for empathy towards the murdering mothers , regardless of or even because of its uncertainty . moreover , the role of forensic medicine and psychiatry in promoting legal reform was small , especially in comparison with england . although punishment became less severe from the end of the eighteenth century , resulting from increasing sympathy for the plight of unmarried mothers , the death penalty for child murder was not abolished in the netherlands until 1854 . the code pnal , used in the netherlands until 1886 , was based on the enlightenment principle of sentencing criminals proportionately to the crime committed , dismissing the personality of the criminal to avoid arbitrariness . this focus on material evidence is also thought to have contributed to the small role of psychiatrists in court cases . in 1886 importantly , this law stated that , unlike in other crimes , a plea for extenuating circumstances was impossible , since these circumstances had already been assumed in the specific description of the crime in the law : the section on child murder mentioned the execution of a decision made under the influence of fear of the discovery of her upcoming delivery. the age of the newborn child was , moreover , neither specified in the french code pnal nor in the new dutch laws introduced in the second half of the nineteenth century . in the netherlands so far only one case of infanticide has been found during which psychiatrists were called in as expert witnesses to establish whether the accused had been suffering from insanity , a condition that could potentially exonerate her . in general , few dutch doctors were invited into the courtroom to assess the mental condition of criminals . in france , for instance , psychiatrists became involved in court cases to establish the accountability of suspects in the 1820s , coining the concept of monomania . nevertheless , despite many french medical studies on puerperal insanity , it seems that the latter notion was rarely applied in cases of child murder before 1901 . in cases of infanticide , eighteenth - century german doctors sometimes described the mental condition of the mother , but did not use the concept of mania puerperalis , although german physicians had published on that topic since the seventeenth century . in contrast to france , there were no court - appointed physicians in the netherlands . donker and faber have suggested that the late application of the psychiatric concept of mania puerperalis in the netherlands was due to the specific formulations of the dutch laws on infanticide , in combination with the emphasis on material evidence like physical proof and witness statements in the dutch legal system . however , they have neither done extensive research to back up this suggestion , nor examined the much broader and important role of forensic medicine as opposed to forensic psychiatry in cases of infanticide . this article , therefore , further explores the cause of the extremely late reference to potential insanity in cases of infanticide , compared to britain , by analysing textbooks of forensic medicine , medical and legal journals , in addition to court cases . i will describe the relationship between body and mind in court cases of infanticide and argue that the emphasis on material evidence and the particular formulation of dutch laws on infanticide were indeed grounds for a late application of the concept of mania puerperalis , as donker and faber have suggested . therefore in the following paragraphs i explore legal records , medical textbooks and articles in medical and legal journals to see whether the possible causes of the scant dutch attention to mental aspects in cases of infanticide ( a general focus on material evidence in the code pnal ; no options for partial accountability ; and the impossibility of pleas for extenuating circumstances from 1854 ) , as suggested in the secondary literature , can be corroborated . equally important are the presence of a vocabulary on emotion , both medical and vernacular , which accorded room to mental aspects . in this particular discourse , body and mind in order to account for the late entrance of psychiatric expertise to dutch cases of infanticide , the first question we need to address is whether the concept of mania puerperalis was known in the netherlands . whereas french , german and english physicians and psychiatrists developed this notion , sometimes in relation to infanticide , this knowledge did not complete its journey to the netherlands , as this section will show . interestingly , the notion was introduced by a dutch doctor who had read foreign texts describing the concept , but for several reasons it was not regarded as important enough to be used on a regular basis by dutch physicians or legal scholars . in these french , german and american medical discussions on the aetiology of puerperal insanity , the crime of infanticide hardly played a role . focusing mostly on physical causes of the mental instabilities associated with pregnancy and the postpartum period , marc suggested that , although more research had to be done into the possible exoneration of the mother accused of infanticide based on her mental conditions , this was certainly an option . in england , the london obstetrician dr robert gooch produced the first detailed account in english of puerperal insanity , described by hilary marland as very much a disorder of the nineteenth century and from 1822 puerperal insanity was used in defence pleas , mediating between the wrath provoked by high levels of child murder and the sympathetic approach towards mothers who committed the crime. in ireland , as well , the concept was often put forward in cases of newborn child murder , and the link was made in the medical literature from the second half of the nineteenth century . first , the american system of state - based criminal law implied that there was neither a uniform legal response to infanticide or insanity , nor national studies into the phenomenon . second , at the end of the nineteenth century , the number of infanticide cases in america decreased due to the creation of foundling homes and homes for unwed mothers , as well as the declining cost of rubber condoms . in the netherlands , mania puerperalis was described in dutch for the first time by dr a. moll in 1822 in a medical journal , summarising the research by gooch , based on an article in a german journal . moll , who wrote the first dutch textbook of forensic medicine , published in 1825 , briefly mentioned mania puerperalis in his chapter on insanity and accountability , classifying it under melancholia , but did not refer to the psychological condition of parturient women in the chapter on infanticide , which was limited to the material and physical traces on the bodies of baby and mother . therefore , contrary to what other dutch historians have assumed , the notion of mania puerperalis or puerperal insanity was introduced in the netherlands in 1822 . nevertheless , in the medical literature few connections were made with insanity pleas in cases of infanticide . although most of the authors of textbooks of forensic medicine did not explicitly mention mania puerperalis , several devoted some lines to the psychic condition of the mother and its relationship to legal accountability . already at the end of the eighteenth century , the aforementioned enlightened doctor petrus camper referred to the emotions experienced by women during pregnancy and delivery . de he s agreed with camper s plea for a more humane treatment of he claimed that , in the netherlands , the advice given by camper fifty years ago , to give heed to the psychic condition of the mother , seems to have disappeared. thus the notion of puerperal mania travelled to the netherlands from france , germany and england . third , a contemporary , omnipresent notion of the inadequacy of dutch forensic medicine might also account for the lack of interest in the application of new research to forensic medicine . in de feyfer s text , the connection between body and mind was central , emotions being inextricably connected to the psyche moreover , he confirmed the neglect by dutch forensic doctors of the mental condition of criminal mothers , pointing to the material aspects of dutch law . this claim was underlined by dr w. koster , who also concluded in his textbook of forensic medicine from 1877 that , in most cases , the forensic doctor only had to unveil the facts relevant to finding whether the baby was violently killed , without mentioning the psychic state of the mother . simon thomas had argued in the dutch journal for criminal law that the emphasis on fear in the 1886 dutch law on infanticide , and the description of special affects in the accompanying explanatory memorandum , did not imply a reference to a psychic condition that made women unaccountable for their actions . thus medical and legal opinions on the existence of puerperal insanity , as well as its relationship to unaccountability , continued to differ at the beginning of the twentieth century . to summarise , we have seen that knowledge of the concept of mania puerperalis existed in the netherlands , introduced by moll . last , it seems that the discussion of emotions in relation to infanticide left considerable room to include psychic components . for example , in an article in a dutch medical journal , the anonymous authors vaguely referred to new research into anthropology and psychology to argue that the term this adjective was intended to leave space for a lenient sentence for the mother , since the unmarried mother , directly after birth , and after the recently suffered labour pains , has not yet felt full love for the new - born baby , and the birth itself so powerfully impacts on the body and mind of the woman , to be able to assume , that the murder committed immediately after birth was committed in a sickly and psychically excited condition . i shall explore this relationship between body , mind and affect further by looking at the role of forensic doctors in dutch court cases of infanticide in order to account for the barriers that prevented psychiatric expertise from travelling to the courtroom , in contrast to medical expertise on the body of the child , which did find its way to court . in all the cases consulted for which sufficient documentation has been preserved , the lung test was executed and , in all of these except for one where the results were inconclusive it was concluded that the baby had been born alive . since neither these signs , nor the other evidence , provided enough certainty , the accused was acquitted . similarly , in a case from 1852 the judge deliberated that the lung test was considered an one explanation for the qualms expressed by judges in regard to the lung test might be that they were reluctant to sentence women accused of infanticide to death , a dilemma they no longer faced in the second half of the nineteenth century . whereas in medicine , as evidenced by textbooks of forensic medicine , the lung test could be promoted wholeheartedly or with some reserve , in the courtroom different criteria applied . to question further experts , professors against professors equally , in texts on forensic medicine authors were concerned with the image of forensic medicine if several doctors disagreed with each other in public court cases . in a case dating from 1884 , the accused was acquitted because the three experts interrogated at the trial contradicted the findings of the post - mortem performed by two other doctors , even though she had made a confession : she had put her fingers in the mouth and throat of the baby , but the experts disagreed over whether that had actually caused the death or whether the child had died because of problems experienced during birth . in a similar case , the doctors disagreed over the cause of death ( bleeding or suffocation ) and therefore it could not be proved that the mother had murdered her baby by pulling a string attached to the baby s neck . paradoxically , although medical expertise was considered a vital element of the evidence sought in cases of infanticide , it was not accepted uncritically by judges , especially in the first half of the nineteenth century , possibly to avoid sentencing the accused women to death . generally , however , in the second half of the nineteenth century , the court agreed with expert opinion when it came to bodily and material evidence . interestingly , forensic doctors connected body and mind in their explanation of fast deliveries , as in the case of maria van dam , a thirty - three - year - old servant accused of infanticide : the inconsistency in her answers [ during interrogation wr ] testifies to the desperation of the prisoner both before , during and after the delivery , causing her to be unable to notice the events and to give a full testimony of them [ ] the emotions of a girl surprised by labour can be deregulated by fear so badly that she is hardly conscious of all that is happening , and can not clearly represent them ; also , these affects influence these parts used during labour this fear especially works on the functioning of the womb which might cause convulsions of the womb , possibly triggering a faster delivery . importantly , this reference to the mind is very much connected to the body and used to explain the fast and sudden delivery , rather than the motive of the accused and her possible unsound mind and lack of responsibility for her actions . the sheer importance of the discourse on affect is shown in the terms used during the trial of jannetje jurgens , the first case in the netherlands where psychiatrists asserted that the female perpetrator probably suffered from a mental condition while committing child murder . both experts furthermore stated that the fact that jannetje had hardly felt any pain during the delivery pointed to her pathological excitement of the nerves , the lack of feeling being one of the symptoms of this nervous condition . clearly , the physical experience of pain during delivery was directly connected to the woman s mental condition . this should not surprise us , considering that from the early days of psychiatry for instance , in the description of monomania the status of the passions or affections had been unclear and the difference between normal and pathological passions was not specified . the strong focus of the dutch law on material evidence , however , meant that the state of mind of the accused was hardly ever the sole focus of the interrogation and therefore rarely served as the only line of defence . this article aimed to provide new source material to study the absence of psychiatric expertise in dutch cases of infanticide , and to explore the use of the concept of travelling knowledge as an approach to the history of forensic medicine . in the case of mania puerperalis , the dutch doctor moll introduced it into the netherlands by referring to the work of the english doctor gooch , having read about it in a german journal . more interestingly , however , travelling knowledge can point to the fields of medicine and law as contact zones inviting , promoting or barring the transmission of knowledge . in the netherlands , the concept of mania puerperalis was not applied to actual court cases of infanticide for several reasons . first , physicians and legal scholars continued to doubt the existence of this form of mania . also , the specific formulation of the law strongly determined what medical evidence was needed in court cases . not only did the code pnal generally emphasise material evidence , the laws on infanticide specifically mentioned fear and therefore additional reference to a mental condition of the accused was not needed . most importantly , i argue , the existing vocabulary on emotion , both vernacular and medical , allowed for an analysis of psychic components , connecting body and mind in the description of the mental state of the mother during and after birth , as well as of the rapidity of her delivery .

interstrand cross - links are among the most deleterious types of damage that can be generated in cellular dna . these lesions are complete blocks to dna transcription and replication because the covalent linkage prevents strand separation . concurrent modification of both strands in the duplex also presents severe challenges to cellular dna repair systems . accordingly , even small numbers of interstrand dna we recently reported a new structural type of interstrand cross - link derived from the reaction of a dna abasic ( ap ) site with a guanine residue on the opposing strand of the duplex ( scheme 1 ) . this cross - link is intriguing because ap sites ( 1 , scheme 2 ) are perhaps the most common type of endogenous damage suffered by cellular dna . ap sites are generated by spontaneous depurination , nitrosative stress , and enzymatic base excision repair processes . as a result , dna from normal mammalian tissue carries steady - state levels of 50 000200 000 ap sites per cell . ap sites are also generated by exposure of dna to a wide variety of mutagens , toxins , and anticancer drugs . cross - links derived from this abundant lesion could have significance in biology and medicine . in our previous work , dg - ap cross - links were observed at 5-cap/5-ag sequences ( duplex a , figure 1 ) in equilibrium yields of 23% , under physiologically relevant conditions . the evidence supported a mechanism involving attack of the exocyclic n - amino group of guanine on the ap aldehyde ( scheme 1 ) . two other canonical nucleobases , adenine and cytosine , also contain exocyclic amino groups that have been shown to react with aldehydes . however , our previous studies provided no indication regarding whether adenine or cytosine can participate in cross - link formation with an ap site in duplex dna . no cross - linking was observed at the adenine residue directly opposing the ap site in duplex a ( figure 1 ) . cross - links involving adenine residues have been observed in dna duplexes containing oxidized abasic sites ; however , it is important to recognize that the structures and reactivities of oxidized abasic sites are distinct from that of the native ap site considered here . dna duplexes used in this study . ap - containing duplexes were generated from the corresponding du - containing duplexes by treatment with udg . locations where cross - links form in substantial yield are indicated with a ( red \ ) . in order to grasp the full potential of ap sites to generate cross - links in duplex dna , we felt it was necessary to examine whether canonical nucleobases other than guanine can forge cross - links with ap sites in dna . dna cross - link involving the reaction of adenine residues with ap sites in double - helical dna . the da - ap cross - links described here formed in remarkably high yields ( 1570% ) under physiologically relevant conditions and we present evidence consistent with a cross - linking mechanism involving attack of the exocyclic n - amino group of adenine on the aldehyde group of the ap site . the widespread occurrence of ap sites in cellular dna combined with the high yields of cross - links observed here raise the possibility that this naturally occurring , dna - templated reaction can generate cross - links in the genetic material of living cells . when considering nucleobase sequences in duplex dna that might support formation of da - ap cross - links , we first recognized that any nucleobase involved in ap - derived cross - link formation likely must be located near the ap site . this is because cross - linking by a distal nucleobase presumably would generate substantial and energetically unfavorable structural distortion in the duplex . with this in mind , we constructed models of ap - containing dna duplexes to determine sequence locations on the opposing strand that would position the n - amino group of adenine in close proximity to the ap site . in these models , we employed the simplifying assumption that ap - containing duplexes retain b - dna - like character . this approach successfully predicted cross - linking sequences in the case of dg - ap cross - links . measurments within these models indicated that the exocyclic n - amino groups of both adenine residues opposing the ap site in a 5-apt/5-aa sequence were located near the ap aldehyde residue ( figure 2 ) . as noted in the introduction , however , our previous studies showed that adenine residues directly opposing an ap site ( e.g. , duplex a ) did not engage in cross - link formation . overall , this analysis led us to the prediction that an adenine residue offset one base to the 3-side of the ap site might have potential to engage the ap - aldehyde in cross - link formation ( figure 2 ) . molecular model illustrating the proximity of the abasic site aldehyde and n - amino group of 2-deoxyadenosine in a 5-apt sequence in b - dna . ap sites were produced at defined locations in dna duplexes by treatment of the corresponding 5-p - labeled , 2-deoxyuridine - containing duplexes with uracil dna glycosylase ( udg ) . efficient formation of ap sites in the dna was confirmed by piperidine treatment to generate the cleavage product 4 ( scheme 2 ; figure 3 , lanes 3 , 7 , and 12 ) . lanes 15 , duplex b ; lanes 69 , duplex c ; and lanes 1014 , duplex d. lanes 1 and 10 contain the p - labeled , uracil - containing oligodeoxynucleotides . lanes 2 , 6 , and 11 contain the p - labeled udg - treated ( abasic - site - containing ) duplexes without incubation . lanes 3 , 7 , and 12 contain the p - labeled , udg - treated ( abasic - site - containing ) duplexes subjected to piperidine workup ( 1 m , 95 c , 25 min ) to cleave the ap site . lanes 4 , 8 , and 13 contain the cross - linking reactions involving incubation of the abasic - site - containing duplexes for 120 h in hepes buffer ( 50 mm , ph 7 ) containing nacl ( 100 mm ) at 37 c . lanes 5 , 9 , and 14 contain the methoxyamine - capping reactions involving incubation of the abasic - site - containing duplexes in hepes buffer ( 50 mm , ph 7 ) containing nacl ( 100 mm ) and ch3onh2hcl ( 2 mm ) at 37 c . the p - labeled oligodeoxynucleotides were resolved by electrophoresis on a 20% denaturing polyacrylamide gel , and the radioactivity in each band quantitatively measured by phosphorimager analysis . the numbers on the left side of the image represent our structural assignment of the bands and referring to the structure numbers in schemes 2 and 3 . incubation of duplex b containing a central 5-gapt/5-aac sequence in hepes buffer ( 50 mm , ph 7 , 100 mm nacl ) at 37 c , followed by denaturing polyacrylamide gel electrophoretic analysis , revealed a 15.1 0.5% yield of a slow - migrating band in the region of the gel where the cross - linked duplex was expected to appear ( figure 3 , lane 4 ) . we also observed bands corresponding to the intact ap - containing oligonucleotide 1 and small amounts of a product inferred to be the elimination product 3 , based upon its gel mobility . duplexes c and d , equipped with 3-overhanging ends , generated slow - migrating bands whose gel mobility was retarded relative to the putative cross - link generated by duplex b ( figure 3 , lanes 8 and 13 ) . these results provided evidence that the slow - migrating bands were indeed cross - linked duplexes containing both full - length strands of the parent duplex and ruled out cross - link formation by the more strongly electrophilic 3-4-hydroxy-2-pentenal-5-phosphate end group 3 . inclusion of the aldehyde - trapping agent methoxyamine in the reactions substantially inhibited formation of the slow - migrating bands ( figure 3 , lanes 5 , 9 , and 14 ) , consistent with involvement of the ap - aldehyde residue in cross - link formation ( 2 , schemes 2 and 3 ) . it is noteworthy that cross - link formation in duplexes b d occurred in good yields ( 1318% ) at physiological ph and did not require conditions of reductive amination ( e.g. , ph 45 and nacnbh3 ) often used to capture the imine products resulting from the reaction of aldehydes and amines . a time course experiment showed that easily detectable amounts of the cross - link were formed in duplex b within a few hours ( figure s1 ) . cross - link formation was a robust reaction that was not highly sensitive to reaction conditions . for example , in duplex b , similar cross - link yields ( 16 3% ) were observed in hepes buffer ( 50 mm , ph 7 ; 100 mm nacl ) , cacodylate ( 50 mm , ph 7 , 100 mm nacl ) , nah2po4/citric acid ( 16.5/1.8 mm , ph 7.0 , 100 mm nacl ) , mops ( 50 mm , ph 7 , 100 mm nacl ) , and bis - tris ( 50 mm , ph 7.4 , 100 mm nacl ) and in the presence of the biological thiol glutathione ( 1 mm , in hepes 50 mm , ph 7 , 100 mm nacl ) ( figure s2 ) . the formation of low molecular weight imines and n - arylglycosides , analogous to those shown in scheme 3 , are reversible processes . this made it interesting to examine the stability of the cross - link generated in duplex b. we found that , once formed , the cross - link in duplex b was stable under a variety of conditions including ph 3 ( sodium acetate , 25 mm , 24 c , 30 min ) , ph 10 ( potassium phthalate , 24 c , 25 mm , 30 min ) or mild piperidine workup ( 0.1 m , 60 c , 15 min ) ( figure s3 ) . on the other hand , the cross - link was decomposed by heat ( 90 c , 15 min ) or treatment with the aldehyde - trapping reagent methoxyamine ( 200 mm , 30 min , 60 c ; figure s3 ) . to identify the site at which the ap - aldehyde was attached to the opposing strand , we carried out hydroxyl radical footprinting of a cross - linked duplex containing the 5-apt/5-aa sequence . the longer duplex e ( figure 1 ) was used to move the dna bands of interest away from a region of the gel where bands were obscured by a salt flare . in this duplex , the strand opposing the ap - containing oligodeoxynucleotide was 5-p - labeled . the cross - link was generated as described above , isolated from the gel , and subjected to cleavage by a mixture of iron - edta - h2o2 . in this type of experiment , the site of cross - linking appears as an interruption in the ladder of strand cleavage products generated by the iron - edta - h2o2 reagent , because cleavages beyond the cross - link yield large , slow - migrating dna fragments that are connected to the opposing strand . in the present case , a clear disruption in the ladder of cleavage products was observed at the 5-adenine residue in the 5-gapt/5-aac sequence ( residue a6 in duplex e , figures 1 and 4 ) . this provided evidence that cross - linking involves attachment of the ap - aldehyde to the adenine residue that is offset one base to the 3-side of the ap site . hydroxyl radical footprinting of duplex i to locate the site of cross - link attachment . duplex i contains the same core sequence as that in duplex b. ( a ) lane 1 is a maxam gilbert g - specific cleavage ( sequencing ) reaction of the labeled oligodeoxynucleotide strand in duplex i. lane 2 is an a+g specific cleavage ( sequencing ) reaction of the labeled oligodeoxynucleotide strand in duplex i. lane 3 is the hydroxyl radical footprinting reaction of the single stranded nonuracil containing oligodeoxynucleotide . lane 4 is the hydroxyl radical footprinting reaction of the slow - migrating , cross - link band generated by incubation of duplex i hepes buffer ( 50 mm , ph 7 ) containing nacl ( 100 mm ) at 37 c . the p - labeled oligodeoxynucleotides were resolved on a 20% denaturing sequencing gel , and the radioactivity in each band quantitatively measured by phosphorimager analysis . gilbert g - specific cleavage ( sequencing ) reaction of the labeled oligodeoxynucleotide strand in duplex i. ( c ) a+g specific cleavage ( sequencing ) reaction of the labeled oligodeoxynucleotide strand in duplex i. ( d ) hydroxyl radical footprinting reaction of the slow - migrating , cross - link band . we next examined the effect of substituting different nucleobases for the key adenine residue in the 5-gapt/5-aac sequence . substitution of this adenine residue in duplex b with a guanine or thymine residue , respectively , gave duplexes f and g that did not produce significant yields of a slow - migrating cross - link band ( figure 5 ) . similarly , duplex h containing 2-aminopurine at this position did not efficiently generate the cross - link ( figure 5 ) . the substitution of 2-aminopurine for the adenine residue amounts to the rather subtle relocation of a single exocyclic amino group from the major groove to the minor groove of the dna duplex ( see structures in figure 1 ) . thus , the failure of duplex h to generate significant yields of cross - link allowed us to infer that the exocyclic n - amino group of da in the 5-gapt/5-aac sequence was intimately involved in the cross - linking reactions described here . substitution of cytosine for adenine at the cross - linking site also did not yield cross - links ( figure s4 ) . cytosine places an exocyclic amino group in the major groove of dna similar to adenine , but the amino group of dc is located 0.7 closer to the sugar phosphate backbone than that of da ( the amino group of da is closer to the helical axis ) . nucleobase replacement experiments reveal that the underlined adenine residue in the 5-gapt/5-aac sequence of duplex b is critical for cross - link formation . replacement of this adenine residue in duplex b with a guanine or thymine residue in duplexes e and f , respectively , abrogates cross - link formation ( lanes 13 and 18 ) . replacement of the crucial adenine residue with 2-aminopurine in duplex g yields only small amounts of a slower - moving band ( lane 8) . lanes 15 , duplex b , lanes 69 , duplex g , lanes 1014 , duplex e , and lanes 1519 , duplex f. lanes 1 , 10 , and 15 contain p - labeled uracil - containing oligodeoxynucleotide duplexes . lanes 2 , 6 , 11 , and 16 contain the p - labeled udg - treated ( abasic - site - containing ) duplexes without incubation . lanes 3 , 7 , 12 , and 17 contain the abasic - site - containing duplexes subjected to piperidine workup ( 1 m , 95 c , 25 min ) to cleave the ap site . lanes 4 , 8 , 13 , and 18 contain the cross - linking reactions involving incubation of the abasic - site - containing duplexes in hepes buffer ( 50 mm , ph 7 ) containing nacl ( 100 mm ) at 37 c . lanes 5 , 9 , 14 , and 19 contain the methoxyamine - capping reactions involving incubation of the abasic - site - containing duplexes incubated in hepes buffer ( 50 mm , ph 7 ) containing nacl ( 100 mm ) , and ch3onh2hcl ( 2 mm ) at 37 c . the p - labeled oligodeoxynucleotides were resolved by electrophoresis on a 20% denaturing polyacrylamide gel and the radioactivity in each band quantitatively measured by phosphorimager analysis . the results described above were broadly consistent with a cross - linking mechanism involving reversible reaction of the exocyclic n - amino group on da with the ap aldehyde to generate hemiaminal , imine , cyclic hydroxyalkylhemiaminal , or enamine linkages at 5-gapt/5-aac sequences in duplex dna ( structures 6 - 9 ) . maldi - ms analysis of the cross - linked duplex b gave a strong [ m - h ] signal at m / z 12718.66 . this value is consistent with the imine 7 , cyclic hydroxylalkylhemiaminal 8 , or enamine 9 ( figure s5 ; calcd [ m h ] = 12719.4 , 20 ppm dev ) . literature precedents describing the properties of structurally related small molecules indicate that the cyclic hydroxylalkylhemiaminal structure 8 likely is favored . lc - ms / ms analysis provided additional insight regarding the chemical structure of the da - ap cross - link . the cross - linked duplex b ( without the 5-p label ) was excised from a 20% denaturing polyacrylamide gel , eluted from the gel slice , digested with a four - enzyme cocktail consisting of nuclease p1 , alkaline phosphatase , and phosphodiesterases i and ii , and subjected to lc - ms / ms analysis using previously reported experimental conditions . selected - ion chromatograms revealed two peaks eluting at 24.1 and 26.1 min displaying the relevant m / z 368252 transition anticipated for the neutral loss of 2-deoxyribose from the putative da - ap cross - link ( figure 6 ) . further cleavage of the m / z 252 ion gave rise to a dominant fragment ion at m / z 136 in ms / ms / ms ( figure s6 ) , which is identical to the ms / ms observed for the [ m + h ] ion of da , suggesting that the initial m / z 368252 transition arises via loss of the cross - linked 2-deoxyribose adduct from da ( figure 6c ) . the presence of multiple peaks in the lc - ms / ms ion chromatogram was consistent with the expectation that the anticipated cross - link remnant resulting from the digestion of 8 will exist as an equilibrating mixture of pyranose and furanose isomers ( scheme s1 ) . lc - esi - ms and ms / ms analysis of the mixture generated by digestion of the cross - linked oligodeoxynucleotide generated by incubation of duplex b in hepes buffer ( 50 mm , ph 7 ) containing nacl ( 100 mm ) at 37 c . ( a ) selected - ion chromatogram monitoring the neutral loss of 2-deoxyribose from the da - ap cross - link . ( b ) ms / ms arising from the fragmentation of the ion at m / z 368 . ( c ) possible structures for the ions observed at m / z 368 , 252 , and 136 in these experiments . we examined cross - link formation in duplex i that contained a central 5capt/5-aag sequence . this experiment was intriguing to us because , in this sequence context , the ap site has the potential to generate cross - links with either the guanine or adenine residue on the opposing strand ( residues colored red in duplex i shown in figure 1 ) . incubation of duplex i in hepes buffer ( 50 mm , ph 7 , 100 mm nacl ) at 37 c produced a remarkable 71.5 3.7% yield of a slow - migrating band in the region of the denaturing gel where the cross - linked duplexes migrate ( figure 7 ) . similar to the experiments described above in the context of the 5-gapt/5-aac sequence , the yield of the slow - moving band in the 5capt/5-aag sequence also was greatly diminished when the aldehyde - capping reagent methoxyamine was added to the reaction mixtures . formation rate and stability of the cross - link in this sequence was similar to that described above for the 5-gapt/5-aac sequence ( figures s7s9 ) . these results along with nanospray tof - ms data ( figure s11 ) confirmed that the slow - migrating band generated in the 5capt/5-aag sequence was an interstrand cross - linked duplex involving the ap - aldehyde residue . footprinting of a cross - linked duplex containing the 5capt/5-aag core sequence indicated that the cross - link attachment was to the underlined adenine residue in the 5capt/5-aag sequence ( figure s10 ) . the footprinting results showed no detectable cross - linking to the dg residue in this sequence . thus , the remarkable cross - link yield in this sequence appears to result from efficient formation of a da - ap cross - link rather than simultaneous formation of dg - ap and da - ap cross - links . interstrand cross - link formation in duplex i. lane 1 , 5-p - labeled uracil - containing precursor of oligodeoxynucleotide duplex i. lane 2 , 5-p - labeled abasic - site - containing oligodeoxynucleotide duplex i without incubation . lane 3 , 5-p - labeled abasic - site - containing oligodeoxynucleotide duplex i cleaved by treatment with piperidine ( 1 m , 95 c , 25 min ) . lane 4 , duplex i incubated in hepes buffer ( 50 mm , ph 7 ) containing nacl ( 100 mm ) at 37 c . lane 5 , duplex i incubated with ch3onh2 ( 2 mm ) in hepes buffer ( 50 mm , ph 7 ) containing nacl ( 100 mm ) at 37 c . the p - labeled oligodeoxynucleotides were resolved on a polyacrylamide gel , and the radioactivity in each band quantitatively measured by phosphorimager analysis . lc - ms / ms analysis of the products generated by nuclease p1 digestion of the cross - linked duplex i revealed the presence of a tetranucleotide with the ap - aldehyde conjugated with the underlined adenine from the 5-capt/5-aag sequence ( figure s12 ) , while the corresponding tetranucleotide with the ap - aldehyde being attached to the guanine residue was not detectable ( data not shown ) . additionally , lc - ms / ms analysis of the products formed from the digestion with a four - enzyme cocktail showed that the da - ap remnant is present at a level that is 2 orders of magnitude higher than its dg - ap counterpart ( figures s1314 ) . these results mesh with the footprinting data described above and indicate that the cross - linking occurs predominantly between the ap - aldehyde and the underlined adenine in the 5-capt/5-aag sequence . the results described here identified a novel cross - link involving reaction of a dna ap site with an adenine residue on the opposing strand of the double helix . it is now clear that both dg and da can engage in cross - linking with an ap site and two distinct cross - linking sequence motifs , 5-cap/5-ag and 5-apt/5-aa , have been defined as locations for the formation of ap - derived cross - links . the difference in the favored locations of da and dg residues in these cross - links , with the cross - linking nucleobase displaced to the 3- and 5-side of the ap site , respectively , is a natural consequence of the helical twist of duplex dna coupled with the fact that the n - amino group of adenine is located in the major groove while the n - amino group of guanine resides in the minor groove ( figure s15 ) . our results were consistent with a mechanism involving attack of the exocyclic n - amino group of adenine on the aldehyde residue of the ap site to generate a hemiaminal intermediate ( 6 , scheme 3 ) . the mass spectrometric data is consistent with subsequent dehydration of 6 to give the imine ( 7 ) , cyclic hydroxyalkylhemiaminal ( 8) , or enamine ( 9 ) product . the detailed chemical structure of the da - ap cross - link remains to be rigorously established using 2d - nmr , but chemical precedents involving structurally related small molecules suggest that the cyclic hydroxyalkylhemiaminal ( 8) likely will be the predominant form . the substantial stability of the da - ap cross - link generated in duplex b is generally consistent with a cyclic hydroxyalkylhemiaminal 8 , in which the reactive imine group of 7 has been masked by intramolecular attack of the hydroxyl group . despite the stability of the cross - link to many of the conditions examined here , the reaction is reversible , and it will be important to determine whether ap - derived cross - links have sufficient stability to block dna processing enzymes such as helicases and polymerases . it may be important to emphasize that the da - ap cross - links described here formed in good yields ( 1370% ) under physiologically relevant conditions ( ph 7 , 100 mm nacl ) and did not require conditions of reductive amination ( e.g. , ph 45 and nacnbh3 ) often used to stabilize and increase the yield of the imine products resulting from the reaction of aldehydes and amines . in the sequences examined to date , the yields of da - ap cross - links were much higher than those observed for the dg - ap cross - links , when both reactions are conducted under the same , physiologically relevant conditions ( ph 7 , in the absence of nacnbh3 ) . the sequence 5-capt/5-aag ( duplex i ) that , in principle , can form both dg - ap and da - ap cross - links , in fact , generates a remarkably high yield ( 70% ) of the da - ap cross - link . it seems likely that , in some duplexes such as i , multiple cross - linked species exist in dynamic equilibrium . the yield of each cross - linked species at equilibrium will be determined by the inherent abilities of each nucleobase to generate imine linkages with the ap aldehyde and by the ability of the dna duplex to position the amino and aldehyde groups in a manner that favors imine formation . thus , formation of these ap - derived cross - links is conceptually related to template - directed synthesis with dynamic combinatorial imine libraries , in which equilibrium sorting processes involving reversible imine formation among a mixture of various aldehydes and amines ultimately generate higher yields of the imine products that form the most stable ensembles with a templating molecule present in the reaction . our results established that the equilibrium yields of the da - ap cross - link can be quite high in some sequence contexts , and future work will systematically explore the effects of sequence variation on cross - link yields . the widespread occurrence of ap sites along with the facile nature of the cross - linking reactions described here make it interesting to consider the possibility that ap - derived cross - links can form in genomic dna . in cells , processes such as repair and strand cleavage ( as shown in scheme 2 ) will compete with the formation of ap - derived cross - links . as a result , the majority of ap sites in genomic dna almost certainly do not go forward to form interstrand dna dna cross - links . nonetheless , because interstrand cross - links are especially noxious lesions , even low yields of endogenous ap - derived cross - links could prove significant in several diverse areas . first , endogenous dna damage is thought to be important in the etiology of both human aging and sporadic cancers . because cross - links present severe challenges to replication , transcription , and repair , endogenously formed cross - links could be especially significant . unrepaired cross - links that block transcription and replication may promote cell death and senescence that contribute to the aging of tissues . at the same time , cellular attempts to repair cross - links are error prone and introduce cancer - causing mutations into the genome . in addition , the realization that ap sites can generate cross - links in duplex dna may help explain why pathways such as nucleotide excision repair and recombination that are typically associated with the repair of more complex lesions are required for eukaryotic cells to survive the introduction of ap sites into their genome . finally , ap - derived cross - links could be relevant to difficulties inherent in the amplification and sequencing of ancient dna . ancient dna samples contain a plethora of lesions including oxidative base damage , ap sites , and strand breaks , but it has been suggested that a high density of interstrand cross - links ultimately limit the ability to amplify and sequence the dna in these samples . the structural nature of the cross - links in ancient dna has not been elucidated , but ap - derived cross - links are good candidates for these blocking lesions that hinder sample processing . overall , these findings establish ap - derived interstrand cross - links as a family of dna lesions that can form in diverse sequence contexts . studies are currently underway to define the structure , occurrence , biochemical properties , and biological consequences of these lesions .

the loss of a coding nucleobase from the structure of dna is a common event that generates an abasic ( ap ) site ( 1 ) . ap sites exist as an equilibrating mixture of a cyclic hemiacetal and a ring - opened aldehyde . aldehydes are electrophilic functional groups that can form covalent adducts with nucleophilic sites in dna . thus , ap sites present a potentially reactive aldehyde as part of the internal structure of dna . here we report evidence that the aldehyde group of ap sites in duplex dna can form a covalent adduct with the n6-amino group of adenine residues on the opposing strand . the resulting interstrand dna dna cross - link occurs at 5-apt/5-aa sequences in remarkably high yields ( 1570% ) under physiologically relevant conditions . this naturally occurring dna - templated reaction has the potential to generate cross - links in the genetic material of living cells .

Introduction Results and Discussion Conclusions

accordingly , even small numbers of interstrand dna we recently reported a new structural type of interstrand cross - link derived from the reaction of a dna abasic ( ap ) site with a guanine residue on the opposing strand of the duplex ( scheme 1 ) . this cross - link is intriguing because ap sites ( 1 , scheme 2 ) are perhaps the most common type of endogenous damage suffered by cellular dna . in our previous work , dg - ap cross - links were observed at 5-cap/5-ag sequences ( duplex a , figure 1 ) in equilibrium yields of 23% , under physiologically relevant conditions . the evidence supported a mechanism involving attack of the exocyclic n - amino group of guanine on the ap aldehyde ( scheme 1 ) . however , our previous studies provided no indication regarding whether adenine or cytosine can participate in cross - link formation with an ap site in duplex dna . no cross - linking was observed at the adenine residue directly opposing the ap site in duplex a ( figure 1 ) . cross - links involving adenine residues have been observed in dna duplexes containing oxidized abasic sites ; however , it is important to recognize that the structures and reactivities of oxidized abasic sites are distinct from that of the native ap site considered here . in order to grasp the full potential of ap sites to generate cross - links in duplex dna , we felt it was necessary to examine whether canonical nucleobases other than guanine can forge cross - links with ap sites in dna . dna cross - link involving the reaction of adenine residues with ap sites in double - helical dna . the da - ap cross - links described here formed in remarkably high yields ( 1570% ) under physiologically relevant conditions and we present evidence consistent with a cross - linking mechanism involving attack of the exocyclic n - amino group of adenine on the aldehyde group of the ap site . the widespread occurrence of ap sites in cellular dna combined with the high yields of cross - links observed here raise the possibility that this naturally occurring , dna - templated reaction can generate cross - links in the genetic material of living cells . when considering nucleobase sequences in duplex dna that might support formation of da - ap cross - links , we first recognized that any nucleobase involved in ap - derived cross - link formation likely must be located near the ap site . with this in mind , we constructed models of ap - containing dna duplexes to determine sequence locations on the opposing strand that would position the n - amino group of adenine in close proximity to the ap site . this approach successfully predicted cross - linking sequences in the case of dg - ap cross - links . overall , this analysis led us to the prediction that an adenine residue offset one base to the 3-side of the ap site might have potential to engage the ap - aldehyde in cross - link formation ( figure 2 ) . ap sites were produced at defined locations in dna duplexes by treatment of the corresponding 5-p - labeled , 2-deoxyuridine - containing duplexes with uracil dna glycosylase ( udg ) . efficient formation of ap sites in the dna was confirmed by piperidine treatment to generate the cleavage product 4 ( scheme 2 ; figure 3 , lanes 3 , 7 , and 12 ) . incubation of duplex b containing a central 5-gapt/5-aac sequence in hepes buffer ( 50 mm , ph 7 , 100 mm nacl ) at 37 c , followed by denaturing polyacrylamide gel electrophoretic analysis , revealed a 15.1 0.5% yield of a slow - migrating band in the region of the gel where the cross - linked duplex was expected to appear ( figure 3 , lane 4 ) . these results provided evidence that the slow - migrating bands were indeed cross - linked duplexes containing both full - length strands of the parent duplex and ruled out cross - link formation by the more strongly electrophilic 3-4-hydroxy-2-pentenal-5-phosphate end group 3 . inclusion of the aldehyde - trapping agent methoxyamine in the reactions substantially inhibited formation of the slow - migrating bands ( figure 3 , lanes 5 , 9 , and 14 ) , consistent with involvement of the ap - aldehyde residue in cross - link formation ( 2 , schemes 2 and 3 ) . a time course experiment showed that easily detectable amounts of the cross - link were formed in duplex b within a few hours ( figure s1 ) . for example , in duplex b , similar cross - link yields ( 16 3% ) were observed in hepes buffer ( 50 mm , ph 7 ; 100 mm nacl ) , cacodylate ( 50 mm , ph 7 , 100 mm nacl ) , nah2po4/citric acid ( 16.5/1.8 mm , ph 7.0 , 100 mm nacl ) , mops ( 50 mm , ph 7 , 100 mm nacl ) , and bis - tris ( 50 mm , ph 7.4 , 100 mm nacl ) and in the presence of the biological thiol glutathione ( 1 mm , in hepes 50 mm , ph 7 , 100 mm nacl ) ( figure s2 ) . this made it interesting to examine the stability of the cross - link generated in duplex b. we found that , once formed , the cross - link in duplex b was stable under a variety of conditions including ph 3 ( sodium acetate , 25 mm , 24 c , 30 min ) , ph 10 ( potassium phthalate , 24 c , 25 mm , 30 min ) or mild piperidine workup ( 0.1 m , 60 c , 15 min ) ( figure s3 ) . on the other hand , the cross - link was decomposed by heat ( 90 c , 15 min ) or treatment with the aldehyde - trapping reagent methoxyamine ( 200 mm , 30 min , 60 c ; figure s3 ) . to identify the site at which the ap - aldehyde was attached to the opposing strand , we carried out hydroxyl radical footprinting of a cross - linked duplex containing the 5-apt/5-aa sequence . the cross - link was generated as described above , isolated from the gel , and subjected to cleavage by a mixture of iron - edta - h2o2 . in this type of experiment , the site of cross - linking appears as an interruption in the ladder of strand cleavage products generated by the iron - edta - h2o2 reagent , because cleavages beyond the cross - link yield large , slow - migrating dna fragments that are connected to the opposing strand . ( a ) lane 1 is a maxam gilbert g - specific cleavage ( sequencing ) reaction of the labeled oligodeoxynucleotide strand in duplex i. lane 2 is an a+g specific cleavage ( sequencing ) reaction of the labeled oligodeoxynucleotide strand in duplex i. lane 3 is the hydroxyl radical footprinting reaction of the single stranded nonuracil containing oligodeoxynucleotide . lane 4 is the hydroxyl radical footprinting reaction of the slow - migrating , cross - link band generated by incubation of duplex i hepes buffer ( 50 mm , ph 7 ) containing nacl ( 100 mm ) at 37 c . substitution of this adenine residue in duplex b with a guanine or thymine residue , respectively , gave duplexes f and g that did not produce significant yields of a slow - migrating cross - link band ( figure 5 ) . the substitution of 2-aminopurine for the adenine residue amounts to the rather subtle relocation of a single exocyclic amino group from the major groove to the minor groove of the dna duplex ( see structures in figure 1 ) . thus , the failure of duplex h to generate significant yields of cross - link allowed us to infer that the exocyclic n - amino group of da in the 5-gapt/5-aac sequence was intimately involved in the cross - linking reactions described here . cytosine places an exocyclic amino group in the major groove of dna similar to adenine , but the amino group of dc is located 0.7 closer to the sugar phosphate backbone than that of da ( the amino group of da is closer to the helical axis ) . nucleobase replacement experiments reveal that the underlined adenine residue in the 5-gapt/5-aac sequence of duplex b is critical for cross - link formation . replacement of this adenine residue in duplex b with a guanine or thymine residue in duplexes e and f , respectively , abrogates cross - link formation ( lanes 13 and 18 ) . the results described above were broadly consistent with a cross - linking mechanism involving reversible reaction of the exocyclic n - amino group on da with the ap aldehyde to generate hemiaminal , imine , cyclic hydroxyalkylhemiaminal , or enamine linkages at 5-gapt/5-aac sequences in duplex dna ( structures 6 - 9 ) . lc - ms / ms analysis provided additional insight regarding the chemical structure of the da - ap cross - link . selected - ion chromatograms revealed two peaks eluting at 24.1 and 26.1 min displaying the relevant m / z 368252 transition anticipated for the neutral loss of 2-deoxyribose from the putative da - ap cross - link ( figure 6 ) . further cleavage of the m / z 252 ion gave rise to a dominant fragment ion at m / z 136 in ms / ms / ms ( figure s6 ) , which is identical to the ms / ms observed for the [ m + h ] ion of da , suggesting that the initial m / z 368252 transition arises via loss of the cross - linked 2-deoxyribose adduct from da ( figure 6c ) . the presence of multiple peaks in the lc - ms / ms ion chromatogram was consistent with the expectation that the anticipated cross - link remnant resulting from the digestion of 8 will exist as an equilibrating mixture of pyranose and furanose isomers ( scheme s1 ) . ( a ) selected - ion chromatogram monitoring the neutral loss of 2-deoxyribose from the da - ap cross - link . this experiment was intriguing to us because , in this sequence context , the ap site has the potential to generate cross - links with either the guanine or adenine residue on the opposing strand ( residues colored red in duplex i shown in figure 1 ) . incubation of duplex i in hepes buffer ( 50 mm , ph 7 , 100 mm nacl ) at 37 c produced a remarkable 71.5 3.7% yield of a slow - migrating band in the region of the denaturing gel where the cross - linked duplexes migrate ( figure 7 ) . these results along with nanospray tof - ms data ( figure s11 ) confirmed that the slow - migrating band generated in the 5capt/5-aag sequence was an interstrand cross - linked duplex involving the ap - aldehyde residue . footprinting of a cross - linked duplex containing the 5capt/5-aag core sequence indicated that the cross - link attachment was to the underlined adenine residue in the 5capt/5-aag sequence ( figure s10 ) . thus , the remarkable cross - link yield in this sequence appears to result from efficient formation of a da - ap cross - link rather than simultaneous formation of dg - ap and da - ap cross - links . lc - ms / ms analysis of the products generated by nuclease p1 digestion of the cross - linked duplex i revealed the presence of a tetranucleotide with the ap - aldehyde conjugated with the underlined adenine from the 5-capt/5-aag sequence ( figure s12 ) , while the corresponding tetranucleotide with the ap - aldehyde being attached to the guanine residue was not detectable ( data not shown ) . additionally , lc - ms / ms analysis of the products formed from the digestion with a four - enzyme cocktail showed that the da - ap remnant is present at a level that is 2 orders of magnitude higher than its dg - ap counterpart ( figures s1314 ) . these results mesh with the footprinting data described above and indicate that the cross - linking occurs predominantly between the ap - aldehyde and the underlined adenine in the 5-capt/5-aag sequence . the results described here identified a novel cross - link involving reaction of a dna ap site with an adenine residue on the opposing strand of the double helix . the difference in the favored locations of da and dg residues in these cross - links , with the cross - linking nucleobase displaced to the 3- and 5-side of the ap site , respectively , is a natural consequence of the helical twist of duplex dna coupled with the fact that the n - amino group of adenine is located in the major groove while the n - amino group of guanine resides in the minor groove ( figure s15 ) . our results were consistent with a mechanism involving attack of the exocyclic n - amino group of adenine on the aldehyde residue of the ap site to generate a hemiaminal intermediate ( 6 , scheme 3 ) . the detailed chemical structure of the da - ap cross - link remains to be rigorously established using 2d - nmr , but chemical precedents involving structurally related small molecules suggest that the cyclic hydroxyalkylhemiaminal ( 8) likely will be the predominant form . the substantial stability of the da - ap cross - link generated in duplex b is generally consistent with a cyclic hydroxyalkylhemiaminal 8 , in which the reactive imine group of 7 has been masked by intramolecular attack of the hydroxyl group . despite the stability of the cross - link to many of the conditions examined here , the reaction is reversible , and it will be important to determine whether ap - derived cross - links have sufficient stability to block dna processing enzymes such as helicases and polymerases . it may be important to emphasize that the da - ap cross - links described here formed in good yields ( 1370% ) under physiologically relevant conditions ( ph 7 , 100 mm nacl ) and did not require conditions of reductive amination ( e.g. in the sequences examined to date , the yields of da - ap cross - links were much higher than those observed for the dg - ap cross - links , when both reactions are conducted under the same , physiologically relevant conditions ( ph 7 , in the absence of nacnbh3 ) . the sequence 5-capt/5-aag ( duplex i ) that , in principle , can form both dg - ap and da - ap cross - links , in fact , generates a remarkably high yield ( 70% ) of the da - ap cross - link . the yield of each cross - linked species at equilibrium will be determined by the inherent abilities of each nucleobase to generate imine linkages with the ap aldehyde and by the ability of the dna duplex to position the amino and aldehyde groups in a manner that favors imine formation . thus , formation of these ap - derived cross - links is conceptually related to template - directed synthesis with dynamic combinatorial imine libraries , in which equilibrium sorting processes involving reversible imine formation among a mixture of various aldehydes and amines ultimately generate higher yields of the imine products that form the most stable ensembles with a templating molecule present in the reaction . our results established that the equilibrium yields of the da - ap cross - link can be quite high in some sequence contexts , and future work will systematically explore the effects of sequence variation on cross - link yields . the widespread occurrence of ap sites along with the facile nature of the cross - linking reactions described here make it interesting to consider the possibility that ap - derived cross - links can form in genomic dna . in cells , processes such as repair and strand cleavage ( as shown in scheme 2 ) will compete with the formation of ap - derived cross - links . as a result , the majority of ap sites in genomic dna almost certainly do not go forward to form interstrand dna dna cross - links . in addition , the realization that ap sites can generate cross - links in duplex dna may help explain why pathways such as nucleotide excision repair and recombination that are typically associated with the repair of more complex lesions are required for eukaryotic cells to survive the introduction of ap sites into their genome . finally , ap - derived cross - links could be relevant to difficulties inherent in the amplification and sequencing of ancient dna . ancient dna samples contain a plethora of lesions including oxidative base damage , ap sites , and strand breaks , but it has been suggested that a high density of interstrand cross - links ultimately limit the ability to amplify and sequence the dna in these samples . the structural nature of the cross - links in ancient dna has not been elucidated , but ap - derived cross - links are good candidates for these blocking lesions that hinder sample processing . overall , these findings establish ap - derived interstrand cross - links as a family of dna lesions that can form in diverse sequence contexts .

