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eighteen sedentary obese subjects with newly diagnosed type 2 diabetes who were not being treated with diabetes medications were randomized to one of three treatment groups : 1 ) placebo ( saline ; four men and two women ; age : 60 3 years ; bmi : 36 1 kg / m ; hba1c : 7.8 0.3% ; fasting plasma glucose concentration : 8.0 0.4 mmol / l ) , 2 ) low - dose ( 30 mg / day ) recombinant methionyl human ( r - met hu ) leptin ( two men and four women ; age : 53 7 years ; bmi : 35 1 kg / m ; hba1c : 8.2 0.4% ; fasting plasma glucose concentration : 8.5 0.6 mmol / l ) , or 3 ) high - dose ( 80 mg / day ) r - met hu leptin ( three men and three women ; age : 54 4 years ; bmi : 36 1 kg / m ; hba1c : 7.8 0.3% ; fasting plasma glucose concentration : 9.5 0.5 mmol / l ) . none of the subjects smoked tobacco , were pregnant or lactating , had significant organ system dysfunction , or took medications known to affect metabolism . all subjects provided written , informed consent before participating in the study , which was approved by the human studies committee of washington university school of medicine . subjects were admitted to the clinical research unit ( cru ) in the afternoon before the clamp procedure . total body mass , fat mass , and fat - free mass were determined by using dual - energy x - ray absorptiometry ( hologic qdr 4500 , waltham , ma ) . at 0600 h the next morning , after an overnight fast , one catheter was inserted into a forearm vein for infusion and a second catheter was inserted into a hand vein , which was heated to 55c by using a thermostatically controlled box , to obtain arterialized blood samples . at 0700 h , a primed , continuous infusion of [ 6,6-h2]glucose was started and maintained until the end of the study , 10.5 h later . at 0900 h , a continuous infusion of [ 1-c]palmitate and a primed , continuous infusion of [ 1,1,2,3,3-h5]glycerol were started and maintained for 5 h ( until the end of stage 1 of the clamp ) . at 1030 h , a two - stage ( 3.5 h each ) euglycemic - hyperinsulinemic clamp was started . during stage 1 ( 3.5 to 7.0 h ) , insulin was infused at a rate of 20 mu / m body surface area ( bsa)/min ( initiated by 80 mu / m bsa / min for 5 min and then 40 mu / m bsa / min for 5 min ) . during stage 2 ( 7.0 to 10.5 h ) , insulin was infused at a rate of 50 mu / m bsa / min ( initiated by 200 mu / m bsa / min for 5 min and then 100 mu / m bsa / min for 5 min ) . these two insulin infusion rates were chosen to evaluate liver and adipose tissue insulin sensitivity ( low - dose insulin infusion to submaximally suppress hepatic glucose production and lipolysis of adipose tissue triglycerides ) and skeletal muscle insulin sensitivity ( high - dose insulin infusion to adequately stimulate muscle glucose uptake ) . euglycemia was maintained at a blood glucose concentration of 5.5 mmol / l by variable rate infusion of 20% dextrose enriched to 2.5% with [ 6,6-h2]glucose . the infusion rates of [ 6,6-h2]glucose , [ 1-c]palmitate , and [ 1,1,2,3,3-h5]glycerol were reduced by 50% during stage 1 , and the infusion rate of [ 6,6-h2]glucose was reduced to 25% during stage 2 to account for changes in endogenous glucose production and lipolysis . blood samples to determine plasma leptin and insulin concentrations and substrate ( glucose , palmitate , and glycerol ) tracer - to - tracee ratios ( ttrs ) were obtained before beginning the tracer infusion and every 10 min during the final 30 min of the basal period and stages 1 and 2 of the clamp . plasma insulin and leptin concentrations were measured by using radioimmunoassay ( linco research , st . plasma glucose , palmitate , and glycerol ttrs were determined by using gas chromatography - mass spectroscopy . substrate rate of appearance ( ra ) in plasma was calculated by dividing the substrate tracer infusion rate by the average plasma substrate ttr . endogenous glucose production rate during the clamp procedure was calculated by subtracting the exogenous glucose infusion rate from glucose ra ; glucose rd was calculated as the sum of endogenous glucose ra and the rate of