the liver is a common site of distant metastasis originating from different neoplasms including gastrointestinal ( pancreatic , stomach , colorectal ) , lung and breast cancers . also primary liver tumours such as cholangiocellular carcinomas ( ccc ) , cancers of the bile ducts , may disseminate into the liver . surgical resection still is the most promising therapy of secondary liver tumours , however , only a minority of patients are candidates for resection , and no adjuvant treatment has been demonstrated to be effective in increasing the survival rate following radical surgery [ 2 , 3 ] . for unresectable disease , several treatments have been tested in the clinical setting ; however , none of them can be currently considered a standard approach . this also applies to systemic chemotherapy , although newer regimens appear to at least improve median survival . locoregional therapies such as hepatic intra - arterial chemotherapy and isolated hepatic perfusion may be offered to patients with unresectable liver metastases in the absence of extrahepatic disease ; however , the efficacy of these treatments is still being determined . both systemic and locoregional chemotherapy might be useful in the neoadjuvant setting to increase the resectability of liver metastases initially not amenable to surgical resection . due to its poor prognosis and unsatisfying treatment options , suitable animal models for secondary liver cancer are required as a prerequisite for studying factors involved in the pathogenesis of the disease as well as for the development and evaluation of new anticancer therapies . various approaches include the use of transgenic or knockout mice [ 5 , 6 ] or mouse models , in which tumour formation is induced chemically . albeit tumours develop in all of these mouse models , tumour formation and progression in mice greatly differ from that in man [ 8 , 9 ] due to physiological differences between the species and differences in cellular and molecular events contributing to cancer development . tumour models established with primary human tumour tissue may overcome some of these limitations . to this aim , immune compromised animals , such as severe combined immunodeficient ( scid ) mice , are grafted either subcutaneously or orthotopically with cultured cells [ 10 , 11 ] or tissue derived from human tumour material [ 1215 ] providing convenient models for evaluation of distinct anticancer strategies , especially those targeting tumour growth . although discussions are ongoing arguing that the orthotopic transplantation model closer resembles the situation in the patient , subcutaneous xenografts still remain the standard for cancer drug screening in the pharmaceutical industry . in both cases , only detailed knowledge about the transplanted tumour cells will facilitate correct interpretation of gained results . thus , in the present study liver metastases derived from various human adenocarcinomas were used to establish subcutaneous xenograft tumours in scid / beige mice . extensive histological analyses were performed to demonstrate that the transplants widely reflect the characteristics of the parental lesion . in addition , gene expression profiling by means of rt - pcr - based microarrays revealed that expression of cancer - related genes appeared to be similar in corresponding original and xenograft tumours as well as in derived cell cultures . therefore , we conclude that the established tumour models and cell cultures may represent valuable tools for the development and analysis of new treatments targeting secondary liver tumours . primary and secondary liver tumours were obtained from patients at the time of liver transplantation or surgical resection of the neoplasm . immediately after surgical resection , tumour samples were transferred into transport medium ( rpmi 1640 , sigma - aldrich , wien , austria ) containing 10% heat - inactivated foetal bovine serum ( fbs ) ( paa , pasching , austria ) , 100 u / ml penicillin - streptomycin ( paa ) , 2.5 g / ml fungizone ( sigma ) , and 100 g / ml gentamycin ( biochrom ag , berlin , germany ) . human tumour samples with an average size of 1 cm were cut into 2 2 mm pieces in the presence of digestion medium ( pbs/2 mg / ml collagenase iii ; 37c ; worthington biochemical corporation , nj , usa ) , transferred into 15 ml tubes ( sarstedt , wiener neustadt , austria ) and further incubated for 1 hour at 37c with continuous shaking at 320 cycles per minute ( thermoshaker htmr 132 ; haep labor consult , bovenden , germany ) . to stop digestion , an equal volume of culture medium ( dmem/10% fbs/50 u / ml penicillin - streptomycin ) was added . the obtained single cell suspension was centrifuged at 180 xg for 5 minutes , and cells were washed twice with pbs . the cell pellet was resuspended in 1 ml of injection medium ( rpmi 1640 phenol red free/1% penicillin - streptomycin ) and 150 - 200 l thereof were inoculated subcutaneously into the left flank of 3 scid / beige mice ( c.b-17/icrhsd - prkcd lyst ; harlan - winkelmann , borchen , germany ) which had been anaesthetised by intraperitoneal injection of ketamine ( 1 mg/10 g body weight ) and xylazin ( 0.039 mg/10 g body weight ) . for individual identification , microchip transponders ( backhome ; virbac , wien , austria ) were implanted subcutaneously . animals were kept under specific pathogen - free conditions in negative pressure containments ( scantainers , scanbur , denmark ) with unlimited access to food and autoclaved tap water . hence , the largest ( a ) and smallest ( b ) superficial diameters of the tumour were determined once a week using a sliding calliper , and then the volume ( v ) was calculated ( v = a b b/2 ) . at 300500 mm , tumours were excised and parts of them , that is , pieces of 2 mm , transplanted into new animals , fixed in formalin or frozen in liquid nitrogen . all animal experiments were performed according to austrian laws governing animal experimentation ( gz 68.205/30-pr/4/2002 ; gz 68.205/59-brgt/2004 ) . tumour pieces either obtained from primary ( akh23 , kfj18 ) or xenografted tumours ( akh10 , kfj6 , kfj9 , kfj10 ) were processed as described above , and obtained single cell suspensions were transferred into cell culture flasks ( sarstedt ) containing culture medium . established cell cultures were characterised by immunocytochemistry using antibodies reacting with human and mouse major histocompatibility complex ( mhc ) class i antigens . briefly , cells were incubated with a r - phycoerythrin conjugated mouse anti - human leukocyte antigen ( hla)-a , b , c ( bd pharmingen , schwechat , austria ) or a fluorescein isothiocyanate ( fitc)conjugated mouse antimouse h-2dd monoclonal antibody ( becton dickinson , heidelberg , germany ) for one hour at 4c in the dark . cells were washed twice , resuspended in pbs , and subjected to facs analysis ( facscalibur , becton dickinson ) . in addition , cells were stained with an antibody directed against a human epithelial - specific antigen ( esa ; serotec , dsseldorf , germany ) followed by detection with fitc - conjugated polyclonal rabbit anti - mouse immunoglobulin ( dakocytomation , glostrup , denmark ) . after characterisation , cells usually with passage numbers 510 were frozen in liquid nitrogen for long - term storage . on demand cells were thawed and expanded for further in vitro analysis or retransplantation into immunodeficient mice . therefore , 15 10 cells were injected subcutaneously into scid / beige mice as described above . in addition to tumour growth in vivo , anchorage independent growth of recultivated tumour - derived cells was analysed by colony formation in a standard soft agar assay . xenograft tumours after the first or second passage in mice were excised and fixed in 4% buffered formalin ( ph 7.0 , sigma - aldrich ) and embedded in paraffin ( histo - comp , sanova , wien , austria ) using automatic embedding equipment ( tissue tek , miles scientific , inc . , ill , three m thick sections of primary and xenograft tumours were routinely stained with haematoxylin and eosin and microscopically analysed . to characterise primary tumours and corresponding xenografts by immunohistochemistry , the following primary antibodies were used : rabbit polyclonal antibody specific for carcinoembryonic antigen ( cea , cd66e ab-2 , neat , labvision neomarkers , cheshire , uk ) , mouse monoclonal antibodies specific for cytokeratin 8/18 ( ck8/18 labvision neomarkers , 1:100 diluted in pbs ) and cytokeratin 20 ( ck20 , dakocytomation ; 1:50 diluted in pbs ) . for detection of ck8/18 , sections were digested with 0.1% protease ( sigma - aldrich ) in pbs for 15 minutes . ck20 was detected after pretreatment with 0.1% proteinase k ( sigma - aldrich ) in pbs . sections were then incubated with 1.5% goat serum ( dakocytomation ) for 30 minutes followed by overnight incubation with the primary antibody at 4c . detection was performed using the vectastain abc - ap kit ( vector laboratories , england , uk ) with new fuchsin ( dakocytomation ) as a substrate followed by counterstaining with mayer 's haemalum ( vwr international gmbh , dresden , germany ) . sections were covered with aquatex ( merck , darmstadt , germany ) and examined by light microscopy ( zeiss axiovert 200 m , carl zeiss gmbh , oberkochen , germany ) . rna was extracted from trypsinised cells or frozen and pulverised tumour samples according to the rneasy mini kit protocol ( qiagen , wien , austria ) and treated afterwards with turbo dnase ( ambion , tex , usa ) according to the manufacturer 's instructions . subsequently , 150 ng of total rna were reverse transcribed using the iscript cdna synthesis kit ( bio - rad laboratories , calif , usa ) . 50 l of cdna template ( 105 ng total input rna ) were amplified using a master mix containing 1x reaction buffer b ( solis biodyne , tartu , estonia ) , 5 mm mgcl2 , 0.2 mm of each dntp ( applied biosystems , calif , usa ) , 300 nm rox reference dye ( invitrogen , lofer , germany ) and 1 unit of hot start firepol polymerase ( solis biodyne , tartu , estonia ) on taqman low density arrays ( applied biosystems ) using the abi prism 7900ht sequence detection system ( applied biosystems ) . the respective human - specific real - time pcr primers and probes are listed in table 1 . according to data base comparisons ( applied biosystems ) , cycling conditions were as follows : 2 minutes at 50c , 10 minutes at 94.5 followed by 40 cycles of 30 seconds at 97c and 1 minute at 59.7c . ct values were determined using the passive reference dye and manual baseline and threshold settings in the sds 2.2 software ( applied biosystems ) . assays with ct values above 33 were excluded from analysis due to variations or inappropriate amplification in duplicate wells . tumour - derived rna was tested in duplicates on three different plates , and their mean values were calculated for further analysis . a calibrator sample consisting of a universal reference rna isolated from 10 different human cancer cell lines ( stratagene , calif , usa ) served as an internal standard for comparison of different assays . differences in gene expression levels of each tumour sample were first normalised to the calibrator sample followed by calculation of differences between original and xenograft tumours according to the 2 method . normalisation of real - time rt - pcr data was performed using the geometric mean ( normalisation factor ) of the included endogenous reference genes gusb ( - glucuronidase ) , actb ( - actin ) and rrna18s ( 18s ribosomal rna ) within the macros - based program qbase ( http://medgen.ugent.be/qbase ) . to identify genes expressed differentially in all xenografts and parental tumours analysed , a wilcoxon paired - samples test was performed ( spss for windows vs. 11.5 ) . , we considered genes to be differentially expressed showing a minimum of 2.5-fold difference between xenograft and original tumours . human secondary liver tumour tissue was obtained from patients at the time of surgery or resection of the neoplasm . in total , tumour samples from 17 patients including liver metastases of colorectal carcinomas ( n = 10 ) , intrahepatic cholangiocellular carcinomas ( n = 6 ) as well as a metastasis of a pancreatic carcinoma were collected . the tumour tissue was digested with collagenase to obtain single cell suspensions which were injected subcutaneously into scid / beige mice . finally , injection of single cells prepared from 10 different samples consisting of liver metastases originating from colorectal ( n = 6 ) , cholangiocellular ( n = 3 ) , and a pancreatic adenocarcinoma resulted in tumour formation . the main characteristics of the original xenografted tumour samples are summarised in table 2 . in order to compare original and xenograft tumours morphologically , representatively for colorectal liver metastases , sections of the original tumour kfj6 and its derived xenograft are shown ( see figures 1(a ) and 1(b ) ) . both original as well as the xenograft tumours revealed irregular tubular structures typical for colon adenocarcinomas . in most of the established xenograft tumours , the tumour akh10 is depicted as an example of an intrahepatic cholangiocellular carcinoma ( see figures 1(c ) and 1(d ) ) . pathohistologically , both xenograft and the parental tumour can be described as a moderately differentiated adenocarcinoma with comparable simple tubular to glandular structures . examination of the liver metastasis akh23 which had originated from a pancreatic adenocarcinoma revealed a solid undifferentiated large cell carcinoma ( see figure 1(e ) ) . tumour cells exhibited anaplastic nuclei and varying amounts of eosinophilic , particular foamy cytoplasm . consistently , subcutaneous implantation of cells derived from tumour akh23 led to formation of a poorly differentiated fast growing anaplastic carcinoma ( see figure 1(f ) ) . to further characterise the established xenograft tumours and their corresponding original counterparts , immunohistological stainings for detection of cea were performed . cea is a glycoprotein expressed in adenocarcinomas of the intestinal tract and in other tumours of epithelial origin such as lung adenocarcinoma , pancreatic adenocarcinoma , and cholangiocellular carcinomas ( cccs ) . additionally , tumours were stained with antibodies specific for ck8/18 , which is expressed in simple and glandular epithelia , and ck20 , which is primarily expressed in colon adenocarcinomas . as summarised in table 3 , immunohistological analyses revealed similar staining patterns within original and xenograft tumour samples with regard to expression of cea , ck8/18 , and ck20 . positive staining exclusively detected in distinct original tumour samples was due to reactions with normal liver cells no more present in the xenograft tumours . representative analyses of original tumours and their corresponding xenografts are shown in figures 24 . immunohistological comparison of the original and xenograft tumour kfj6 in both samples revealed expression of cea in the cytoplasm and membranes of luminal cells ( see figures 2(a ) and 2(b ) ) . additionally , expression of ck8/18 ( see figures 2(c ) and 2(d ) ) and ck20 ( see figures 2(e ) and 2(f ) ) was detected . original and xenograft tumours ( see figure 3 ) both reacted with antibodies specific for cea ( see figures 3(a ) and 3(b ) ) and ck8/18 ( see figures 3(c ) and 3(d ) ) but did not show expression of ck20 ( see figures 3(e ) and 3(f ) ) . the original pancreatic adenocarcinoma - derived liver metastasis akh23 ( see figure 4 ) revealed single cells expressing cea ( see figure 4(a ) ) , whereas the corresponding xenograft tumour appeared negative for cea expression . a robust staining in both samples was obtained when expression of ck8/18 ( see figures 4(c ) and 4(d ) ) was analysed . in contrast , neither original nor xenograft tumour - derived sections revealed expression of ck20 ( see figures 4(e ) and 4(f ) ) . based on these findings , we conclude that the investigated human tumours retained their typical morphological and histological characteristics after xenotransplantation into mice . in order to compare the established xenograft tumour models with the respective original tumour counterparts on a molecular basis , relative expression levels of a number of cancer - relevant genes ( see table 1 ) were determined in the respective corresponding tumour samples using taqman low density expression arrays . interassay specific differences were first normalised to an arbitrarily chosen calibrator ( reference rna ) , and then the ratio of gene expression levels in an original tumour versus the corresponding xenograft tumour was determined . genes were considered to be differentially expressed when a 2.5-fold minimal difference between original and xenograft tumour samples was obtained . table 4 summarises data acquired for a representative selection of different original tumours in comparison to their respective xenografts . interestingly , genes encoding cell cycle regulators and proto - oncogenes , such as bcl-2 , cyclin d1 , cdc25b , cyclin - dependent kinase inhibitor 1b , erb - b2 , k - ras , met and myc as well as epidermal growth factor receptor ( egfr ) and a - catenin encoding gene ( ctnnb1 ) , showed comparable expression levels in all the investigated original and xenograft tumours . expression of the proto - oncogene wnt-1 was neither detected in original nor in xenograft tumour tissue . in contrast , genes encoding cytokines such as interleukin 8 ( il-8 ) and 6 ( il-6 ) , its receptor il6-r , cyclooxygenase ( cox)-2 , vascular endothelial growth factor ( vegf)-c as well as matrix metalloproteinase ( mmp ) 11 appeared to be differentially expressed in some of the analysed samples ( see table 4 ) . in particular , il-6 , cox-2 , and vegf - c expression was nearly exclusively detected in original tumour samples . expression of il-6 receptor ( il-6r ) was either found to be equal in original and xenograft tumours or significantly increased ( 4- to 18-fold ) in some of the original tumours ( akh10 , kfj18 , kfj21 ) . similarly , il-8 appeared to be 12- to 100-fold higher expressed in original tumour samples compared to the corresponding xenograft tissue . analysis of mmp-11 expression revealed a 4- to 22-fold difference between original and xenograft tumours . although few more differences were encountered concerning expression of serpin and vegf ( akh23 ) , statistical analysis of results obtained for all investigated original and xenograft tumour samples revealed significant differences exclusively for the expression of il-8 ( p = .017 ) and mmp-11 ( p = .018 ) . finally , gene expression levels of original and xenograft tumour samples exemplarily were compared to those of their derived cell cultures ( see table 5 ) . immunocytochemical characterisation of established cell cultures confirmed their human and epithelial origin , respectively ( data not shown ) . again , the most striking differences in expression levels were observed for il6-r and mmp encoding genes . il6r - expression levels were about 5-fold decreased in tumour - derived cell cultures compared to the corresponding tissue . demonstrative differences in mmp-1 expression were observed for akh23-derived cells , which showed a > 300-fold higher amount of mrna compared to the parental tumour . in contrast , mmp-11 ( 10-fold ) and vegf ( 4-fold ) expression levels were found to be higher in akh23 original tumour tissue . tumour mouse models as well as tumour - derived cell lines are a prerequisite for the development and evaluation of new and existing tumour therapies . although a number of xenograft models have been published for colorectal carcinomas and pancreatic adenocarcinomas in most cases , these were established from cultured cell lines available for example from atcc . in these examples , it is not clear how long - term cultivation of these ( mostly poorly characterised ) cells affects tumour formation and biology . therefore , we decided to establish xenografts directly from patient tumours and subsequently analyse both tissues in detail to demonstrate that the generated model closely reflects the original malignancy . in the present study , we report the establishment and detailed characterisation of human xenograft tumour models derived from secondary liver cancer , that is , tumour metastases originating from colorectal , cholangiocellular , and pancreatic cancers . xenografts were established directly from tumour biopsies omitting culturing of isolated cells , which may cause development of tumours that do not share the characteristics of the respective original due to the selection and expansion of specific cell clones . the applied method of enzymatic digestion of whole tumour samples followed by injection of a mixture of tumour and stromal cells was shown to overcome this obstacle . with respect to xenografts derived from colorectal carcinomas , the applied method resulted in a take rate of 60% and 50% , respectively , when cholangiocellular carcinoma - derived cells were injected . retrospective analysis of xenograft tumour growth with clinical data of the respective patient did not reveal any significant correlation . instead , the condition of the primary tumour sample , for example , the presence of large necrotic areas appeared to be critical . pathohistological examination of the established xenografts and comparison to their respective original tumours demonstrated that the typical morphology of the tumours was retained after xenotransplantation . moreover , immunohistological analyses showed that each of the established xenograft tumours retained the typical tumour - specific antigen profile observed in the original tumour sample . cell cultures established either from original or xenograft tumour tissues were shown to be of epithelial origin and not contaminated with murine cells ( data not shown ) . although the respective tumour transplants could be passaged in mice for extended periods ( up to 30 times ) without major changes in growth behaviour and morphology ( data not shown ) , a cryoconservation protocol was established facilitating storage of samples at early passages to avoid development of histopathological alterations over time . retransplantation experiments with tumour samples frozen for different time spans ( 3 , 6 , and 12 months ) revealed an average take rate of 70% to 100% in both scid / beige and nude mice . molecular characterisation based on quantitative gene expression analyses using human specific primers and probes revealed that in most of the corresponding original and xenograft tumour samples expression of oncogenes and genes involved in cell cycle regulation appeared not to be affected by the xenografting process . major differences within original and xenograft tumour samples as well as their derived cell cultures were detected regarding genes encoding cytokines ( il-8 , il-6 ) and matrix metalloproteinases ( mmp-1 , mmp-11 ) . this finding can be explained by the fact that these molecules are rather expressed by inflammatory cells ( monocytes , neutrophils ) , stromal fibroblasts , and endothelial cells than by the tumour cells themselves . a high level il-8 expression , however , was also reported in cultured colon carcinoma cells , where it was associated with the metastatic behaviour of these cells . consistently , we have shown il-8 expression in cultured xenograft - derived colon carcinoma cells ( e.g. , kfj10 ) , and their metastatic potential was demonstrated by colony formation in soft agar assays ( data not shown ) . matrix metalloproteinases ( mmps ) are a family of extracellular matrix degrading enzymes , which have their physiological role in tissue remodelling processes such as embryonic development or wound healing . in cancer , mmps are described to be involved in tumour invasion , metastasis , and angiogenesis [ 23 , 24 ] . mmp-1 , also known as interstitial collagenase , is expressed in a wide variety of cells such as stromal fibroblasts , endothelial cells , macrophages , and epithelial cells . either equal expression levels were found in original and xenograft tumours or expression was exclusively detected in original tumours . a weak or lacking mmp-1 expression in some of the xenograft tumours could not be linked to an individual tumour type . original tumours representing liver metastases showed higher mmp-1 levels , reflecting the potential of tumour cells to invade and metastasise from their original site to distant organs . accordingly , akh23 primary tumour - derived cells exhibiting a markedly high mmp-1 expression level demonstrated a very aggressive growth behaviour when injected into immunodeficient mice . injection of 5 10 cells in this case resulted in growth of tumours of up to 1000 mm within 35 days whereas in average xenografted cells took 60 to 80 days to reach this tumour volume ( data not shown ) . mmp-11 in comparison to mmp-1 is described to be specifically expressed in stromal fibroblasts surrounding tumour cells . thus , the determined reduced expression level of mmp-11 in xenograft tumours most probably is due to the absence of human stroma cells in the murine environment . interestingly , expression of cox-2 ( ptgs2 ) and vegf - c , both known to regulate angiogenesis and lymphangiogenesis , was detected in original tumour samples but , in contrast to vegf - a , was beyond detection limits in most of the xenograft tissues . recently , it has been described that these two genes are coexpressed in human colorectal carcinoma cells and can be significantly associated with lymph node metastasis and prognosis . further investigation of the mechanisms of down regulation of expression of lymphangiogenesis inducing factors in xenografted tumours may give insight into metastatic progression of crc . the developed carefully characterised human xenograft tumours derived from secondary liver tumours share assertive characteristics with their respective original human counterparts . in addition , the established cell cultures offer the possibility to evaluate new therapeutic strategies in vitro before their use in vivo in the corresponding tumour mouse models . these valuable tools might be used for the development and preclinical evaluation of new therapeutic drugs as well as of alternative methods such as expression targeted retroviral vectors or liver specific therapeutic nanoparticles generated for an application in cancer gene therapy .

to develop and evaluate new therapeutic strategies for the treatment of human cancers , well - characterised preclinical model systems are a prerequisite . to this aim , we have established xenotransplantation mouse models and corresponding cell cultures from surgically obtained secondary human liver tumours . established xenograft tumours were patho- and immunohistologically characterised , and expression levels of cancer - relevant genes were quantified in paired original and xenograft tumours and the derivative cell cultures applying rt - pcr - based array technology . most of the characteristic morphological and immunohistochemical features of the original tumours were shown to be maintained . no differences were found concerning expression of genes involved in cell cycle regulation and oncogenesis . interestingly , cytokine and matrix metalloproteinase encoding genes appeared to be expressed differentially . thus , the established models are closely reflecting pathohistological and molecular characteristics of the selected human tumours and may therefore provide useful tools for preclinical analyses of new antitumour strategies in vivo .

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

also primary liver tumours such as cholangiocellular carcinomas ( ccc ) , cancers of the bile ducts , may disseminate into the liver . surgical resection still is the most promising therapy of secondary liver tumours , however , only a minority of patients are candidates for resection , and no adjuvant treatment has been demonstrated to be effective in increasing the survival rate following radical surgery [ 2 , 3 ] . due to its poor prognosis and unsatisfying treatment options , suitable animal models for secondary liver cancer are required as a prerequisite for studying factors involved in the pathogenesis of the disease as well as for the development and evaluation of new anticancer therapies . various approaches include the use of transgenic or knockout mice [ 5 , 6 ] or mouse models , in which tumour formation is induced chemically . albeit tumours develop in all of these mouse models , tumour formation and progression in mice greatly differ from that in man [ 8 , 9 ] due to physiological differences between the species and differences in cellular and molecular events contributing to cancer development . to this aim , immune compromised animals , such as severe combined immunodeficient ( scid ) mice , are grafted either subcutaneously or orthotopically with cultured cells [ 10 , 11 ] or tissue derived from human tumour material [ 1215 ] providing convenient models for evaluation of distinct anticancer strategies , especially those targeting tumour growth . thus , in the present study liver metastases derived from various human adenocarcinomas were used to establish subcutaneous xenograft tumours in scid / beige mice . extensive histological analyses were performed to demonstrate that the transplants widely reflect the characteristics of the parental lesion . in addition , gene expression profiling by means of rt - pcr - based microarrays revealed that expression of cancer - related genes appeared to be similar in corresponding original and xenograft tumours as well as in derived cell cultures . therefore , we conclude that the established tumour models and cell cultures may represent valuable tools for the development and analysis of new treatments targeting secondary liver tumours . primary and secondary liver tumours were obtained from patients at the time of liver transplantation or surgical resection of the neoplasm . the obtained single cell suspension was centrifuged at 180 xg for 5 minutes , and cells were washed twice with pbs . hence , the largest ( a ) and smallest ( b ) superficial diameters of the tumour were determined once a week using a sliding calliper , and then the volume ( v ) was calculated ( v = a b b/2 ) . at 300500 mm , tumours were excised and parts of them , that is , pieces of 2 mm , transplanted into new animals , fixed in formalin or frozen in liquid nitrogen . tumour pieces either obtained from primary ( akh23 , kfj18 ) or xenografted tumours ( akh10 , kfj6 , kfj9 , kfj10 ) were processed as described above , and obtained single cell suspensions were transferred into cell culture flasks ( sarstedt ) containing culture medium . established cell cultures were characterised by immunocytochemistry using antibodies reacting with human and mouse major histocompatibility complex ( mhc ) class i antigens . cells were washed twice , resuspended in pbs , and subjected to facs analysis ( facscalibur , becton dickinson ) . xenograft tumours after the first or second passage in mice were excised and fixed in 4% buffered formalin ( ph 7.0 , sigma - aldrich ) and embedded in paraffin ( histo - comp , sanova , wien , austria ) using automatic embedding equipment ( tissue tek , miles scientific , inc . , ill , three m thick sections of primary and xenograft tumours were routinely stained with haematoxylin and eosin and microscopically analysed . to characterise primary tumours and corresponding xenografts by immunohistochemistry , the following primary antibodies were used : rabbit polyclonal antibody specific for carcinoembryonic antigen ( cea , cd66e ab-2 , neat , labvision neomarkers , cheshire , uk ) , mouse monoclonal antibodies specific for cytokeratin 8/18 ( ck8/18 labvision neomarkers , 1:100 diluted in pbs ) and cytokeratin 20 ( ck20 , dakocytomation ; 1:50 diluted in pbs ) . tumour - derived rna was tested in duplicates on three different plates , and their mean values were calculated for further analysis . differences in gene expression levels of each tumour sample were first normalised to the calibrator sample followed by calculation of differences between original and xenograft tumours according to the 2 method . normalisation of real - time rt - pcr data was performed using the geometric mean ( normalisation factor ) of the included endogenous reference genes gusb ( - glucuronidase ) , actb ( - actin ) and rrna18s ( 18s ribosomal rna ) within the macros - based program qbase ( http://medgen.ugent.be/qbase ) . to identify genes expressed differentially in all xenografts and parental tumours analysed , a wilcoxon paired - samples test was performed ( spss for windows vs. 11.5 ) . , we considered genes to be differentially expressed showing a minimum of 2.5-fold difference between xenograft and original tumours . human secondary liver tumour tissue was obtained from patients at the time of surgery or resection of the neoplasm . finally , injection of single cells prepared from 10 different samples consisting of liver metastases originating from colorectal ( n = 6 ) , cholangiocellular ( n = 3 ) , and a pancreatic adenocarcinoma resulted in tumour formation . the main characteristics of the original xenografted tumour samples are summarised in table 2 . in order to compare original and xenograft tumours morphologically , representatively for colorectal liver metastases , sections of the original tumour kfj6 and its derived xenograft are shown ( see figures 1(a ) and 1(b ) ) . both original as well as the xenograft tumours revealed irregular tubular structures typical for colon adenocarcinomas . in most of the established xenograft tumours , the tumour akh10 is depicted as an example of an intrahepatic cholangiocellular carcinoma ( see figures 1(c ) and 1(d ) ) . pathohistologically , both xenograft and the parental tumour can be described as a moderately differentiated adenocarcinoma with comparable simple tubular to glandular structures . examination of the liver metastasis akh23 which had originated from a pancreatic adenocarcinoma revealed a solid undifferentiated large cell carcinoma ( see figure 1(e ) ) . to further characterise the established xenograft tumours and their corresponding original counterparts , immunohistological stainings for detection of cea were performed . cea is a glycoprotein expressed in adenocarcinomas of the intestinal tract and in other tumours of epithelial origin such as lung adenocarcinoma , pancreatic adenocarcinoma , and cholangiocellular carcinomas ( cccs ) . additionally , tumours were stained with antibodies specific for ck8/18 , which is expressed in simple and glandular epithelia , and ck20 , which is primarily expressed in colon adenocarcinomas . as summarised in table 3 , immunohistological analyses revealed similar staining patterns within original and xenograft tumour samples with regard to expression of cea , ck8/18 , and ck20 . positive staining exclusively detected in distinct original tumour samples was due to reactions with normal liver cells no more present in the xenograft tumours . representative analyses of original tumours and their corresponding xenografts are shown in figures 24 . immunohistological comparison of the original and xenograft tumour kfj6 in both samples revealed expression of cea in the cytoplasm and membranes of luminal cells ( see figures 2(a ) and 2(b ) ) . original and xenograft tumours ( see figure 3 ) both reacted with antibodies specific for cea ( see figures 3(a ) and 3(b ) ) and ck8/18 ( see figures 3(c ) and 3(d ) ) but did not show expression of ck20 ( see figures 3(e ) and 3(f ) ) . in contrast , neither original nor xenograft tumour - derived sections revealed expression of ck20 ( see figures 4(e ) and 4(f ) ) . based on these findings , we conclude that the investigated human tumours retained their typical morphological and histological characteristics after xenotransplantation into mice . in order to compare the established xenograft tumour models with the respective original tumour counterparts on a molecular basis , relative expression levels of a number of cancer - relevant genes ( see table 1 ) were determined in the respective corresponding tumour samples using taqman low density expression arrays . interassay specific differences were first normalised to an arbitrarily chosen calibrator ( reference rna ) , and then the ratio of gene expression levels in an original tumour versus the corresponding xenograft tumour was determined . genes were considered to be differentially expressed when a 2.5-fold minimal difference between original and xenograft tumour samples was obtained . table 4 summarises data acquired for a representative selection of different original tumours in comparison to their respective xenografts . interestingly , genes encoding cell cycle regulators and proto - oncogenes , such as bcl-2 , cyclin d1 , cdc25b , cyclin - dependent kinase inhibitor 1b , erb - b2 , k - ras , met and myc as well as epidermal growth factor receptor ( egfr ) and a - catenin encoding gene ( ctnnb1 ) , showed comparable expression levels in all the investigated original and xenograft tumours . expression of the proto - oncogene wnt-1 was neither detected in original nor in xenograft tumour tissue . in contrast , genes encoding cytokines such as interleukin 8 ( il-8 ) and 6 ( il-6 ) , its receptor il6-r , cyclooxygenase ( cox)-2 , vascular endothelial growth factor ( vegf)-c as well as matrix metalloproteinase ( mmp ) 11 appeared to be differentially expressed in some of the analysed samples ( see table 4 ) . in particular , il-6 , cox-2 , and vegf - c expression was nearly exclusively detected in original tumour samples . expression of il-6 receptor ( il-6r ) was either found to be equal in original and xenograft tumours or significantly increased ( 4- to 18-fold ) in some of the original tumours ( akh10 , kfj18 , kfj21 ) . similarly , il-8 appeared to be 12- to 100-fold higher expressed in original tumour samples compared to the corresponding xenograft tissue . analysis of mmp-11 expression revealed a 4- to 22-fold difference between original and xenograft tumours . although few more differences were encountered concerning expression of serpin and vegf ( akh23 ) , statistical analysis of results obtained for all investigated original and xenograft tumour samples revealed significant differences exclusively for the expression of il-8 ( p = .017 ) and mmp-11 ( p = .018 ) . finally , gene expression levels of original and xenograft tumour samples exemplarily were compared to those of their derived cell cultures ( see table 5 ) . immunocytochemical characterisation of established cell cultures confirmed their human and epithelial origin , respectively ( data not shown ) . again , the most striking differences in expression levels were observed for il6-r and mmp encoding genes . il6r - expression levels were about 5-fold decreased in tumour - derived cell cultures compared to the corresponding tissue . in contrast , mmp-11 ( 10-fold ) and vegf ( 4-fold ) expression levels were found to be higher in akh23 original tumour tissue . tumour mouse models as well as tumour - derived cell lines are a prerequisite for the development and evaluation of new and existing tumour therapies . therefore , we decided to establish xenografts directly from patient tumours and subsequently analyse both tissues in detail to demonstrate that the generated model closely reflects the original malignancy . in the present study , we report the establishment and detailed characterisation of human xenograft tumour models derived from secondary liver cancer , that is , tumour metastases originating from colorectal , cholangiocellular , and pancreatic cancers . xenografts were established directly from tumour biopsies omitting culturing of isolated cells , which may cause development of tumours that do not share the characteristics of the respective original due to the selection and expansion of specific cell clones . with respect to xenografts derived from colorectal carcinomas , the applied method resulted in a take rate of 60% and 50% , respectively , when cholangiocellular carcinoma - derived cells were injected . retrospective analysis of xenograft tumour growth with clinical data of the respective patient did not reveal any significant correlation . instead , the condition of the primary tumour sample , for example , the presence of large necrotic areas appeared to be critical . pathohistological examination of the established xenografts and comparison to their respective original tumours demonstrated that the typical morphology of the tumours was retained after xenotransplantation . moreover , immunohistological analyses showed that each of the established xenograft tumours retained the typical tumour - specific antigen profile observed in the original tumour sample . cell cultures established either from original or xenograft tumour tissues were shown to be of epithelial origin and not contaminated with murine cells ( data not shown ) . retransplantation experiments with tumour samples frozen for different time spans ( 3 , 6 , and 12 months ) revealed an average take rate of 70% to 100% in both scid / beige and nude mice . molecular characterisation based on quantitative gene expression analyses using human specific primers and probes revealed that in most of the corresponding original and xenograft tumour samples expression of oncogenes and genes involved in cell cycle regulation appeared not to be affected by the xenografting process . major differences within original and xenograft tumour samples as well as their derived cell cultures were detected regarding genes encoding cytokines ( il-8 , il-6 ) and matrix metalloproteinases ( mmp-1 , mmp-11 ) . this finding can be explained by the fact that these molecules are rather expressed by inflammatory cells ( monocytes , neutrophils ) , stromal fibroblasts , and endothelial cells than by the tumour cells themselves . consistently , we have shown il-8 expression in cultured xenograft - derived colon carcinoma cells ( e.g. , kfj10 ) , and their metastatic potential was demonstrated by colony formation in soft agar assays ( data not shown ) . matrix metalloproteinases ( mmps ) are a family of extracellular matrix degrading enzymes , which have their physiological role in tissue remodelling processes such as embryonic development or wound healing . in cancer , mmps are described to be involved in tumour invasion , metastasis , and angiogenesis [ 23 , 24 ] . mmp-1 , also known as interstitial collagenase , is expressed in a wide variety of cells such as stromal fibroblasts , endothelial cells , macrophages , and epithelial cells . either equal expression levels were found in original and xenograft tumours or expression was exclusively detected in original tumours . a weak or lacking mmp-1 expression in some of the xenograft tumours could not be linked to an individual tumour type . thus , the determined reduced expression level of mmp-11 in xenograft tumours most probably is due to the absence of human stroma cells in the murine environment . interestingly , expression of cox-2 ( ptgs2 ) and vegf - c , both known to regulate angiogenesis and lymphangiogenesis , was detected in original tumour samples but , in contrast to vegf - a , was beyond detection limits in most of the xenograft tissues . further investigation of the mechanisms of down regulation of expression of lymphangiogenesis inducing factors in xenografted tumours may give insight into metastatic progression of crc . the developed carefully characterised human xenograft tumours derived from secondary liver tumours share assertive characteristics with their respective original human counterparts . in addition , the established cell cultures offer the possibility to evaluate new therapeutic strategies in vitro before their use in vivo in the corresponding tumour mouse models . these valuable tools might be used for the development and preclinical evaluation of new therapeutic drugs as well as of alternative methods such as expression targeted retroviral vectors or liver specific therapeutic nanoparticles generated for an application in cancer gene therapy .

biologists and bioinformaticians have made extensive use of protein interaction information to interpret experimental results in the context of a global protein interaction network and to test new hypotheses . protein interaction databases have played a major role in capturing this information from the literature and in presenting it in a structured format to interested users . nevertheless no single database covers the entire interaction information reported in the literature and , to achieve the largest possible coverage , users or online resources are forced to combine data downloaded from different databases with different data models and ontologies ( 14 ) . to facilitate the exchange and integration of molecular interactions by data providers , databases and data users , the molecular interaction group of the human proteome organization protein standard initiative , has proposed a data representation standard ( current version psi - mi 2.5 ) ( 5 ) . this standard has been adopted by the major protein protein interaction ( ppi ) databases and has formed the basis for the emergence of the international molecular exchange ( imex ) consortium ( http://imex.sourceforge.net/ ) ( 6 ) . imex follows the model of similar initiatives in different domains of biological data , such as the nucleotide sequence exchange between embl ( 7 ) , genbank ( 8) and ddbj ( 9 ) , and aims at distributing the curation workload between participating databases thus avoiding work duplication and increasing literature coverage . the databases adhering to this consortium have developed and adopted a common curation manual ( http://imex.sourceforge.net/doc/imex-curationmanual.doc ) , describing both the information that should be captured by the member databases and how the information should be represented . the consortium currently comprises four active members : dip ( 10 ) , intact ( 11 ) , matrixdb ( 12 ) and mint ( 13 ) . the psi - mi controlled vocabulary , a major component of the psi - mi and imex data representation standard , defines interactions in a broad sense . two proteins are said to interact if they score positive in any of the many experimental procedures used to detect molecular interactions , without implying that they make direct physical contact . however , the experimental evidence is clearly defined in associated database records leaving users free to assess confidence for a given piece of experimental evidence . that is , each entry is annotated with the supporting methods , some of which are understood to provide evidence of direct physical contact between the partner proteins such as x - ray crystallography , nuclear magnetic resonance , biochemical assays carried out on purified proteins , and those for which the presence of bridging molecules can not be excluded such as , pull - down , co - immunoprecipitation and two hybrid assays . a recent major advance in the protein interaction field is the community definition of the minimal information required for reporting a molecular interaction experiment ( mimix ) ( 14 ) . authors reporting protein interaction information in their manuscripts are asked to follow this guidelines , ensuring that this minimal information is unambiguously described with controlled vocabularies and cross - references to major public databases . the checklist contains information such as the name and organism of the proteins , their experimental role ( bait , prey ) , or the detection method . mint , as a member of the imex consortium , is one of the major ppi repositories . the database content has grown ( more than 19 000 experimental evidences have been added since the last report in 2006 ) , the curation policy has been updated to meet psi standards , and the web interface has become more user friendly with the development of new query and graphic tools . in addition each interaction is now annotated with a score ranging from 0 to 1 reflecting the quality and quantity of experimental information supporting the interaction . over the past three years in concert with other databases , mainly intact and dip , the annotation policy has been reviewed to meet the standards of psi - mi and the guidelines of the imex consortium . one major advance of the psi - mi standard has been the replacement of the physical interaction term with two new definitions that permit to discriminate between interactions that have a clear experimental evidence for direct contact between the two partners ( physical association ) and those where the direct contact is not demonstrated ( association ) . each article is curated in its entirety , following the psi - mi recommendations , and all the interactions and the experimental details of their supporting evidences are captured in the database entries . there has recently been some vociferous debate between some data providers and databases regarding the role of database curators with respect to assessment of data quality or reliability ( 15 ) ( thorneycroft et al . to clarify , the mint curation process does not involve any judgment of the accuracy of the published evidence and curators do not make any assessment or ad - hoc reliability ranking of the different interactions reported in a peer reviewed article . this supporting evidence can then be used by database users to filter the data according to their own reliability standards . in order to facilitate the navigation of the results of a query , the mint database associate to each interaction a reliability score ( described below ) that takes into account the experimental support . mint relies on the work of two professional curators and , as a member of the imex consortium , has been assigned the task of curating four journals : febs letters , embo journal , embo reports and more recently the febs journal . all ppi described in an article published by one of these journals are added to mint according to the imex manual . furthermore , as many high - throughput datasets are published by journals not covered by the imex consortium , these articles are curated in rotation by the three member databases . whereas most entries are curated after publication , mint has begun collaborating with febs letter and febs journal on an editorial procedure that , as recommended by the mimix guidelines , involves pre - publication participation of the manuscript authors in the curation process ( 16 ) . the journal editorial offices submit accepted articles to mint curators who , in concert with authors , process the protein interaction information as database entries . the processed information is returned to the journal publisher as a structured digital abstract ( sda ) , where all the interactions described in the article are summarized in a short structured sentence that uses a controlled vocabulary and is appended at the end of the traditional abstract . this sda can be easily parsed by automatic software and , in the online version of the manuscript , they are hyperlinked to relevant databases . as a consequence of limited support ( i.e. small curation team ) and of the curation depth required by imex , mint does not cover all the protein interactions reported in the literature . to increase the coverage in domains of particular interest to our experimental group , mint also contains entries that are not fully imex compliant , albeit adhering to the psi - mi model and controlled vocabularies . light curation and has allowed an increase in the number of articles curated per time unit . for example mint has a very high coverage of the experimental evidence supporting interactions mediated by modular domains such as sh3 , sh2 or 14 - 3 - 3 domains , similarly most interactions between viral and host proteins are annotated in mint . many of the articles supporting these interactions were curated to a lower level of detail than recommended by imex , and only the information required by the mimix guidelines were captured by curators . the differences between imex curation and light ( mimix based ) curation is summarized in table 1 . table 1.differences between imex and light curationannotationimex curationlight curationadditional information / examplespublicationreferencepmid / d.o.i.interactionfigurefigure , tableinteractioninteraction typedirect , physical , enzymatic reactionexperimentdetection methodco - immunoprecipitation , two - hybridexperimentbiosourcetaxid , cell type , tissueinteractorauthor given nameinteractorcross referenceuniprotkb , refseqinteractororganismtaxidinteractorexperimental rolebait / preyinteractorbiological roleenzyme , enzyme target western blotinteractorparticipant identificationinteractorexpression levelendogenous / over - expressed purificationinteractorsample processinteractortaginteractorbinding siterange , domaininteractormodificationphosphorylation , resulting / required position , amino - acidsinteractormutation differences between imex and light curation it is important to understand that entries curated according to the light curation model are as accurate as the imex ones . the same controlled vocabularies , proposed by the psi - mi consortium , are used and in both cases , for instance , the annotator makes a distinction between direct interactions and physical associations , where the experimental evidence can not prove direct association between the partners . most of these light curation entries are annotated by experimentalists that are specialists in the given biological domain and are reviewed by one of the professional curators . the differences between the two curation models do not affect most users , mainly looking for high quality data , but should be taken into consideration when the analysis requires more details about the experimental setup . the entries that are not annotated according to the imex manual are clearly labeled in the web - interface and in the exported files . in addition , a light curator may choose to capture from an article only those interactions related to his topic of interest , for instance interactions involving a specific domain , and skip additional interaction information that may be present in the same article . this label may be used by users that only want to focus on imex data , and by curators who may later complete those entries . in 2009 the oldest interactions in mint , for which these policies had not been followed , have been blocked and hidden to the users . once an interaction is curated and validated in mint it is automatically imported , according to its properties , by one or more sister databases . homomint ( http://mint.bio.uniroma2.it/homomint ) ( 17 ) is a database of human interactions that are either experimentally verified or inferred from model organisms . each time an interaction between human proteins is deposited into mint , it is automatically imported into homomint . the interactions between proteins of model organisms ( for instance rat , mouse , yeast , worm or fly ) are imported into homomint after mapping to the human orthologs . orthologs are retrieved from ensembl compara through the biomart webservice ( http://www.ensembl.org/biomart/martview ) ( 18 ) . the information about the species in which the interaction was experimentally demonstrated is maintained in the homomint entry . the database of domain peptide interactions , domino ( http://mint.bio.uniroma2.it/domino ) ( 19 ) , focuses on interactions mediated by domains ( sh3 , sh2 , 14 - 3 - 3 , pdz , etc . ) . wherever the domain mediating an interaction is specified in the mint entry , the entry is automatically imported into domino . in addition domino contains interactions between domains and peptides that are not present in proteins ( for instance peptides selected by phage display ) and offers a different interface , including the domino viewer applet in which the modular composition of the proteins is displayed . finally , all virus virus and virus host interactions in mint are automatically transferred to virusmint ( http://mint.bio.uniroma2.it/virusmint ) ( 20 ) . virusmint has a specialized interface which focuses on virus interactomes , completed with host interactions connecting the viral networks . this is partly due to experimental false positives , especially in high throughput experiments and partly to the different sensitivity and specificity of the diverse experimental setups . thus , as it remains difficult for the final user to assess the quality of each binary interaction , we have developed a scoring system to facilitate the evaluation of the reliability of each single interaction , with particular focus on direct physical interactions ( 21 ) . the mint scoring system reflects the quantity and quality of independent supporting evidence stored in the database . cumulative evidence as the sum of all the supporting evidence weighted by coefficients that reflects the confidence in the specific approach . this is based on : the size of the experiment : experiments are defined large scale if the article reporting them describes more than 50 interactions , otherwise they are defined small scale . as only the 0.01% of the stored articles report more than 50 interactions we considered this a reasonable threshold to distinguish between large and small scale experiments. the type of experiment supporting the interaction. it emphasizes evidence of direct interaction ( i.e. two - hybrid ) with respect to experimental support that does not provide unequivocal evidence of direct interaction ( i.e. in vivo co - immunoprecipitation). the number of interaction partners detected in a single purification. the sequence similarity of ortholog proteins , for interactions mapped to the human proteome in homomint. the number of different publications supporting the interaction . the resulting score ranges between 0 and 1 and only well supported interactions obtain a value close to 1 . more details and updates about the score are available at http://mint.bio.uniroma2.it/mint/doc/mint-confidence-score.html . the size of the experiment : experiments are defined large scale if the article reporting them describes more than 50 interactions , otherwise they are defined small scale . as only the 0.01% of the stored articles report more than 50 interactions we considered this a reasonable threshold to distinguish between large and small scale experiments . the type of experiment supporting the interaction . it emphasizes evidence of direct interaction ( i.e. two - hybrid ) with respect to experimental support that does not provide unequivocal evidence of direct interaction ( i.e. in vivo co - immunoprecipitation ) . the number of interaction partners detected in a single purification . the sequence similarity of ortholog proteins , for interactions mapped to the human proteome in homomint . the number of different publications supporting the interaction . the scoring system , as illustrated in figure 1 , is an effective tool for filtering interactions . in panel ( a ) we have displayed all the proteins that , according to the mint database interact with the proteins participating in the egfr pathway as defined by the reactome database ( 22 ) . panel ( b ) shows the network that is obtained after removing interactions that are below a certain confidence threshold . interestingly , the remaining interactions can be easily recognized by any biologist familiar with this pathway ( e.g. efgr - grb2 , efgr - shc1 ) . the network in panel ( b ) is obtained by removing interactions below a certain confidence threshold ( 0.72 ) . the network in panel ( b ) is obtained by removing interactions below a certain confidence threshold ( 0.72 ) . mint can be queried online using the web interface available at http://mint.bio.uniroma2.it / mint/. the list of molecules that have been shown to interact with a chosen query protein may be displayed either as text on an html page or as a graph in the viewer applet . in the html output page ( figure 2 ) , we have recently added new columns to provide an overview of the type of evidence supporting the interaction . we distinguish between experimental evidence for direct interactions , associations ( we group here both psi - mi terms association and physical association ) , enzymatic reactions and co - localizations . the number of experiments supporting the association of the two proteins in a larger complex and the number of high throughput experiments are indicated in the last two columns . proteins partners of the e3 ubiquitin - protein ligase cbl . in the right frame interactors the total number of evidences is provided as well as the number of direct interactions , physical associations ( the two psi - mi terms physical association and association are grouped here ) , colocalizations and enzymatic reactions . even if enzymatic reactions are considered according to psi - mi standard as a subtype of direct interactions , there are counted here only as enzymatic reaction . the number of evidences by high throughput experiment and the number of evidences in which the two proteins are part of a larger complex are indicated in the last two columns . each figure in the different columns is linked to the description of the evidences supporting the interaction . by clicking the 22 link on the first row one obtains the information in the the left frame where the evidences for sh3kbp1 as a partner of cbl are grouped by publication and detection method . proteins partners of the e3 ubiquitin - protein ligase cbl . in the right frame interactors the total number of evidences is provided as well as the number of direct interactions , physical associations ( the two psi - mi terms physical association and association are grouped here ) , colocalizations and enzymatic reactions . even if enzymatic reactions are considered according to psi - mi standard as a subtype of direct interactions , there are counted here only as enzymatic reaction . the number of evidences by high throughput experiment and the number of evidences in which the two proteins are part of a larger complex are indicated in the last two columns . each figure in the different columns is linked to the description of the evidences supporting the interaction . by clicking the 22 link on the first row one obtains the information in the the left frame where the evidences for sh3kbp1 as a partner of cbl are grouped by publication and detection method . in the viewer applet the network can be extended by clicking on the + symbol in the small circles ( it is possible to undo this operation by right clicking on the same symbol ) . a protein can be removed from the displayed graph by right - clicking on it . the size of the nodes and the distance between them can be controlled through the slide bars in the upper part of the graphic frame . a third slide bar allows the user to hide interactions whose scores are below a chosen threshold . in the graphic display algorithm , the node repulsion force is proportional to the number of partners . as a consequence , two proteins with many interactors ( hubs ) will tend to lie further away in the graph display when compared with proteins with fewer partners . this graph display rule facilitates the identification of hubs in the graph . as an additional feature , to help identify interaction partners in complex graphs , the action of clicking on a node brings all its partners forward and all the other nodes in the network decrease in size . only bait prey partners are represented in the viewer ( spoke model ) , but all components of the same complex are also brought forward during this operation . any network displayed by the viewer can be exported as a list of protein pairs along with their confidence score ( button score ) , or in either psi - mitab and psi - mi xml format , both described later in this document . the network exported in a standard psi - mi format may be easily imported and analyzed in visualization software such as cytoscape ( 23 ) . both the html page and the viewer applet are connected to relevant information in other pages of the mint website . hyperlinks are provided to the source of the descriptions [ this information is imported from the uniprot knowledge base ( 24 ) ] or the full description of the interactions and the experiments by which they are supported . a visit to the mint web - site typically starts by searching for a gene name , a protein name or a cross - reference [ to uniprotkb , refseq ( 25 ) , etc . ] . if the protein is present in the mint database , the query returns an html page describing the protein on the left frame and listing the interactors in the right frame as described in the previous paragraph . in addition to the information about the protein imported from uniprot , a list of ortholog proteins available in mint is appended . if the protein is human or has a human ortholog , it is possible to switch to homomint , where the network of experimentally verified human protein interactions is extended with the interactions transferred from model organisms . an additional page describes all the experimental details and provides a link to sister databases whenever the interaction is relevant for the specific topic . sister databases offer several advantages , including additional data ( for instance inferred interactions , or interactions between non - natural peptides and proteins ) and an interface adapted to the specific needs of the specialized database ( display of the modular structure of the proteins , visualization of viral - host networks ) . finally , a new tool . this tool permits interrogation of the database with a list of proteins and returns the entire network of interactions connecting them . the search is performed on a list of proteins cross - references ( for instance a list of uniprot accession numbers ) , and the user can choose whether to include in the network additional proteins that connect the query proteins ( figure 3 ) . since the algorithm underlying this tool is demanding up to 100 proteins can be submitted . the node size has been reduced to the minimum with the scrollbar to provide a lighter view of the network . the node size has been reduced to the minimum with the scrollbar to provide a lighter view of the network . all the entries in mint can be freely downloaded in several formats from a public ftp site , and are programmatically accessible through a web - service . the former version of the psi - mi xml format ( 1.0 ) has been deprecated and it is no longer available for downloading . the model is normalized , meaning that the experiments and interactors are not repeated in the interaction descriptions but are first listed and then referenced by the interactions . the normalized model results in lighter files . the psi - mi xml format , in its complex structure , is not human readable but allows a complete description of molecular interactions and their experimental evidences . one of the most relevant advantages is the possibility to associate more than two proteins with an interaction , allowing the correct representation of purified complexes , without misleading binary extensions . additionally , features such as mutations , modifications or binding sites can be annotated with their sequence . the ftp site for psi - mi xml export contains two directories : pmids : a single file is generated for each publication in mint.datasets : containing xml files with the complete dataset or separate files describing interaction entries for each of the main model organisms ( human , yeast , fly and worm ) or a group of phylogenetically related organisms ( e.g. mammals ) . psi - mi xml files for large datasets containing more than 1000 interactions , such as those derived from manuscripts reporting high throughput experiments or datasets for intensively studied organisms , are split in smaller files and zipped together . datasets : containing xml files with the complete dataset or separate files describing interaction entries for each of the main model organisms ( human , yeast , fly and worm ) or a group of phylogenetically related organisms ( e.g. mammals ) . files in the psi - mitab format contain the same information , but in a tab - delimited format that can be opened in a spreadsheet software and it is easier to parse . the columns of version 2.6 are the 15 columns of version 2.5 ( see http://code.google.com/p/psimi/wiki/psimitabformat ) extended with 16 new columns ( read discussion at http://code.google.com/p/psimi/issues/detail?id=2 ) . an expansion strategy is applied in order to represent a complex of three or more proteins in a binary file format . there are two models that can be adopted to achieve this goal . on one hand , matrix model , one can represent the complex as the ensemble of all the possible binary interactions between the protein members . alternatively , spoke model , one member of the complex ( bait ) can be associated to all the remaining members ( prey ) . none of those models faithfully describes the topology of a complex , and the resulting protein pairs are not meant to represent the interaction between the protein in the complex . if the interaction on a row results from the expansion of a complex , the expansion mode ( spoke , matrix ) is specified in this field . the field is empty if the binary interaction is not the result of an expansion procedure . this allows the user to filter rows where the interaction is not supported by an experiment that is evidence for a binary interaction , or to reconstruct the complexes ( for instance by grouping rows by interaction identifier and looking at the experimental role of the components ) . ppi databases , adhering to psi mi , have commonly agreed to use the spoke model as a method to represent n - ary interaction data in a binary format during the psi - mi 2009 spring meeting . this means that if the experiment consists of the identification of many preys with a single bait ( i.e. as in tap tag technology ) , all possible bait prey interactions are represented . if , on the other hand , the role of the proteins in the experiment supporting the existence of the complex is neutral ( i.e. in the case of a complex identified by purification by co - sedimentation ) , one protein is chosen as an arbitrary bait . since we feel that this policy may be misleading for some user cases , we provide two files . exploded according to the spoke model . there is no overlap between those two files , which can be appended one to the other to obtain the full dataset . this issue is not relevant for xml files in which the representation of complexes is possible . in the last additional column , caution interaction , we export the caution annotations described in the previous paragraphs for instance if an interaction is curated with mimix standards rather than fully adhering to the imex curation manual , or if a publication has been only partially curated . finally , we provide a tab - delimited format , similar to mitab , where all the proteins forming a complex are described in a single line ( complexes are not exploded ) . in this format we list the baits or enzymes ( for enzymatic reactions ) in the first column , and the preys or enzyme targets in the second . web services allow the access to the data computationally and have become increasingly more popular in the bioinformatics community . the hupo - psi workgroup has defined a standard web service to access molecular databases : the psicquic interface . documentation about the psicquic interface , the psicquic providers and the reference implementation are available at http://code.google.com / p / psicquic/. italian association for cancer research ( airc ) , telethon and the enfin fp7 network of excellence .

mint ( http://mint.bio.uniroma2.it/mint ) is a public repository for molecular interactions reported in peer - reviewed journals . since its last report , mint has grown considerably in size and evolved in scope to meet the requirements of its users . the main changes include a more precise definition of the curation policy and the development of an enhanced and user - friendly interface to facilitate the analysis of the ever - growing interaction dataset . mint has adopted the psi - mi standards for the annotation and for the representation of molecular interactions and is a member of the imex consortium .

INTRODUCTION ANNOTATION POLICY LIGHT CURATION SISTER DATABASES A SCORING SYSTEM FOR INTERACTION CONFIDENCE WEB INTERFACE DATA AVAILABILITY FUNDING

biologists and bioinformaticians have made extensive use of protein interaction information to interpret experimental results in the context of a global protein interaction network and to test new hypotheses . nevertheless no single database covers the entire interaction information reported in the literature and , to achieve the largest possible coverage , users or online resources are forced to combine data downloaded from different databases with different data models and ontologies ( 14 ) . to facilitate the exchange and integration of molecular interactions by data providers , databases and data users , the molecular interaction group of the human proteome organization protein standard initiative , has proposed a data representation standard ( current version psi - mi 2.5 ) ( 5 ) . this standard has been adopted by the major protein protein interaction ( ppi ) databases and has formed the basis for the emergence of the international molecular exchange ( imex ) consortium ( http://imex.sourceforge.net/ ) ( 6 ) . imex follows the model of similar initiatives in different domains of biological data , such as the nucleotide sequence exchange between embl ( 7 ) , genbank ( 8) and ddbj ( 9 ) , and aims at distributing the curation workload between participating databases thus avoiding work duplication and increasing literature coverage . the consortium currently comprises four active members : dip ( 10 ) , intact ( 11 ) , matrixdb ( 12 ) and mint ( 13 ) . the psi - mi controlled vocabulary , a major component of the psi - mi and imex data representation standard , defines interactions in a broad sense . two proteins are said to interact if they score positive in any of the many experimental procedures used to detect molecular interactions , without implying that they make direct physical contact . however , the experimental evidence is clearly defined in associated database records leaving users free to assess confidence for a given piece of experimental evidence . a recent major advance in the protein interaction field is the community definition of the minimal information required for reporting a molecular interaction experiment ( mimix ) ( 14 ) . the checklist contains information such as the name and organism of the proteins , their experimental role ( bait , prey ) , or the detection method . mint , as a member of the imex consortium , is one of the major ppi repositories . the database content has grown ( more than 19 000 experimental evidences have been added since the last report in 2006 ) , the curation policy has been updated to meet psi standards , and the web interface has become more user friendly with the development of new query and graphic tools . over the past three years in concert with other databases , mainly intact and dip , the annotation policy has been reviewed to meet the standards of psi - mi and the guidelines of the imex consortium . one major advance of the psi - mi standard has been the replacement of the physical interaction term with two new definitions that permit to discriminate between interactions that have a clear experimental evidence for direct contact between the two partners ( physical association ) and those where the direct contact is not demonstrated ( association ) . each article is curated in its entirety , following the psi - mi recommendations , and all the interactions and the experimental details of their supporting evidences are captured in the database entries . to clarify , the mint curation process does not involve any judgment of the accuracy of the published evidence and curators do not make any assessment or ad - hoc reliability ranking of the different interactions reported in a peer reviewed article . this supporting evidence can then be used by database users to filter the data according to their own reliability standards . in order to facilitate the navigation of the results of a query , the mint database associate to each interaction a reliability score ( described below ) that takes into account the experimental support . mint relies on the work of two professional curators and , as a member of the imex consortium , has been assigned the task of curating four journals : febs letters , embo journal , embo reports and more recently the febs journal . all ppi described in an article published by one of these journals are added to mint according to the imex manual . furthermore , as many high - throughput datasets are published by journals not covered by the imex consortium , these articles are curated in rotation by the three member databases . whereas most entries are curated after publication , mint has begun collaborating with febs letter and febs journal on an editorial procedure that , as recommended by the mimix guidelines , involves pre - publication participation of the manuscript authors in the curation process ( 16 ) . the processed information is returned to the journal publisher as a structured digital abstract ( sda ) , where all the interactions described in the article are summarized in a short structured sentence that uses a controlled vocabulary and is appended at the end of the traditional abstract . this sda can be easily parsed by automatic software and , in the online version of the manuscript , they are hyperlinked to relevant databases . small curation team ) and of the curation depth required by imex , mint does not cover all the protein interactions reported in the literature . to increase the coverage in domains of particular interest to our experimental group , mint also contains entries that are not fully imex compliant , albeit adhering to the psi - mi model and controlled vocabularies . for example mint has a very high coverage of the experimental evidence supporting interactions mediated by modular domains such as sh3 , sh2 or 14 - 3 - 3 domains , similarly most interactions between viral and host proteins are annotated in mint . many of the articles supporting these interactions were curated to a lower level of detail than recommended by imex , and only the information required by the mimix guidelines were captured by curators . table 1.differences between imex and light curationannotationimex curationlight curationadditional information / examplespublicationreferencepmid / d.o.i.interactionfigurefigure , tableinteractioninteraction typedirect , physical , enzymatic reactionexperimentdetection methodco - immunoprecipitation , two - hybridexperimentbiosourcetaxid , cell type , tissueinteractorauthor given nameinteractorcross referenceuniprotkb , refseqinteractororganismtaxidinteractorexperimental rolebait / preyinteractorbiological roleenzyme , enzyme target western blotinteractorparticipant identificationinteractorexpression levelendogenous / over - expressed purificationinteractorsample processinteractortaginteractorbinding siterange , domaininteractormodificationphosphorylation , resulting / required position , amino - acidsinteractormutation differences between imex and light curation it is important to understand that entries curated according to the light curation model are as accurate as the imex ones . the same controlled vocabularies , proposed by the psi - mi consortium , are used and in both cases , for instance , the annotator makes a distinction between direct interactions and physical associations , where the experimental evidence can not prove direct association between the partners . most of these light curation entries are annotated by experimentalists that are specialists in the given biological domain and are reviewed by one of the professional curators . the differences between the two curation models do not affect most users , mainly looking for high quality data , but should be taken into consideration when the analysis requires more details about the experimental setup . the entries that are not annotated according to the imex manual are clearly labeled in the web - interface and in the exported files . this label may be used by users that only want to focus on imex data , and by curators who may later complete those entries . homomint ( http://mint.bio.uniroma2.it/homomint ) ( 17 ) is a database of human interactions that are either experimentally verified or inferred from model organisms . orthologs are retrieved from ensembl compara through the biomart webservice ( http://www.ensembl.org/biomart/martview ) ( 18 ) . the information about the species in which the interaction was experimentally demonstrated is maintained in the homomint entry . the database of domain peptide interactions , domino ( http://mint.bio.uniroma2.it/domino ) ( 19 ) , focuses on interactions mediated by domains ( sh3 , sh2 , 14 - 3 - 3 , pdz , etc . ) wherever the domain mediating an interaction is specified in the mint entry , the entry is automatically imported into domino . in addition domino contains interactions between domains and peptides that are not present in proteins ( for instance peptides selected by phage display ) and offers a different interface , including the domino viewer applet in which the modular composition of the proteins is displayed . finally , all virus virus and virus host interactions in mint are automatically transferred to virusmint ( http://mint.bio.uniroma2.it/virusmint ) ( 20 ) . virusmint has a specialized interface which focuses on virus interactomes , completed with host interactions connecting the viral networks . this is partly due to experimental false positives , especially in high throughput experiments and partly to the different sensitivity and specificity of the diverse experimental setups . thus , as it remains difficult for the final user to assess the quality of each binary interaction , we have developed a scoring system to facilitate the evaluation of the reliability of each single interaction , with particular focus on direct physical interactions ( 21 ) . this is based on : the size of the experiment : experiments are defined large scale if the article reporting them describes more than 50 interactions , otherwise they are defined small scale . as only the 0.01% of the stored articles report more than 50 interactions we considered this a reasonable threshold to distinguish between large and small scale experiments. the type of experiment supporting the interaction. it emphasizes evidence of direct interaction ( i.e. more details and updates about the score are available at http://mint.bio.uniroma2.it/mint/doc/mint-confidence-score.html . the size of the experiment : experiments are defined large scale if the article reporting them describes more than 50 interactions , otherwise they are defined small scale . as only the 0.01% of the stored articles report more than 50 interactions we considered this a reasonable threshold to distinguish between large and small scale experiments . the number of different publications supporting the interaction . the scoring system , as illustrated in figure 1 , is an effective tool for filtering interactions . panel ( b ) shows the network that is obtained after removing interactions that are below a certain confidence threshold . interestingly , the remaining interactions can be easily recognized by any biologist familiar with this pathway ( e.g. the network in panel ( b ) is obtained by removing interactions below a certain confidence threshold ( 0.72 ) . the network in panel ( b ) is obtained by removing interactions below a certain confidence threshold ( 0.72 ) . mint can be queried online using the web interface available at http://mint.bio.uniroma2.it / mint/. in the html output page ( figure 2 ) , we have recently added new columns to provide an overview of the type of evidence supporting the interaction . we distinguish between experimental evidence for direct interactions , associations ( we group here both psi - mi terms association and physical association ) , enzymatic reactions and co - localizations . the number of experiments supporting the association of the two proteins in a larger complex and the number of high throughput experiments are indicated in the last two columns . proteins partners of the e3 ubiquitin - protein ligase cbl . in the right frame interactors the total number of evidences is provided as well as the number of direct interactions , physical associations ( the two psi - mi terms physical association and association are grouped here ) , colocalizations and enzymatic reactions . even if enzymatic reactions are considered according to psi - mi standard as a subtype of direct interactions , there are counted here only as enzymatic reaction . the number of evidences by high throughput experiment and the number of evidences in which the two proteins are part of a larger complex are indicated in the last two columns . each figure in the different columns is linked to the description of the evidences supporting the interaction . by clicking the 22 link on the first row one obtains the information in the the left frame where the evidences for sh3kbp1 as a partner of cbl are grouped by publication and detection method . proteins partners of the e3 ubiquitin - protein ligase cbl . in the right frame interactors the total number of evidences is provided as well as the number of direct interactions , physical associations ( the two psi - mi terms physical association and association are grouped here ) , colocalizations and enzymatic reactions . even if enzymatic reactions are considered according to psi - mi standard as a subtype of direct interactions , there are counted here only as enzymatic reaction . the number of evidences by high throughput experiment and the number of evidences in which the two proteins are part of a larger complex are indicated in the last two columns . each figure in the different columns is linked to the description of the evidences supporting the interaction . the size of the nodes and the distance between them can be controlled through the slide bars in the upper part of the graphic frame . as an additional feature , to help identify interaction partners in complex graphs , the action of clicking on a node brings all its partners forward and all the other nodes in the network decrease in size . only bait prey partners are represented in the viewer ( spoke model ) , but all components of the same complex are also brought forward during this operation . any network displayed by the viewer can be exported as a list of protein pairs along with their confidence score ( button score ) , or in either psi - mitab and psi - mi xml format , both described later in this document . the network exported in a standard psi - mi format may be easily imported and analyzed in visualization software such as cytoscape ( 23 ) . both the html page and the viewer applet are connected to relevant information in other pages of the mint website . hyperlinks are provided to the source of the descriptions [ this information is imported from the uniprot knowledge base ( 24 ) ] or the full description of the interactions and the experiments by which they are supported . a visit to the mint web - site typically starts by searching for a gene name , a protein name or a cross - reference [ to uniprotkb , refseq ( 25 ) , etc . ] if the protein is present in the mint database , the query returns an html page describing the protein on the left frame and listing the interactors in the right frame as described in the previous paragraph . in addition to the information about the protein imported from uniprot , a list of ortholog proteins available in mint is appended . an additional page describes all the experimental details and provides a link to sister databases whenever the interaction is relevant for the specific topic . sister databases offer several advantages , including additional data ( for instance inferred interactions , or interactions between non - natural peptides and proteins ) and an interface adapted to the specific needs of the specialized database ( display of the modular structure of the proteins , visualization of viral - host networks ) . this tool permits interrogation of the database with a list of proteins and returns the entire network of interactions connecting them . the search is performed on a list of proteins cross - references ( for instance a list of uniprot accession numbers ) , and the user can choose whether to include in the network additional proteins that connect the query proteins ( figure 3 ) . the node size has been reduced to the minimum with the scrollbar to provide a lighter view of the network . the node size has been reduced to the minimum with the scrollbar to provide a lighter view of the network . all the entries in mint can be freely downloaded in several formats from a public ftp site , and are programmatically accessible through a web - service . the former version of the psi - mi xml format ( 1.0 ) has been deprecated and it is no longer available for downloading . the psi - mi xml format , in its complex structure , is not human readable but allows a complete description of molecular interactions and their experimental evidences . one of the most relevant advantages is the possibility to associate more than two proteins with an interaction , allowing the correct representation of purified complexes , without misleading binary extensions . the ftp site for psi - mi xml export contains two directories : pmids : a single file is generated for each publication in mint.datasets : containing xml files with the complete dataset or separate files describing interaction entries for each of the main model organisms ( human , yeast , fly and worm ) or a group of phylogenetically related organisms ( e.g. psi - mi xml files for large datasets containing more than 1000 interactions , such as those derived from manuscripts reporting high throughput experiments or datasets for intensively studied organisms , are split in smaller files and zipped together . datasets : containing xml files with the complete dataset or separate files describing interaction entries for each of the main model organisms ( human , yeast , fly and worm ) or a group of phylogenetically related organisms ( e.g. files in the psi - mitab format contain the same information , but in a tab - delimited format that can be opened in a spreadsheet software and it is easier to parse . the columns of version 2.6 are the 15 columns of version 2.5 ( see http://code.google.com/p/psimi/wiki/psimitabformat ) extended with 16 new columns ( read discussion at http://code.google.com/p/psimi/issues/detail?id=2 ) . alternatively , spoke model , one member of the complex ( bait ) can be associated to all the remaining members ( prey ) . none of those models faithfully describes the topology of a complex , and the resulting protein pairs are not meant to represent the interaction between the protein in the complex . if the interaction on a row results from the expansion of a complex , the expansion mode ( spoke , matrix ) is specified in this field . the field is empty if the binary interaction is not the result of an expansion procedure . this allows the user to filter rows where the interaction is not supported by an experiment that is evidence for a binary interaction , or to reconstruct the complexes ( for instance by grouping rows by interaction identifier and looking at the experimental role of the components ) . ppi databases , adhering to psi mi , have commonly agreed to use the spoke model as a method to represent n - ary interaction data in a binary format during the psi - mi 2009 spring meeting . this means that if the experiment consists of the identification of many preys with a single bait ( i.e. if , on the other hand , the role of the proteins in the experiment supporting the existence of the complex is neutral ( i.e. exploded according to the spoke model . this issue is not relevant for xml files in which the representation of complexes is possible . in the last additional column , caution interaction , we export the caution annotations described in the previous paragraphs for instance if an interaction is curated with mimix standards rather than fully adhering to the imex curation manual , or if a publication has been only partially curated . finally , we provide a tab - delimited format , similar to mitab , where all the proteins forming a complex are described in a single line ( complexes are not exploded ) . in this format we list the baits or enzymes ( for enzymatic reactions ) in the first column , and the preys or enzyme targets in the second . documentation about the psicquic interface , the psicquic providers and the reference implementation are available at http://code.google.com / p / psicquic/. italian association for cancer research ( airc ) , telethon and the enfin fp7 network of excellence .

figure 1 represents the schematic diagram of our solid - state , label - free plasmonic biosensor fabrication for mir detection . chemically synthesized gold nanoprisms ( figure 1a ) , which displayed their lspr at 797 nm upon attachment to solid substrate immersed in pbs buffer , were selected as nanoantennas for our biosensor fabrication because ( 1 ) their lspr peak position ( in the 700900 nm wavelength range ) is particularly suitable for biomolecule detection because of negligible background scattering and adsorption of endogeneous chromophores from physiological media such as blood and plasma ; ( 2 ) they have strong electromagnetic ( em ) field enhancement at the sharp tips ; ( 3 ) they exhibits a strong lspr response to small changes in their surrounding environment ; ( 4 ) their atomically smooth surface allows formation of a self - assembled monolayer ( sam ) of receptors with both a tightly packed lower layer of alkylthiols and a more loosely packed upper layer that provide the required space for duplex formation with complementary mir strands ; ( 5 ) gold is nontoxic and stable under extreme physiological conditions ; and ( 6 ) the gold sulfur bond is very stable with thiol - modified receptors making a strong covalent bond with the gold surface . details describing the synthesis of gold nanoprisms and their attachment onto the silanized glass substrate are provided in supporting information . recently , we have shown that a molecular sensor fabricated using an 35 nm average edge - length gold nanoprisms displayed an unprecedentedly large 21 nm reversible shift upon a minor 0.6 nm increase in the thickness of the local dielectric environment . therefore , gold nanoprisms of this size and geometry are unique and should provide extremely high sensitivity if plasmonic biosensors are fabricated using them , which is the scope of this letter . this letter provides the first example of lspr - based mirs sensing in physiological media . design of plasmonic biosensors and detecting mir - x in various physiological media . ( a ) chemically synthesized and freshly prepared gold nanoprisms were covalently attached onto a 3-mercaptopropyltriethoxysilane - functionalized glass coverslip ( substrate ) . ( b ) surface of gold nanoprisms was chemically modified with a 1.0 m 1:1 mixture of sh - c6-ssdna - x and peg6-sh in pbs buffer ( ph 7.4 ) to prepare the plasmonic biosensor . ( c ) incubation of sensor in mir - x solution and formation of dna duplex . ( d ) schematic of the extinction spectrum of the biosensor collected in pbs buffer after modification with a 1.0 m 1:1 mixture of sh - c6-ssdna - x and peg6-sh ( blue curve ) . the extinction spectrum was again collected after incubation in mir - x solution and careful rinsing with pbs buffer to determine the new peak position ( red curve ) . the extent of lspr dipole peak shift ( lspr ) depends on the concentration of mir - x used during the incubation in ( c ) , which ranged from 100 nm to 50 fm . ( e ) plot of lspr versus log of mir - x concentrations used to determine the limit of detection . our targets were mir-21 and mir-10b , because we have shown by locked nuclei acid based in situ hybridization that they are overexpressed in pancreatic cells ( pccs ) within the tumor mass and that circulating mir-10b may serve as biomarker for diagnosis of pdac . we designed the sensing strategy based on the hybridization between complementary probes ( -c6-ssdna - x , x = 21 and 10b ) attached to gold nanoprisms and their target mirs . the introduction of spacers in - between the dna probes was included to reduce steric hindrance between the probes and the mirs and therefore to enhance the hybridization and ultimately the sensitivity . as shown in figure 1b , poly(ethylene glycol)6-thiols ( peg6-sh ) were used as spacers because they avoid nonspecific adsorption of extraneous materials onto the nanoprism s surface and are not reactive toward mirs or other biological constituents present in plasma . previously , we demonstrated that functionalization of a nanoantenna s surface with an equal mole ratio of receptor and spacer provided the best sensitivity and lowest limit of detection ( lod ) . therefore , a 1:1 ratio of hs c6-ssdna - x : peg6-sh was used to prepare the plasmonic biosensors ( figure 1b ) . all the mirs and oligonucleotides sequences used in these studies are provided in supporting information , table s1 . as illustrated in figure 2a , we used uv vis spectroscopy to monitor the changes in lspr of the gold nanoprisms at different functionalization steps . the functionalization of substrate - bound nanoprisms with 1:1 ratio of hs c6-ssdna-21:peg6-sh resulted in an 20.5 3.2 nm red - shift of lspr as a result of the increase in local refractive index , which suggested the attachment of both molecular species onto the nanoprism s surface . these plasmonic biosensors were utilized for mir detection by incubating mir-21 ( obtained from sigma - aldrich , u.s.a . ) with concentration ranging from 100 nm to 50 fm in pbs buffer , 40% bovine plasma , or 40% human plasma . the lspr response of gold nanoprisms for each mir-21 concentration was measured where the highest 18.8 1.9 nm lspr red shift was observed for 100 nm mir-21 ( figure 2a , blue ) in pbs buffer . we hypothesize that the lspr red - shift is due to hybridization between ssdna-21 and mir-21 . it was found that the magnitude of the lspr shift was concentration dependent , where 50 fm mir-21 caused a 3.7 0.3 nm lspr red shift in pbs buffer . figure 2b illustrates the magnitude of the lspr shift ( lspr ) upon dna / rna duplex formation for various mir-21 concentrations in the three different media . evidently higher concentrations of mir-21 induced a larger number of ssdna-21 strands to convert to dna / rna duplexes and consequently a larger change in the local refractive index around the nanoprisms , which results in a larger value of lspr . ( a ) monitoring lspr dipole peak ( lspr ) changes by uv visible absorption spectra of gold nanoprisms during various functionalization steps : before ( black , lspr = 800 nm ) and after functionalized with a 1 m/1 m ratio of hs - c6-ssdna-21/peg6-sh ( red , lspr = 821 nm ) , and after incubation with 100 nm mir-21 solution in pbs buffer ( blue , lspr = 839 nm ) . ( b ) average lspr peak shift ( lspr ) of gold nanoprisms functionalized with a 1 m/1 m ratio of hs - c6-ssdna-21/peg6-sh after incubation in different concentrations of mir-21 in pbs buffer ( red triangles ) , 40% human plasma ( black squares ) , and 40% bovine plasma ( blue diamonds ) . the lspr were calculated by taking the difference between the lspr peak position of the plasmonic biosensor after and before the hybridization with mir-21 in the various media . ( c ) average lspr of the plasmonic biosensor functionalized with a 1 m/1 m ratio of hs - c6-ssdna-10b / peg6-sh after hybridization with different mir-10b concentrations in pbs buffer ( red triangles ) , in 40% human plasma ( black squares ) , and in 40% bovine plasma ( blue diamonds ) . all extinction spectra recorded after mir - x incubation were measured in pbs buffer after rinsing with pbs buffer . the highest red shift of 18.8 1.9 nm we observed for 100 nm mir-21 incubation with our plasmonic biosensor is significant considering only an 5% change in the refractive index upon ssdna / rna duplex formation . we believe such a high sensitivity of our plasmonic biosensors is because of the unique lspr properties of our gold nanoprisms and the possibility of electron delocalization as the ssdna forms the duplex and becomes double - strand . the atomically flat surface , extremely small height ( 8 nm ) , and sharp tips of our nanoprisms display strong em field enhancement near their surface and therefore are expected to be extremely sensitive to small changes of their local dielectric environment . moreover , transformation of ssdna into double - strand dna significantly changes the refractive index because of the high charge density and polarizability of the dnas . the duplex dna is capable of long - range charge transfer and alters the electron density around the nanoprisms thus influencing their lspr properties . this interesting phenomenon requires further scientific study , which is currently under our investigation . our sensing mechanism is based on the hypothesis that the attachment of complementary target mirs to our plasmonic biosensor will shift the lspr to higher wavelength ( figure 1c ) . the total shift ( lspr ) depended on the mir concentration ( figure 1d ) and could be used to determine the limit of detection ( lod ) ( figure 1e ) . the lods calculated for mir-21 in three different media were found to be in the range of 2335 fm , which was more than 1000 and 3 fold lower than with the label - free microring resonator ( 150 fmol ) and the nanopore based ( 100 fm ) mir sensors , respectively . importantly , these techniques detected mirs in pbs buffer whereas we have demonstrated here for the first time a sensing approach in physiological media . utilizing our same direct hybridization - based detection approach , plasmonic biosensors were constructed with of c6-ssdna-10 , while keeping other parameters constant . the lod for mir-10b in the above media was determined over a concentration range from 100 nm to 50 fm . the average lspr and lods for mir-10b in three diverse media are shown in figure 2c . the principle underlying the actions of plasmonic biosensors is based on the successful hybridization between mirs and ssdna attached to nanoprisms , where a higher number of duplex formations will result in a larger change in the refractive index surrounding the nanoprisms resulting in larger lspr and higher sensitivity . therefore , it would be expected that functionalization of gold nanoprisms with 100% hs c6-ssdna - x ( without the peg6-thiol spacer ) should reduce the lod values because of steric hindrance and low attachment of mirs . to investigate this , gold nanoprisms were functionalized with 100% c6-ssdna-21 resulting in an 15.0 1.8 nm lspr red shift ( figure 3a ) . the sensor was then incubated in different concentrations of mir-21 prepared in 40% human plasma . as illustrated in figure 3a , an 9.6 1.1 nm red shift was observed for a 100 nm mir-21 concentrations and the lowest concentration that can be repeatedly detected was 10 pm from a lspr of 3.4 0.5 nm . figure 3b shows the lspr versus concentration plot . evidently , functionalization of the nanoprism s surface with 100% c6-ssdna-21 resulted in a 200-fold increase in detection limit in comparison to the 1:1 ratio c6-ssdna-21/peg6-sh mixed functionalization ( supporting information table s2 ) . these experimental data further highlight our rationale for using spacers that increase the likelihood of hybridization . we believe fully covered gold nanoprisms were obtained when 100% c6-ssdna-21 was used for functionalization , which creates steric hindrance and does not allow the maximum number of mir-21 strands to come into close proximity with c6-ssdna-21 for hybridization . therefore , not all the c6-ssdna-21 attached on the gold nanoprisms surface was hybridized with mir-21 strands resulting in low sensing response . thus , if we introduce a spacer between the c6-ssdna-21 , it will allow the maximum -ssdna-21 strands to be freely available for hybridization without any interference and ultimately enhance the sensitivity of the biosensor . accordingly , for the remaining of our investigation , we used a 1:1 mixed c6-ssdna - x : peg6-sh to functionalize the gold nanoprisms . ( a ) uv visible extinction spectra monitoring the lspr dipole peak ( lspr ) of gold nanoprisms attached to silanized glass substrate before ( black , lspr = 796 nm ) and after ( red , lspr = 811 nm ) functionalization with 1 m of hs - c6-ssdna-21 without peg6-sh spacers and after incubation in 100 nm mir-21 solution in 40% human plasma ( blue , lspr = 822 nm ) . ( b ) average lspr of these hs - c6-ssdna-21 functionalized gold nanoprisms upon hybridization with different mir-21 concentrations in 40% human plasma . the lspr were calculated by taking the difference between the lspr peak position of the nanoprisms after and before the incubation with mir-21 in pbs buffer . in order to confirm the hybridization of mir - x with c6-ssdna - x that resulted in the lspr , the enzyme rnase h was used to selectively cleave the dna : rna duplex and potentially reverse the lspr . initially , the plasmonic biosensor for mir-21 was incubated in a 100 nm solution of mir-21 , which resulted in red - shift of lspr potentially reflecting hybridization . the biosensor was then immersed in 15 units of rnase h solution for 2 h. afterward the lspr showed a blue shift and reverted back to its original position before mir-21 incubation ( figure 4a ) . when the 1:1 ratio c6-ssdna-21:peg6-sh mixed functionalized biosensor was incubated with rnase h solution alone overnight , no noticeable change in lspr value was observed ( supporting information figure s7 ) . these experimental results validate our previous observation that the lspr blue shift was due to the cleavage of heteroduplex done by rnase h. the biosensors were rinsed with rnase free water to pbs buffer and again incubated in 100 nm mir-21 solution for rehybridization where an 14 nm red shift of the lspr was observed . these experiments confirm our working hypothesis that hybridization between the nanoprism s surface ligands ( c6-ssdna - x ) and the mir - x resulted in changes in the local dielectric environment around the nanoprisms causing wavelength shift . as shown in figure 4b , the lspr responses were identical for several cycles due to hybridization and dehybridization of mir-21 over a period of 6 days . the same experiments were done for the mir-10b biosensor and similar results were observed , underscoring the long - term stability of the sensors and their potential for being developed into cost - effective point of care diagnostic tools . ( a ) uv visible extinction spectra of gold nanoprisms functionalized with a 1 m/1 m ratio of hs - c6-ssdna-21/peg6-sh ( red , lspr = 818 nm ) attached to silanized glass , after incubation with 100 nm of mir-21 ( blue , lspr = 832 nm ) , after treatment with 15 units of rnase h for 2 h ( red dotted , lspr = 818 nm ) , and after rehybridized with 100 nm of mir-21 ( blue dotted , lspr = 832 nm ) . ( b ) changes in lspr dipole peak position ( lspr ) of gold nanoprisms functionalized with a 1 m/1 m ratio of hs - c6-ssdna-21/peg6-sh upon hybridization and dehybridization with mir-21 for several cycles . the lspr peak shifts back and forth upon sensor regeneration with rnase h by cleaving dna / rna duplex and rehybridization after incubation into 100 nm mir-21 in 40% human plasma . after each of the dehybridization steps , the plasmonic biosensors were thoroughly rinsed with pbs buffer to completely remove enzyme rnase h. the hybridization takes place at the 5 end of c6-ssdna-21 and the 3 end of mir-21 , which evidently increased the refractive index . additionally such hybridization would also increase the thickness of the local dielectric environment of the nanoprisms . together , a significantly large lspr was generated for both mir-21 and mir-10b . atomic force microscopy ( afm ) analysis was conducted to characterize our plasmonic biosensors and also to verify the change in surface area caused by mir-21 incubation with mixed c6-ssdna-21 and peg6-sh - functioanlized gold nanoprisms . after analyzing 40 different nanoprisms ( figure 5 and supporting information figure s8 ) the exact same area of four different sections of the sensor - an average 2.4 10 m increase in surface area was observed by afm . thus , attachment of mirs to plasmonic biosensors has increased the thickness of local dielectric environment around the gold nanoprisms and influenced their lspr properties . ultrasensitive refractive index - induced lspr response of nanoprisms allows us to fabricate label - free plasmonic biosensor . atomic force microscopy images of gold nanoprisms bound to silanized glass substrate ( a ) after functionalization with 1:1 ratio of hs - c6-ssdna-21/peg6-sh ( b ) and after hybridization with100 nm mir-21 in 40% human plasma ( c ) . ( d ) changes in the surface area of gold nanoprisms after each functionalization steps . the successful implementation of plasmonic biosensors for use with real biological samples mandates documentation of their specificity toward target mirs in that patient samples containing multiple mir species . the mix functionalized ( c6-ssdna-21 and peg6-sh ) biosensors were incubated overnight in 40% human plasma solution containing 100 nm each of mir-16 , mir-122 , mir-126 , and mir-141 because these mirs are commonly present in human plasma . the lspr response was measured before and after incubation ( supporting information figure s9 ) and resulted in an 2.5 0.3 nm lspr red shift , which is within the instrument noise level and/or minor nonspecific adsorption of extraneous materials present in human plasma . in another control experiment , gold nanoprisms attached as before to glass substrate were functionalized with 100% peg6-sh by incubation in a 1 m aqueous solution , and after rinsing with large amounts of water , incubated in a 40% human plasma solution of 100 nm mir-21 for 12 h. this procedure resulted in only an 0.9 0.7 nm lspr red shift ( supporting information figure s10 ) , confirming that the plasmonic biosensors we designed are highly specific toward the target mirs . pancreatic cancer is the fourth - leading cause of cancer death in the united states with an annual mortality of nearly 40 000 and a dismal five - year survival rate of 6% . pdac is characterized by chemotherapeutic resistance and by the absence of an effective screening procedure for early disease . it is generally accepted that early diagnosis could reduce mortality rates substantially and thus a noninvasive early pdac test must be developed . several mirs ( such as mir-21 , -10b , -103 , -155 , -196a , 210 , and -221 ) were found to be overexpressed in pdac . given their resistance to degradation , plasma mirs have the potential to serve as biomarkers for the noninvasive diagnosis of pdac . previously , nanopore sensors were used to detect mirs in lung cancer patients , but to the best of our knowledge no sensors have been developed to date to detect pdac - related mirs in human plasma . utilizing our plasmonic biosensors we detected mir-21 and mir-10b in plasma from pdac patients . total plasma rnas including mirs were extracted from 100 l of each plasma sample using a trizol kit with a final elution volume of 28 l . next , 14 l volumes were used for mir quantification by the plasmonic biosensor and the remaining 14 l were used in the qrt - pcr assay . the plasmonic biosensors were fabricated as described before for both mir-21 and mir-10b detection . the extracted human mir-21 or mir-10b samples were diluted in pbs buffer and incubated with the biosensors were for 12 h , followed by rinsing with pbs buffer and measurement of the lspr response in pbs buffer . the observed lspr shift for each mir-21 and mir-10b sample was converted into the corresponding concentration using the calibration curve derived for mir-21 or 10b under optimized conditions and compared with the value from normal control subjects ( figure 6a , c ) . the concentrations of mir-21 and mir-10b determined from plasmonic biosensors were also compared with the values obtained from the qrt - pcr assay ( figure 6b and supporting information figure s16 ) . importantly , for the first time through a label - free technique we have shown that mir-10b concentration is nearly 4-fold higher than the mir-21 level in patient samples . inasmuch as both mir-21 and mir-10b are overexpressed in pdac , it is possible that mir-10b is released more efficiently by pancreatic cancer cells than mir-21 , allowing it to achieve higher levels in the circulation . it is therefore possible that mir-10b levels are also increased within the pancreatic tumor microenvironment , where it could be acting to enhance pdac biological aggressiveness . ( a ) the average lspr peak shifts of gold nanoprisms functionalized with a 1:1 ratio of hs - c6-ssdna-21/peg6-sh upon hybridization with mir-21 from the total rnas extracted from plasma samples of pdac patients ( blue diamonds ) and normal control subjects ( blue squares ) . the respective lspr peak shifts were converted to concentrations using the calibration curve established for mir-21 in pbs buffer as shown in figure 2b [ pdac patients ( red triangles ) , and for normal control subjects ( red circles ) ] . ( b ) comparison of mir-21 concentration for six pdac patients determined using plasmonic biosensors ( blue diamond ) and qrt - pcr ( red square ) . ( c ) similar experiments were conducted to detect mir-10b where the lspr peak shifts and concentrations for pdac patients are shown in blue diamonds and red triangles , respectively . the lspr peak shifts ( blue squares ) and concentrations ( red circles ) for normal controls are shown for comparison . ( d ) the average lspr peak shifts ( blue diamonds ) and concentration ( red triangles ) for the mir-21 in plasma samples from pdac patients without any purification . we also detected mir-21 levels directly in human plasma samples collected from pdac patients without rnas extraction . thus , human plasma ( 50 l / sample ) from six pancreatic cancer patients were diluted in pbs buffer followed by incubation with our plasmonic biosensors for 12 h. the lspr response of each sample was measured through uv vis spectroscopy and showed a steady increase in concentration from sample 6 to 1 ( figure 6d ) . both plasmonic biosensor and qrt - pcr results indicated that mir-10b levels were higher in pdac patients compared to normal control subjects and that the levels of mir-21 and mir-10b can be quantified with high accuracy using our gold nanoprism - based plasmonic biosensor without any modification , amplification , or labeling . importantly , the mir-21 concentration in extracted samples was at least 2-fold lower than in the pure plasma samples . we believe this is due to loss of mirs during the rna extraction process , which requires multiple steps for rna purification . therefore , the most widely used qrt - pcr method to determine the concentration of mirs in patients may not accurately represent the actual concentration . this limitation and imprecise quantification can be avoided by using our newly developed plasmonic biosensors , which can provide a unique opportunity as potential diagnostic and prognostic markers in pdac , other cancers , and potentially other disease states . we have designed , fabricated , and characterized a plasmonic biosensor that was able to detect pdac relevant mirs in human plasma without using rnas extraction , which opens a new avenue for the direct detection and quantification of mir levels in clinical samples without any form of sample preparation . to our knowledge , this is the first lspr - based , label - free , direct hybridization method for mir detection , which eliminates all the current drawbacks such as labeling , tagging , amplification , use of highly toxic chemical , and further modification of the sensor . furthermore , it vastly simplifies the detection approach without requiring detailed knowledge of the electron or energy transfer processes involved as in other more complicated techniques . additionally , this ultrasensitive , plasmonic - based , direct hybridization - controlled detection approach is applicable to any type of mirs that are relevant to various diseases . it was found that our plasmonic biosensor can be regenerated through several cycles and is stable for several days without compromising its sensitivity and selectivity , which should enable the development of simple , cost - effective tools for the early detection of mirs and thus facilitate the early diagnosis of various cancers . finally , the large em - field enhancement at the nanoprism s sharp tips will enhance the raman scattering intensity of the analytes . in theory , therefore , nanoprisms can be used to design effective substrates for surface - enhanced raman spectroscopy - based detection and quantification of multiple mirs simultaneously through integration of their spectral characteristic with the lspr shifts . all synthetic dna probes and micrornas were purchased from sigma - aldrich ( u.s.a . ) . pbs buffer prepared with rnase - free water was used to dilute oligonucleotides and mirs solutions . patient plasma was obtained from the indiana university simon cancer center solid tissue bank ( indianapolis , indiana ) . the gold nanoprism - based mir sensors were designed using our published procedure with modification . the attachment of gold nanoprisms on silanized glass substrates is described in the supporting information file . the substrate - bound nanoprisms were incubated in pbs buffer solution containing 1 m each of hs c6-ssdna - x and peg6-sh overnight and rinsed with pbs buffer . the initial lspr peak position of each sensing platform was determined using uv visible spectroscopy in pbs buffer and then was incubated in the different concentrations of mir solutions , for exmaple , either in pbs buffer , 40% bovine plasma , or 40% human plasma for 12 h at room temperature . the plasmonic biosensors were thoroughly washed with pbs buffer to remove any nonspecifically adsorbed species . the mir bound biosensor was then placed in pbs buffer for 10 min before the lspr peak position was determined . for uv vis extinction spectra measurement , one particular solvent was chosen to avoid the solvent dielectric constant effect , which is known to shift the lspr peak . total rna was isolated from plasma samples that were obtained from the indiana university simon cancer center solid tissue bank ( indianapolis , in , u.s.a . ) using trizol - ls reagent ( life technologies , carlsbad , ca , u.s.a . ) . cdna was generated using 10 ng of rna and mir-10b , mir-21 , or mir-4255p rt primers and a mir reverse transcription kit ( life technologies ) as per the manufacturer s recommendations . expression levels as determined by qpcr were normalized to mir-425 - 5p , since this mir was expressed at similar levels in all samples and exhibited < 1 cycle threshold ( ct ) difference across all samples . after normalization to mir-425 - 5p ( ct ) , the ct values for mirs in controls were averaged and subtracted from the ct values of each individual sample ( ct ) . the gold nanoprism - based mir sensors were designed using our published procedure with modification . the attachment of gold nanoprisms on silanized glass substrates is described in the supporting information file . the substrate - bound nanoprisms were incubated in pbs buffer solution containing 1 m each of hs c6-ssdna - x and peg6-sh overnight and rinsed with pbs buffer . the initial lspr peak position of each sensing platform was determined using uv visible spectroscopy in pbs buffer and then was incubated in the different concentrations of mir solutions , for exmaple , either in pbs buffer , 40% bovine plasma , or 40% human plasma for 12 h at room temperature . the plasmonic biosensors were thoroughly washed with pbs buffer to remove any nonspecifically adsorbed species . the mir bound biosensor was then placed in pbs buffer for 10 min before the lspr peak position was determined . for uv vis extinction spectra measurement , one particular solvent was chosen to avoid the solvent dielectric constant effect , which is known to shift the lspr peak . total rna was isolated from plasma samples that were obtained from the indiana university simon cancer center solid tissue bank ( indianapolis , in , u.s.a . ) using trizol - ls reagent ( life technologies , carlsbad , ca , u.s.a . ) . cdna was generated using 10 ng of rna and mir-10b , mir-21 , or mir-4255p rt primers and a mir reverse transcription kit ( life technologies ) as per the manufacturer s recommendations . expression levels as determined by qpcr were normalized to mir-425 - 5p , since this mir was expressed at similar levels in all samples and exhibited < 1 cycle threshold ( ct ) difference across all samples . after normalization to mir-425 - 5p ( ct ) , the ct values for mirs in controls were averaged and subtracted from the ct values of each individual sample ( ct ) .

micrornas ( mirs ) are small noncoding rnas that regulate mrna stability and/or translation . because of their release into the circulation and their remarkable stability , mir levels in plasma and other biological fluids can serve as diagnostic and prognostic disease biomarkers . however , quantifying mirs in the circulation is challenging due to issues with sensitivity and specificity . this letter describes for the first time the design and characterization of a regenerative , solid - state localized surface plasmon resonance ( lspr ) sensor based on highly sensitive nanostructures ( gold nanoprisms ) that obviates the need for labels or amplification of the mirs . our direct hybridization approach has enabled the detection of subfemtomolar concentration of mir - x ( x = 21 and 10b ) in human plasma in pancreatic cancer patients . our lspr - based measurements showed that the mir levels measured directly in patient plasma were at least 2-fold higher than following rna extraction and quantification by reverse transcriptase - polymerase chain reaction . through lspr - based measurements we have shown nearly 4-fold higher concentrations of mir-10b than mir-21 in plasma of pancreatic cancer patients . we propose that our highly sensitive and selective detection approach for assaying mirs in plasma can be applied to many cancer types and disease states and should allow a rational approach for testing the utility of mirs as markers for early disease diagnosis and prognosis , which could allow for the design of effective individualized therapeutic approaches .

Fabrication of the Plasmonic Biosensor for miRs Detection Detection of miR Levels in in Plasma from Pancreatic Cancer Patients Conclusion Materials and Methods Fabrication of LSPR-Based miR Sensors and Detection Total RNA Extraction and Quantification of MicroRNA by qRT-PCR Supplementary Material

figure 1 represents the schematic diagram of our solid - state , label - free plasmonic biosensor fabrication for mir detection . chemically synthesized gold nanoprisms ( figure 1a ) , which displayed their lspr at 797 nm upon attachment to solid substrate immersed in pbs buffer , were selected as nanoantennas for our biosensor fabrication because ( 1 ) their lspr peak position ( in the 700900 nm wavelength range ) is particularly suitable for biomolecule detection because of negligible background scattering and adsorption of endogeneous chromophores from physiological media such as blood and plasma ; ( 2 ) they have strong electromagnetic ( em ) field enhancement at the sharp tips ; ( 3 ) they exhibits a strong lspr response to small changes in their surrounding environment ; ( 4 ) their atomically smooth surface allows formation of a self - assembled monolayer ( sam ) of receptors with both a tightly packed lower layer of alkylthiols and a more loosely packed upper layer that provide the required space for duplex formation with complementary mir strands ; ( 5 ) gold is nontoxic and stable under extreme physiological conditions ; and ( 6 ) the gold sulfur bond is very stable with thiol - modified receptors making a strong covalent bond with the gold surface . recently , we have shown that a molecular sensor fabricated using an 35 nm average edge - length gold nanoprisms displayed an unprecedentedly large 21 nm reversible shift upon a minor 0.6 nm increase in the thickness of the local dielectric environment . therefore , gold nanoprisms of this size and geometry are unique and should provide extremely high sensitivity if plasmonic biosensors are fabricated using them , which is the scope of this letter . this letter provides the first example of lspr - based mirs sensing in physiological media . design of plasmonic biosensors and detecting mir - x in various physiological media . ( b ) surface of gold nanoprisms was chemically modified with a 1.0 m 1:1 mixture of sh - c6-ssdna - x and peg6-sh in pbs buffer ( ph 7.4 ) to prepare the plasmonic biosensor . ( c ) incubation of sensor in mir - x solution and formation of dna duplex . the extent of lspr dipole peak shift ( lspr ) depends on the concentration of mir - x used during the incubation in ( c ) , which ranged from 100 nm to 50 fm . ( e ) plot of lspr versus log of mir - x concentrations used to determine the limit of detection . our targets were mir-21 and mir-10b , because we have shown by locked nuclei acid based in situ hybridization that they are overexpressed in pancreatic cells ( pccs ) within the tumor mass and that circulating mir-10b may serve as biomarker for diagnosis of pdac . we designed the sensing strategy based on the hybridization between complementary probes ( -c6-ssdna - x , x = 21 and 10b ) attached to gold nanoprisms and their target mirs . as shown in figure 1b , poly(ethylene glycol)6-thiols ( peg6-sh ) were used as spacers because they avoid nonspecific adsorption of extraneous materials onto the nanoprism s surface and are not reactive toward mirs or other biological constituents present in plasma . as illustrated in figure 2a , we used uv vis spectroscopy to monitor the changes in lspr of the gold nanoprisms at different functionalization steps . the functionalization of substrate - bound nanoprisms with 1:1 ratio of hs c6-ssdna-21:peg6-sh resulted in an 20.5 3.2 nm red - shift of lspr as a result of the increase in local refractive index , which suggested the attachment of both molecular species onto the nanoprism s surface . it was found that the magnitude of the lspr shift was concentration dependent , where 50 fm mir-21 caused a 3.7 0.3 nm lspr red shift in pbs buffer . figure 2b illustrates the magnitude of the lspr shift ( lspr ) upon dna / rna duplex formation for various mir-21 concentrations in the three different media . evidently higher concentrations of mir-21 induced a larger number of ssdna-21 strands to convert to dna / rna duplexes and consequently a larger change in the local refractive index around the nanoprisms , which results in a larger value of lspr . ( a ) monitoring lspr dipole peak ( lspr ) changes by uv visible absorption spectra of gold nanoprisms during various functionalization steps : before ( black , lspr = 800 nm ) and after functionalized with a 1 m/1 m ratio of hs - c6-ssdna-21/peg6-sh ( red , lspr = 821 nm ) , and after incubation with 100 nm mir-21 solution in pbs buffer ( blue , lspr = 839 nm ) . ( b ) average lspr peak shift ( lspr ) of gold nanoprisms functionalized with a 1 m/1 m ratio of hs - c6-ssdna-21/peg6-sh after incubation in different concentrations of mir-21 in pbs buffer ( red triangles ) , 40% human plasma ( black squares ) , and 40% bovine plasma ( blue diamonds ) . the lspr were calculated by taking the difference between the lspr peak position of the plasmonic biosensor after and before the hybridization with mir-21 in the various media . ( c ) average lspr of the plasmonic biosensor functionalized with a 1 m/1 m ratio of hs - c6-ssdna-10b / peg6-sh after hybridization with different mir-10b concentrations in pbs buffer ( red triangles ) , in 40% human plasma ( black squares ) , and in 40% bovine plasma ( blue diamonds ) . all extinction spectra recorded after mir - x incubation were measured in pbs buffer after rinsing with pbs buffer . we believe such a high sensitivity of our plasmonic biosensors is because of the unique lspr properties of our gold nanoprisms and the possibility of electron delocalization as the ssdna forms the duplex and becomes double - strand . moreover , transformation of ssdna into double - strand dna significantly changes the refractive index because of the high charge density and polarizability of the dnas . the total shift ( lspr ) depended on the mir concentration ( figure 1d ) and could be used to determine the limit of detection ( lod ) ( figure 1e ) . the lods calculated for mir-21 in three different media were found to be in the range of 2335 fm , which was more than 1000 and 3 fold lower than with the label - free microring resonator ( 150 fmol ) and the nanopore based ( 100 fm ) mir sensors , respectively . importantly , these techniques detected mirs in pbs buffer whereas we have demonstrated here for the first time a sensing approach in physiological media . utilizing our same direct hybridization - based detection approach , plasmonic biosensors were constructed with of c6-ssdna-10 , while keeping other parameters constant . the principle underlying the actions of plasmonic biosensors is based on the successful hybridization between mirs and ssdna attached to nanoprisms , where a higher number of duplex formations will result in a larger change in the refractive index surrounding the nanoprisms resulting in larger lspr and higher sensitivity . therefore , it would be expected that functionalization of gold nanoprisms with 100% hs c6-ssdna - x ( without the peg6-thiol spacer ) should reduce the lod values because of steric hindrance and low attachment of mirs . we believe fully covered gold nanoprisms were obtained when 100% c6-ssdna-21 was used for functionalization , which creates steric hindrance and does not allow the maximum number of mir-21 strands to come into close proximity with c6-ssdna-21 for hybridization . accordingly , for the remaining of our investigation , we used a 1:1 mixed c6-ssdna - x : peg6-sh to functionalize the gold nanoprisms . ( a ) uv visible extinction spectra monitoring the lspr dipole peak ( lspr ) of gold nanoprisms attached to silanized glass substrate before ( black , lspr = 796 nm ) and after ( red , lspr = 811 nm ) functionalization with 1 m of hs - c6-ssdna-21 without peg6-sh spacers and after incubation in 100 nm mir-21 solution in 40% human plasma ( blue , lspr = 822 nm ) . ( b ) average lspr of these hs - c6-ssdna-21 functionalized gold nanoprisms upon hybridization with different mir-21 concentrations in 40% human plasma . the lspr were calculated by taking the difference between the lspr peak position of the nanoprisms after and before the incubation with mir-21 in pbs buffer . in order to confirm the hybridization of mir - x with c6-ssdna - x that resulted in the lspr , the enzyme rnase h was used to selectively cleave the dna : rna duplex and potentially reverse the lspr . these experimental results validate our previous observation that the lspr blue shift was due to the cleavage of heteroduplex done by rnase h. the biosensors were rinsed with rnase free water to pbs buffer and again incubated in 100 nm mir-21 solution for rehybridization where an 14 nm red shift of the lspr was observed . these experiments confirm our working hypothesis that hybridization between the nanoprism s surface ligands ( c6-ssdna - x ) and the mir - x resulted in changes in the local dielectric environment around the nanoprisms causing wavelength shift . the same experiments were done for the mir-10b biosensor and similar results were observed , underscoring the long - term stability of the sensors and their potential for being developed into cost - effective point of care diagnostic tools . ( b ) changes in lspr dipole peak position ( lspr ) of gold nanoprisms functionalized with a 1 m/1 m ratio of hs - c6-ssdna-21/peg6-sh upon hybridization and dehybridization with mir-21 for several cycles . the lspr peak shifts back and forth upon sensor regeneration with rnase h by cleaving dna / rna duplex and rehybridization after incubation into 100 nm mir-21 in 40% human plasma . after each of the dehybridization steps , the plasmonic biosensors were thoroughly rinsed with pbs buffer to completely remove enzyme rnase h. the hybridization takes place at the 5 end of c6-ssdna-21 and the 3 end of mir-21 , which evidently increased the refractive index . thus , attachment of mirs to plasmonic biosensors has increased the thickness of local dielectric environment around the gold nanoprisms and influenced their lspr properties . atomic force microscopy images of gold nanoprisms bound to silanized glass substrate ( a ) after functionalization with 1:1 ratio of hs - c6-ssdna-21/peg6-sh ( b ) and after hybridization with100 nm mir-21 in 40% human plasma ( c ) . ( d ) changes in the surface area of gold nanoprisms after each functionalization steps . the successful implementation of plasmonic biosensors for use with real biological samples mandates documentation of their specificity toward target mirs in that patient samples containing multiple mir species . the lspr response was measured before and after incubation ( supporting information figure s9 ) and resulted in an 2.5 0.3 nm lspr red shift , which is within the instrument noise level and/or minor nonspecific adsorption of extraneous materials present in human plasma . in another control experiment , gold nanoprisms attached as before to glass substrate were functionalized with 100% peg6-sh by incubation in a 1 m aqueous solution , and after rinsing with large amounts of water , incubated in a 40% human plasma solution of 100 nm mir-21 for 12 h. this procedure resulted in only an 0.9 0.7 nm lspr red shift ( supporting information figure s10 ) , confirming that the plasmonic biosensors we designed are highly specific toward the target mirs . pdac is characterized by chemotherapeutic resistance and by the absence of an effective screening procedure for early disease . given their resistance to degradation , plasma mirs have the potential to serve as biomarkers for the noninvasive diagnosis of pdac . previously , nanopore sensors were used to detect mirs in lung cancer patients , but to the best of our knowledge no sensors have been developed to date to detect pdac - related mirs in human plasma . next , 14 l volumes were used for mir quantification by the plasmonic biosensor and the remaining 14 l were used in the qrt - pcr assay . importantly , for the first time through a label - free technique we have shown that mir-10b concentration is nearly 4-fold higher than the mir-21 level in patient samples . inasmuch as both mir-21 and mir-10b are overexpressed in pdac , it is possible that mir-10b is released more efficiently by pancreatic cancer cells than mir-21 , allowing it to achieve higher levels in the circulation . ( d ) the average lspr peak shifts ( blue diamonds ) and concentration ( red triangles ) for the mir-21 in plasma samples from pdac patients without any purification . we also detected mir-21 levels directly in human plasma samples collected from pdac patients without rnas extraction . thus , human plasma ( 50 l / sample ) from six pancreatic cancer patients were diluted in pbs buffer followed by incubation with our plasmonic biosensors for 12 h. the lspr response of each sample was measured through uv vis spectroscopy and showed a steady increase in concentration from sample 6 to 1 ( figure 6d ) . both plasmonic biosensor and qrt - pcr results indicated that mir-10b levels were higher in pdac patients compared to normal control subjects and that the levels of mir-21 and mir-10b can be quantified with high accuracy using our gold nanoprism - based plasmonic biosensor without any modification , amplification , or labeling . importantly , the mir-21 concentration in extracted samples was at least 2-fold lower than in the pure plasma samples . we believe this is due to loss of mirs during the rna extraction process , which requires multiple steps for rna purification . therefore , the most widely used qrt - pcr method to determine the concentration of mirs in patients may not accurately represent the actual concentration . this limitation and imprecise quantification can be avoided by using our newly developed plasmonic biosensors , which can provide a unique opportunity as potential diagnostic and prognostic markers in pdac , other cancers , and potentially other disease states . we have designed , fabricated , and characterized a plasmonic biosensor that was able to detect pdac relevant mirs in human plasma without using rnas extraction , which opens a new avenue for the direct detection and quantification of mir levels in clinical samples without any form of sample preparation . to our knowledge , this is the first lspr - based , label - free , direct hybridization method for mir detection , which eliminates all the current drawbacks such as labeling , tagging , amplification , use of highly toxic chemical , and further modification of the sensor . furthermore , it vastly simplifies the detection approach without requiring detailed knowledge of the electron or energy transfer processes involved as in other more complicated techniques . additionally , this ultrasensitive , plasmonic - based , direct hybridization - controlled detection approach is applicable to any type of mirs that are relevant to various diseases . it was found that our plasmonic biosensor can be regenerated through several cycles and is stable for several days without compromising its sensitivity and selectivity , which should enable the development of simple , cost - effective tools for the early detection of mirs and thus facilitate the early diagnosis of various cancers . in theory , therefore , nanoprisms can be used to design effective substrates for surface - enhanced raman spectroscopy - based detection and quantification of multiple mirs simultaneously through integration of their spectral characteristic with the lspr shifts . the attachment of gold nanoprisms on silanized glass substrates is described in the supporting information file . the initial lspr peak position of each sensing platform was determined using uv visible spectroscopy in pbs buffer and then was incubated in the different concentrations of mir solutions , for exmaple , either in pbs buffer , 40% bovine plasma , or 40% human plasma for 12 h at room temperature . the initial lspr peak position of each sensing platform was determined using uv visible spectroscopy in pbs buffer and then was incubated in the different concentrations of mir solutions , for exmaple , either in pbs buffer , 40% bovine plasma , or 40% human plasma for 12 h at room temperature .

biologic inflammation in its multifaceted subsistence lends itself to delineation by big laboratory data it in this age of data warehouse extension . many life maintaining biological interactions function as multicomponent weight balance , equilibrium standing for health . settling on one or the other side to vacate fine adjustment may go on to result in final targeting be it for health maintenance or to develop pathological transformation . the paths to excess are paved with stop- and go - signaling : activation signals can be held back with the hazard to cause overshoot in the other direction of the balance . to such intrinsically complex regulatory framework of a single system adds up the interaction between different systems involving health maintaining cross - reactivities or expanding pathological effects . thus a large amount of metabolites , proteins , intermediate and terminal enzymes interact simultaneously to maintain physiological wellbeing or they will thwart equilibrium . when attempting diagnosis , medical laboratories test for single disease - related leading analytes / markers and they go for an appropriate choice to give patients and physicians a representative picture to tailor therapy . we here describe some approaches to sort out the relevant results for patient care in precision medicine . in this analysis we exclude the doctors ' choice on categories on order sheets of the appropriate lab assays at the outset , assuming that big data accumulate over time during medical checkups unrelated to a single morbus . metabotyping and high - resolution omics data has the promise to picture diseases based on metabolite 's profile or ratios of selected analytes and might develop into a relevant component of diagnosis and treatment of single nosological entities . mechatronic engineering designs , testing and operation of machinery and equipment , in which there is a high level of functional integration of mechanical systems with electronics and computer control in laboratory equipment brings information gain from metabotyping upfront . biologists of the swiss federal institute of technology work on real - time simultaneous analysis of hundreds of analytes measured with the same instrument , results of which might be funneled into biocomputing circuits . mathematical models are then used to quantitatively relate metabolomics , expression , and proteomics data to the functional network output related to fluxes ( figure 1 ) . the usability of such waves of it based information , particularly if used in health - monitoring systems , will need original / innovative approaches for secure storage . we here attempt to envision the inflammation portion of the whole body metabolism as an envelope containing interactive signal circuits which interact at the frontend of genetic , transcriptional , and proteomic backgrounds and react to inflammation inducing forces : the current view of senescence being brought forward , at least in part , by inflammatory mechanisms has coined the neologistic term of inflammaging , none the least of these being senescence ( inflammaging ) [ 36 ] . our update can be read as a background to discern digital memories eventually leading to biological computer science . each single patient produces data in the long run with her or his standard data entry description : accession number , sample number , patient i d , sex , birthday , clinic , ward , doctor , order comment collection source , ( repeat ) collection date , and sample comment . dialog boxes upon receipt of samples in the lab may help to reduce data flow but can not ban it . analysis of most disease - related metabolites , including glycoproteidic biomarkers for disease diagnosis , is based on elisa , eclia , and enzyme - substrate colorimetric / light extinction / electrochemoluminescence ( ecl ) technology . mass spectrometry with improved resolution is now often preceded by multidimensional chromatographic separation schemes which enlarges the spectrum of possible analytes . a uniform distribution of the number of acquired ms / ms , protein , and peptide identifications undergo proteomic runs which allow protein identification on large scales estimated up to as high as ~14 000 proteins and ~250 000 unique peptides . a large spectrum glycoprotein profiling in plasma , serum , other bodily fluids or tissues is thus possible . transplantation of donor organs into patients in need has exceeded hla - compatibility requirements since abo blood type system incompatible donor / recipient pairs are becoming routine . infectious and inflammatory complications remain within limits in such settings and their prophylaxis includes tight lab test controls . subclinical inflammation , reliably diagnosed using c - reactive protein ( crp ) serum level cutoff 10 mg / l , constitutes a risk factor for the development of interstitial fibrosis and seems to reflect not only inflammation but general well being : crp is a subtle separator for hand grip strength , physical performance , and decline in older populations . serum pattern recognition compounds , among them crp , bind to apoptotic cells and nucleoprotein autoantigens and fc receptors to ultimately inhibit plasmacytoid dendritic cell interferon responses which are elicited by autoantibody immune complexes . the monomeric form of crp als inhibits renal cell - directed complement activation mediated by properdin . transplantation medicine largely contributing to big data is going to include abo - glycan microarray results which now allow detailed characterization of donor - specific antibodies necessary for effective transplant management of solid organs [ 1416 ] . in health care , the use of big data , meant to use the large number of digitalized single analyses accumulated daily , a computational medicine , that is , ehealth or electronic health or medical record ( her and emr ) , expands at the expense of health care professionals to read its message . data warehouses containing big data need continuous validation , data management for updates , and most importantly an analysis to convert such resource into clinically relevant information for medical care . mass spectrometry and bioinformatics it solutions may establish profiles which help to diagnose frequent and rare disease alike . the designation of given lab analyses as biomarker is currently moving towards genome sequencing to reveal genetic risks for future illness ( figure 2 ) obviously a component of personal big data ; patients on their own will thus access to their data bank . multibiomarker diagnosis and disease activity scoring , often a difficult curse to work , is being bundled by the clinician intending to focus on patients ' complaint and clinical signs and symptoms pattern clustered together by cross - reactive algorithms [ 20 , 21 ] . for practical reason , we here lend the four major fields of laboratory medicine in switzerland , under the auspices of a federal organization , foederatio medicorum analyticorum helveticorum ( famh ) : clinical chemistry , immunology , haematology , and microbiology with genetics wrapping up each of these ( figure 2 ) . to a large extent , this distinction of laboratory analyses by category corresponds to international habits defined by the international federation of clinical chemistry ( ifcc ) . the substantial expansion of the analyses performed boosts each of these specialities alike , enriching the respective fields with unprecedented wealth of data . this brings medical doctors to dilemmas on the appropriate choice of information for patient care in the present context focusing on inflammation . to turn big data into appropriate data thus computational methods may throw a bridge from bench to bedside and vice versa [ 7 , 25 ] and web - based platforms would allow seamless interaction between warehouse and patient - centered information as recently put forward through an eu project . academic as well as business intelligence literature counsels are available for assisting transformation of big data into a selection of data assembled for care of single disease entities ( http://www.sas.com/ and http://www.aacc.org/ ) . structuring of such information for particular disease states is crucial and platforms for gene sets might improve understanding different biological data types to reach meaningful outputs . web mapping and power - grid including care for people on a medical device that depends on electricity may be included . software solutions for medical laboratories , that is , laboratory data management systems provide for a several decenny old experience on a local , intralaboratory scale . long - term archival of data implies repeat migration from one media / storage environment to updated systems on regular schedule to prevent hacking . whilst encryption is useful for privacy / intimacy , our golden age of surveillance likes coding as a ( secrete ) language system . the multilayer dimensionality of big data warehouses needs data - driven algorithms necessary to reach their basic goal : to translate big data into clinically useful evidence . logistic regression models , cox analysis , and kaplan meier curves can sort out analytes which would predict clinical evolution , for example , kidney - associated morbidity [ 3032 ] ; our own studies are being in line with researchers in scandinavia . platelet count and icu survival , actually completely unrelated parameters , can be used for predictive modelling purpose . one of the possibilities to unify assignments of terms for distinct analyses currently successful on an international level are the logical observation identifiers names and codes ( loinc ) which have been created under the auspices of the regenstrief inc . , the usage of loinc codes remains subject to variations in the way they are used and semantic , taxonomic interoperability might turn out to be contradictory in some places . therefore , loinc committees enforce detailed guidance on best practices for mapping from local to international loinc codes and for using loinc codes in data exchange . experiences using data warehouse produced collaboratively between academic medical centers and private practice throwing bridges to ehr do reveal potential to improve utilization of clinical pathology testing . thus , in the us , objective electronic laboratory reporting has now been promoted as a public health priority with two coding systems endorsed : loinc for lab test orders and systemized nomenclature of medicine - clinical terms ( snomed ct ) for test results , the former being in use more commonly . loinc is now de rigueur in france : assistance public des hpitaux de paris , aphp , has created a biomedical observation dictionary mapped to loinc which is bound to integrate this language into the entire biomedical production chain . since its outset in 2010 , participation of 120 laboratories including 50.000 codes now ensures interoperability in the entire french ehr system . loinc codes comprise categories to inform the data storage software on ( i ) the analyte , ( ii ) measured property , that is , enzyme activity or concentration , ( iii ) time ( span ) of sample collection , ( iv ) system used , for example , urine plasma serum , liquor , and ( v ) scale , that is , nominal and ordinal . one single analyte , for example , complement hemolytic activity activated through the classical pathway , ch50 , comprises as much as 7 different loinc codes ( table 1 ) , depending on which starting material was employed for ch50 analysis . newborn screening , including such analyses with high - stake health implications necessitates rapid / effective communication between many people and organizations and increasingly depend on big data registries which can aggregate results from national programs and help harmonize inherited metabolic disorder to an international level . the march of dimes recommends screening newborns for 29 conditions , such as phenylketonuria , hypothyroidism , galactosemia , and sickle - cell anemia ( http://www.marchofdimes.com/ ) . a prenatal screening program is taking shape and involves pregnancy - associated plasma protein a ( papp - a ) and pregnancy hormone hcg and pregnancies with hypertensive disorders might be detected early on using analysis of cell - free fetal dna , cell - free total dna , and biochemical markers . such a bayes estimator for single analyses with their variance , confidence interval can be derived from a posterior distribution ; the minimum square error also called squared error risk is defined by mse = e[((x ) ) ] . some of the key features of a bayesian analysis as a powerful package for molecular sequence variation have been delineated 10 years ago . this novel task of doctors on scooping the right selection of relevant data requires a minimum understanding of what it can and can not do for the benefit of patient care ( table 2 ) . the european informatics institute ( embl - ebi ) based on the consolidated apache lucene technology might inspire search engine development to direct scalable search paths towards medical diagnosis . the number of clinically relevant chemical analyses offered for diagnostic purpose at swiss university hospitals and private industry amounts up to roughly 180 . henceforward , such overseeable data built up during the last century currently expands to big data produced by automated workflow using intelligent robotics with throughputs of 315 million clinical chemistry assays / year . as learned from internet searches , unilabs processes 40.000 medical analyses / day and synlab offers > 4000 different analyses to its clients . a laboratory automate , for example , the cobas machine ( roche diagnostics , rotkreuz , switzerland ) with its large panel of possible analyses , may serve as an example of ever growing lab service function . competing industries , for example , abbott ( abbott diagnostics , abbott park , il , usa ) , siemens ( siemens healthcare , erlangen , germany ) , hitachi ( hitachi , mountain view , usa ) , capillarys sebia ( paris , france ) , kiestra ( bd , franklin lakes , nj , usa ) , bruker ( bruker , billerica , ma , usa ) , biomrieux ( lyon , france ) , are continuously updating their offers in order to increase capacity , that is , number of analyses / time throughput . the analytes of the clinical chemistry lab section can be subdivided into provision of information for whole body pathology and into organ - specific lab assays . selected groups of analyses are assembled as suggestion to clinicians in order to investigate single organs : endocrinology , liver function , gastroenterology , nephrology , vitamins . each of these specialities of medicine sees its own lab definition increased in number of different analyses such as we have recently used cystatin c and its ratio to creatinine to improve significance of interpretation in kidney insufficiency . the inflammation parameters in clinical chemistry , occasionally with biomarker status , are acute phase proteins , that is , crp , serum amyloid a , fibrinogen , tryptase , haptoglobin , procalcitonin , interleukin-6 , and again crp used to classify disease stage of rheumatoid arthritis and now even to estimate extent of fitness and senescence . two lines to enriching haematological patient findings emerged recently : ( i ) intelligent picture readings of blood and bone marrow films making possible telehaematology [ 4648 ] and continuous flow analysis of single cells sorted according to their clonal origin . picturing blood films does not lend itself to electronic storage in big data banks in contrast to findings coming out from forward and sideward scatter beamer flow cytometer cell analysers , such as sysmex xe-5000 ( kobe , japan ) , abbot sapphire ( abbott diagnostics division , santa clara , ca , usa ) , siemens advia ( siemens healthcare , erlangen , germany ) , beckman coulter ( beckman coulter eurocenter , geneva , switzerland ) , and amnis flowsight ( seattle , wa , usa ) . complete blood counts ( cbc ) , hemostasis assays now forming part of sysmex machines , bone marrow , and progress in stem cell therapeutics are all prone to be integrated into big data banks . the diagnostic value of both microscopic and automated neutrophil left - shift parameters as indicators of inflammatory disease is limited but confirmed routine automated coagulation assays and pharmacomonitoring of many drugs with lc ms / ms machines are fit for integration into big data banks and miniaturization of assay principles is contributing to this trend . blood group typing , the classical way , ( still ) uses haemagglutination systems with monoclonal antibodies by and large on automated platforms . results from haemagglutination are now fully completed and soon will be replaced , at least in part , by genotyping procedures [ 5356 ] . multiple data release prone to be sections in big data registries are now being making precision medicine even more precise . in fact , inexpensive molecular typing of histoblood types paired with powerful bioinformatics has enabled mass - scale information but bears the risk that personalized red blood cell matching for transfusion becomes less precise . small- and large - order haemogram blocks for blood microarray technology can be used to more precisely delineate anti - abh antibodies , a progress which will make solid organ transplantation across abo barriers more successful thanks to using bioinformatics . stem cell transfusion and cord blood based therapy are making inclusion of hla types and gwas whole genome typing into registries which are based on big data informatics ( http://www.hpscreg.eu/ ) . analytical approaches of the immune system branches into cellular and humoral patient samples which are tested frequently ( figure 2 ) . the order form of our institution lists up to 200 analyses offered to the clinician , not including the large field of tests to rule out allergic diseases . care for patients suffering from allergic diseases recognizes the usefulness of a systemwide profiling approach , which associates big lab data with the biological approach to asthma and allergy . cellular immunological analysis overlaps with hematological tests of the myeloid compartment , but they make their own data box with lymphocyte subsets and their cd marker pattern . close to 400 cd markers have been identified up to now and their number might grow . contribution of the complement system to big data is considerable ; with three activation pathways ~48 proteins and their fragments , 9 protein complexes and ~12 receptors , measured w / v and/or by functional activity , the complement system contributes to warehouses on its own . extraction of a selection of relevant data for patient care , data from data warehouses to which complement levels contribute involves interdisciplinary algorithm flow charts . these are focusing on diagnostic and therapeutic needs in precision medicine and so far are based on care for patients with immunological and microbial diseases . however , complement analysis should also be seen in perspective with other types of analyses . with the seniorlab study ( isrctn registry number 53778569 ) we put w / v concentrations of c4 and c3 and immunoglobulin ( ig ) levels into perspective with serum vitamin d levels and senescence . immunoassays were used to quantitate c4 and c3 and ig in 1470 apparently healthy subjects > 60 yrs . low levels of 25(oh)d were positively associated with igg2 and c4 ( the lower vitamin d , the lower c4 , figure 3 ) yet inversely related to levels of igg1 and iga and c3 [ 59 , 60 ] . as can be seen in table 1 , a single one and the very same analysis can account for 7 different loinc codes depending on the material in which measurements are made and depending on w / v versus functional performance measured . acute phase complement proteins related to c - reactive protein ( crp ) evolve in parallel during inflammatory states and are now known to play a role in type 2 diabetes , lipid metabolism , and atherosclerosis ( figure 4 ) . these insights suggest that complement system - related algorithms destined to extract significant patient data from big data must be seen related to noncomplement analyses performed in the routine laboratory . on the immune cell level , high - throughput sequencing has sparked information on tcr repertoire diversity informative on functional capacity of the adaptive immune system . nothing but the diversity of tcrs is mirrored by a receptors ' dispersity based on different peptide - sequence which might reach > 1000 . given these large numbers , high - throughput sequencing is required to achieve sufficient sequencing depth to estimate clonal abundance . big data warehouses will certainly have to make reference to the age group of study subjects . thus , we have seen that il-6 levels were lower and tnf - alpha reference intervals were higher in healthy newborns and toddlers than the adult reference intervals . biomarkers relating to particular disease states , for example , prostate - specific antigen ( psa ) or those of oxidized lipoproteins , genetically determined come to increase the size of big data in personalized medicine , their heritability being under scrutiny with twin pair studies . personal ehr health profiles captured by individuals themselves ( e.g. , from smart phones and wearable devices ) will contribute to the next wave in big data upload to the cloud and propagation across social networks make encryption prevent access to information by insurance companies , ransomware hackers , and state writ guardians . host , microbiomes , and pathogenic microbes are analyzable with a big data array of laboratory criteria making bioinformatics an indispensable pillar of big data in inflammation exploration . bacterial , viral , fungal , and parasitic infections and diseases due to microbial toxins are among the most common and medically important causes of inflammation with different pathogens eliciting varied responses ranging from mild and short - term to severe and long - term . foreign bodies , catheters , splinters , sutures , and dirt may elicit inflammation and inflammation tissue and laboratory data may circumscribe hypersensitivity and autoimmune disease induced inflammation under the control of cytokines produced by t lymphocytes mainly . on hosts ' side the predisposition to provide for a favourable environment for infectious agents , an array of receptor molecules ( fy a / b for malaria , cho recognition on pmnl , fibronectin on catheters , figure 4 ) can be appreciated to then enter bmld ( biosafety in microbiological and biomedical laboratories ) banking . nothing but the human gut containing a myriad of different bacteria and other microorganisms such as archaea , viruses , and fungi , making the microbiome expand to big data sets , an enter - system . on the side of microbes , the bmld provides important information on the clinical presentation the infectious agent will cause if successfully attacking the host . as an example , the strain k 157 of e. coli is predictive of hus and other strains of e. coli , such as o157:h7 , o104:h4 , o121 , o26 , o103 , o111 , o145 , and o104:h21 , produce potentially lethal toxins . this is but one example of the enormous extent of data which microbiology occupies the space of a data warehouse . with the maldi tof system , fast typing has entered the practice since a decade which facilitates updates of bmld boxes . virulence factors can be spotted in staphylococcus aureus using whole genome sequencing combined with dna microarray hybridization prone to increased big data informatics . as well laboratory assays differ between specimen analyses in acute disease and the assay approach used for specimens taken during convalesce . the current example here is guideline updates considering the ongoing zika virus and other flavivirus epidemics ( e.g. , dengue , yellow fever , st . louis encephalitis , and west nile virus ) which enforce usage of rt - pcr , also for chikungunya viruses . proposed test algorithms start out with both molecular and antibody testing to minimize the risk for cross - reactivity vulnerable meandering ( memorandum cdc ) ( http://www.cdc.gov/zika/pdfs/denvchikvzikv-testing-algorithm.pdf ) . whilst big genetic data validation overlaps and completes each of the preceding subchapters ( figure 2 ) , genetic studies of many diseases are now allowing closer insights into human pathology . ngss can be used to detect genetic anomalies of essentially any size scale , from snps to very large rearrangements ; all of todays ' genetic diagnostic tests could in principle be supplanted by ngs , including rna analysis , because transcriptome ( rna - seq ) sequencing is possible and now boosts with crisp - cas9 technology . on the leading edge of a revolution in medicine the complete dna sequence , properly encrypted , will become a permanent part of individual 's ehr utilized by health care professionals to make decisions about drug prescriptions , diagnostics , and disease prevention . molecular geneticist validation reviews all variants called on a 10-gene panel with 30 variants per case without additional it support , but it will be swamped when this would be have to be done for a 100-gene panel . as an example phenotypes are now closely linkable to genetic findings and to the age of the patient , younger age at diagnosis associated with extensive / aggressive crohn 's disease and ulcerative colitis . dissection of genotype - phenotype relations using immunochip array designed to capture up to 200 different loci associated with common autoimmune diseases can be focused on nod2 , mhc , and mst1 3p21 to allow establishment of a genetic risk score and predictive modelling . the genetic diseases encountered in medical practice are the tip of the iceberg , that is , those with less extreme genotypic glitches that permit full embryonic development and live birth . genetic variants of complement genes , for example , cfigly119arg , such as recently evaluated in the eugenda cohort may be associated with age - related macular degeneration . how many mutations remain hidden ? with the determination of the complete sequence of the human genome , dna imprints not only for disease but also for the gray zone between health and disease are under scrutiny . as an example , training of legasthenics could use genomic insights to improve its efficacy . as dr . collins puts it it will take decades , if not centuries to understand the instructions of the genetic language , and , one might add , improve and make big data information more meaningful . ransomware attacks hackers blocking hospital and private practice computers have now been reported here and there . computer systems , including those needed for lab work , can be set out of function relatively easily which makes big data clouds vulnerable to an extent to which some workers keep copies on separate hardware aside ; desktop virtualization systems , for example , citrix , are improved for hacker risk reduction and saving data apart on separate servers might do the rest to prevent big data hacking . two motive forces are thriving attempts to reduce the number of health parameters ( i ) to bring customers of the medical lab to a reasonable and patient driven block of analyses asked for and ( ii ) to optimize financial sources doctors often ignoring the financial consequences of their test - ordering behavior . study results from attempts to reduce the number of ordered tests become known and an expert panel may bring some good ideas but will never be able to brake medical revolution continuing to creep on us , especially in the field of laboratory analyses . in conclusion , multidisciplinary efforts are required to master the mighty offer of data and to make the information improve patient care .

we work on the assumption that four major specialities or sectors of medical laboratory assays , comprising clinical chemistry , haematology , immunology , and microbiology , embraced by genome sequencing techniques , are routinely in use . medical laboratory markers for inflammation serve as model : they are allotted to most fields of medical lab assays including genomics . incessant coding of assays aligns each of them in the long lists of big data . as exemplified with the complement gene family , containing c2 , c3 , c8a , c8b , cfh , cfi , and itgb2 , heritability patterns / risk factors associated with diseases with genetic glitch of complement components are unfolding . the c4 component serum levels depend on sufficient vitamin d whilst low vitamin d is inversely related to igg1 , iga , and c3 linking vitamin sufficiency to innate immunity . whole genome sequencing of microbial organisms may distinguish virulent from nonvirulent and antibiotic resistant from nonresistant varieties of the same species and thus can be listed in personal big data banks including microbiological pathology ; the big data warehouse continues to grow .

1. Introduction 2. Metabotyping May Circumscribe Inflammation in the Lab 3. Medical Laboratory Copes with Big Data 4. An Attempt to Categorize 5. Calibration, Steps, Hierarchy, and How to Scale 6. Attempts to Constrain Big Data in Clinical Settings

biologic inflammation in its multifaceted subsistence lends itself to delineation by big laboratory data it in this age of data warehouse extension . the paths to excess are paved with stop- and go - signaling : activation signals can be held back with the hazard to cause overshoot in the other direction of the balance . to such intrinsically complex regulatory framework of a single system adds up the interaction between different systems involving health maintaining cross - reactivities or expanding pathological effects . in this analysis we exclude the doctors ' choice on categories on order sheets of the appropriate lab assays at the outset , assuming that big data accumulate over time during medical checkups unrelated to a single morbus . biologists of the swiss federal institute of technology work on real - time simultaneous analysis of hundreds of analytes measured with the same instrument , results of which might be funneled into biocomputing circuits . mathematical models are then used to quantitatively relate metabolomics , expression , and proteomics data to the functional network output related to fluxes ( figure 1 ) . we here attempt to envision the inflammation portion of the whole body metabolism as an envelope containing interactive signal circuits which interact at the frontend of genetic , transcriptional , and proteomic backgrounds and react to inflammation inducing forces : the current view of senescence being brought forward , at least in part , by inflammatory mechanisms has coined the neologistic term of inflammaging , none the least of these being senescence ( inflammaging ) [ 36 ] . our update can be read as a background to discern digital memories eventually leading to biological computer science . each single patient produces data in the long run with her or his standard data entry description : accession number , sample number , patient i d , sex , birthday , clinic , ward , doctor , order comment collection source , ( repeat ) collection date , and sample comment . dialog boxes upon receipt of samples in the lab may help to reduce data flow but can not ban it . analysis of most disease - related metabolites , including glycoproteidic biomarkers for disease diagnosis , is based on elisa , eclia , and enzyme - substrate colorimetric / light extinction / electrochemoluminescence ( ecl ) technology . a uniform distribution of the number of acquired ms / ms , protein , and peptide identifications undergo proteomic runs which allow protein identification on large scales estimated up to as high as ~14 000 proteins and ~250 000 unique peptides . subclinical inflammation , reliably diagnosed using c - reactive protein ( crp ) serum level cutoff 10 mg / l , constitutes a risk factor for the development of interstitial fibrosis and seems to reflect not only inflammation but general well being : crp is a subtle separator for hand grip strength , physical performance , and decline in older populations . transplantation medicine largely contributing to big data is going to include abo - glycan microarray results which now allow detailed characterization of donor - specific antibodies necessary for effective transplant management of solid organs [ 1416 ] . in health care , the use of big data , meant to use the large number of digitalized single analyses accumulated daily , a computational medicine , that is , ehealth or electronic health or medical record ( her and emr ) , expands at the expense of health care professionals to read its message . data warehouses containing big data need continuous validation , data management for updates , and most importantly an analysis to convert such resource into clinically relevant information for medical care . the designation of given lab analyses as biomarker is currently moving towards genome sequencing to reveal genetic risks for future illness ( figure 2 ) obviously a component of personal big data ; patients on their own will thus access to their data bank . for practical reason , we here lend the four major fields of laboratory medicine in switzerland , under the auspices of a federal organization , foederatio medicorum analyticorum helveticorum ( famh ) : clinical chemistry , immunology , haematology , and microbiology with genetics wrapping up each of these ( figure 2 ) . to a large extent , this distinction of laboratory analyses by category corresponds to international habits defined by the international federation of clinical chemistry ( ifcc ) . the substantial expansion of the analyses performed boosts each of these specialities alike , enriching the respective fields with unprecedented wealth of data . this brings medical doctors to dilemmas on the appropriate choice of information for patient care in the present context focusing on inflammation . to turn big data into appropriate data thus computational methods may throw a bridge from bench to bedside and vice versa [ 7 , 25 ] and web - based platforms would allow seamless interaction between warehouse and patient - centered information as recently put forward through an eu project . academic as well as business intelligence literature counsels are available for assisting transformation of big data into a selection of data assembled for care of single disease entities ( http://www.sas.com/ and http://www.aacc.org/ ) . the multilayer dimensionality of big data warehouses needs data - driven algorithms necessary to reach their basic goal : to translate big data into clinically useful evidence . logistic regression models , cox analysis , and kaplan meier curves can sort out analytes which would predict clinical evolution , for example , kidney - associated morbidity [ 3032 ] ; our own studies are being in line with researchers in scandinavia . platelet count and icu survival , actually completely unrelated parameters , can be used for predictive modelling purpose . one of the possibilities to unify assignments of terms for distinct analyses currently successful on an international level are the logical observation identifiers names and codes ( loinc ) which have been created under the auspices of the regenstrief inc . , the usage of loinc codes remains subject to variations in the way they are used and semantic , taxonomic interoperability might turn out to be contradictory in some places . experiences using data warehouse produced collaboratively between academic medical centers and private practice throwing bridges to ehr do reveal potential to improve utilization of clinical pathology testing . thus , in the us , objective electronic laboratory reporting has now been promoted as a public health priority with two coding systems endorsed : loinc for lab test orders and systemized nomenclature of medicine - clinical terms ( snomed ct ) for test results , the former being in use more commonly . since its outset in 2010 , participation of 120 laboratories including 50.000 codes now ensures interoperability in the entire french ehr system . loinc codes comprise categories to inform the data storage software on ( i ) the analyte , ( ii ) measured property , that is , enzyme activity or concentration , ( iii ) time ( span ) of sample collection , ( iv ) system used , for example , urine plasma serum , liquor , and ( v ) scale , that is , nominal and ordinal . one single analyte , for example , complement hemolytic activity activated through the classical pathway , ch50 , comprises as much as 7 different loinc codes ( table 1 ) , depending on which starting material was employed for ch50 analysis . newborn screening , including such analyses with high - stake health implications necessitates rapid / effective communication between many people and organizations and increasingly depend on big data registries which can aggregate results from national programs and help harmonize inherited metabolic disorder to an international level . the march of dimes recommends screening newborns for 29 conditions , such as phenylketonuria , hypothyroidism , galactosemia , and sickle - cell anemia ( http://www.marchofdimes.com/ ) . a prenatal screening program is taking shape and involves pregnancy - associated plasma protein a ( papp - a ) and pregnancy hormone hcg and pregnancies with hypertensive disorders might be detected early on using analysis of cell - free fetal dna , cell - free total dna , and biochemical markers . such a bayes estimator for single analyses with their variance , confidence interval can be derived from a posterior distribution ; the minimum square error also called squared error risk is defined by mse = e[((x ) ) ] . some of the key features of a bayesian analysis as a powerful package for molecular sequence variation have been delineated 10 years ago . the european informatics institute ( embl - ebi ) based on the consolidated apache lucene technology might inspire search engine development to direct scalable search paths towards medical diagnosis . henceforward , such overseeable data built up during the last century currently expands to big data produced by automated workflow using intelligent robotics with throughputs of 315 million clinical chemistry assays / year . a laboratory automate , for example , the cobas machine ( roche diagnostics , rotkreuz , switzerland ) with its large panel of possible analyses , may serve as an example of ever growing lab service function . competing industries , for example , abbott ( abbott diagnostics , abbott park , il , usa ) , siemens ( siemens healthcare , erlangen , germany ) , hitachi ( hitachi , mountain view , usa ) , capillarys sebia ( paris , france ) , kiestra ( bd , franklin lakes , nj , usa ) , bruker ( bruker , billerica , ma , usa ) , biomrieux ( lyon , france ) , are continuously updating their offers in order to increase capacity , that is , number of analyses / time throughput . the analytes of the clinical chemistry lab section can be subdivided into provision of information for whole body pathology and into organ - specific lab assays . each of these specialities of medicine sees its own lab definition increased in number of different analyses such as we have recently used cystatin c and its ratio to creatinine to improve significance of interpretation in kidney insufficiency . the inflammation parameters in clinical chemistry , occasionally with biomarker status , are acute phase proteins , that is , crp , serum amyloid a , fibrinogen , tryptase , haptoglobin , procalcitonin , interleukin-6 , and again crp used to classify disease stage of rheumatoid arthritis and now even to estimate extent of fitness and senescence . picturing blood films does not lend itself to electronic storage in big data banks in contrast to findings coming out from forward and sideward scatter beamer flow cytometer cell analysers , such as sysmex xe-5000 ( kobe , japan ) , abbot sapphire ( abbott diagnostics division , santa clara , ca , usa ) , siemens advia ( siemens healthcare , erlangen , germany ) , beckman coulter ( beckman coulter eurocenter , geneva , switzerland ) , and amnis flowsight ( seattle , wa , usa ) . complete blood counts ( cbc ) , hemostasis assays now forming part of sysmex machines , bone marrow , and progress in stem cell therapeutics are all prone to be integrated into big data banks . the diagnostic value of both microscopic and automated neutrophil left - shift parameters as indicators of inflammatory disease is limited but confirmed routine automated coagulation assays and pharmacomonitoring of many drugs with lc ms / ms machines are fit for integration into big data banks and miniaturization of assay principles is contributing to this trend . multiple data release prone to be sections in big data registries are now being making precision medicine even more precise . in fact , inexpensive molecular typing of histoblood types paired with powerful bioinformatics has enabled mass - scale information but bears the risk that personalized red blood cell matching for transfusion becomes less precise . small- and large - order haemogram blocks for blood microarray technology can be used to more precisely delineate anti - abh antibodies , a progress which will make solid organ transplantation across abo barriers more successful thanks to using bioinformatics . stem cell transfusion and cord blood based therapy are making inclusion of hla types and gwas whole genome typing into registries which are based on big data informatics ( http://www.hpscreg.eu/ ) . analytical approaches of the immune system branches into cellular and humoral patient samples which are tested frequently ( figure 2 ) . care for patients suffering from allergic diseases recognizes the usefulness of a systemwide profiling approach , which associates big lab data with the biological approach to asthma and allergy . cellular immunological analysis overlaps with hematological tests of the myeloid compartment , but they make their own data box with lymphocyte subsets and their cd marker pattern . contribution of the complement system to big data is considerable ; with three activation pathways ~48 proteins and their fragments , 9 protein complexes and ~12 receptors , measured w / v and/or by functional activity , the complement system contributes to warehouses on its own . with the seniorlab study ( isrctn registry number 53778569 ) we put w / v concentrations of c4 and c3 and immunoglobulin ( ig ) levels into perspective with serum vitamin d levels and senescence . immunoassays were used to quantitate c4 and c3 and ig in 1470 apparently healthy subjects > 60 yrs . low levels of 25(oh)d were positively associated with igg2 and c4 ( the lower vitamin d , the lower c4 , figure 3 ) yet inversely related to levels of igg1 and iga and c3 [ 59 , 60 ] . as can be seen in table 1 , a single one and the very same analysis can account for 7 different loinc codes depending on the material in which measurements are made and depending on w / v versus functional performance measured . acute phase complement proteins related to c - reactive protein ( crp ) evolve in parallel during inflammatory states and are now known to play a role in type 2 diabetes , lipid metabolism , and atherosclerosis ( figure 4 ) . these insights suggest that complement system - related algorithms destined to extract significant patient data from big data must be seen related to noncomplement analyses performed in the routine laboratory . on the immune cell level , high - throughput sequencing has sparked information on tcr repertoire diversity informative on functional capacity of the adaptive immune system . big data warehouses will certainly have to make reference to the age group of study subjects . biomarkers relating to particular disease states , for example , prostate - specific antigen ( psa ) or those of oxidized lipoproteins , genetically determined come to increase the size of big data in personalized medicine , their heritability being under scrutiny with twin pair studies . , from smart phones and wearable devices ) will contribute to the next wave in big data upload to the cloud and propagation across social networks make encryption prevent access to information by insurance companies , ransomware hackers , and state writ guardians . host , microbiomes , and pathogenic microbes are analyzable with a big data array of laboratory criteria making bioinformatics an indispensable pillar of big data in inflammation exploration . bacterial , viral , fungal , and parasitic infections and diseases due to microbial toxins are among the most common and medically important causes of inflammation with different pathogens eliciting varied responses ranging from mild and short - term to severe and long - term . foreign bodies , catheters , splinters , sutures , and dirt may elicit inflammation and inflammation tissue and laboratory data may circumscribe hypersensitivity and autoimmune disease induced inflammation under the control of cytokines produced by t lymphocytes mainly . on hosts ' side the predisposition to provide for a favourable environment for infectious agents , an array of receptor molecules ( fy a / b for malaria , cho recognition on pmnl , fibronectin on catheters , figure 4 ) can be appreciated to then enter bmld ( biosafety in microbiological and biomedical laboratories ) banking . nothing but the human gut containing a myriad of different bacteria and other microorganisms such as archaea , viruses , and fungi , making the microbiome expand to big data sets , an enter - system . on the side of microbes , the bmld provides important information on the clinical presentation the infectious agent will cause if successfully attacking the host . as an example , the strain k 157 of e. coli is predictive of hus and other strains of e. coli , such as o157:h7 , o104:h4 , o121 , o26 , o103 , o111 , o145 , and o104:h21 , produce potentially lethal toxins . this is but one example of the enormous extent of data which microbiology occupies the space of a data warehouse . with the maldi tof system , fast typing has entered the practice since a decade which facilitates updates of bmld boxes . virulence factors can be spotted in staphylococcus aureus using whole genome sequencing combined with dna microarray hybridization prone to increased big data informatics . as well laboratory assays differ between specimen analyses in acute disease and the assay approach used for specimens taken during convalesce . louis encephalitis , and west nile virus ) which enforce usage of rt - pcr , also for chikungunya viruses . whilst big genetic data validation overlaps and completes each of the preceding subchapters ( figure 2 ) , genetic studies of many diseases are now allowing closer insights into human pathology . ngss can be used to detect genetic anomalies of essentially any size scale , from snps to very large rearrangements ; all of todays ' genetic diagnostic tests could in principle be supplanted by ngs , including rna analysis , because transcriptome ( rna - seq ) sequencing is possible and now boosts with crisp - cas9 technology . on the leading edge of a revolution in medicine the complete dna sequence , properly encrypted , will become a permanent part of individual 's ehr utilized by health care professionals to make decisions about drug prescriptions , diagnostics , and disease prevention . as an example phenotypes are now closely linkable to genetic findings and to the age of the patient , younger age at diagnosis associated with extensive / aggressive crohn 's disease and ulcerative colitis . dissection of genotype - phenotype relations using immunochip array designed to capture up to 200 different loci associated with common autoimmune diseases can be focused on nod2 , mhc , and mst1 3p21 to allow establishment of a genetic risk score and predictive modelling . the genetic diseases encountered in medical practice are the tip of the iceberg , that is , those with less extreme genotypic glitches that permit full embryonic development and live birth . genetic variants of complement genes , for example , cfigly119arg , such as recently evaluated in the eugenda cohort may be associated with age - related macular degeneration . with the determination of the complete sequence of the human genome , dna imprints not only for disease but also for the gray zone between health and disease are under scrutiny . collins puts it it will take decades , if not centuries to understand the instructions of the genetic language , and , one might add , improve and make big data information more meaningful . ransomware attacks hackers blocking hospital and private practice computers have now been reported here and there . computer systems , including those needed for lab work , can be set out of function relatively easily which makes big data clouds vulnerable to an extent to which some workers keep copies on separate hardware aside ; desktop virtualization systems , for example , citrix , are improved for hacker risk reduction and saving data apart on separate servers might do the rest to prevent big data hacking . two motive forces are thriving attempts to reduce the number of health parameters ( i ) to bring customers of the medical lab to a reasonable and patient driven block of analyses asked for and ( ii ) to optimize financial sources doctors often ignoring the financial consequences of their test - ordering behavior . study results from attempts to reduce the number of ordered tests become known and an expert panel may bring some good ideas but will never be able to brake medical revolution continuing to creep on us , especially in the field of laboratory analyses .

biosolid samples - digested mesophilic sludge samples ( biosolids ) were obtained from a large urban wwtp located in rio de janeiro ( rj ) , brazil . the wwtp receives urban sewage with a mean inflow rate of 1.600 l s. the wwtp utilises an aerobic process ( conventional activated sludge ) , mad and dewatering in its treatment process . from february - july 2011 samples were collected monthly ( with 15 days intervals , approximately ) , except in march , when four biosolid samples were provided by the wastewater sanitation company ( cedae ) . samples were collected in sterile plastic bags , kept at 4c and transported to the laboratory for immediate analysis . viruses and the ic - rva g1p ( genbank accession gu831596 ) and nov gii/4 strain ( genbank accession dq997040 ) , both of which were isolated from a positive faecal suspension ( 10% ) and identified during acute gastroenteritis outbreaks in brazil , were used in the spiked experiments . the adv serotype 5 , which was propagated in cell culture ( hep-2 ) and the hav strain ( haf-203 ) , which was propagated in rhesus kidney cell cultures ( frhk-4 ) , were used in all of the experiments ( villar et al . a pp7 bacteriophage ( atcc 15692-b2 ) was kindly provided by dr vernica rajal ( salta university , argentina ) and included as an ic . its replication was performed by culture in pseudomonas aeruginosa ( atcc 15692 ) using a previously described protocol ( rajal et al . virus stock solutions were quantified according to real - time pcr protocols ( table i ) to determine the viral concentrations used to spike the biosolid samples . table i viruses analysed , polymerase chain reactions ( pcrs ) assays , primers ' sequence , genome region and referencesviruses and qpcr assayprimers and probesequences 5'-3'genome regionreferencesrva and pp7 multiplex rt - qpcrnsp3 faccatctwcacrtraccctctatgagnsp3fumian et al . ( 2010)nsp3 rggtcacataacgcccctatagcnsp3 probevic - agttaaaagctaacactgtcaaa247 fgttatgaaccaatgtggccgttatreplicase320 rcgggatgcctctgaaaaaag274 probefam - tcggtggtcaacgaggaactggaac - tamrahadv qpcraq1 fgccacggtggggtttctaaactthexonheim et al . ( 2003)cog2rtcgacgccatcttcattcacaring2-probefam - tgggagggcgatcgcaatct - tamrahav rt - qpcrforward primerctgcaggttcagggttcttaaatc5 ' non - coding regionvillar et al . ( 2006)reverse primergagagccctggaagaaagaagaprobefam - actcatttttcacgctttctg a : international union of biochemistry code ( w : a / t ; r : a / g ; b : c / g / t ; y : c / t ; n : a / c / g / t ) ; adv : adenoviruses ; hav : hepatitis a virus ; nov gii : norovirus genogroup ii ; orf : open reading frame ; qpcr : quantitative pcr ; rt : reverse transcription ; rva : rotavirus species a. a : international union of biochemistry code ( w : a / t ; r : a / g ; b : c / g / t ; y : c / t ; n : a / c / g / t ) ; adv : adenoviruses ; hav : hepatitis a virus ; nov gii : norovirus genogroup ii ; orf : open reading frame ; qpcr : quantitative pcr ; rt : reverse transcription ; rva : rotavirus species a. virus concentration methods - viruses were concentrated using two different techniques . ( 1998 ) , consists of an ultracentrifugation - based method , with minor modifications . briefly , 5 g [ dry matter ( d.m . ) ] of biosolid were suspended in 15 ml of 0.25 n glycine buffer ( ph 9.5 ) and incubated on ice for 30 min . the solution was neutralised by adding 10 ml of 2 x phosphate - buffered saline ( pbs ) ( ph 7.2 ) . the mixture was centrifuged ( 12,000 g for 15 min , 4c ) and the supernatant was ultracentrifuged ( beckman ultracentrifuge , equipped with a type 35 rotor ) at 100,000 g for 1 h at 4c . ( 2007 ) , consists of a simplified method similar to that used by the us epa ( 2003 ) . briefly , 10% beef extract solution ( lp029b , oxoid ltd basingstoke , hants , england ) , ph 7.2 at 1:10 ( v / v ) or ( w / v ) was added to 5 g ( d.m . ) of sewage sludge . the sample was magnetically stirred ( 500 rpm ) for 20 min at room temperature . the supernatant was recovered and filtered through low protein binding membrane filters ( millipore , 0.22 m pore size ) to decontaminate it . all of the concentrated samples were stored at -70c until molecular biology analysis was performed . spike experiments and inhibition tests - the biosolid samples were autoclaved at 121c for 30 min for decontamination . - determined according to us epa ( 2003 ) ] of biosolid sample and the virus titres ( final concentrations ) seeded into the samples were determined by a qpcr assay . the viral titres spiked in each method were as follows ( gc ml : method 1 : 1.1 x 10 ( adv ) , 3.2 x 10 ( rva ) , 1.4 x 10 ( nov ) , 2 x 10 ( hav ) and 4.2 x 10 ( pp7 ) ; method 2 : 2.7 x 10 ( adv ) , 3.8 x 10 [ standard deviation ( sd ) 2.8 x 10 ] ( rva ) , 3.4 x 10 ( sd 2.3 x 10 ( nov ) , 9 x 10 ( sd 1 x 10 ( hav ) and 4.2 x 10 ( pp7 ) . viruses were adsorbed onto sludge flocs by adjusting the ph to 3.5 0.1 with hcl ( 1 n ) , as described by sano et al . the biosolid samples were then centrifuged ( 10,000 g , 15 min , 4c ) and the supernatant ( 1 ml ) and pellet generated in each experiment were analysed with a qpcr assay to determine the virus recovery yield . procedures were performed in triplicate and repeated on different dates . in all of the experiments , extracted nucleic acids were diluted in rna / dnase free water using tenfold serial dilutions ( 1:10 and 1:100 ) to verify inhibitors of the pcr reactions . pp7 , which was used as an ic for the biosolid samples , was spiked with high concentrations of viral particles ( ranging from 10 - 10 . the initial viral titres used in the spiked experiments were diluted to test the limits of virus detection when using the concentration methods and qpcr assays . viral genomic extraction and the reverse transcription ( rt ) reaction - nucleic acids were extracted from 140 l of the eluate to obtain a final volume of 60 l using the qiaamp viral rna kit ( qiagen , inc , valencia , ca ) according to the manufacturer 's instructions . cdna synthesis was conducted by rt using a random primer ( pdn6 , 50a260 units , amersham biosciences , chalfont st giles , buckinghamshire , uk ) for rv , nov gii , hav and pp7 . louis , mo ) and 10 l of rna were mixed briefly , heated at 97c for 7 min and chilled for 4 min . the components of the mixture and their final concentrations for the 50-l rt reaction were as follows : 2.5 mm of each deoxynucleoside triphosphate ( gibco brl , life technologies , inc , grand island , ny ) , 1.5 mm mgcl2 , 200 u of superscript iii reverse transcriptase ( invitrogen ) and 1 l of pdn6 . the rt reaction mixture was incubated in a thermal cycler ( ptc-100 programmable thermal controller , mj research , inc , watertown , ma ) at 25c for 5 min , 50c for 60 min and 70c for 20 min . qpcr - the sequence of primers and probes , region of amplification on the genome and references to qpcr protocols for virus quantification can be found in table i. to avoid false - positive results , quality control measures such as the use of separate rooms and the inclusion of negative controls in each set of amplifications were adopted . inhibition tests were performed by diluting nucleic acids ( 10-fold serial dilutions : 1:10 and 1:100 ) in all of the analysed samples . a standard curve ( 10 - 10 copies per reaction ) was generated for all of the viruses using 10-fold serial dilutions of pcr2.1 vectors ( invitrogen , usa ) containing the target region . the qpcr reaction was performed in a final volume of 25 l by using 12.5 l of the universal pcr master mix ( applied biosystems , ca , usa ) and 5 l of the dna / cdna under the following incubation conditions : 50c for 2 min to activate ung , 95c for 10 min for initial denaturation , 40 - 45 cycles at 95c for 15 s and then 50 - 60c for 1 min , depending on virus type . amplification data were collected and analysed using applied biosystems 7500 software version 2.0 ( applied biosystems , foster city , ca ) . a positive result was considered when the sample signals crossed the threshold line , presenting a characteristic sigmoid curve . the number of viral particles was determined by adjusting the values according to the volumes used for each step of the procedure ( extraction , cdna synthesis and qpcr reaction ) . the amount of genome copies ( gc ) detected using each methodology was reported in g ( d.m . ) of concentrated sample . the total number of viral particles spiked in the samples ( per ml ) vs. the total number of viral particles recovered ( per g dry matter ) was considered to estimate the virus recovery efficiency ( % ) . virus recovery efficiency - three different virus concentration methods were tested to evaluate the recovery efficiencies for each type of virus . the mean values ( gc ml of the viruses detected in the supernatant of the spiked experiments ( i.e. , viruses not adsorbed onto sludge flocs ) are shown in table ii . all of the viruses analysed were adsorbed onto sludge flocs in percentages higher than 99% ( table ii ) . negative results were obtained for all of the viruses analysed in the negative control samples . table ii percentage of viruses adsorbed on sludge flocsvirusesviruses spiked onto sludge samples ( gc ml ) mean/sdviruses in supernatant ( gc ml ) mean/sdviruses adsorbed on sludge flocs ( % ) adv4 x 10/5.9 x 10 6.7 x 10/5.4 x 10 99.99rva3.5 x 10/2.8 x 10 8.5 x 10/8.5 x 10 99.97nov3.6 x 10/1.9 x 10 2.5 x 10/1.5 x 1099.93hav2.7 x 10/6.3 x 10 2.6 x 10/4.5 x 10 99.90pp74.2 x 10/04.2 x 10/4.0 x 10 99.0 a : methods were performed in triplicate ; adv : adenoviruses ; gc : genome copies ; hav : hepatitis a virus ; nov gii : norovirus genogroup ii ; rva : rotavirus species ; sd : standard deviation . a : methods were performed in triplicate ; adv : adenoviruses ; gc : genome copies ; hav : hepatitis a virus ; nov gii : norovirus genogroup ii ; rva : rotavirus species ; sd : standard deviation . higher mean viral loads ( vls ) ( gc g d.m . ) from each method performed , including the inhibition tests , are shown in fig . 1 . in general , higher mean vls were recovered when the nucleic acids were diluted at least once ( 1:10 ) , except for the rva in method 1 ( fig . 1mean numbers in log10 units of viruses recovered [ genome copies ( gc ) g-1 ] from biosolid samples . ten - fold serial dilution of nucleic acids are shown in paren thesis ( 1:10 and 1:100 ) on the x axis . adv : adenoviruses ; bars : minimum and maximum value ; hav : hepatitis a virus ; method 1 : ultracentrifuga tion ; method 2 : beef extract ; nov gii : norovirus genogroup ii ; rva : rotavirus species a ; square in black : mean value . pp7 was recovered from all of the analysed samples and the mean vls detected by methods 1 and 2 were as follows : 5.2 x 10 gc g sd 1.1 x 10 and 2.8 x 10 gc g sd 2.3 x 10 , respectively . when the nucleic acids were diluted 1:100 in method 2 , a higher mean recovery rate was obtained for pp7 : 1.3 x 10 sd 1.1 x 10 gc g ( fig . 2recovery efficiency ( % ) from each method performed in trip licate used for detecting enteric viruses . adv : adenoviruses ; hav : hepatitis a virus ; method 1 : ultracentrifugation ; method 2 : beef ex tract ; nov gii : norovirus genogroup ii ; rva : rotavirus species a. the mean maximum value ( gc g obtained from each procedure performed in triplicate ( including the dilution of nucleic acids to 1:10 or 1:100 ) was considered in the analysis of the virus mean recovery rates ( % ) for each virus type ( fig . hav showed the worst results in both methods , with detection limits higher than 10 gc ml . for nov , the detection limits were 1.4 x 10 gc ml and 4.8 x 10 gc ml for methods 1 and 2 , respectively . the detection limits for adv were 1.1 x 10 gc ml and 1.5 x 10 gc ml for methods 1 and 2 , respectively . finally , for rva the detection limit was 3 x 10 gc ml for both methods . natural occurrence of enteric viruses - table iii shows the vl for adv , rva , nov and hav from 11 biosolid samples collected from an activated sludge process . elution with beef extract ( method 2 ) showed a better recovery for adv , which was detected in 90% of the analysed samples . the second most detected viruses were rva and nov ( 45% ) ( table iii ) . rva was detected at higher concentrations by the ultracentrifugation method ( table iii ) and nov gii was only detected in may , june and july , when higher concentrations of the virus were obtained by the beef extract method ( table iii ) . hav was only detected in two samples at relatively high concentrations ( table iii ) . pp7 was detected in all of the spiked field samples , with lower concentrations obtained from samples collected in february and march ( table iii ) . table iii results of viral genome loads g-1 ( dry matter ) detected in anaerobically digested sludge samples using two concentration methods sampling dateadvrvanovhavpp7method121212121228 feb6.8 x 100.9 x 100000001.0 x 10 nd02 mar04.2 x 1003.8 x 1000003.8 x 10 2.6 x 10 04 mar2.1 x 106.8 x 101.6 x 10 000002.0 x 10 1.4 x 10 14 mar1.4 x 101.1 x 10 7.8 x 106.2 x 1000003.7 x 10 1.8 x 10 28 mar04.3 x 100000001.4 x 10 3.7 x 10 11 apr000004.8 x 10 001.3 x 10 4.8 x 10 25 apr1.0 x 102.4 x 100000001.3 x 10 9.4 x 10 16 may02.7 x 10 006.4 x 101.2 x 10 001.3 x 10 5.4 x 10 13 jun01.9 x 10 001.4 x 101.5 x 10 02.0 x 10 3.1 x 10 5.6 x 10 27 jun2.4 x 10 3.6 x 10 2.5 x 10 02.2 x 102.3 x 10 4.8 x 10 0nd9.2 x 10 11 jul01.6 x 10 07.2 x 101.8 x 105.2 x 10 001.0 x 10 ndpositive / total ( n)5/1110/113/113/114/115/111/111/1110/109/9 a : diluted nucleic acids ( 1:10 ) ; adv : adenoviruses ; hav : hepatitis a virus ; method 1 : ultracentrifugation ; method 2 : beef extract ; nd : not done ; nov gii : norovirus genogroup ii ; rva : rotavirus species . a : diluted nucleic acids ( 1:10 ) ; adv : adenoviruses ; hav : hepatitis a virus ; method 1 : ultracentrifugation ; method 2 : beef extract ; nd : not done ; nov gii : norovirus genogroup ii ; rva : rotavirus species . samples with negative results were submitted to a second round of experiments based on the dilution of the nucleic acids ( 1:10 and 1:100 ) . the 1:10 dilution enabled detection of nov in one sample that had been negative when tested with method 1 ( sampling date : april 11th ) ( table iii ) . this dilution also enabled detection of rva in a sample that had been negative when tested with method 2 ( sampling date : june 27th ) ( table iii ) . however , all of the samples were still negative for hav when this dilution was made ( 1:10 ) . ph measurements showed a mean result of 6.7 ( sd 0.5 ) in the biosolid samples ; however , samples collected in february had a lower ph value , at 5.5 . viruses are charged particles that can be highly concentrated in biosolids due to aggregation and adhesion to sludge solids ( sidhu & toze 2009 ) . several methods have been tested for detecting viruses in sewage sludge samples ( monpoeho et al . 2004 , belguith et al . 2006 , guzmn et al . 2007 ) ; however , few comparisons between the different viruses have been made regarding recovery yields of the different viral groups , specifically hav and other enteric viruses ( sidhu & toze 2009 ) . in some cases , the negative results can be attributed to the inefficiency of the methods used . the methods chosen for this study have been recommended by the resolution of the national council of the environment ( conama 2006 ) , which provides guidelines for monitoring enteric viruses in biosolids . these guidelines recommend protocols that use beef extract or those based on ultracentrifugation for virus concentration . pcr is also recommended for the detection of some viral groups , including rv and hav ( conama 2006 ) . the primers and probes used in taqman qpcr assays target more conservative regions of the virus genome and have been considered suitable for detecting enteric viruses in several environmental matrices ( villar et al . 2010 , 2011 , prado et al . 2011a , simmons & xagoraraki 2011 ) . however , there is some difficulty in amplifying targeted nucleic acids in biosolid samples due to the presence of a variety of inhibitors , such as humic and fulvic acids , fats , proteins , organic and inorganic compounds , including polyphenols and heavy metals that form complexes with nucleic acids and inhibit amplification enzymes ( sano et al . the results obtained in this study indicate that different virus recovery rates are the result of the different virus concentration methods used . the mean recovery rates of the beef extract method were 6.2% and 6.3% for adv and nov , respectively , and are similar to the results obtained by sano et al . ( 2003 ) , who detected a 7% recovery rate for poliovirus from sewage sludge by rt - pcr . ultracentrifugation was considered the best alternative method for detecting rva and hav in the spiked experiments , but not suitable for naturally contaminated biosolid samples . it is possible that in the spiked experiments , the original organic matter was compromised due to autoclaving , contributing to an elevated recovery efficiency rate for the concentration methods . however , the mean recovery rate of adv was very low when using glycine buffer followed by ultracentrifugation . 2010 ) has reported that beef extract and glycine buffer can concentrate different inhibitor compounds responsible for causing different results when using a qpcr assay . nevertheless , it is difficult to determine which compound affected the qpcr efficiency because ultracentrifugation was used as the final step to concentrate the viruses . the pellet that is generated may contain viruses and other substances , such as suspended solids of the final eluate . adv had a higher level of adsorption in this particulate matter because the virus size affects the interaction mechanisms with the colloidal particles of the environmental matrices ( dowd et al . moreover , the presence of suspended solids in the final eluate can affect the results of the nucleic acid extraction . several strategies can be adopted to minimise or predict the interference of environmental inhibitors in pcr reactions ( viau & peccia 2009 , rock et al . 2010 ) , but the dilution of nucleic acids appears to be the simplest of these strategies , specifically by avoiding the addition of other reagents . nevertheless , in contrast to the results obtained from the spiked experiments , nucleic acids from the field samples diluted at 1:100 showed negative results , which indicates that the vl of these samples may have been diluted below the detectable limits of the assays . the use of an ic ( pp7 bacteriophage ) to monitor the stages of detection provides an interesting solution for avoiding false negative results . when high concentrations of pp7 were seeded in the field samples , the vls recovered were low in some samples . the negative results obtained for some of the enteric viruses in these samples may be attributed to variable compositions of the biosolids . experiments using anaerobically digested sludge samples detected at least one virus in each of the analysed samples . comparatively , adv was the most detected virus , demonstrating its widespread dissemination in treated sewage sludge . these results support the hypothesis that adv could be a good indicator for evaluating the presence of enteric viruses in sewage sludge samples ( bofill - mas et al . ( 2006 ) reported similar concentrations for adv , other investigations have found higher adv genome loads in sewage sludge treated by mad , with concentrations varying from 10 - 10 gc g ( d.m . ) rva can be detected at higher levels ( 45% ) in biosolid samples ; recent studies have shown a large dissemination of rva ( 90% ) in wastewaters from rj ( fumian et al . however , researchers hypothesise that rv is poorly adsorbed in solid fractions of sludge ( arraj et al . 2005 , sidhu & toze 2009 ) , possibly explaining the lower frequency of detection and vls when compared with the amount of adv found in these samples . interestingly , nov gii was predominantly detected in the colder months , suggesting a higher burden and circulation of these viruses during this period and corroborating previous results concerning the peak occurrences of nov in brazilian sewage samples ( victoria et al . hav was poorly recovered from the sewage sludge samples , corroborating data from other studies ( schlindwein et al . the lower frequency of hav detection in biosolids can be expected because improvements in sanitary and socioeconomic conditions may be preventing the circulation of these viruses in the community and consequently , in wastewaters , as recently verified in studies conducted in rj ( prado et al . however , another explanation is related to the low detection limits of the methods used to detect hav in biosolids , as demonstrated in this study and by other authors ( jebri et al . therefore , the detection of hav in biosolids can be underestimated , primarily when low vls are circulating in these environments . although viral infectivity is not determined by methods based on nucleic acid amplification , studies have confirmed that a great proportion of viral genomes detected by molecular methods correspond with viable infectious particles detected in sewage sludge or biosolids ( schlindwein et al . moreover , the aggregation of viruses onto sludge flocs could prevent viral inactivation ( sidhu & toze 2009 ) . the concentration method using beef extract elution followed by qpcr assay seems suitable for detecting adv in biosolids . however , other concentration methods should be investigated for the detection of other viruses , especially hav , to avoid inaccuracies related to potential contamination and quantification of viruses in biosolids , ensuring a reliable public health risk analysis .

the presence of enteric viruses in biosolids can be underestimated due to the inefficient methods ( mainly molecular methods ) used to recover the viruses from these matrices . therefore , the goal of this study was to evaluate the different methods used to recover adenoviruses ( adv ) , rotavirus species a ( rva ) , norovirus genogroup ii ( nov gii ) and the hepatitis a virus ( hav ) from biosolid samples at a large urban wastewater treatment plant in brazil after they had been treated by mesophilic anaerobic digestion . quantitative polymerase chain reaction ( pcr ) was used for spiking experiments to compare the detection limits of feasible methods , such as beef extract elution and ultracentrifugation . tests were performed to detect the inhibition levels and the bacteriophage pp7 was used as an internal control . the results showed that the inhibitors affected the efficiency of the pcr reaction and that beef extract elution is a suitable method for detecting enteric viruses , mainly adv from biosolid samples . all of the viral groups were detected in the biosolid samples : adv ( 90% ) , rva , nov gii ( 45% ) and hav ( 18% ) , indicating the viruses ' resistance to the anaerobic treatment process . this is the first study in brazil to detect the presence of rva , adv , nov gii and hav in anaerobically digested sludge , highlighting the importance of adequate waste management .

MATERIALS AND METHODS RESULTS DISCUSSION

biosolid samples - digested mesophilic sludge samples ( biosolids ) were obtained from a large urban wwtp located in rio de janeiro ( rj ) , brazil . the wwtp receives urban sewage with a mean inflow rate of 1.600 l s. the wwtp utilises an aerobic process ( conventional activated sludge ) , mad and dewatering in its treatment process . from february - july 2011 samples were collected monthly ( with 15 days intervals , approximately ) , except in march , when four biosolid samples were provided by the wastewater sanitation company ( cedae ) . viruses and the ic - rva g1p ( genbank accession gu831596 ) and nov gii/4 strain ( genbank accession dq997040 ) , both of which were isolated from a positive faecal suspension ( 10% ) and identified during acute gastroenteritis outbreaks in brazil , were used in the spiked experiments . the adv serotype 5 , which was propagated in cell culture ( hep-2 ) and the hav strain ( haf-203 ) , which was propagated in rhesus kidney cell cultures ( frhk-4 ) , were used in all of the experiments ( villar et al . a pp7 bacteriophage ( atcc 15692-b2 ) was kindly provided by dr vernica rajal ( salta university , argentina ) and included as an ic . virus stock solutions were quantified according to real - time pcr protocols ( table i ) to determine the viral concentrations used to spike the biosolid samples . ( 2006)reverse primergagagccctggaagaaagaagaprobefam - actcatttttcacgctttctg a : international union of biochemistry code ( w : a / t ; r : a / g ; b : c / g / t ; y : c / t ; n : a / c / g / t ) ; adv : adenoviruses ; hav : hepatitis a virus ; nov gii : norovirus genogroup ii ; orf : open reading frame ; qpcr : quantitative pcr ; rt : reverse transcription ; rva : rotavirus species a. a : international union of biochemistry code ( w : a / t ; r : a / g ; b : c / g / t ; y : c / t ; n : a / c / g / t ) ; adv : adenoviruses ; hav : hepatitis a virus ; nov gii : norovirus genogroup ii ; orf : open reading frame ; qpcr : quantitative pcr ; rt : reverse transcription ; rva : rotavirus species a. virus concentration methods - viruses were concentrated using two different techniques . the mixture was centrifuged ( 12,000 g for 15 min , 4c ) and the supernatant was ultracentrifuged ( beckman ultracentrifuge , equipped with a type 35 rotor ) at 100,000 g for 1 h at 4c . briefly , 10% beef extract solution ( lp029b , oxoid ltd basingstoke , hants , england ) , ph 7.2 at 1:10 ( v / v ) or ( w / v ) was added to 5 g ( d.m . ) all of the concentrated samples were stored at -70c until molecular biology analysis was performed . the viral titres spiked in each method were as follows ( gc ml : method 1 : 1.1 x 10 ( adv ) , 3.2 x 10 ( rva ) , 1.4 x 10 ( nov ) , 2 x 10 ( hav ) and 4.2 x 10 ( pp7 ) ; method 2 : 2.7 x 10 ( adv ) , 3.8 x 10 [ standard deviation ( sd ) 2.8 x 10 ] ( rva ) , 3.4 x 10 ( sd 2.3 x 10 ( nov ) , 9 x 10 ( sd 1 x 10 ( hav ) and 4.2 x 10 ( pp7 ) . the biosolid samples were then centrifuged ( 10,000 g , 15 min , 4c ) and the supernatant ( 1 ml ) and pellet generated in each experiment were analysed with a qpcr assay to determine the virus recovery yield . in all of the experiments , extracted nucleic acids were diluted in rna / dnase free water using tenfold serial dilutions ( 1:10 and 1:100 ) to verify inhibitors of the pcr reactions . pp7 , which was used as an ic for the biosolid samples , was spiked with high concentrations of viral particles ( ranging from 10 - 10 . viral genomic extraction and the reverse transcription ( rt ) reaction - nucleic acids were extracted from 140 l of the eluate to obtain a final volume of 60 l using the qiaamp viral rna kit ( qiagen , inc , valencia , ca ) according to the manufacturer 's instructions . the components of the mixture and their final concentrations for the 50-l rt reaction were as follows : 2.5 mm of each deoxynucleoside triphosphate ( gibco brl , life technologies , inc , grand island , ny ) , 1.5 mm mgcl2 , 200 u of superscript iii reverse transcriptase ( invitrogen ) and 1 l of pdn6 . qpcr - the sequence of primers and probes , region of amplification on the genome and references to qpcr protocols for virus quantification can be found in table i. to avoid false - positive results , quality control measures such as the use of separate rooms and the inclusion of negative controls in each set of amplifications were adopted . inhibition tests were performed by diluting nucleic acids ( 10-fold serial dilutions : 1:10 and 1:100 ) in all of the analysed samples . a standard curve ( 10 - 10 copies per reaction ) was generated for all of the viruses using 10-fold serial dilutions of pcr2.1 vectors ( invitrogen , usa ) containing the target region . the qpcr reaction was performed in a final volume of 25 l by using 12.5 l of the universal pcr master mix ( applied biosystems , ca , usa ) and 5 l of the dna / cdna under the following incubation conditions : 50c for 2 min to activate ung , 95c for 10 min for initial denaturation , 40 - 45 cycles at 95c for 15 s and then 50 - 60c for 1 min , depending on virus type . the number of viral particles was determined by adjusting the values according to the volumes used for each step of the procedure ( extraction , cdna synthesis and qpcr reaction ) . the total number of viral particles spiked in the samples ( per ml ) vs. the total number of viral particles recovered ( per g dry matter ) was considered to estimate the virus recovery efficiency ( % ) . the mean values ( gc ml of the viruses detected in the supernatant of the spiked experiments ( i.e. all of the viruses analysed were adsorbed onto sludge flocs in percentages higher than 99% ( table ii ) . negative results were obtained for all of the viruses analysed in the negative control samples . table ii percentage of viruses adsorbed on sludge flocsvirusesviruses spiked onto sludge samples ( gc ml ) mean/sdviruses in supernatant ( gc ml ) mean/sdviruses adsorbed on sludge flocs ( % ) adv4 x 10/5.9 x 10 6.7 x 10/5.4 x 10 99.99rva3.5 x 10/2.8 x 10 8.5 x 10/8.5 x 10 99.97nov3.6 x 10/1.9 x 10 2.5 x 10/1.5 x 1099.93hav2.7 x 10/6.3 x 10 2.6 x 10/4.5 x 10 99.90pp74.2 x 10/04.2 x 10/4.0 x 10 99.0 a : methods were performed in triplicate ; adv : adenoviruses ; gc : genome copies ; hav : hepatitis a virus ; nov gii : norovirus genogroup ii ; rva : rotavirus species ; sd : standard deviation . a : methods were performed in triplicate ; adv : adenoviruses ; gc : genome copies ; hav : hepatitis a virus ; nov gii : norovirus genogroup ii ; rva : rotavirus species ; sd : standard deviation . 1mean numbers in log10 units of viruses recovered [ genome copies ( gc ) g-1 ] from biosolid samples . adv : adenoviruses ; bars : minimum and maximum value ; hav : hepatitis a virus ; method 1 : ultracentrifuga tion ; method 2 : beef extract ; nov gii : norovirus genogroup ii ; rva : rotavirus species a ; square in black : mean value . pp7 was recovered from all of the analysed samples and the mean vls detected by methods 1 and 2 were as follows : 5.2 x 10 gc g sd 1.1 x 10 and 2.8 x 10 gc g sd 2.3 x 10 , respectively . 2recovery efficiency ( % ) from each method performed in trip licate used for detecting enteric viruses . adv : adenoviruses ; hav : hepatitis a virus ; method 1 : ultracentrifugation ; method 2 : beef ex tract ; nov gii : norovirus genogroup ii ; rva : rotavirus species a. the mean maximum value ( gc g obtained from each procedure performed in triplicate ( including the dilution of nucleic acids to 1:10 or 1:100 ) was considered in the analysis of the virus mean recovery rates ( % ) for each virus type ( fig . hav showed the worst results in both methods , with detection limits higher than 10 gc ml . for nov , the detection limits were 1.4 x 10 gc ml and 4.8 x 10 gc ml for methods 1 and 2 , respectively . the detection limits for adv were 1.1 x 10 gc ml and 1.5 x 10 gc ml for methods 1 and 2 , respectively . natural occurrence of enteric viruses - table iii shows the vl for adv , rva , nov and hav from 11 biosolid samples collected from an activated sludge process . elution with beef extract ( method 2 ) showed a better recovery for adv , which was detected in 90% of the analysed samples . the second most detected viruses were rva and nov ( 45% ) ( table iii ) . rva was detected at higher concentrations by the ultracentrifugation method ( table iii ) and nov gii was only detected in may , june and july , when higher concentrations of the virus were obtained by the beef extract method ( table iii ) . pp7 was detected in all of the spiked field samples , with lower concentrations obtained from samples collected in february and march ( table iii ) . table iii results of viral genome loads g-1 ( dry matter ) detected in anaerobically digested sludge samples using two concentration methods sampling dateadvrvanovhavpp7method121212121228 feb6.8 x 100.9 x 100000001.0 x 10 nd02 mar04.2 x 1003.8 x 1000003.8 x 10 2.6 x 10 04 mar2.1 x 106.8 x 101.6 x 10 000002.0 x 10 1.4 x 10 14 mar1.4 x 101.1 x 10 7.8 x 106.2 x 1000003.7 x 10 1.8 x 10 28 mar04.3 x 100000001.4 x 10 3.7 x 10 11 apr000004.8 x 10 001.3 x 10 4.8 x 10 25 apr1.0 x 102.4 x 100000001.3 x 10 9.4 x 10 16 may02.7 x 10 006.4 x 101.2 x 10 001.3 x 10 5.4 x 10 13 jun01.9 x 10 001.4 x 101.5 x 10 02.0 x 10 3.1 x 10 5.6 x 10 27 jun2.4 x 10 3.6 x 10 2.5 x 10 02.2 x 102.3 x 10 4.8 x 10 0nd9.2 x 10 11 jul01.6 x 10 07.2 x 101.8 x 105.2 x 10 001.0 x 10 ndpositive / total ( n)5/1110/113/113/114/115/111/111/1110/109/9 a : diluted nucleic acids ( 1:10 ) ; adv : adenoviruses ; hav : hepatitis a virus ; method 1 : ultracentrifugation ; method 2 : beef extract ; nd : not done ; nov gii : norovirus genogroup ii ; rva : rotavirus species . a : diluted nucleic acids ( 1:10 ) ; adv : adenoviruses ; hav : hepatitis a virus ; method 1 : ultracentrifugation ; method 2 : beef extract ; nd : not done ; nov gii : norovirus genogroup ii ; rva : rotavirus species . this dilution also enabled detection of rva in a sample that had been negative when tested with method 2 ( sampling date : june 27th ) ( table iii ) . ph measurements showed a mean result of 6.7 ( sd 0.5 ) in the biosolid samples ; however , samples collected in february had a lower ph value , at 5.5 . viruses are charged particles that can be highly concentrated in biosolids due to aggregation and adhesion to sludge solids ( sidhu & toze 2009 ) . 2007 ) ; however , few comparisons between the different viruses have been made regarding recovery yields of the different viral groups , specifically hav and other enteric viruses ( sidhu & toze 2009 ) . in some cases , the negative results can be attributed to the inefficiency of the methods used . the methods chosen for this study have been recommended by the resolution of the national council of the environment ( conama 2006 ) , which provides guidelines for monitoring enteric viruses in biosolids . pcr is also recommended for the detection of some viral groups , including rv and hav ( conama 2006 ) . the primers and probes used in taqman qpcr assays target more conservative regions of the virus genome and have been considered suitable for detecting enteric viruses in several environmental matrices ( villar et al . however , there is some difficulty in amplifying targeted nucleic acids in biosolid samples due to the presence of a variety of inhibitors , such as humic and fulvic acids , fats , proteins , organic and inorganic compounds , including polyphenols and heavy metals that form complexes with nucleic acids and inhibit amplification enzymes ( sano et al . the results obtained in this study indicate that different virus recovery rates are the result of the different virus concentration methods used . the mean recovery rates of the beef extract method were 6.2% and 6.3% for adv and nov , respectively , and are similar to the results obtained by sano et al . ultracentrifugation was considered the best alternative method for detecting rva and hav in the spiked experiments , but not suitable for naturally contaminated biosolid samples . it is possible that in the spiked experiments , the original organic matter was compromised due to autoclaving , contributing to an elevated recovery efficiency rate for the concentration methods . nevertheless , it is difficult to determine which compound affected the qpcr efficiency because ultracentrifugation was used as the final step to concentrate the viruses . the pellet that is generated may contain viruses and other substances , such as suspended solids of the final eluate . moreover , the presence of suspended solids in the final eluate can affect the results of the nucleic acid extraction . nevertheless , in contrast to the results obtained from the spiked experiments , nucleic acids from the field samples diluted at 1:100 showed negative results , which indicates that the vl of these samples may have been diluted below the detectable limits of the assays . when high concentrations of pp7 were seeded in the field samples , the vls recovered were low in some samples . the negative results obtained for some of the enteric viruses in these samples may be attributed to variable compositions of the biosolids . experiments using anaerobically digested sludge samples detected at least one virus in each of the analysed samples . these results support the hypothesis that adv could be a good indicator for evaluating the presence of enteric viruses in sewage sludge samples ( bofill - mas et al . ( 2006 ) reported similar concentrations for adv , other investigations have found higher adv genome loads in sewage sludge treated by mad , with concentrations varying from 10 - 10 gc g ( d.m . ) rva can be detected at higher levels ( 45% ) in biosolid samples ; recent studies have shown a large dissemination of rva ( 90% ) in wastewaters from rj ( fumian et al . interestingly , nov gii was predominantly detected in the colder months , suggesting a higher burden and circulation of these viruses during this period and corroborating previous results concerning the peak occurrences of nov in brazilian sewage samples ( victoria et al . the lower frequency of hav detection in biosolids can be expected because improvements in sanitary and socioeconomic conditions may be preventing the circulation of these viruses in the community and consequently , in wastewaters , as recently verified in studies conducted in rj ( prado et al . however , another explanation is related to the low detection limits of the methods used to detect hav in biosolids , as demonstrated in this study and by other authors ( jebri et al . therefore , the detection of hav in biosolids can be underestimated , primarily when low vls are circulating in these environments . although viral infectivity is not determined by methods based on nucleic acid amplification , studies have confirmed that a great proportion of viral genomes detected by molecular methods correspond with viable infectious particles detected in sewage sludge or biosolids ( schlindwein et al . the concentration method using beef extract elution followed by qpcr assay seems suitable for detecting adv in biosolids . however , other concentration methods should be investigated for the detection of other viruses , especially hav , to avoid inaccuracies related to potential contamination and quantification of viruses in biosolids , ensuring a reliable public health risk analysis .

knowledge of the role rna plays in biological processes is ever increasing , and critical to rna function are its structure , folding , dynamics , and interactions with other molecules . biomolecular simulation methods , including molecular dynamics ( md ) , are promising tools that augment experimental approaches by providing a detailed depiction of rna structure and interactions on time scales ranging from pico- to microseconds . crucial to the usefulness of md methods are the reliability of the force fields . due to the lag in the development of nucleic acid force fields compared to protein force fields , many question the accuracy and validity of the current rna force fields . this lag can be attributed to the high charge state of nucleic acids , the subtle balance between interactions within the rna and with the solvent environment , the high conformational variability in the rna backbone , and the lack of adequately converged sampling data . state of the art conventional md simulations can not be trusted to parametrize force fields because convergence errors could be as large as or equal to those in the force field , leading to corrections for misdiagnosed problems that could instead simply be the result of incomplete sampling . assessing force fields by standard md simulation of rna motifs like tetraloops has proven challenging because efficient sampling is limited by their large size . therefore , smaller tetranucleotides are being used as test systems for force field development and assessment . though generating the full conformational ensemble is more tractable than it is for tetraloops , tetranucleotides display significant conformational variability . the complexity of elucidating the full conformational ensemble of tetranucleotide models is demonstrated by the lack of convergence of conventional md simulations , even after microseconds of simulation time . convergence of population distributions is more readily achieved by using enhanced sampling or an ensemble method such as remd , which has been widely applied to study protein dynamics , and , to a lesser extent , nucleic acid dynamics . this is beginning to change , as recent studies by chen and garcia and khrov et al . have used remd in combination with their respective force field modifications to fold hairpins and tetraloops to their native structure . traditional remd involves multiple independent simulations run at different temperatures ( t - remd ) which periodically attempt an exchange in temperature space . replicas at low temperature have an opportunity to exchange to a higher temperature where energy barriers may be more easily crossed ; in this manner the effective sampling space of each replica is enhanced . however , this method has its drawbacks : t - remd may not substantially enhance sampling if the system is too large and the number of replicas needed to bridge a temperature range is prohibitive , or if increased temperature does not facilitate the conformational transition of interest . an alternative is hamiltonian replica exchange ( h - remd ) where part of the hamiltonian is scaled or altered , for example , with a weighted biasing potential with the opposite sign of the dihedral term ( targeting protein backbone dihedrals ) or with various sets of umbrella sampling - like restraints for different elements of the ensemble . a benefit is that one replica may be simulated with the original hamiltonian which , given sufficient sampling and exchange , provides the complete unbiased ensemble . not only is convergence crucial for force field development , but it is also even more important for accuracy and comparison with experiment . generating a formal definition of convergence greatly depends on which system properties one wishes to converge in a simulation . many authors have determined metrics of convergence for replica exchange simulations , and most , if not all , replica exchange studies discuss the extent to which their simulations are converged . abraham and gready proposed that within replica exchange simulations there is interplay between thermodynamic efficiency of sampling the ensemble properties , and mixing efficiency of replicas traversing the temperature or hamiltonian range of interest . several studies analyze peptide melting curves , rmsd to known experimental structures , radius of gyration , and principal component projections as ensemble properties , ideally between independent sets of remd simulations which start from different conformations . quantitative measures of convergence in biomolecular simulation include autocorrelation functions of potential energy , a decorrelation time defined by lyman and zuckerman , and kullback previously , we illustrated the importance of generating a converged ensemble with the r(gacc ) tetranucleotide ; however , in 2 s of t - remd per replica with 24 replicas spanning 277396 k , the ensemble was not yet completely converged . even with longer simulation , now at 3.8 s of t - remd per replica or an aggregate sampling of over 90 s , a well - converged ensemble is elusive ( supporting information figures s1 and s2 ) . differences in the populations of conformations in the ensemble are significant and warrant further exploration of sampling methods , including h - remd and its extension to multidimensional replica exchange ( m - remd ) to see if complete sampling can be obtained more quickly . in t - remd and h - remd , exchanges occur in only one dimension ; i.e. , only one part of the hamiltonian is being altered between replicas . however , two or more exchange dimensions can be coupled ; this approach is referred to as multidimensional remd ( m - remd ) . not only are there more replicas in the simulation simultaneously sampling conformational space , but also different conformational properties of the system can be enhanced . m - remd has been implemented in the md engines sander and pmemd in a development version of amber ( to be released with amber 14 ) and will be briefly described here . the current implementation allows for the use of any number of hamiltonian dimensions ( i.e. , changes in the system topology that do not involve changing the number of atoms , as well as changes to input parameters ) and/or temperature dimensions . in addition , the code has been designed to be extensible to facilitate the addition of other dimension types . a schematic of the m - remd simulation setup used in this work is shown in figure 1 . each plane represents a different hamiltonian , and each arrow represents a different temperature . the unbiased hamiltonian , shown in yellow , can be recovered at the temperature of interest . exchange in temperature dimension between 285.7 and 290.2 k is followed by an exchange in hamiltonian space between h0 and h1 . this figure represents four out of 24 temperatures and three out of eight hamiltonian dimensions used in this work . m - remd in sander / pmemd makes use of the existing t - remd and h - remd exchange subroutines . exchanges are attempted in each dimension in turn , so that the first exchange is attempted in the first dimension , the second exchange is attempted in the second dimension , and so on . this is illustrated in the following pseudocode : here % denotes the modulo operation , n_exchange is the number of the exchange being attempted , remd_dimension is the total number of replica dimensions , and remd_types [ ] is an array of size n_dimension containing an index corresponding to the type of exchange to be performed in that dimension . as with t - remd or h - remd , , with each line defining the input file , input coordinates , topology file , etc . for each replica . in m this file follows the same fortran namelist format that amber md input files use ; there is a title followed by a & multirem namelist for each replica dimension with format : exchange type ( exch_type ) is currently restricted at present to either temperature or hamiltonian . a replica list consists of a comma - separated list of integers corresponding to positions in the group file , where replica 1 is the first entry in the group file ; this defines which replicas are allowed to exchange within that dimension . m - remd is enabled in a simulation by specifying -remd - file < dimension file > on the command line . as with the current implementations of t - remd and h - remd in amber , exchanges in temperature space are accomplished by swapping temperatures , and exchanges in hamiltonian space are accomplished by swapping coordinates . this means that if any dimension is temperature , trajectories will have to be sorted if data are to be processed at a single temperature . this requires that every trajectory file contain information describing the overall dimensionality of the run ( i.e. , how many dimensions there are and of what type ) and each trajectory frame contain information on where it is located in each dimension . similarly , the restart files used to checkpoint the simulation must contain the same information . because netcdf files are by their nature extensible , adding additional information to the format does not break existing parsers that have not yet been set up to recognize it ; the additional information is not used . primarily because of this , it was decided that only the amber netcdf trajectory and restart formats would be updated for m - remd , and thus all m - remd runs require the use of the amber netcdf trajectory and restart formats . only three additional pieces of data were required to be added to the netcdf trajectory / restart formats : an integer containing the number of replica dimensions ( n ) , an integer array ( of dimension n ) containing the type of each dimension , and an array ( of dimension f n , where f is the total number of frames ) containing the indices of a replica in each dimension for each frame . cpptraj from ambertools 13.0 has been modified to process the new information from these trajectories . postprocessing of all remd simulations was performed using ptraj and specially modified development versions of cpptraj . cluster analysis was performed using two methods : the average - linkage hierarchical agglomerative and dbscan clustering methods . for both algorithms , coordinate rmsd was used as the distance metric . the average - linkage agglomerative algorithm clustered on heavy atoms of residues 14 used a critical distance value of 2.3 and a variable sieve value to ensure a 5000 frame initial pass through the trajectory . dbscan clustering was performed on a subset of atoms described using the amber mask syntax ( : 1@n2,o6,c1,p,:2@h2,n6,c1,p,:3@o2,h5,c1,p,:4@o2,h5,c1,p ) , used a distance cutoff of 0.9 between clusters and a minimum of 25 points required to form a cluster . again , a variable sieve was employed to ensure at least a 5000 frame first pass through the trajectory . principal component analysis was performed using cpptraj . to compare principal components obtained from two independent runs , the covariance matrix was calculated from the combined trajectories of the two independent simulations for a given remd type ; each frame was first rms - fit to an overall average structure to remove global translational and rotational motion . the eigenvectors and eigenvalues were then obtained from diagonalization of the combined covariance matrix , after which coordinates from each independent trajectory were projected along eigenvectors of interest to obtain projection values for given modes . the kullback leibler divergence analysis over time was performed using a development version of cpptraj . at each frame t , a histogram for each data set being compared ( pt and qt ) was constructed from data at all previous frames ( 0 t ) using a gaussian kernel density estimator with 400 bins and a bandwidth estimated from the normal distribution approximation for the entire data set . the kullback leibler divergence at frame t ( kl(t ) ) was then calculated:1to ensure that the kullback leibler divergence was properly defined , the sum over each histogram was normalized to 1.0 and frames in which a bin had density in one histogram but no density in the other histogram were ignored . the gacc rna , r(gacc ) , system setup , building , and equilibration protocol used here was previously described in henriksen et al . the amber ff12sb force field parameters were used that include the base amber ff99 force field and the parmbsc0 corrections for / nucleic acid backbone torsions and xol3 torsion corrections for rna . the r(gacc ) structures used in temperature replica - exchange ( t - remd ) simulations were directly taken from the previous work and were solvated with 2497 tip3p waters and 3 na ions . equilibration was performed at each of the 24 replicas target temperature ( temperatures ranged from 277 to 396 k ) . the previous t - remd runs were extended from 2 s per replica to 3.8 s per replica . all ( nonreplica exchange ) molecular dynamics ( md ) simulations were carried out with the pmemd.cuda.mpi module of the amber12 suite of programs . all replica exchange molecular dynamics ( md ) simulations were carried out with the development version of the pmemd.cuda.mpi and pmemd.mpi modules based on the amber12 suite of programs ; this code will eventually be released as part of amber14 . temperature was regulated using the langevin thermostat with a collision frequency of 2 ps using the ig=-1 option to randomly set the random number seeds at each restart , avoiding synchronization effects . an integration time step of 2 fs was used . the nonbonded direct space cutoff was set to 8.0 , and default amber12 particle mesh ewald settings were used for reciprocal space calculations . simulations and analysis were performed both locally at the university of utah s center for high performance computing and also on the ncsa blue waters and xsede keeneland and stampede computational resources . h - remd simulations were performed with eight replicas ; this drastically cuts back on the number of replicas in the simulation compared to t - remd , which was possible because decent exchange probabilities between the hamiltonians could be obtained . for the h - remd , we uniformly scaled the dihedral force constant ( dfc ) of all dihedrals by a constant , from 1.0 ( full force field ) to 0.3 ( lowest torsion barriers in this work ) by 0.1 intervals : ( 1.0 , 0.9 , 0.8 , 0.7 , 0.6 , 0.5 , 0.4 , 0.3 ) leading to eight replicas . support for such parameter / topology file modifications will be included in the parmed and cpptraj tools distributed with ambertools 14 . when the torsion barrier heights are decreased , interconversion between minima and overall sampling of structural populations is enhanced . on the basis of our system benchmarks , scaling from full dfc to 30% dfc by 10% intervals gave acceptable potential energy overlap between replicas and led to an exchange acceptance of 57% to 26% ( supporting information figure s3 ) . simulations were performed using the development version of amber14 and ambertools14 suite of programs in which h - remd and m - remd are implemented . for the initial h - remd simulations , the equilibrated structure at 300 to generate starting structures for the subsequent and independent h - remd simulation , the restart structures from the original h - remd simulation after 510 ns were assigned new velocities and simulated for 1 ns in the nvt ensemble . with fewer replicas , convergence of the h - remd distribution requires long simulations per replica , or long wall - clock time , to aggregate a similar amount of sampling . to accumulate sampling on the same order as the m - remd and t - remd , the dfc was scaled from a factor of 1.0 ( full dfc ) to 0.3 over 192 replicas using an interval of 0.0036 . starting structures for the 192 replica h - remd were identical to the 8 replica h - remd run 2 structures and were each used 24 times . an exchange acceptance rate from 96% to 99% was observed . to further enhance sampling , the temperature and hamiltonian dimensions were combined into a two - dimensional m - remd run . by combining the temperature and hamiltonian dimensions , we can reach the converged ensemble for a solvated r(gacc ) tetranucleotide much faster than using either t - remd or h - remd alone . for the m - remd simulations , the temperature range used was the same as in the previously published t - remd simulation , totaling 24 t - remd replicas from 277 to 396 k , which , when coupled with the 8 h - remd replicas , leads to a total of 192 replicas . each equilibrated starting structure from t - remd was copied eight times , and each copy was assigned a different hamiltonian . to generate the starting structures for an independent m - remd simulation , the initial r(gacc ) structures equilibrated at their target temperatures figure 2 shows histograms of the mass - weighted heavy atom rmsd of each individual h - remd and m - remd simulation to an a - form reference structure . overlapping histograms indicate that separate simulations are sampling the same rmsd space , which is a necessary but not sufficient condition for convergence . the large error bars between the two independent h - remd runs in figure 2 indicates the simulations have sampled somewhat different populations . error bars between the two m - remd simulations are smaller , and indicate that the simulations , which start from different structure sets , ultimately sample more similar rmsd space than the h - remd simulations , as well as the t - remd simulation ( shown in supporting information figure s2 ) . to characterize the populations , cluster analysis on the 300 k trajectories was performed using cpptraj ( sample scripts are available in the supporting information ) . representative structures and cluster populations are shown in figure 3 and supporting information table 1 and figure s4 . the two h - remd simulations disagree about the populations of the major peak at 5.0 ( the intercalated - anti and inverted - syn structures in figure 3 ) , as well as the structures at 4.0 ( 1_3-basepair ) and 6.0 ( intercalated - syn ) . in contrast , the smaller error bars between independent runs of m - remd indicate much better convergence of the populations between the two simulations ( shown more clearly in supporting information table 1 and figures s5 , s6 , and s7 ) . this also indicates that the higher temperature replicas are critical to resolve structures in the unconverged regions of the h - remd simulation . population analysis showing the number of structures at specific rmsd values from an a - form reference structure . mass weighted rmsd histograms of the unbiased replicas at 300 k are averaged ( shown in black ) between two runs ( shown in red and blue ) . representative structures from dbscan cluster analysis at 300 k. the top six most populated clusters in most remd simulations are shown above . for the r(gacc ) sequence the coloring is g1 ( red ) , a2 ( green ) , c3 ( cyan ) , and c4 ( magenta ) . cluster populations for m - remd are shown in black , h - remd in red , and t - remd in blue . m - remd and h - remd cluster populations represent the average of two independent simulations . cluster names were assigned on the basis of analysis of the 277 k t - remd trajectory . poorer apparent convergence between the h - remd runs is supported by the lower correlation between cluster populations of the two independent runs compared to m - remd , shown in figure 4 . independent simulations are clustered together , and the cluster population from each independent run is reported . if the simulations are sampling the same conformational space despite the difference in their starting structure conditions , they are better converged , and the cluster populations from the two independent runs will be the same . the cluster populations from the h - remd simulations have a correlation coefficient of 0.93 , whereas the populations from the m - remd simulations have a correlation coefficient of 0.99 . to test the robustness of the correlation , the 95% confidence intervals were calculated for the slope ( ) and intercept ( ) of the linear fits for the h - remd and m - remd simulations , shown as the shaded area in figure 4 . the correlation was also calculated with the most populated cluster left out ( supporting information figure s8 ) . the difference between the two sets is more apparent in the h - remd simulations , again indicating that the independent simulations remain unconverged . correlation of cluster populations between independent h - remd runs and independent m - remd runs . linear fit and correlation coefficients for h - remd and m - remd are shown , where red fits all clusters . the shaded area represents a 95% confidence interval where the slope and y - intercept bounds are denoted by and , respectively . principal components , which describe the overall dynamics of the system , are less easily converged than other metrics , particularly those components corresponding to the lowest frequency motions . to ascertain whether the dynamics of two independent runs appear to be converged , we looked at the overlap of histograms of principal component projections calculated from cartesian coordinates obtained from each simulation ( see computational methods : principal component analysis for more details ) . if the replicas have sufficiently diffused through temperature and hamiltonian dimensions , the different initial structure sets should sample the same conformational space resulting in a close principal component overlap . a script that can be used to perform this calculation with cpptraj figure 5 shows the overlap in the first five principal components for the h - remd and m - remd simulations . the independent h - remd simulations diverge , particularly in the first low frequency mode ( black solid line vs black dashed line ) , which is representative of the majority of fluctuations in the system . conversely , the m - remd principal component histograms show excellent overlap , indicating similarity between the overall dynamics of each independent m - remd run . principal component projection of top five modes onto ( left ) h - remd run 1 ( solid lines ) and h - remd run 2 ( dashed lines ) show little overlap in the low frequency modes , whereas ( right ) m - remd run 1 and m - remd run 2 show the overlap in the low frequency modes . to quantitatively examine rates of convergence , we performed kullback leibler divergence analysis ( kl divergence ) over time on histograms of principal component ( pc ) projections for the unbiased 300 k trajectories from h - remd and m - remd , as well as for the histograms of mass weighted rmsd to an a - form reference structure . the results are a measure of the difference between probability distributions sampled from each simulation as a function of simulation length , yielding a time - dependent measure of ensemble differences . low divergence values indicate the ensemble at time x is not significantly different from the final ensemble , whereas high divergence values indicate the ensemble at time x is very different from the final , or most converged ensemble . figure 6 shows the kl divergence of the first three principal components for the h - remd and m - remd simulations . m - remd converges to the final ensemble quickly , reaching a divergence of almost zero after 250 ns . in contrast , the h - remd remains unconverged ( particularly in the first pc ) even after more than 1 s per replica . this pattern is echoed in the kl divergence analysis of the mass weighted rmsd to an a - form reference for the m - remd and h - remd simulations , where the h - remd simulations are clearly not converged whereas the m - remd simulations appear well - converged . leibler divergence . left : first three principal components from the h - remd and m - remd simulations . h - remd principal components 1 , 2 , and 3 are maroon , red , and orange . m - remd principal components 1 , 2 , and 3 are blue , purple , and cyan . right : mass - weighted heavy atom rmsd to an a - form reference from the m - remd ( blue ) and h - remd ( red ) simulations . in addition to poorer convergence , even with equivalent aggregate md simulation times ( i.e. , using 192 h - remd replicas to match the sampling of the m - remd simulation ) , h - remd tends to underpopulate the peak at 4.0 ( the 1_3-basepair structure ) and show significant differences between resulting ensembles ( supporting information figure s9 ) . for example , the h - remd simulations yield several low - populated clusters that differ from more populated clusters solely due to glycosidic x flips and shifts to more optimal -stacking geometries . the t - remd samples few of these extended structures , whereas the m - remd cluster structures contain a mix of both t - remd - like structures and lower population h - remd - like structures ( supporting information table s1 and figure s4 ) . sampling of alternate x - flipped/-stacked structures in the h - remd simulations is likely a direct result of our choice to bias the dihedral force constant . in knocking down the dihedral barriers , the nonbonded terms of the force field have a greater effect on the structure . at high biasing levels , less energy is required to overcome unfavorable torsional barriers to form favorable nonbonded contacts . conformations which contain flipped x dihedrals , for example , are seen more often in the h - remd simulations compared to t - remd simulations . when the temperature is added into the sampling with m - remd , the higher temperature replicas are less likely to become trapped because they can more easily break these favorable nonbonded interactions . the choice of bias and the resulting overemphasis of nonbonded interactions may result in the nonconvergence of the h - remd simulations . if specific structures ( e.g. , the x - flipped structures ) are favored at high biases , rearranging to a canonical x value is prohibitive in the unbiased replica where the dihedral barriers are at their full heights . supporting information figure s9 illustrates this point ; through clustering , a x - flipped structure becomes representative of one of the four major clusters in h - remd . this phenomenon could possibly be remedied by increasing the number of replicas at low biasing levels or by providing more time at low to no bias for x dihedrals to repopulate canonical values . a summary of the populations sampled by each remd method is shown in figure 7 . here the relative differences in population at the 3.0 and 5.0 peak reflect differences in the nmr minor and intercalated - anti conformations , two of the highest populated clusters . if only h - remd was used to study r(gacc ) , the 1_3-basepair conformation , represented by the peak at 4.0 , would most likely not be seen because this structure seems to be the most difficult to converge on the basis of the m - remd and t - remd simulations . the efficiency of the m - remd also becomes obvious ; with the same amount of aggregate sampling , the 1_3-basepair conformation is not sampled in h - remd ( black ) , whereas the two independent m - remd simulations appear more converged in this region than any other simulations performed here . are the normalized populations ( y - axis ) of structures at particular mass - weighted rmsd values ( x - axis ) of all atoms in residues 14 to a reference structure ( a - form rna ) for the various remd simulations . an additional concern raised in review and in presentation of these results is the influence of salt on the conformational ensemble and the potential for biasing the population due to the inclusion of only net - neutralizing na ions . to address this , additional m - remd simulations equivalent to the previously discussed simulations were performed in 100 mm nacl and also 100 mm kcl using the joung / cheatham ion parameters . as shown in figure s10 and described in the supporting information , the change in salt concentration and ion identity appears to have a minimal effect on the rmsd population histograms suggesting a nearly equivalent conformational ensemble comparing net - neutralizing na to 100 mm nacl or 100 mm kcl . as previously mentioned , rigorous analysis of simulation convergence is necessary to properly validate force fields , so after having apparently converged our simulations , we are now in a unique position to truly interrogate our current parameters . though we generate a very well - converged ensemble for r(gacc ) , it is apparent that our current force field does not adequately reproduce the experimental nmr data seen for this tetranucleotide . an a - form major conformation was observed via nmr , as well as a minor conformation characterized by a flipped c4 base . in our converged m - remd simulations , the populations of the nmr major and minor conformations were 18% and 24% , respectively . this structure has been seen in previous simulations , yet there is no experimental evidence showing that it should be this highly populated . the structure is characterized by intercalated stacking and increased number of hydrogen bonds . on the basis of these observations , we are in the process of both comparing to other existing rna force fields and attempting to refine various force field terms , such as altering charges , dihedral terms , and stacking interactions to help guide revisions to the amber force fields . in summary , we have demonstrated that apparent convergence for very flexible systems can be more quickly achieved by combining temperature and dihedral force constant biasing in m - remd . by itself , the h - remd simulation did not converge in an adequate time frame ; overcoming the high - bias replica s preference for stacked and x flipped structures at low or unbiased replicas was prohibitively slow . by addition of the temperature dimension , a well - converged ensemble , determined by various metrics presented here , was achieved within 300 ns of sampling ( per the 192 replicas ) . as m - remd tends to utilize more replicas , the time to solution in wall - clock hours improves if sufficient access to large - scale , high performance computational resources is available . in the case of amber simulations , speed - up is especially impressive with access to large - scale gpu resources such as ncsa blue waters and the xsede stampede and keeneland resources where these simulations were performed . this method will be used to investigate the ensembles of other ( or all ) tetranucleotide sequences , providing a comprehensive look at the underlying structure preference of current force fields .

a necessary step to properly assess and validate the performance of force fields for biomolecules is to exhaustively sample the accessible conformational space , which is challenging for large rna structures . given questions regarding the reliability of modeling rna structure and dynamics with current methods , we have begun to use rna tetranucleotides to evaluate force fields . these systems , though small , display considerable conformational variability and complete sampling with standard simulation methods remains challenging . here we compare and discuss the performance of known variations of replica exchange molecular dynamics ( remd ) methods , specifically temperature remd ( t - remd ) , hamiltonian remd ( h - remd ) , and multidimensional remd ( m - remd ) methods , which have been implemented in amber s accelerated gpu code . using two independent simulations , we show that m - remd not only makes very efficient use of emerging large - scale gpu clusters , like blue waters at the university of illinois , but also is critically important in generating the converged ensemble more efficiently than either t - remd or h - remd . with 57.6 s aggregate sampling of a conformational ensemble with m - remd methods , the populations can be compared to nmr data to evaluate force field reliability and further understand how putative changes to the force field may alter populations to be in more consistent agreement with experiment .

Introduction Computational Methods Results Discussion Conclusions

biomolecular simulation methods , including molecular dynamics ( md ) , are promising tools that augment experimental approaches by providing a detailed depiction of rna structure and interactions on time scales ranging from pico- to microseconds . crucial to the usefulness of md methods are the reliability of the force fields . due to the lag in the development of nucleic acid force fields compared to protein force fields , many question the accuracy and validity of the current rna force fields . this lag can be attributed to the high charge state of nucleic acids , the subtle balance between interactions within the rna and with the solvent environment , the high conformational variability in the rna backbone , and the lack of adequately converged sampling data . state of the art conventional md simulations can not be trusted to parametrize force fields because convergence errors could be as large as or equal to those in the force field , leading to corrections for misdiagnosed problems that could instead simply be the result of incomplete sampling . though generating the full conformational ensemble is more tractable than it is for tetraloops , tetranucleotides display significant conformational variability . traditional remd involves multiple independent simulations run at different temperatures ( t - remd ) which periodically attempt an exchange in temperature space . an alternative is hamiltonian replica exchange ( h - remd ) where part of the hamiltonian is scaled or altered , for example , with a weighted biasing potential with the opposite sign of the dihedral term ( targeting protein backbone dihedrals ) or with various sets of umbrella sampling - like restraints for different elements of the ensemble . not only is convergence crucial for force field development , but it is also even more important for accuracy and comparison with experiment . many authors have determined metrics of convergence for replica exchange simulations , and most , if not all , replica exchange studies discuss the extent to which their simulations are converged . quantitative measures of convergence in biomolecular simulation include autocorrelation functions of potential energy , a decorrelation time defined by lyman and zuckerman , and kullback previously , we illustrated the importance of generating a converged ensemble with the r(gacc ) tetranucleotide ; however , in 2 s of t - remd per replica with 24 replicas spanning 277396 k , the ensemble was not yet completely converged . even with longer simulation , now at 3.8 s of t - remd per replica or an aggregate sampling of over 90 s , a well - converged ensemble is elusive ( supporting information figures s1 and s2 ) . differences in the populations of conformations in the ensemble are significant and warrant further exploration of sampling methods , including h - remd and its extension to multidimensional replica exchange ( m - remd ) to see if complete sampling can be obtained more quickly . in t - remd and h - remd , exchanges occur in only one dimension ; i.e. however , two or more exchange dimensions can be coupled ; this approach is referred to as multidimensional remd ( m - remd ) . not only are there more replicas in the simulation simultaneously sampling conformational space , but also different conformational properties of the system can be enhanced . m - remd has been implemented in the md engines sander and pmemd in a development version of amber ( to be released with amber 14 ) and will be briefly described here . m - remd in sander / pmemd makes use of the existing t - remd and h - remd exchange subroutines . this is illustrated in the following pseudocode : here % denotes the modulo operation , n_exchange is the number of the exchange being attempted , remd_dimension is the total number of replica dimensions , and remd_types [ ] is an array of size n_dimension containing an index corresponding to the type of exchange to be performed in that dimension . as with t - remd or h - remd , , with each line defining the input file , input coordinates , topology file , etc . as with the current implementations of t - remd and h - remd in amber , exchanges in temperature space are accomplished by swapping temperatures , and exchanges in hamiltonian space are accomplished by swapping coordinates . primarily because of this , it was decided that only the amber netcdf trajectory and restart formats would be updated for m - remd , and thus all m - remd runs require the use of the amber netcdf trajectory and restart formats . only three additional pieces of data were required to be added to the netcdf trajectory / restart formats : an integer containing the number of replica dimensions ( n ) , an integer array ( of dimension n ) containing the type of each dimension , and an array ( of dimension f n , where f is the total number of frames ) containing the indices of a replica in each dimension for each frame . to compare principal components obtained from two independent runs , the covariance matrix was calculated from the combined trajectories of the two independent simulations for a given remd type ; each frame was first rms - fit to an overall average structure to remove global translational and rotational motion . the r(gacc ) structures used in temperature replica - exchange ( t - remd ) simulations were directly taken from the previous work and were solvated with 2497 tip3p waters and 3 na ions . all replica exchange molecular dynamics ( md ) simulations were carried out with the development version of the pmemd.cuda.mpi and pmemd.mpi modules based on the amber12 suite of programs ; this code will eventually be released as part of amber14 . simulations and analysis were performed both locally at the university of utah s center for high performance computing and also on the ncsa blue waters and xsede keeneland and stampede computational resources . h - remd simulations were performed with eight replicas ; this drastically cuts back on the number of replicas in the simulation compared to t - remd , which was possible because decent exchange probabilities between the hamiltonians could be obtained . for the h - remd , we uniformly scaled the dihedral force constant ( dfc ) of all dihedrals by a constant , from 1.0 ( full force field ) to 0.3 ( lowest torsion barriers in this work ) by 0.1 intervals : ( 1.0 , 0.9 , 0.8 , 0.7 , 0.6 , 0.5 , 0.4 , 0.3 ) leading to eight replicas . simulations were performed using the development version of amber14 and ambertools14 suite of programs in which h - remd and m - remd are implemented . for the initial h - remd simulations , the equilibrated structure at 300 to generate starting structures for the subsequent and independent h - remd simulation , the restart structures from the original h - remd simulation after 510 ns were assigned new velocities and simulated for 1 ns in the nvt ensemble . to accumulate sampling on the same order as the m - remd and t - remd , the dfc was scaled from a factor of 1.0 ( full dfc ) to 0.3 over 192 replicas using an interval of 0.0036 . starting structures for the 192 replica h - remd were identical to the 8 replica h - remd run 2 structures and were each used 24 times . to further enhance sampling , the temperature and hamiltonian dimensions were combined into a two - dimensional m - remd run . by combining the temperature and hamiltonian dimensions , we can reach the converged ensemble for a solvated r(gacc ) tetranucleotide much faster than using either t - remd or h - remd alone . for the m - remd simulations , the temperature range used was the same as in the previously published t - remd simulation , totaling 24 t - remd replicas from 277 to 396 k , which , when coupled with the 8 h - remd replicas , leads to a total of 192 replicas . each equilibrated starting structure from t - remd was copied eight times , and each copy was assigned a different hamiltonian . to generate the starting structures for an independent m - remd simulation , the initial r(gacc ) structures equilibrated at their target temperatures figure 2 shows histograms of the mass - weighted heavy atom rmsd of each individual h - remd and m - remd simulation to an a - form reference structure . overlapping histograms indicate that separate simulations are sampling the same rmsd space , which is a necessary but not sufficient condition for convergence . the large error bars between the two independent h - remd runs in figure 2 indicates the simulations have sampled somewhat different populations . error bars between the two m - remd simulations are smaller , and indicate that the simulations , which start from different structure sets , ultimately sample more similar rmsd space than the h - remd simulations , as well as the t - remd simulation ( shown in supporting information figure s2 ) . the two h - remd simulations disagree about the populations of the major peak at 5.0 ( the intercalated - anti and inverted - syn structures in figure 3 ) , as well as the structures at 4.0 ( 1_3-basepair ) and 6.0 ( intercalated - syn ) . in contrast , the smaller error bars between independent runs of m - remd indicate much better convergence of the populations between the two simulations ( shown more clearly in supporting information table 1 and figures s5 , s6 , and s7 ) . cluster populations for m - remd are shown in black , h - remd in red , and t - remd in blue . m - remd and h - remd cluster populations represent the average of two independent simulations . poorer apparent convergence between the h - remd runs is supported by the lower correlation between cluster populations of the two independent runs compared to m - remd , shown in figure 4 . if the simulations are sampling the same conformational space despite the difference in their starting structure conditions , they are better converged , and the cluster populations from the two independent runs will be the same . the cluster populations from the h - remd simulations have a correlation coefficient of 0.93 , whereas the populations from the m - remd simulations have a correlation coefficient of 0.99 . to test the robustness of the correlation , the 95% confidence intervals were calculated for the slope ( ) and intercept ( ) of the linear fits for the h - remd and m - remd simulations , shown as the shaded area in figure 4 . the difference between the two sets is more apparent in the h - remd simulations , again indicating that the independent simulations remain unconverged . correlation of cluster populations between independent h - remd runs and independent m - remd runs . linear fit and correlation coefficients for h - remd and m - remd are shown , where red fits all clusters . to ascertain whether the dynamics of two independent runs appear to be converged , we looked at the overlap of histograms of principal component projections calculated from cartesian coordinates obtained from each simulation ( see computational methods : principal component analysis for more details ) . if the replicas have sufficiently diffused through temperature and hamiltonian dimensions , the different initial structure sets should sample the same conformational space resulting in a close principal component overlap . a script that can be used to perform this calculation with cpptraj figure 5 shows the overlap in the first five principal components for the h - remd and m - remd simulations . the independent h - remd simulations diverge , particularly in the first low frequency mode ( black solid line vs black dashed line ) , which is representative of the majority of fluctuations in the system . conversely , the m - remd principal component histograms show excellent overlap , indicating similarity between the overall dynamics of each independent m - remd run . principal component projection of top five modes onto ( left ) h - remd run 1 ( solid lines ) and h - remd run 2 ( dashed lines ) show little overlap in the low frequency modes , whereas ( right ) m - remd run 1 and m - remd run 2 show the overlap in the low frequency modes . to quantitatively examine rates of convergence , we performed kullback leibler divergence analysis ( kl divergence ) over time on histograms of principal component ( pc ) projections for the unbiased 300 k trajectories from h - remd and m - remd , as well as for the histograms of mass weighted rmsd to an a - form reference structure . figure 6 shows the kl divergence of the first three principal components for the h - remd and m - remd simulations . m - remd converges to the final ensemble quickly , reaching a divergence of almost zero after 250 ns . in contrast , the h - remd remains unconverged ( particularly in the first pc ) even after more than 1 s per replica . this pattern is echoed in the kl divergence analysis of the mass weighted rmsd to an a - form reference for the m - remd and h - remd simulations , where the h - remd simulations are clearly not converged whereas the m - remd simulations appear well - converged . left : first three principal components from the h - remd and m - remd simulations . h - remd principal components 1 , 2 , and 3 are maroon , red , and orange . m - remd principal components 1 , 2 , and 3 are blue , purple , and cyan . right : mass - weighted heavy atom rmsd to an a - form reference from the m - remd ( blue ) and h - remd ( red ) simulations . , using 192 h - remd replicas to match the sampling of the m - remd simulation ) , h - remd tends to underpopulate the peak at 4.0 ( the 1_3-basepair structure ) and show significant differences between resulting ensembles ( supporting information figure s9 ) . for example , the h - remd simulations yield several low - populated clusters that differ from more populated clusters solely due to glycosidic x flips and shifts to more optimal -stacking geometries . the t - remd samples few of these extended structures , whereas the m - remd cluster structures contain a mix of both t - remd - like structures and lower population h - remd - like structures ( supporting information table s1 and figure s4 ) . sampling of alternate x - flipped/-stacked structures in the h - remd simulations is likely a direct result of our choice to bias the dihedral force constant . in knocking down the dihedral barriers , the nonbonded terms of the force field have a greater effect on the structure . conformations which contain flipped x dihedrals , for example , are seen more often in the h - remd simulations compared to t - remd simulations . when the temperature is added into the sampling with m - remd , the higher temperature replicas are less likely to become trapped because they can more easily break these favorable nonbonded interactions . if only h - remd was used to study r(gacc ) , the 1_3-basepair conformation , represented by the peak at 4.0 , would most likely not be seen because this structure seems to be the most difficult to converge on the basis of the m - remd and t - remd simulations . the efficiency of the m - remd also becomes obvious ; with the same amount of aggregate sampling , the 1_3-basepair conformation is not sampled in h - remd ( black ) , whereas the two independent m - remd simulations appear more converged in this region than any other simulations performed here . to address this , additional m - remd simulations equivalent to the previously discussed simulations were performed in 100 mm nacl and also 100 mm kcl using the joung / cheatham ion parameters . as previously mentioned , rigorous analysis of simulation convergence is necessary to properly validate force fields , so after having apparently converged our simulations , we are now in a unique position to truly interrogate our current parameters . though we generate a very well - converged ensemble for r(gacc ) , it is apparent that our current force field does not adequately reproduce the experimental nmr data seen for this tetranucleotide . in our converged m - remd simulations , the populations of the nmr major and minor conformations were 18% and 24% , respectively . on the basis of these observations , we are in the process of both comparing to other existing rna force fields and attempting to refine various force field terms , such as altering charges , dihedral terms , and stacking interactions to help guide revisions to the amber force fields . in summary , we have demonstrated that apparent convergence for very flexible systems can be more quickly achieved by combining temperature and dihedral force constant biasing in m - remd . by itself , the h - remd simulation did not converge in an adequate time frame ; overcoming the high - bias replica s preference for stacked and x flipped structures at low or unbiased replicas was prohibitively slow . as m - remd tends to utilize more replicas , the time to solution in wall - clock hours improves if sufficient access to large - scale , high performance computational resources is available . in the case of amber simulations , speed - up is especially impressive with access to large - scale gpu resources such as ncsa blue waters and the xsede stampede and keeneland resources where these simulations were performed .

national and international reports , including one published recently by the institute of medicine , describe the potential for advanced practice registered nurses ( aprns ) to contribute to the provision of high - quality healthcare as part of comprehensive healthcare reform [ 2 , 3 ] . preparing aprns for practice and fostering the role of aprns in a variety of educational , clinical , and research settings are necessary steps toward achieving this vision . given the current economic and political climate in the united states , however , success may be elusive . at present , a shrinking number of nurse educators carry an increasingly large responsibility for educating a declining number of aprns [ 4 , 5 ] . in many settings , outdated regulations , policies , and biases prevent aprns from practicing to the fullest extent of their education , skills , and competencies [ 68 ] . some us - based physician organizations have mounted campaigns aimed at discrediting aprn education and practice and decrying the potential of aprns to provide cost - effective and clinically efficient care [ 9 , 10 ] . while barriers to practice are significant , innovative approaches to clinical education and curricular transformation offer promise to nursing administrators , nursing educators , and practicing aprns who are committed to preparing a highly qualified aprn workforce that will serve future generations of americans . the rapid development and establishment of the practice doctorate has generated cautious enthusiasm among many nurse educators who are eager to help aprns achieve their fullest potential in clinical practice . the purpose of this paper is to describe challenges in providing aprn clinical education and to propose achievable strategies for educating future aprns to participate fully in transforming the united states healthcare system . we argue that the time is right to identify and implement educational practices that will lead to the optimal development of clinical skills , knowledge , and practice acumen and help meet the goals endorsed by national nursing organizations and set forth in the future of nursing report published in 2011 . while the iom report is extraordinarily thorough , its scope does not include suggestions for specific strategies for improving aprn clinical education , a gap this paper seeks to fill . advanced practice registered nurses include nurse practitioners ( nps ) , certified nurse - midwives ( cnms ) , certified registered nurse anesthetists ( crnas ) , and clinical nurse specialists ( cnss ) . only 3.8% of the 2.4 million us registered nurses ( rns ) are nps , 0.3% are cnms , 1.1% are crnas , and 0.9% ( down from 1.2% in 2004 ) are cnss . while the nurse anesthetist was the first advanced practice role to emerge in the late 19th century , formal aprns education programs did not start until the 20th century . the first nurse - midwifery program began in 1932 at the maternity care association in new york , and in 1954 , rutgers university offered the first cns graduate program with a specialty in psychiatric and mental health . the role of the nurse practitioner then developed in the 1960s with the increase in federal funding for advanced nursing education in order to fill the need for primary care providers . since the various roles have emerged , aprns consistently provide high - quality , cost - effective patient care in a variety of healthcare settings . today , the majority of aprns are employed in primary care settings , with most providing women 's health , obstetrics , and mental health services . one hallmark of aprn practice is the provision of care directed at illness prevention , health promotion , and improved patient care outcomes . aprn practice represents one aspect of the nursing profession 's ongoing efforts to provide high - quality healthcare to diverse populations . overcoming barriers to aprn practice in today 's healthcare environment will lead to improvements in health care for many , especially among traditionally underserved populations . we define many challenges associated with providing effective aprn clinical education , particularly in clinical practice settings . our analysis of the challenges in table 1 led us to identify innovative educational and programmatic strategies with potential to improve aprn education . the strategies we present include both internal ( those related to educational institutions ) and external ( those related to social , political , and interprofessional practice issues ) factors . for the purpose of this paper , we defined internal challenges as those existing within the profession and/or within educational organizations responsible for preparing aprns for practice . when considering these internal challenges , we discovered , not surprisingly , that the literature was dominated by information about the critical role of the growing nursing and nursing faculty shortages . clearly , not enough qualified nursing faculty are available to meet the nation 's need for increased numbers of aprns , and the projections describing future shortfalls are bleak [ 15 , 16 ] . while the nursing faculty shortage has been well described in the literature , some aspects of it are germane in a discussion about aprn education , especially given the relatively large numbers of potential students unable to gain admission because of limited faculty resources . educational organizations find it increasingly difficult to attract qualified aprns willing to serve in faculty roles . the demand for aprns in both educational institutions and in a variety of practice settings has increased simultaneously , but educational institutions are disadvantaged by their inability to offer competitive compensation packages . constrained budgets result in compressed salaries throughout higher education systems , increasing the gap between salaries available in practice and those offered for teaching positions . when aprns do pursue education at the phd level , they often graduate only to face the reality of the tenure process in research - driven educational institutions . emphasis on the role of faculty in conducting research and generating research - related revenue limits the availability of phd - prepared aprn faculty to participate in direct clinical supervision of aprn students . one result is that the primary responsibility for aprn clinical education falls to faculty not eligible for tenure and whose salaries are typically lower than those available for aprns in clinical practice . educational institutions without established faculty practice plans face additional barriers for supporting and retaining faculty who need to practice to maintain certification and licensure , in addition to teaching and meeting tenure criteria . as many schools of nursing transition to the doctorate of nursing practice ( dnp ) , existing advanced practitioner faculty without a doctorate may find that they are underqualified . institutional requirements for supervisory committees of doctoral students may require faculty to hold equivalent doctorates , and supervision of dnp students may increase faculty workloads . phd - prepared nursing faculty may lack the advanced practice qualifications to teach specialty content in aprn programs . smaller educational institutions may not have the institutional structures or additional faculty necessary to support the development of dnp programs . while the development of dnp preparation and practice offers much promise for preparing the future workforce , the transition process may temporarily exacerbate the shortage of available clinical faculty and result in decreased numbers of aprn graduates . it is too soon to tell whether these transitional challenges will affect the quality of aprn clinical education . the net result may be additional reductions in the available supply of aprns at precisely the time when they are most needed to address the challenges of healthcare reform in the us . the number of annual graduates from aprn programs has fallen from a peak in 1998 . this decline is multifaceted , relating to a variety of barriers facing nurses who might otherwise pursue graduate education . as many as 17% of graduate nursing programs are highly selective , and there are insufficient openings for qualified applicants . program costs present challenges to potential applicants whose educational plans are altered by the recent economic downturn in the us as well as by declines in available employer tuition - reimbursement programs ; in 2009 , 15% of masters of nursing programs cited affordability as a commonly stated reason for students not enrolling . program location can be a deterrent to nurses who are place bound by responsibilities to support family and provide income . although the need for more aprns in rural communities is critical , aprn programs are less accessible to nurses in rural areas , where there are fewer nurses , and nurses must contend with lower salaries and longer commutes . in some areas , there are vacancies in some nursing programs , while others may turn away qualified applicants . additionally , there are significant shortages of hispanic , native american , and men in nursing and in aprn programs . the result is a professional nursing community that does not reflect the diversity of the us population . since world war ii , educational programs offering associate degrees have proliferated and graduates of those programs have become registered nurses ( adns ) in increasing numbers . in turn , this internal challenge has influenced the shortage of aprns , given that nurses prepared in adn programs are less likely than bachelor 's prepared nurses to obtain graduate degrees . if adns do pursue graduate education , time to completion of an aprn program expands , given the requirement for adns to complete bachelor 's education before entering a graduate nursing program . such problems clearly bring the aprn supply needs back to nurse educators and leaders at all levels . the primary challenge facing aprn education from outside educational institutions is the limited number of available clinical sites and preceptors . to increase the number of aprns prepared to practice independently and to the fullest extent of their scope of practice , nursing education programs must increase both the number and quality of available preceptors and sites . since many existing faculty practice settings are inadequate to meet this need , educational institutions must rely on cooperative , volunteer community preceptors . there is a shortage of aprn preceptors , particularly in acute care or hospital - based specialties ( i.e. , cnms , neonatal nurse practitioners ( nnps ) , and acute care nurse practitioners ) . for example , certified nurse - midwives ( cnms ) must provide education to cnm students . while there is a great need for aprn graduates to serve rural areas , federal funding through the medicare program supports resident education , but not aprn preparation . in many academic medical centers , aprns are employed for medical student and resident education , further reducing the field of potential preceptors for aprn students . nursing educational institutions are concentrated in large urban areas near hospitals and may compete with other nursing educational institutions for clinical sites and preceptors . state regulations and specialty certification agencies place additional requirements on educational institutions that further limit the capacity to prepare aprn students . direct supervision of students limits the number of students individual preceptors may have at any given time . the requirement for low student - faculty ratios in clinical courses makes aprn education expensive . for example , the national task force on quality nurse practitioner education recommends faculty - to - student ratios of 1 : 6 in situations where there is indirect clinical supervision . requirements for supervised student clinical practice in most aprn programs are typically established at a minimum of 500 hours , and the dnp requires at least 1000 hours of clinical practice . this increase in dnp student practice hours will increase the need for qualified and willing preceptors . the limited availability of national funding poses a significant external challenge to successful aprn education . the current prioritization for medical education and residency training through federal support makes increasing funding for nursing education difficult . furthermore , current research funding priorities by the national institute of nursing research do not support the investigation of nursing education issues , nor do they support research about the implementation of innovative practice education models at the graduate level . in many research organizations , nursing faculty pursuing academic careers and tenure are discouraged from pursuing clinical education research as a funded line of inquiry . among potential aprn preceptors , there may be a lack of willingness to precept aprn students due to a lack of incentives beyond the ideals of serving the profession . most educational institutions are unable to compensate preceptors financially for their teaching roles and are limited in the nonfinancial benefits they may provide preceptors such as faculty titles and access to educational resources . potential preceptors may see the challenges to practitioner productivity or the additional time commitments of being a preceptor as disincentives to assuming the role . the lack of formal preparation and support for the teaching role may further discourage aprns from being a preceptor . while direct or graduate entry training is increasingly used as a mechanism for increasing the supply of aprn graduates , potential preceptors may be resistant to training students with little or no health care experience . the final challenge to increasing the preparation of aprns is closely tied to the profession 's relationship with the citizens who are served . nursing continues to be a profession dominated by caucasian women , a limitation that affects the profession 's negotiation of relationships with other more male - dominated professions . in addition to the chronic underrepresentation of men , diverse populations , and rural inhabitants in the nursing workforce , advanced practice nursing continues to contend with an identity crisis among the us population as a whole , who suffer from a knowledge deficit regarding the skills and abilities of aprns . historically , nurses work at the direction of physicians , and cultural and occupational patterns that reinforce this dependent relationship are slow to change . while it is not clear the american medical association 's efforts to counter the iom 's future of nursing report will be entirely successful , the lack of support for full - scope aprn practice from this influential organization is disappointing to those with a vision for the provision of collaborative care in an efficient and effective interprofessional model . negotiating a new position in health care for nurses and aprns will continue to be complicated by gender politics as well as power positioning . the iom report presents an unparalleled challenge to nursing educators , that is , to foster the development of an significant innovation and change are needed to accomplish this vision and to increase the number of aprn graduates . while some of what is required must be implemented on a nation - wide scale , there is strong potential for nursing education programs to implement local and regional strategies that will increase the numbers of aprn graduates prepared to practice at the fullest extent of their education and licensure . in preparing this discussion of strategies and solutions described in table 2 , we considered our own experience as educators in graduate nursing programs and explored recommendations from multiple authors describing approaches that have been successful in enhancing the education of aprns . taken individually , each of these strategies has the potential to help programs make incremental improvements in the recruitment , retention , and preparation of graduate nursing students . in combination , these strategies offer the promise of helping nursing education affect transformation in the preparation and practice of aprns . for the purposes of this paper , internal strategies are those that can be undertaken within nursing education programs and the universities that house them , while external are those that reflect some level of engagement with other organizations including other nursing education programs and healthcare organizations . as noted above and in the iom report , the expansion of advanced nursing education programs is hampered by a faculty shortage that represents the convergence of multiple factors . these include supply - side problems related to the nursing shortage itself as well as to competitive factors that reflect , among other things , the relatively high cost of graduate nursing education when compared to the earning potential of nurse educators . like prelicensure nursing education , advanced practice nursing education is resource intensive , requiring sophisticated laboratory settings , computer equipment , and high faculty - to - student ratios . one approach with potential to aid in the nursing faculty shortage and to make more clinical resources available for aprn education involves internal efforts by educational institutions to develop and strengthen collaborative partnerships . the american association of colleges of nursing and the robert wood johnson foundation recommend that educational organizations work with one another as well as with hospitals and healthcare organizations to develop innovative capacity expanding approaches for preparing nurses and nurse educators and to foster the expansion of nursing education programs . these programs are likely to be costly , but if the benefits can be well - described , educational institutions , hospitals , and healthcare organizations may be willing to invest in their success . as one example of innovative collaboration between university programs , siewert and her colleagues from the university of iowa college of nursing report on collaborative efforts with the university of missouri at kansas city that allows for dual enrollment of neonatal nurse practitioner students and helps to optimize faculty resources and enhance student learning opportunities at both institutions . an innovative array of academic and service partnerships linking bassett medical center in cooperstown , new york , with educational programs at the state university of new york institute for technology in utica , new york now offers tuition support for advanced practice nursing preparation with an emphasis on improving care in a large rural community . these programs and others like them offer much promise in addressing faculty shortages and other challenges while offering innovative contemporary aprn education to place - bound students . in almost every aspect , curriculum , teaching , and learning nursing programs have traditionally been content driven , but the needs of students and faculty are changing along with those of the workplace . at the core of these new and revised curricula is an emphasis on integrating established educational and professional competencies with educational strategies that encourage problem solving and that enhance students ' critical thinking abilities . such curricula will encourage the simultaneous development of innovative learning activities , ensure effective student evaluations , and provide clinical experiences that emphasize the optimization of student practice outcomes . competency - based education may have additional advantages including the development of more learner competence , confidence , and compassion [ 34 , 35 ] . problem - based learning can be integrated within a competency - based framework or as a stand - alone strategy to enhance the development of critical thinking and hypothesis - testing skills [ 36 , 37 ] . problem - based learning ( also known by other terms with slightly different applications , including case- , practice- , or concept - based learning ) helps students ground learning in relevant clinical experiences [ 38 , 39 ] . as students engage closely with faculty in exploring new concepts and identifying new solutions , the process of discovery can lead to the development of improved clinical judgment . the use of simulation in nursing education is becoming increasingly popular for its ability to enhance the critical thinking of advanced practice nursing students and because it provides a useful evaluative tool for faculty . through the use of high - fidelity computerized simulation models , aprn students safely develop new knowledge and skills about high - risk , low - volume practices . other simulation activities involving scripted patients or rotation through skill - based practice stations in laboratory settings also offer enhanced opportunity for student learning and faculty participation . clinical simulation activities can add greater value by linking aprn students with medicine , pharmacy , and rehabilitation students across the health sciences . interprofessional education offers the potential to enhance efficiency in the provision of clinical education for all students and fosters collaborative practice beyond the educational period . success has been demonstrated when aprn education has been integrated with specialty and generalist physician practice in a mental health practice setting , as described by roberts and her colleagues and likely has much potential to improve education and patient care in a variety of other settings . while mistrust by physicians of the aprn role threatens to constrain the development of collaborative educational models , the promise of interprofessional education also has the potential to unite aprn and physician practice . such efforts to integrate education and training hold much promise for the us healthcare system as a whole . distance education helps create opportunities for otherwise place - bound nurses to pursue graduate studies to become aprns by extending the reach of nursing education programs beyond traditional boundaries . improvements in online course management software and evidence - based distance teaching pedagogical approaches provide a foundation for the asynchronous delivery of high - quality and engaging course content . the use of streaming media and a wide range of unified communication technologies ( e.g. , video cameras , instant messaging , web - connected whiteboards , etc . ) enhance faculty - student and student - student engagement . despite the obvious challenges of providing adequate supervision for aprn students who may be completing coursework from remote areas and with little direct faculty contact , the rewards of accessing optimal professional education using distance education technologies can be great for place - bound students living in underserved communities . to help these programs and students to succeed , educational programs can develop innovative faculty hiring agreements , hiring aprns who live in the students ' home communities to provide supervision for didactic learning experiences as well as for clinical practice and evaluation . the education and support these faculty members may require can be provided in part by professional development or continuing education programming . not all responsibility for enhancing advanced practice nursing lies with classroom or faculty - driven learning activities . as the number of available clinical sites and preceptors has declined , the need to consider effective alternatives for aprn clinical education nursing education programs must aggressively pursue alternative clinical learning sites and experiences if they want to assure that students participate in appropriate patient - centered learning activities . the development of partnerships with a broad range of community organizations and providers can create mutual benefits and provide additional learning opportunities for aprn students . while faculty may believe that an ideal clinical placement would pair students with preceptors in one - to - one relationships with clients arriving at set appointment times , there may be great value in developing partnerships with agencies and individuals who provide care in different models and settings . the development of community partnerships with a service - learning framework can provide aprn students with innovative opportunities to engage in health promotion , physical and mental health assessments , and intervention with individuals who might not otherwise receive healthcare services in a given setting . for example , assignment of students to a correctional facility could offer students the opportunity to engage with individuals in need of health assessment or behavioral intervention , even in the absence of a formally organized on - site health clinic . assigning students to work with clients through a variety of community agencies can enhance learning opportunities for aprn students and improve care for individuals seeking nonhealthcare services such as meal delivery or day care . facilitating student engagement in homeless centers can provide a variety of learning opportunities while serving to increase student understanding of social conditions and mental illness . these innovative learning opportunities can provide students with opportunities to build personally meaningful collateral skills even when the emphasis is on accomplishing practice - related learning objectives [ 50 , 51 ] . in 2004 , connolly and her colleagues described the innovative creation of a collaborative approach to nursing education . although writing about associate degree nursing education , key concepts have the potential for application in advanced practice education . these include the introduction of interprofessional collaboration that links nursing , medicine , and allied health personnel education within single community health settings , allowing the development of knowledge and skills that are essential to advanced practice nursing . academic health centers that integrate faculty practice opportunities with clinical education experience opportunities may well provide ideal environments for aprn education . not all graduate nursing programs are situated on campuses that house such centers , however . heller and goldwater suggest that the development of innovative patient - driven programs , designed to improve access , may also offer enhanced clinical education opportunities for advanced practice students . their experience with the development of a mobile clinic offering primary care services by aprns and their supervising faculty , dubbed the wellmobile , illustrates a comprehensive and innovative approach to clinical care . in addition to providing a structured environment that places emphasis on the clinical education of aprn students , the wellmobile also offered students the opportunity to develop strong business and management skills . although they can be costly and somewhat difficult to coordinate and offer , domestic and international healthcare missions do offer aprn students and faculty innovative opportunities to provide care to the underserved . while many available international opportunities are useful for student enrichment alone , with secure funding , careful planning , and rigorous attention to the management of learning and evaluation , successful programs can extend clinical education beyond local limits . participation in mission - driven clinical experiences offers students opportunities to provide care for vulnerable populations and can serve as cultural immersion experiences , enriching students ' cultural competence . they may also provide opportunities for students to develop skills in leadership and practice inquiry , cornerstones of dnp practice . finally , funding must be made available to support the vision that advanced practice nurses will assume a large measure of responsibility for the success of healthcare reform in the united states . improvement in the healthcare system requires the collaborative effort of many disciplines . at present , the current system of medical education and graduate training is not aligned with the delivery system reforms essential for increasing the value of health care in the united states . [ 54 , page 103 ] the current system of funding graduate medical education does not provide sufficient resources to support the education of nurses in clinical practice settings . while it is typical for medical residents to be supported with salaries , stipends , living allowances , and even resources such as equipment and textbooks , responsibility for aprn clinical education rests solely with the students themselves . educating an effective nursing workforce is a responsibility that must be shared by nursing programs , academic institutions , and government agencies with support from policy makers who will stand firm in sponsoring a coherent and appropriate approach to the education of a collaborative workforce . it will not be sufficient to simply provide increases in available loans or to improve loan repayment programs ; for aprn clinical education to be on par with medical education , nursing classroom and clinical education must receive full financial support . further , there must be improvements in medicare compensation for services provided by aprns , including those related to performance as clinical preceptors and research mentors . funding for improved and financially supported residency programs for aprns could come from federal programs that accept a mandate to provide healthcare services to all citizens or that compensate physicians at greater rates than aprns for the provision of equal services . the institute of medicine report on the future of nursing calls for increasing the supply of highly educated and clinically skilled aprns who can practice to the fullest possible extent of their scope of practice . clearly , aprns have the potential to contribute to the provision of high - quality healthcare as part of comprehensive healthcare reform in the united states . if this vision is to be accomplished , however , numerous challenges inherent in the current aprn educational process and barriers in the practice environment must be overcome . this paper has identified challenges that specifically hinder the clinical education of aprns and proposed strategies and solutions to help educational institutions address them . in preparing this paper , we considered our personal experience and explored the literature describing innovative approaches and strategies that have been successful for others . these approaches to aprn clinical education can affect a radical transformation in the preparation of aprns and help ensure the healthcare needs of us citizens are met by a diverse and collaborative workforce of professionals united in a vision to optimize the practice potential of all practitioners . it is imperative that nurse educators work with all stakeholders to improve the education of aprns through the identification and implementation of best practice clinical education strategies designed to overcome the current barriers to the provision of high - quality clinical experiences .

nursing education programs may face significant difficulty as they struggle to prepare sufficient numbers of advanced practice registered nurses to fulfill the vision of helping to design an improved us healthcare system as described in the institute of medicine 's future of nursing report . this paper describes specific challenges and provides strategies to improve advanced practice nursing clinical education in order to ensure that a sufficient number of aprns are available to work in educational , practice , and research settings . best practices are identified through a review of classic and current nursing literature . strategies include intensive interprofessional collaborations and radical curriculum revisions such as increased use of simulation and domestic and international service work . nurse educators must work with all stakeholders to create effective and lasting change .

1. Introduction 2. Background 3. Internal Challenges 4. External Challenges 5. Strategies and Solutions 6. Conclusions

national and international reports , including one published recently by the institute of medicine , describe the potential for advanced practice registered nurses ( aprns ) to contribute to the provision of high - quality healthcare as part of comprehensive healthcare reform [ 2 , 3 ] . preparing aprns for practice and fostering the role of aprns in a variety of educational , clinical , and research settings are necessary steps toward achieving this vision . given the current economic and political climate in the united states , however , success may be elusive . at present , a shrinking number of nurse educators carry an increasingly large responsibility for educating a declining number of aprns [ 4 , 5 ] . in many settings , outdated regulations , policies , and biases prevent aprns from practicing to the fullest extent of their education , skills , and competencies [ 68 ] . some us - based physician organizations have mounted campaigns aimed at discrediting aprn education and practice and decrying the potential of aprns to provide cost - effective and clinically efficient care [ 9 , 10 ] . while barriers to practice are significant , innovative approaches to clinical education and curricular transformation offer promise to nursing administrators , nursing educators , and practicing aprns who are committed to preparing a highly qualified aprn workforce that will serve future generations of americans . the rapid development and establishment of the practice doctorate has generated cautious enthusiasm among many nurse educators who are eager to help aprns achieve their fullest potential in clinical practice . the purpose of this paper is to describe challenges in providing aprn clinical education and to propose achievable strategies for educating future aprns to participate fully in transforming the united states healthcare system . we argue that the time is right to identify and implement educational practices that will lead to the optimal development of clinical skills , knowledge , and practice acumen and help meet the goals endorsed by national nursing organizations and set forth in the future of nursing report published in 2011 . while the iom report is extraordinarily thorough , its scope does not include suggestions for specific strategies for improving aprn clinical education , a gap this paper seeks to fill . advanced practice registered nurses include nurse practitioners ( nps ) , certified nurse - midwives ( cnms ) , certified registered nurse anesthetists ( crnas ) , and clinical nurse specialists ( cnss ) . only 3.8% of the 2.4 million us registered nurses ( rns ) are nps , 0.3% are cnms , 1.1% are crnas , and 0.9% ( down from 1.2% in 2004 ) are cnss . while the nurse anesthetist was the first advanced practice role to emerge in the late 19th century , formal aprns education programs did not start until the 20th century . the first nurse - midwifery program began in 1932 at the maternity care association in new york , and in 1954 , rutgers university offered the first cns graduate program with a specialty in psychiatric and mental health . the role of the nurse practitioner then developed in the 1960s with the increase in federal funding for advanced nursing education in order to fill the need for primary care providers . today , the majority of aprns are employed in primary care settings , with most providing women 's health , obstetrics , and mental health services . one hallmark of aprn practice is the provision of care directed at illness prevention , health promotion , and improved patient care outcomes . we define many challenges associated with providing effective aprn clinical education , particularly in clinical practice settings . our analysis of the challenges in table 1 led us to identify innovative educational and programmatic strategies with potential to improve aprn education . the strategies we present include both internal ( those related to educational institutions ) and external ( those related to social , political , and interprofessional practice issues ) factors . for the purpose of this paper , we defined internal challenges as those existing within the profession and/or within educational organizations responsible for preparing aprns for practice . clearly , not enough qualified nursing faculty are available to meet the nation 's need for increased numbers of aprns , and the projections describing future shortfalls are bleak [ 15 , 16 ] . while the nursing faculty shortage has been well described in the literature , some aspects of it are germane in a discussion about aprn education , especially given the relatively large numbers of potential students unable to gain admission because of limited faculty resources . the demand for aprns in both educational institutions and in a variety of practice settings has increased simultaneously , but educational institutions are disadvantaged by their inability to offer competitive compensation packages . one result is that the primary responsibility for aprn clinical education falls to faculty not eligible for tenure and whose salaries are typically lower than those available for aprns in clinical practice . as many schools of nursing transition to the doctorate of nursing practice ( dnp ) , existing advanced practitioner faculty without a doctorate may find that they are underqualified . institutional requirements for supervisory committees of doctoral students may require faculty to hold equivalent doctorates , and supervision of dnp students may increase faculty workloads . phd - prepared nursing faculty may lack the advanced practice qualifications to teach specialty content in aprn programs . while the development of dnp preparation and practice offers much promise for preparing the future workforce , the transition process may temporarily exacerbate the shortage of available clinical faculty and result in decreased numbers of aprn graduates . it is too soon to tell whether these transitional challenges will affect the quality of aprn clinical education . the net result may be additional reductions in the available supply of aprns at precisely the time when they are most needed to address the challenges of healthcare reform in the us . the number of annual graduates from aprn programs has fallen from a peak in 1998 . this decline is multifaceted , relating to a variety of barriers facing nurses who might otherwise pursue graduate education . as many as 17% of graduate nursing programs are highly selective , and there are insufficient openings for qualified applicants . program costs present challenges to potential applicants whose educational plans are altered by the recent economic downturn in the us as well as by declines in available employer tuition - reimbursement programs ; in 2009 , 15% of masters of nursing programs cited affordability as a commonly stated reason for students not enrolling . although the need for more aprns in rural communities is critical , aprn programs are less accessible to nurses in rural areas , where there are fewer nurses , and nurses must contend with lower salaries and longer commutes . additionally , there are significant shortages of hispanic , native american , and men in nursing and in aprn programs . since world war ii , educational programs offering associate degrees have proliferated and graduates of those programs have become registered nurses ( adns ) in increasing numbers . in turn , this internal challenge has influenced the shortage of aprns , given that nurses prepared in adn programs are less likely than bachelor 's prepared nurses to obtain graduate degrees . such problems clearly bring the aprn supply needs back to nurse educators and leaders at all levels . the primary challenge facing aprn education from outside educational institutions is the limited number of available clinical sites and preceptors . to increase the number of aprns prepared to practice independently and to the fullest extent of their scope of practice , nursing education programs must increase both the number and quality of available preceptors and sites . there is a shortage of aprn preceptors , particularly in acute care or hospital - based specialties ( i.e. , cnms , neonatal nurse practitioners ( nnps ) , and acute care nurse practitioners ) . in many academic medical centers , aprns are employed for medical student and resident education , further reducing the field of potential preceptors for aprn students . nursing educational institutions are concentrated in large urban areas near hospitals and may compete with other nursing educational institutions for clinical sites and preceptors . state regulations and specialty certification agencies place additional requirements on educational institutions that further limit the capacity to prepare aprn students . direct supervision of students limits the number of students individual preceptors may have at any given time . for example , the national task force on quality nurse practitioner education recommends faculty - to - student ratios of 1 : 6 in situations where there is indirect clinical supervision . requirements for supervised student clinical practice in most aprn programs are typically established at a minimum of 500 hours , and the dnp requires at least 1000 hours of clinical practice . the limited availability of national funding poses a significant external challenge to successful aprn education . the current prioritization for medical education and residency training through federal support makes increasing funding for nursing education difficult . furthermore , current research funding priorities by the national institute of nursing research do not support the investigation of nursing education issues , nor do they support research about the implementation of innovative practice education models at the graduate level . in many research organizations , nursing faculty pursuing academic careers and tenure are discouraged from pursuing clinical education research as a funded line of inquiry . most educational institutions are unable to compensate preceptors financially for their teaching roles and are limited in the nonfinancial benefits they may provide preceptors such as faculty titles and access to educational resources . potential preceptors may see the challenges to practitioner productivity or the additional time commitments of being a preceptor as disincentives to assuming the role . the final challenge to increasing the preparation of aprns is closely tied to the profession 's relationship with the citizens who are served . in addition to the chronic underrepresentation of men , diverse populations , and rural inhabitants in the nursing workforce , advanced practice nursing continues to contend with an identity crisis among the us population as a whole , who suffer from a knowledge deficit regarding the skills and abilities of aprns . historically , nurses work at the direction of physicians , and cultural and occupational patterns that reinforce this dependent relationship are slow to change . while it is not clear the american medical association 's efforts to counter the iom 's future of nursing report will be entirely successful , the lack of support for full - scope aprn practice from this influential organization is disappointing to those with a vision for the provision of collaborative care in an efficient and effective interprofessional model . the iom report presents an unparalleled challenge to nursing educators , that is , to foster the development of an significant innovation and change are needed to accomplish this vision and to increase the number of aprn graduates . while some of what is required must be implemented on a nation - wide scale , there is strong potential for nursing education programs to implement local and regional strategies that will increase the numbers of aprn graduates prepared to practice at the fullest extent of their education and licensure . in preparing this discussion of strategies and solutions described in table 2 , we considered our own experience as educators in graduate nursing programs and explored recommendations from multiple authors describing approaches that have been successful in enhancing the education of aprns . taken individually , each of these strategies has the potential to help programs make incremental improvements in the recruitment , retention , and preparation of graduate nursing students . in combination , these strategies offer the promise of helping nursing education affect transformation in the preparation and practice of aprns . for the purposes of this paper , internal strategies are those that can be undertaken within nursing education programs and the universities that house them , while external are those that reflect some level of engagement with other organizations including other nursing education programs and healthcare organizations . as noted above and in the iom report , the expansion of advanced nursing education programs is hampered by a faculty shortage that represents the convergence of multiple factors . these include supply - side problems related to the nursing shortage itself as well as to competitive factors that reflect , among other things , the relatively high cost of graduate nursing education when compared to the earning potential of nurse educators . like prelicensure nursing education , advanced practice nursing education is resource intensive , requiring sophisticated laboratory settings , computer equipment , and high faculty - to - student ratios . one approach with potential to aid in the nursing faculty shortage and to make more clinical resources available for aprn education involves internal efforts by educational institutions to develop and strengthen collaborative partnerships . the american association of colleges of nursing and the robert wood johnson foundation recommend that educational organizations work with one another as well as with hospitals and healthcare organizations to develop innovative capacity expanding approaches for preparing nurses and nurse educators and to foster the expansion of nursing education programs . these programs are likely to be costly , but if the benefits can be well - described , educational institutions , hospitals , and healthcare organizations may be willing to invest in their success . as one example of innovative collaboration between university programs , siewert and her colleagues from the university of iowa college of nursing report on collaborative efforts with the university of missouri at kansas city that allows for dual enrollment of neonatal nurse practitioner students and helps to optimize faculty resources and enhance student learning opportunities at both institutions . an innovative array of academic and service partnerships linking bassett medical center in cooperstown , new york , with educational programs at the state university of new york institute for technology in utica , new york now offers tuition support for advanced practice nursing preparation with an emphasis on improving care in a large rural community . in almost every aspect , curriculum , teaching , and learning nursing programs have traditionally been content driven , but the needs of students and faculty are changing along with those of the workplace . such curricula will encourage the simultaneous development of innovative learning activities , ensure effective student evaluations , and provide clinical experiences that emphasize the optimization of student practice outcomes . competency - based education may have additional advantages including the development of more learner competence , confidence , and compassion [ 34 , 35 ] . problem - based learning ( also known by other terms with slightly different applications , including case- , practice- , or concept - based learning ) helps students ground learning in relevant clinical experiences [ 38 , 39 ] . as students engage closely with faculty in exploring new concepts and identifying new solutions , the process of discovery can lead to the development of improved clinical judgment . the use of simulation in nursing education is becoming increasingly popular for its ability to enhance the critical thinking of advanced practice nursing students and because it provides a useful evaluative tool for faculty . through the use of high - fidelity computerized simulation models , aprn students safely develop new knowledge and skills about high - risk , low - volume practices . clinical simulation activities can add greater value by linking aprn students with medicine , pharmacy , and rehabilitation students across the health sciences . interprofessional education offers the potential to enhance efficiency in the provision of clinical education for all students and fosters collaborative practice beyond the educational period . success has been demonstrated when aprn education has been integrated with specialty and generalist physician practice in a mental health practice setting , as described by roberts and her colleagues and likely has much potential to improve education and patient care in a variety of other settings . such efforts to integrate education and training hold much promise for the us healthcare system as a whole . distance education helps create opportunities for otherwise place - bound nurses to pursue graduate studies to become aprns by extending the reach of nursing education programs beyond traditional boundaries . the use of streaming media and a wide range of unified communication technologies ( e.g. to help these programs and students to succeed , educational programs can develop innovative faculty hiring agreements , hiring aprns who live in the students ' home communities to provide supervision for didactic learning experiences as well as for clinical practice and evaluation . the education and support these faculty members may require can be provided in part by professional development or continuing education programming . not all responsibility for enhancing advanced practice nursing lies with classroom or faculty - driven learning activities . as the number of available clinical sites and preceptors has declined , the need to consider effective alternatives for aprn clinical education nursing education programs must aggressively pursue alternative clinical learning sites and experiences if they want to assure that students participate in appropriate patient - centered learning activities . the development of community partnerships with a service - learning framework can provide aprn students with innovative opportunities to engage in health promotion , physical and mental health assessments , and intervention with individuals who might not otherwise receive healthcare services in a given setting . for example , assignment of students to a correctional facility could offer students the opportunity to engage with individuals in need of health assessment or behavioral intervention , even in the absence of a formally organized on - site health clinic . assigning students to work with clients through a variety of community agencies can enhance learning opportunities for aprn students and improve care for individuals seeking nonhealthcare services such as meal delivery or day care . facilitating student engagement in homeless centers can provide a variety of learning opportunities while serving to increase student understanding of social conditions and mental illness . in 2004 , connolly and her colleagues described the innovative creation of a collaborative approach to nursing education . although writing about associate degree nursing education , key concepts have the potential for application in advanced practice education . these include the introduction of interprofessional collaboration that links nursing , medicine , and allied health personnel education within single community health settings , allowing the development of knowledge and skills that are essential to advanced practice nursing . academic health centers that integrate faculty practice opportunities with clinical education experience opportunities may well provide ideal environments for aprn education . not all graduate nursing programs are situated on campuses that house such centers , however . heller and goldwater suggest that the development of innovative patient - driven programs , designed to improve access , may also offer enhanced clinical education opportunities for advanced practice students . in addition to providing a structured environment that places emphasis on the clinical education of aprn students , the wellmobile also offered students the opportunity to develop strong business and management skills . although they can be costly and somewhat difficult to coordinate and offer , domestic and international healthcare missions do offer aprn students and faculty innovative opportunities to provide care to the underserved . while many available international opportunities are useful for student enrichment alone , with secure funding , careful planning , and rigorous attention to the management of learning and evaluation , successful programs can extend clinical education beyond local limits . finally , funding must be made available to support the vision that advanced practice nurses will assume a large measure of responsibility for the success of healthcare reform in the united states . improvement in the healthcare system requires the collaborative effort of many disciplines . at present , the current system of medical education and graduate training is not aligned with the delivery system reforms essential for increasing the value of health care in the united states . while it is typical for medical residents to be supported with salaries , stipends , living allowances , and even resources such as equipment and textbooks , responsibility for aprn clinical education rests solely with the students themselves . educating an effective nursing workforce is a responsibility that must be shared by nursing programs , academic institutions , and government agencies with support from policy makers who will stand firm in sponsoring a coherent and appropriate approach to the education of a collaborative workforce . it will not be sufficient to simply provide increases in available loans or to improve loan repayment programs ; for aprn clinical education to be on par with medical education , nursing classroom and clinical education must receive full financial support . further , there must be improvements in medicare compensation for services provided by aprns , including those related to performance as clinical preceptors and research mentors . the institute of medicine report on the future of nursing calls for increasing the supply of highly educated and clinically skilled aprns who can practice to the fullest possible extent of their scope of practice . clearly , aprns have the potential to contribute to the provision of high - quality healthcare as part of comprehensive healthcare reform in the united states . if this vision is to be accomplished , however , numerous challenges inherent in the current aprn educational process and barriers in the practice environment must be overcome . this paper has identified challenges that specifically hinder the clinical education of aprns and proposed strategies and solutions to help educational institutions address them . in preparing this paper , we considered our personal experience and explored the literature describing innovative approaches and strategies that have been successful for others . these approaches to aprn clinical education can affect a radical transformation in the preparation of aprns and help ensure the healthcare needs of us citizens are met by a diverse and collaborative workforce of professionals united in a vision to optimize the practice potential of all practitioners . it is imperative that nurse educators work with all stakeholders to improve the education of aprns through the identification and implementation of best practice clinical education strategies designed to overcome the current barriers to the provision of high - quality clinical experiences .

hypertension , nearly universal in hemodialysis patients , is a leading risk factor for increased morbidity and mortality in this population . a wide range of blood pressure ( bp ) measurements both during and between hemodialysis sessions poses a challenge in hypertension management . compared to individual bp measurements obtained in the hemodialysis unit , the average of bp measurements obtained during the interdialytic period provides better prognostic information for adverse clinical outcomes , including mortality . because hemodialysis unit measurements are directly available to nephrologists , it is important to 1 ) recognize the associations between intradialytic bp patterns and overall interdialytic bp burden and 2 ) further explore the nature of these relationships . intradialytic hypertension ( ih ) , an increase in bp from pre to post - hemodialysis , is a recurrent and persistent phenomenon in a subset of hemodialysis patients that has been shown to be an independent risk factor for increased morbidity and mortality [ 46 ] . recent observational evidence demonstrates that ih patients have higher ambulatory systolic bp measured during the 44-hour interdialytic period compared to hypertensive hemodialysis controls . there has been no formal analysis of how the interdialytic bp patterns differ between these groups . as interdialytic bp measurements are the most reliable bp measurements to predict outcomes in hemodialysis patients , it is critical to identify what factors influence bp most strongly during the interdialytic period , particularly in high - risk patients . furthermore , while hypotheses exist to explain the intradialytic increases in bp including acute increases in vasoconstrictor peptides [ 79 ] , changes in cardiac performance related to improvement in extracellular volume status [ 10 , 11 ] , or greater endothelial cell dysfunction , none of these variables have been examined in the context of how they influence the bp during the interdialytic period . in this retrospective analysis of a previously published case - control study , we sought to identify and characterize quantitative differences in ambulatory interdialytic bp patterns between ih patients and hemodialysis controls using linear mixed models . based on qualitative inspection of the bp patterns , we hypothesized that the bp slopes of these two patient groups would be significantly different during the initial 24 hours after hd , but similar throughout the remainder of the interdialytic period . we furthermore sought to explore how clinical and demographic variables that influenced the bp patterns during the first 24 hours after dialysis . full details of this study s methods are previously published [ 2 , 12 ] . using consecutive sampling , we screened esrd patients receiving thrice weekly in - center maintenance hemodialysis at university of texas southwestern - affiliated units . inclusion criteria were : 1 ) age 1880 years ; 2 ) hemodialysis vintage 1 month ; 3 ) ability to achieve the dry weight as determined clinically by the patient s individual nephrologist ; 4 ) clinical hypertension as defined by bp exceeding the recommended guidelines set forth by the national kidney foundation ( pre - dialysis systolic bp 140 mmhg or post - dialysis systolic bp > 130 mmhg ) . subjects were defined as case subjects if they demonstrated an increase in systolic bp 10 mmhg from pre to post - hemodialysis in 4/6 screening treatments . subjects were defined as control subjects if they demonstrated a decrease in systolic bp 10 mmhg from pre to post hemodialysis in 4/6 screening treatments . the subjects had signed written , informed consent prior to any study procedures taking place . the study was approved by the university of texas southwestern medical center institutional review board , and all procedures were in accordance with the declaration of helsinki . the study was part of a registered trial . before , during , and after a midweek hemodialysis treatment , brachial artery bp was measured using a standard automated hemodialysis unit sphygmomanometer in the seated position with feet on the floor and the patient s back supported by a chair . following treatment , while the subjects were still in the hemodialysis unit , interdialytic bp measurement was initiated with a spacelabs 90207 ambulatory bp monitor . blood pressure was measured every 30 minutes during daytime ( 6 am 10 pm ) and hourly at night for 44 hours until the next hemodialysis treatment . immediately before and after the same midweek hemodialysis treatment , a nurse obtained blood from the patient s hemodialysis access and centrifuged at 1000 rpm for 10 minutes . samples were shipped to a local laboratory where albumin was measured using spectrophotometry and sodium was measured by direction - selective potentiometry . remaining serum was stored in a 80 degree celsius freezer , and endo - thelin-1 ( et-1 ) was analyzed with elisa in batch at study conclusion . endothelial cell function was assessed with measurement of brachial artery vasodilation in response to shear stress . on a midweek , non - dialysis day , ultrasound of the brachial artery was obtained with an 11-mhz pulsed doppler ultrasound probe at an insonation angle of 60 degree ( philips ie33 , bothell , wa ) , and mean blood flow velocity and brachial artery diameter were measured . two minutes after deflation , repeat measurements of brachial artery diameter were repeated to determine flow - mediated dilation ( fmd ) . images were digitized and analyzed in a blinded fashion with an automatic wall tracking system ( vascular analysis tools , medical imaging analysis ) . the final fmd was normalized for peak sheer stress [ 17 , 18 ] and expressed as a percentage change from the baseline diameter . images were repeated after replacing the forearm occlusion step with oral intake of a single 0.4 mg tab of sublingual nitroglycerin . on the same midweek , non - dialysis day that fmd was measured , a pulse transducer device ( cardiovascular engineering , inc . ) carotid - femoral pulse wave velocity was measured as distance / time using the foot - foot method . continuous variables are reported as means with standard deviations or median with interquartile range when data is not normally distributed . between - group comparisons in baseline characteristics were performed with unpaired t - tests for continuous variables . for categorical variables , between - group comparisons were performed with chi - square test or fisher s exact test when the frequency was less than 5 for an individual group . the primary outcome is the systolic bp slope defined as the hourly change of systolic bp in the interdialytic period which was analyzed with mixed effect regression analysis using time ( hour ) modeled for systolic bp . a slope was determined for each of the following 3 predefined intervals : hours 124 after dialysis , hours 2544 after dialysis , and hours 144 after dialysis . we then explored mixed effect models to assess the relationship between bp slope during hours 124 with several clinical variables ( presence of diabetes , age , gender , race , number of antihypertensive medications , tobacco use , fmd , intradialytic change in et-1 , dialysis shift , percentage of interdialytic weight gain , pulse wave velocity , ultrafiltration rate , dialysate to serum sodium gradient , change in intradialytic systolic bp during screening as well as in the treatment preceding ambulatory bp measurement ) . for these analyses , time was a random effect variable , and others were fixed effect variables modeled for systolic bp . full details of this study s methods are previously published [ 2 , 12 ] . using consecutive sampling , we screened esrd patients receiving thrice weekly in - center maintenance hemodialysis at university of texas southwestern - affiliated units . inclusion criteria were : 1 ) age 1880 years ; 2 ) hemodialysis vintage 1 month ; 3 ) ability to achieve the dry weight as determined clinically by the patient s individual nephrologist ; 4 ) clinical hypertension as defined by bp exceeding the recommended guidelines set forth by the national kidney foundation ( pre - dialysis systolic bp 140 mmhg or post - dialysis systolic bp > 130 mmhg ) . subjects were defined as case subjects if they demonstrated an increase in systolic bp 10 mmhg from pre to post - hemodialysis in 4/6 screening treatments . subjects were defined as control subjects if they demonstrated a decrease in systolic bp 10 mmhg from pre to post hemodialysis in 4/6 screening treatments . the subjects had signed written , informed consent prior to any study procedures taking place . the study was approved by the university of texas southwestern medical center institutional review board , and all procedures were in accordance with the declaration of helsinki . before , during , and after a midweek hemodialysis treatment , brachial artery bp was measured using a standard automated hemodialysis unit sphygmomanometer in the seated position with feet on the floor and the patient s back supported by a chair . following treatment , while the subjects were still in the hemodialysis unit , interdialytic bp measurement was initiated with a spacelabs 90207 ambulatory bp monitor . blood pressure was measured every 30 minutes during daytime ( 6 am 10 pm ) and hourly at night for 44 hours until the next hemodialysis treatment . immediately before and after the same midweek hemodialysis treatment , a nurse obtained blood from the patient s hemodialysis access and centrifuged at 1000 rpm for 10 minutes . samples were shipped to a local laboratory where albumin was measured using spectrophotometry and sodium was measured by direction - selective potentiometry . remaining serum was stored in a 80 degree celsius freezer , and endo - thelin-1 ( et-1 ) was analyzed with elisa in batch at study conclusion . endothelial cell function was assessed with measurement of brachial artery vasodilation in response to shear stress . on a midweek , non - dialysis day , ultrasound of the brachial artery was obtained with an 11-mhz pulsed doppler ultrasound probe at an insonation angle of 60 degree ( philips ie33 , bothell , wa ) , and mean blood flow velocity and brachial artery diameter were measured . two minutes after deflation , repeat measurements of brachial artery diameter were repeated to determine flow - mediated dilation ( fmd ) . images were digitized and analyzed in a blinded fashion with an automatic wall tracking system ( vascular analysis tools , medical imaging analysis ) . the final fmd was normalized for peak sheer stress [ 17 , 18 ] and expressed as a percentage change from the baseline diameter . images were repeated after replacing the forearm occlusion step with oral intake of a single 0.4 mg tab of sublingual nitroglycerin . on the same midweek , non - dialysis day that fmd was measured , a pulse transducer device ( cardiovascular engineering , inc . ) was used to obtain arterial tonometry and simultaneous electrocardiogram . carotid - femoral pulse wave velocity was measured as distance / time using the foot - foot method . continuous variables are reported as means with standard deviations or median with interquartile range when data is not normally distributed . between - group comparisons in baseline characteristics were performed with unpaired t - tests for continuous variables . for categorical variables , between - group comparisons were performed with chi - square test or fisher s exact test when the frequency was less than 5 for an individual group . the primary outcome is the systolic bp slope defined as the hourly change of systolic bp in the interdialytic period which was analyzed with mixed effect regression analysis using time ( hour ) modeled for systolic bp . a slope was determined for each of the following 3 predefined intervals : hours 124 after dialysis , hours 2544 after dialysis , and hours 144 after dialysis . we then explored mixed effect models to assess the relationship between bp slope during hours 124 with several clinical variables ( presence of diabetes , age , gender , race , number of antihypertensive medications , tobacco use , fmd , intradialytic change in et-1 , dialysis shift , percentage of interdialytic weight gain , pulse wave velocity , ultrafiltration rate , dialysate to serum sodium gradient , change in intradialytic systolic bp during screening as well as in the treatment preceding ambulatory bp measurement ) . for these analyses , time was a random effect variable , and others were fixed effect variables modeled for systolic bp . in the parent case - control study , there were 460 patients screened and 59 that were enrolled ( 401 were ineligible or not interested ) . a total of nine subjects withdrew ( four controls and five cases ) from that study , and we did not have complete hourly bp measurements for one of the remaining controls . the only difference between groups was noted for angiotensin converting enzyme inhibitor ( ace - i ) use , which 64% of the ih subjects were taking and 33% of the controls were taking ( p=0.05 ) . there was no statistically significant difference between groups regarding angiotensin - receptor blocker use ( 8% vs. 21% , p=0.2 ) . serum sodium and albumin measurements obtained prior to the dialysis treatment immediately preceding the ambulatory blood pressure measurements were similar between groups . there were not any differences in dialysate to serum sodium gradients between the two groups . there were no differences in monthly lab parameters drawn routinely at the dialysis unit . during the 2-week screening period used to identify case subjects and controls , the mean ( standard deviation ) pre - dialysis systolic bp were 155.0 ( 15.7 ) mmhg in the controls and 144.0 ( 9.7 ) mmhg in the ih patients ( p=0.005 ) . post - dialysis systolic bp were 128.3 ( 11.0 ) and 159.0 ( 9.3 ) mmhg in the two groups , respectively ( p<0.001 ) ; and the changes in systolic bp from pre to post dialysis were 26.7 ( 12.5 ) and + 15.0 ( 9.1 ) mmhg in the two groups ( p<0.001 ) . during the single hemodialysis treatment occurring immediately prior to the ambulatory bp measurements , all control subjects had decreases in systolic bp from pre to post - dialysis . among the ih subjects , there were 13 subjects with bp increases and 12 subjects with bp decreases during this treatment . a comparison of measurements obtained in the treatment immediately prior to the ambulatory bp monitoring as well as other measurements of endothelial cell function and fluid changes are shown in table 2 . while pre - dialysis systolic bps for this single treatment were similar , post - dialysis systolic bp and change in systolic bp from pre to post dialysis were different between these groups . the average systolic bp during the 44 hour interdialytic time period was 143.1 ( 16.5 ) mmhg in the controls and 155.4 ( 14.2 ) mmhg in the ih subjects ( p=0.008 ) . for the first 24 hours , the average systolic bp was 138.0 ( 21.2 ) mmhg in the controls and 152.7 ( 22.8 ) mmhg in the ih subjects ( p=0.02 ) . during the remainder of the interdialytic time period ( hours 2544 ) , the average systolic bp was 150.8 ( 22.3 ) mmhg and 156.5 ( 20.8 ) mmhg , respectively ( p=0.4 ) . the systolic bp slope for the controls during the entire 44-hour interdialytic period was + 0.6 mmhg / hr ( p<0.0001 compared to zero slope , figure 1 ) . in the controls , the systolic bp slope for hours 124 and 2544 were + 0.6 mmhg / hr ( p=0.001 compared to zero slope ) and + 0.4 mmhg / hr ( p=0.09 compared to zero slope ) , respectively . within the control group there was no difference between these time periods ( 0.1 mmhg / hr , p=0.4 ) . in the ih subjects , the systolic bp slope during the entire 44 hour interdialytic time period was + 0.1 mmhg / hr ( p=0.1 compared to zero slope ) . the slope for hours 124 and 2544 were 0.3 mmhg / hr ( p=0.2 compared to zero slope ) and + 0.3 mmhg / hr ( p=0.3 compared to zero slope ) , respectively . within the ih subjects , there was a difference between these two periods ( + 0.6 mmhg / hr when comparing hours 2544 to hours 124 , p=0.001 ) . the between - group differences for controls and ih subjects slopes for hours 124 and 2544 were 0.8 mmhg / hr ( p=0.002 ) and 0.1 there were no differences in the 124 hour systolic bp slope among the ih patients who had intradialytic bp increases or decreases in the treatment immediately prior to the ambulatory blood pressure monitoring ( + 0.2 mmhg / hr for decrease vs increase , p=0.7 ) . we explored demographic and potentially clinically relevant variables to assess if they had associations with interdialytic bp slope during hours 124 in univariate analyses . the change in systolic bp from pre to post dialysis averaged during the screening period was associated with interdialytic systolic bp slope in the control group with a trend for such an association in the ih subjects ( table 3 ) . however , there was no significant association between the change in systolic bp during the treatment prior to measurement of ambulatory bp and the ambulatory bp slope . african american race also had a significant association with slope in the controls , but not the ih subjects . the results of the remaining variables are shown in table 3 . in the parent case - control study , there were 460 patients screened and 59 that were enrolled ( 401 were ineligible or not interested ) . a total of nine subjects withdrew ( four controls and five cases ) from that study , and we did not have complete hourly bp measurements for one of the remaining controls . the only difference between groups was noted for angiotensin converting enzyme inhibitor ( ace - i ) use , which 64% of the ih subjects were taking and 33% of the controls were taking ( p=0.05 ) . there was no statistically significant difference between groups regarding angiotensin - receptor blocker use ( 8% vs. 21% , p=0.2 ) . serum sodium and albumin measurements obtained prior to the dialysis treatment immediately preceding the ambulatory blood pressure measurements were similar between groups . there were not any differences in dialysate to serum sodium gradients between the two groups . during the 2-week screening period used to identify case subjects and controls , the mean ( standard deviation ) pre - dialysis systolic bp were 155.0 ( 15.7 ) mmhg in the controls and 144.0 ( 9.7 ) mmhg in the ih patients ( p=0.005 ) . post - dialysis systolic bp were 128.3 ( 11.0 ) and 159.0 ( 9.3 ) mmhg in the two groups , respectively ( p<0.001 ) ; and the changes in systolic bp from pre to post dialysis were 26.7 ( 12.5 ) and + 15.0 ( 9.1 ) mmhg in the two groups ( p<0.001 ) . during the single hemodialysis treatment occurring immediately prior to the ambulatory bp measurements , all control subjects had decreases in systolic bp from pre to post - dialysis . among the ih subjects , there were 13 subjects with bp increases and 12 subjects with bp decreases during this treatment . a comparison of measurements obtained in the treatment immediately prior to the ambulatory bp monitoring as well as other measurements of endothelial cell function and fluid changes are shown in table 2 . while pre - dialysis systolic bps for this single treatment were similar , post - dialysis systolic bp and change in systolic bp from pre to post dialysis the average systolic bp during the 44 hour interdialytic time period was 143.1 ( 16.5 ) mmhg in the controls and 155.4 ( 14.2 ) mmhg in the ih subjects ( p=0.008 ) . for the first 24 hours , the average systolic bp was 138.0 ( 21.2 ) mmhg in the controls and 152.7 ( 22.8 ) mmhg in the ih subjects ( p=0.02 ) . during the remainder of the interdialytic time period ( hours 2544 ) , the average systolic bp was 150.8 ( 22.3 ) mmhg and 156.5 ( 20.8 ) mmhg , respectively ( p=0.4 ) . the systolic bp slope for the controls during the entire 44-hour interdialytic period was + 0.6 mmhg / hr ( p<0.0001 compared to zero slope , figure 1 ) . in the controls , the systolic bp slope for hours 124 and 2544 were + 0.6 mmhg / hr ( p=0.001 compared to zero slope ) and + 0.4 mmhg / hr ( p=0.09 compared to zero slope ) , respectively . within the control group there was no difference between these time periods ( 0.1 mmhg / hr , p=0.4 ) . in the ih subjects , the systolic bp slope during the entire 44 hour interdialytic time period was + 0.1 mmhg / hr ( p=0.1 compared to zero slope ) . the slope for hours 124 and 2544 were 0.3 mmhg / hr ( p=0.2 compared to zero slope ) and + 0.3 mmhg / hr ( p=0.3 compared to zero slope ) , respectively . within the ih subjects , there was a difference between these two periods ( + 0.6 mmhg / hr when comparing hours 2544 to hours 124 , p=0.001 ) . the between - group differences for controls and ih subjects slopes for hours 124 and 2544 were 0.8 mmhg / hr ( p=0.002 ) and 0.1 there were no differences in the 124 hour systolic bp slope among the ih patients who had intradialytic bp increases or decreases in the treatment immediately prior to the ambulatory blood pressure monitoring ( + 0.2 we explored demographic and potentially clinically relevant variables to assess if they had associations with interdialytic bp slope during hours 124 in univariate analyses . the change in systolic bp from pre to post dialysis averaged during the screening period was associated with interdialytic systolic bp slope in the control group with a trend for such an association in the ih subjects ( table 3 ) . however , there was no significant association between the change in systolic bp during the treatment prior to measurement of ambulatory bp and the ambulatory bp slope . african american race also had a significant association with slope in the controls , but not the ih subjects . the principal finding of this study was that we found quantitatively distinct interdialytic bp patterns in ih patients compared to our hypertensive hemodialysis controls . most hemodialysis patients have decreases in bp during dialysis followed by a persistent rise in systolic bp during the interdialytic period . the interdialytic bp pattern in our ih patients was characterized by a trend for decreasing bp for the initial 24 hours after dialysis , and it was followed by a more typical increase in bp for the remainder of the interdialytic period . we did not identify any additional clinical variables related to baseline characteristics or intradialytic hemodynamic changes that consistently influenced this bp slope . as published previously , while the average systolic bp measured from hour 2544 was similar between the two groups , there is a significant difference between the two groups for the average systolic bp measured during the initial 24 hours after dialysis . this study demonstrates the significance of the initial 24 hour time period following hd in defining the overall ambulatory bp burden in patients with ih compared to other hemodialysis patients . specifically , the elevated post - hd bp in patients with ih is not transient and takes many hours to normalize . furthermore , we provide a formal quantitative analysis supporting that the bp patterns , defined by the change in systolic bp each hour , are distinct during this 24 hour time period among patients with different intradialytic bp characteristics . this suggests that the mechanisms responsible for increased bp likely differ between these two groups of patients not only during dialysis , but also throughout a large part of the interdialytic time period . extracellular volume status is an important determinant of bp in hemodialysis patients , in general . cohort data of hemodialysis patients shows that the average response to a hemodialysis treatment is a two - slope decrease in bp with an acute decrease in bp during the first hour of the treatment followed by a more blunted decrease in bp throughout the remainder of the treatment . greater ultrafiltration volume or rate during dialysis is associated with more a negative slope , or greater decline in bp , during the latter portion of the treatment . during the interdialytic time period , bp increases with time , with faster increases in bp seen with greater amount of interdialytic weight gain . controls in our study demonstrated an expected increase in bp throughout the entire interdialytic time period . systolic bp in this group increased 0.6 mmhg each hour after dialysis during both the initial 24 hours after dialysis and the entire 44-hour interdialytic time period . the slope did not change in controls from hours 124 after dialysis to hours 2544 . in contrast , the bp slope in the ih patients showed a trend towards a decrease with each hour ( 0.3 mmhg / hr ) during the first 24 hours , and this change was different from not only the same time period in controls ( p=0.002 , figure 1 ) , but also when compared to hours 2544 in the ih patients ( p=0.001 ) . we conclude from this study that intradialytic hypertension patients respond to acute volume expansion differently than the hypertensive controls based on the between group differences in ambulatory bp slope with similar interdialytic weight gain ( and percentage of interdialytic weight gain ) . exploratory analyses showed no association in either group with systolic bp slope and the variables interdialytic weight gain , ultrafiltration rate , or dialysate to serum sodium gradient . despite similarities in acute change in extracellular volume between our patient groups , we can not exclude that chronic volume overload did not play a role in the different ambulatory bp patterns . extracellular volume overload has been proposed to be a mechanism responsible for intradialytic hypertension in general [ 10 , 11 , 22 , 23 ] , and it remains possible that there were differences in chronic volume overload between these two groups . we acknowledge the important findings of others that the degree of chronic extracellular expansion does influence interdialytic bp patterns in hemodialysis patients such that more volume overloaded patients have more blunted increases in bp between dialysis treatments [ 22 , 24 ] . chronic volume expansion might be expected to influence not only the interdialytic bp via a persistent elevation of vascular resistance , but it might also influence intradialytic bp due to more rapid intravascular refilling as ultrafiltration takes place during dialysis . others have found vascular resistance to increase during dialysis in ih patients in general , and it is unknown how acute intravascular refilling would influence acute changes in vascular resistance . it is conceivable that if our ih patients were more chronically overloaded with a baseline state of increased vascular resistance , some stimulus of increased vascular resistance during dialysis ( whether related to volume changes or not ) , would result in a prolonged period of heightened vascular resistance before it returns back down to the baseline value . it would be of interest to have simultaneous measurements of extracellular volume , intravascular volume , cardiac output , and vascular resistance in a similar study to specifically address this . our prior work has identified a greater endothelial cell dysfunction in ih patients compared to hypertensive hemodialysis controls [ 12 , 15 ] . specifically , there is lower baseline fmd and lower levels of circulating endothelial progenitor cells . no prior studies have looked at the quantitative relationship between impaired endothelial cell function and interdialytic bp pattern among patients with ih . in this study , we performed exploratory analyses to determine how markers of endothelial cell function were associated with bp slopes . we did not find any strong association of intradialytic change in et-1 with the systolic bp slope . so , while intradialytic increases in et-1 have been proposed to explain increases in bp during hd in ih patients , [ 79 , 15 ] there is no evidence to conclude from our study that the magnitude of an increase in et-1 affects bp for prolonged periods of time . we also found that neither baseline fmd or pwv had an association with bp slope . we did find associations between screening intradialytic bp change and systolic bp in the controls in a univariate model with a trend for such an association in the ih patients . conversely , there was no association with change in systolic bp from pre to post dialysis and bp slope when considering the treatment immediately before this generates the hypothesis that the ambulatory bp pattern may not just be a response to what happened during the prior treatment , but may be more likely related to whatever mechanisms make an individual prone to intradialytic hypertension in the first place . this was supported by the similarity in bp slopes for the ih patients with increases and decreases in intradialytic bp in the treatment immediately prior to the ambulatory bp monitoring . we acknowledge that we can not confirm these findings from our exploratory analysis without multiple ambulatory bp measurements obtained at different points in time and a larger sample size . there are numerous other hypotheses for the etiology of ih that warrant further consideration in the context of how ambulatory bp might affected . silva et al . found that higher dialysate bicarbonate concentration was associated with lower post - dialysis cardiac index and bp , while higher dialysate to serum potassium gradient was associated with more preserved bp and cardiac index . there were no differences in dialysate bicarbonate , dialysate potassium , or dialysate to serum potassium gradient in our study to support these as primary mechanisms responsible for the intradialytic bp differences we saw . using a lower dialysate sodium has been shown to prevent bp increases in ih patients , but we found no difference in serum to dialysate sodium gradient between groups and no association with this gradient on the interdialytic bp slope . others have found that intradialytic exercise results in a higher intradialytic bp than in controls , but the bp decreases by the end of dialysis . although specific targeted exercise regimens are not part of our protocol , we did not have ascertainment of relative activity during dialysis in our study to fully address this possibility . as stated above , the role of chronic volume overload and other unidentified vasoconstrictors on the intradialytic changes in vascular resistance remain to be further explored . limitations to this study include a lack of extracellular volume measurements from bioimpedance spectroscopy to establish whether one group was chronically more volume overloaded than the other . one study using multifrequency bioimpedance spectroscopy suggests that volume overload is a characteristic of ih based on measurements obtained in a cross sectional study . we also did not have weight measurements between hd treatments to determine if the rate of weight gain was different between these groups . while we know that percentage of weight gain was similar when considering the entire interdialytic period , we can not confirm if there were differences in weight gain on the non - hd day . furthermore , we did not have reliable ascertainment of residual renal function to compare this between groups or assess the possible effect this may have had on interdialytic bp patterns . the absence of associations of most variables tested with the ambulatory bp slope may have been limited by inadequate sample size . it is possible that associations exist that would have required a larger study to uncover these relationships . finally , we are basing our conclusions on a single set of ambulatory bp measurements in a relatively small sample of patients . we found in this study that interdialytic bp patterns differ significantly between patients with ih and hypertensive hemodialysis controls . there is an absence of the expected systolic bp increase during the initial 24 hours after dialysis in patients with ih . furthermore , this period of time defines the overall higher bp burden in patients with compared to other hemodialysis patients as both the average bp and the bp slope are similar in the two groups during the latter portion of the interdialytic time period . our study further establishes how intradialytic bp patterns can provide information on bp during the interdialytic time period .

background / aimsintradialytic hypertension ( ih ) patients have higher mortality risk than other hemodialysis patients and have been shown to have higher ambulatory blood pressure ( bp ) . we hypothesized that interdialytic bp patterns would differ in ih patients and hypertensive hemodialysis controls.methodswe consecutively screened hemodialysis patients at our university - affliated units . based on pre and post - hd bp measurements during the prior 2 week period , we identified ih patients and demographically matched hemodialysis controls . we measured ambulatory interdialytic bp , fow - mediated vasodilation , and intradialytic endothelin-1 ( et-1 ) . using linear mixed - models , we compared bp slopes during the following intervals : 124 hours post - dialysis , 2544 hours post - dialysis , and 144 hours post-dialysis.resultsthere were 25 case subjects with ih and 24 controls . systolic bp during hours 144 , 124 , and 2544 were 143.1 ( 16.5 ) , 138.0 ( 21.2 ) , and 150.8 ( 22.3 ) mmhg in controls . for ih subjects , they were 155.4 ( 14.2 ) , 152.7 ( 22.8 ) , and 156.5 ( 20.8 ) mmhg ( p=0.008 , 0.02 , 0.4 ) . in controls , the slopes were + 0.6 , + 0.6 , and + 0.4 mmhg / hr . in ih subjects , they were + 0.1 , 0.3 , and + 0.3 mmhg / hr . the ih 124 hour slope differed from the ih 2544 hour slope ( p=0.001 ) and the control 124 hour slope ( p=0.002 ) . the change in et-1 from pre to post dialysis was 0.5 ( 1.5 ) pg / ml in controls and 1.0 ( 2.3 ) pg / ml in ih patients ( p=0.4 ) . in a univariate model , there was an association with screening bp and bp slope ( p=0.002 for controls and p=0.1 for ih patients).conclusionsinterdialytic bp patterns differ in ih patients and hemodialysis controls . the elevated post dialysis blood pressure persists for many hours in ih patients contributing to the overall increased bp burden .

Introduction Materials and Methods Study Population Blood Pressure Measurements Blood Measurements Flow Mediated Vasodilation Pulse wave velocity Statistical Analysis Results Patient Characteristics Intradialytic Blood Pressure and Measurements of Endothelial Cell Function Interdialytic Blood Pressure Patterns Discussion Conclusion

a wide range of blood pressure ( bp ) measurements both during and between hemodialysis sessions poses a challenge in hypertension management . compared to individual bp measurements obtained in the hemodialysis unit , the average of bp measurements obtained during the interdialytic period provides better prognostic information for adverse clinical outcomes , including mortality . because hemodialysis unit measurements are directly available to nephrologists , it is important to 1 ) recognize the associations between intradialytic bp patterns and overall interdialytic bp burden and 2 ) further explore the nature of these relationships . intradialytic hypertension ( ih ) , an increase in bp from pre to post - hemodialysis , is a recurrent and persistent phenomenon in a subset of hemodialysis patients that has been shown to be an independent risk factor for increased morbidity and mortality [ 46 ] . recent observational evidence demonstrates that ih patients have higher ambulatory systolic bp measured during the 44-hour interdialytic period compared to hypertensive hemodialysis controls . as interdialytic bp measurements are the most reliable bp measurements to predict outcomes in hemodialysis patients , it is critical to identify what factors influence bp most strongly during the interdialytic period , particularly in high - risk patients . in this retrospective analysis of a previously published case - control study , we sought to identify and characterize quantitative differences in ambulatory interdialytic bp patterns between ih patients and hemodialysis controls using linear mixed models . based on qualitative inspection of the bp patterns , we hypothesized that the bp slopes of these two patient groups would be significantly different during the initial 24 hours after hd , but similar throughout the remainder of the interdialytic period . inclusion criteria were : 1 ) age 1880 years ; 2 ) hemodialysis vintage 1 month ; 3 ) ability to achieve the dry weight as determined clinically by the patient s individual nephrologist ; 4 ) clinical hypertension as defined by bp exceeding the recommended guidelines set forth by the national kidney foundation ( pre - dialysis systolic bp 140 mmhg or post - dialysis systolic bp > 130 mmhg ) . subjects were defined as case subjects if they demonstrated an increase in systolic bp 10 mmhg from pre to post - hemodialysis in 4/6 screening treatments . subjects were defined as control subjects if they demonstrated a decrease in systolic bp 10 mmhg from pre to post hemodialysis in 4/6 screening treatments . remaining serum was stored in a 80 degree celsius freezer , and endo - thelin-1 ( et-1 ) was analyzed with elisa in batch at study conclusion . on a midweek , non - dialysis day , ultrasound of the brachial artery was obtained with an 11-mhz pulsed doppler ultrasound probe at an insonation angle of 60 degree ( philips ie33 , bothell , wa ) , and mean blood flow velocity and brachial artery diameter were measured . a slope was determined for each of the following 3 predefined intervals : hours 124 after dialysis , hours 2544 after dialysis , and hours 144 after dialysis . we then explored mixed effect models to assess the relationship between bp slope during hours 124 with several clinical variables ( presence of diabetes , age , gender , race , number of antihypertensive medications , tobacco use , fmd , intradialytic change in et-1 , dialysis shift , percentage of interdialytic weight gain , pulse wave velocity , ultrafiltration rate , dialysate to serum sodium gradient , change in intradialytic systolic bp during screening as well as in the treatment preceding ambulatory bp measurement ) . inclusion criteria were : 1 ) age 1880 years ; 2 ) hemodialysis vintage 1 month ; 3 ) ability to achieve the dry weight as determined clinically by the patient s individual nephrologist ; 4 ) clinical hypertension as defined by bp exceeding the recommended guidelines set forth by the national kidney foundation ( pre - dialysis systolic bp 140 mmhg or post - dialysis systolic bp > 130 mmhg ) . subjects were defined as case subjects if they demonstrated an increase in systolic bp 10 mmhg from pre to post - hemodialysis in 4/6 screening treatments . subjects were defined as control subjects if they demonstrated a decrease in systolic bp 10 mmhg from pre to post hemodialysis in 4/6 screening treatments . remaining serum was stored in a 80 degree celsius freezer , and endo - thelin-1 ( et-1 ) was analyzed with elisa in batch at study conclusion . a slope was determined for each of the following 3 predefined intervals : hours 124 after dialysis , hours 2544 after dialysis , and hours 144 after dialysis . we then explored mixed effect models to assess the relationship between bp slope during hours 124 with several clinical variables ( presence of diabetes , age , gender , race , number of antihypertensive medications , tobacco use , fmd , intradialytic change in et-1 , dialysis shift , percentage of interdialytic weight gain , pulse wave velocity , ultrafiltration rate , dialysate to serum sodium gradient , change in intradialytic systolic bp during screening as well as in the treatment preceding ambulatory bp measurement ) . during the 2-week screening period used to identify case subjects and controls , the mean ( standard deviation ) pre - dialysis systolic bp were 155.0 ( 15.7 ) mmhg in the controls and 144.0 ( 9.7 ) mmhg in the ih patients ( p=0.005 ) . post - dialysis systolic bp were 128.3 ( 11.0 ) and 159.0 ( 9.3 ) mmhg in the two groups , respectively ( p<0.001 ) ; and the changes in systolic bp from pre to post dialysis were 26.7 ( 12.5 ) and + 15.0 ( 9.1 ) mmhg in the two groups ( p<0.001 ) . during the single hemodialysis treatment occurring immediately prior to the ambulatory bp measurements , all control subjects had decreases in systolic bp from pre to post - dialysis . among the ih subjects , there were 13 subjects with bp increases and 12 subjects with bp decreases during this treatment . while pre - dialysis systolic bps for this single treatment were similar , post - dialysis systolic bp and change in systolic bp from pre to post dialysis were different between these groups . the average systolic bp during the 44 hour interdialytic time period was 143.1 ( 16.5 ) mmhg in the controls and 155.4 ( 14.2 ) mmhg in the ih subjects ( p=0.008 ) . for the first 24 hours , the average systolic bp was 138.0 ( 21.2 ) mmhg in the controls and 152.7 ( 22.8 ) mmhg in the ih subjects ( p=0.02 ) . during the remainder of the interdialytic time period ( hours 2544 ) , the average systolic bp was 150.8 ( 22.3 ) mmhg and 156.5 ( 20.8 ) mmhg , respectively ( p=0.4 ) . the systolic bp slope for the controls during the entire 44-hour interdialytic period was + 0.6 mmhg / hr ( p<0.0001 compared to zero slope , figure 1 ) . in the controls , the systolic bp slope for hours 124 and 2544 were + 0.6 mmhg / hr ( p=0.001 compared to zero slope ) and + 0.4 mmhg / hr ( p=0.09 compared to zero slope ) , respectively . within the control group there was no difference between these time periods ( 0.1 mmhg / hr , p=0.4 ) . in the ih subjects , the systolic bp slope during the entire 44 hour interdialytic time period was + 0.1 mmhg / hr ( p=0.1 compared to zero slope ) . the slope for hours 124 and 2544 were 0.3 mmhg / hr ( p=0.2 compared to zero slope ) and + 0.3 mmhg / hr ( p=0.3 compared to zero slope ) , respectively . within the ih subjects , there was a difference between these two periods ( + 0.6 mmhg / hr when comparing hours 2544 to hours 124 , p=0.001 ) . the between - group differences for controls and ih subjects slopes for hours 124 and 2544 were 0.8 mmhg / hr ( p=0.002 ) and 0.1 there were no differences in the 124 hour systolic bp slope among the ih patients who had intradialytic bp increases or decreases in the treatment immediately prior to the ambulatory blood pressure monitoring ( + 0.2 mmhg / hr for decrease vs increase , p=0.7 ) . the change in systolic bp from pre to post dialysis averaged during the screening period was associated with interdialytic systolic bp slope in the control group with a trend for such an association in the ih subjects ( table 3 ) . however , there was no significant association between the change in systolic bp during the treatment prior to measurement of ambulatory bp and the ambulatory bp slope . african american race also had a significant association with slope in the controls , but not the ih subjects . during the 2-week screening period used to identify case subjects and controls , the mean ( standard deviation ) pre - dialysis systolic bp were 155.0 ( 15.7 ) mmhg in the controls and 144.0 ( 9.7 ) mmhg in the ih patients ( p=0.005 ) . post - dialysis systolic bp were 128.3 ( 11.0 ) and 159.0 ( 9.3 ) mmhg in the two groups , respectively ( p<0.001 ) ; and the changes in systolic bp from pre to post dialysis were 26.7 ( 12.5 ) and + 15.0 ( 9.1 ) mmhg in the two groups ( p<0.001 ) . during the single hemodialysis treatment occurring immediately prior to the ambulatory bp measurements , all control subjects had decreases in systolic bp from pre to post - dialysis . among the ih subjects , there were 13 subjects with bp increases and 12 subjects with bp decreases during this treatment . while pre - dialysis systolic bps for this single treatment were similar , post - dialysis systolic bp and change in systolic bp from pre to post dialysis the average systolic bp during the 44 hour interdialytic time period was 143.1 ( 16.5 ) mmhg in the controls and 155.4 ( 14.2 ) mmhg in the ih subjects ( p=0.008 ) . for the first 24 hours , the average systolic bp was 138.0 ( 21.2 ) mmhg in the controls and 152.7 ( 22.8 ) mmhg in the ih subjects ( p=0.02 ) . during the remainder of the interdialytic time period ( hours 2544 ) , the average systolic bp was 150.8 ( 22.3 ) mmhg and 156.5 ( 20.8 ) mmhg , respectively ( p=0.4 ) . the systolic bp slope for the controls during the entire 44-hour interdialytic period was + 0.6 mmhg / hr ( p<0.0001 compared to zero slope , figure 1 ) . in the controls , the systolic bp slope for hours 124 and 2544 were + 0.6 mmhg / hr ( p=0.001 compared to zero slope ) and + 0.4 mmhg / hr ( p=0.09 compared to zero slope ) , respectively . within the control group there was no difference between these time periods ( 0.1 mmhg / hr , p=0.4 ) . in the ih subjects , the systolic bp slope during the entire 44 hour interdialytic time period was + 0.1 mmhg / hr ( p=0.1 compared to zero slope ) . the slope for hours 124 and 2544 were 0.3 mmhg / hr ( p=0.2 compared to zero slope ) and + 0.3 mmhg / hr ( p=0.3 compared to zero slope ) , respectively . within the ih subjects , there was a difference between these two periods ( + 0.6 mmhg / hr when comparing hours 2544 to hours 124 , p=0.001 ) . the between - group differences for controls and ih subjects slopes for hours 124 and 2544 were 0.8 mmhg / hr ( p=0.002 ) and 0.1 there were no differences in the 124 hour systolic bp slope among the ih patients who had intradialytic bp increases or decreases in the treatment immediately prior to the ambulatory blood pressure monitoring ( + 0.2 we explored demographic and potentially clinically relevant variables to assess if they had associations with interdialytic bp slope during hours 124 in univariate analyses . the change in systolic bp from pre to post dialysis averaged during the screening period was associated with interdialytic systolic bp slope in the control group with a trend for such an association in the ih subjects ( table 3 ) . however , there was no significant association between the change in systolic bp during the treatment prior to measurement of ambulatory bp and the ambulatory bp slope . african american race also had a significant association with slope in the controls , but not the ih subjects . the principal finding of this study was that we found quantitatively distinct interdialytic bp patterns in ih patients compared to our hypertensive hemodialysis controls . most hemodialysis patients have decreases in bp during dialysis followed by a persistent rise in systolic bp during the interdialytic period . the interdialytic bp pattern in our ih patients was characterized by a trend for decreasing bp for the initial 24 hours after dialysis , and it was followed by a more typical increase in bp for the remainder of the interdialytic period . this study demonstrates the significance of the initial 24 hour time period following hd in defining the overall ambulatory bp burden in patients with ih compared to other hemodialysis patients . specifically , the elevated post - hd bp in patients with ih is not transient and takes many hours to normalize . furthermore , we provide a formal quantitative analysis supporting that the bp patterns , defined by the change in systolic bp each hour , are distinct during this 24 hour time period among patients with different intradialytic bp characteristics . cohort data of hemodialysis patients shows that the average response to a hemodialysis treatment is a two - slope decrease in bp with an acute decrease in bp during the first hour of the treatment followed by a more blunted decrease in bp throughout the remainder of the treatment . in contrast , the bp slope in the ih patients showed a trend towards a decrease with each hour ( 0.3 mmhg / hr ) during the first 24 hours , and this change was different from not only the same time period in controls ( p=0.002 , figure 1 ) , but also when compared to hours 2544 in the ih patients ( p=0.001 ) . exploratory analyses showed no association in either group with systolic bp slope and the variables interdialytic weight gain , ultrafiltration rate , or dialysate to serum sodium gradient . we acknowledge the important findings of others that the degree of chronic extracellular expansion does influence interdialytic bp patterns in hemodialysis patients such that more volume overloaded patients have more blunted increases in bp between dialysis treatments [ 22 , 24 ] . it is conceivable that if our ih patients were more chronically overloaded with a baseline state of increased vascular resistance , some stimulus of increased vascular resistance during dialysis ( whether related to volume changes or not ) , would result in a prolonged period of heightened vascular resistance before it returns back down to the baseline value . it would be of interest to have simultaneous measurements of extracellular volume , intravascular volume , cardiac output , and vascular resistance in a similar study to specifically address this . our prior work has identified a greater endothelial cell dysfunction in ih patients compared to hypertensive hemodialysis controls [ 12 , 15 ] . we did not find any strong association of intradialytic change in et-1 with the systolic bp slope . so , while intradialytic increases in et-1 have been proposed to explain increases in bp during hd in ih patients , [ 79 , 15 ] there is no evidence to conclude from our study that the magnitude of an increase in et-1 affects bp for prolonged periods of time . we did find associations between screening intradialytic bp change and systolic bp in the controls in a univariate model with a trend for such an association in the ih patients . conversely , there was no association with change in systolic bp from pre to post dialysis and bp slope when considering the treatment immediately before this generates the hypothesis that the ambulatory bp pattern may not just be a response to what happened during the prior treatment , but may be more likely related to whatever mechanisms make an individual prone to intradialytic hypertension in the first place . this was supported by the similarity in bp slopes for the ih patients with increases and decreases in intradialytic bp in the treatment immediately prior to the ambulatory bp monitoring . found that higher dialysate bicarbonate concentration was associated with lower post - dialysis cardiac index and bp , while higher dialysate to serum potassium gradient was associated with more preserved bp and cardiac index . using a lower dialysate sodium has been shown to prevent bp increases in ih patients , but we found no difference in serum to dialysate sodium gradient between groups and no association with this gradient on the interdialytic bp slope . while we know that percentage of weight gain was similar when considering the entire interdialytic period , we can not confirm if there were differences in weight gain on the non - hd day . we found in this study that interdialytic bp patterns differ significantly between patients with ih and hypertensive hemodialysis controls . there is an absence of the expected systolic bp increase during the initial 24 hours after dialysis in patients with ih . furthermore , this period of time defines the overall higher bp burden in patients with compared to other hemodialysis patients as both the average bp and the bp slope are similar in the two groups during the latter portion of the interdialytic time period .