infused glucose . hepatic insulin sensitivity was assessed by using the hepatic insulin sensitivity index , calculated as the product of basal endogenous glucose ra and plasma insulin concentration ( 13 ) . after completion of the clamp procedure , subjects were randomized to treatment with subcutaneous low - dose r - met hu leptin ( 15-mg bid ) , high - dose r - met hu leptin ( 40-mg bid ) , or placebo ( saline , 2-ml bid ) for 14 days , given by a research nurse during twice - daily home visits . the low dose was intended to provide a modest ( threefold ) increase in plasma leptin concentrations , whereas the high dose was intended to provide a pharmacological exposure ( 200-fold above baseline ) . subjects were instructed to maintain their usual dietary and physical activity habits and were weighed daily by the research nurse . an increase in dietary intake was encouraged in any subject who demonstrated a trend toward a decrease in body weight . on day 14 of treatment , subjects were readmitted to the cru , where the body composition analyses and the clamp procedure were repeated . anova with repeated measures was used to compare between ( placebo vs. low - dose and high - dose leptin ) and within ( before vs. after treatment ) group differences . based on our own data evaluating the reproducibility of the effect of insulin on glucose and fatty acid kinetics during a euglycemic - hyperinsulinemic clamp in obese , insulin - resistant subjects ( 14 ) , we estimated that six subjects in each group would allow us to detect a 25 , 30 , and 35% between - group difference in insulin - mediated effects of glucose ra , palmitate ra , and glucose rd , respectively , at the 0.05 -level and a -value of 0.20 ( i.e. , 80% power ) . eighteen sedentary obese subjects with newly diagnosed type 2 diabetes who were not being treated with diabetes medications were randomized to one of three treatment groups : 1 ) placebo ( saline ; four men and two women ; age : 60 3 years ; bmi : 36 1 kg / m ; hba1c : 7.8 0.3% ; fasting plasma glucose concentration : 8.0 0.4 mmol / l ) , 2 ) low - dose ( 30 mg / day ) recombinant methionyl human ( r - met hu ) leptin ( two men and four women ; age : 53 7 years ; bmi : 35 1 kg / m ; hba1c : 8.2 0.4% ; fasting plasma glucose concentration : 8.5 0.6 mmol / l ) , or 3 ) high - dose ( 80 mg / day ) r - met hu leptin ( three men and three women ; age : 54 4 years ; bmi : 36 1 kg / m ; hba1c : 7.8 0.3% ; fasting plasma glucose concentration : 9.5 0.5 mmol / l ) . none of the subjects smoked tobacco , were pregnant or lactating , had significant organ system dysfunction , or took medications known to affect metabolism . all subjects provided written , informed consent before participating in the study , which was approved by the human studies committee of washington university school of medicine . subjects were admitted to the clinical research unit ( cru ) in the afternoon before the clamp procedure . total body mass , fat mass , and fat - free mass were determined by using dual - energy x - ray absorptiometry ( hologic qdr 4500 , waltham , ma ) . at 0600 h the next morning , after an overnight fast , one catheter was inserted into a forearm vein for infusion and a second catheter was inserted into a hand vein , which was heated to 55c by using a thermostatically controlled box , to obtain arterialized blood samples . at 0700 h , a primed , continuous infusion of [ 6,6-h2]glucose was started and maintained until the end of the study , 10.5 h later . at 0900 h , a continuous infusion of [ 1-c]palmitate and a primed , continuous infusion of [ 1,1,2,3,3-h5]glycerol were started and maintained for 5 h ( until the end of stage 1 of the clamp ) . at 1030 h , a two - stage ( 3.5 h each ) euglycemic - hyperinsulinemic clamp was started . during stage 1 ( 3.5 to 7.0 h ) , insulin was infused at a rate of 20 mu / m body surface area ( bsa)/min ( initiated by 80 mu / m bsa / min for 5 min and then 40 mu / m bsa / min for 5 min ) . during stage 2 ( 7.0 to 10.5 h ) , insulin was infused at a rate of 50 mu / m bsa / min ( initiated by 200 mu / m bsa / min for 5 min and then 100 mu / m bsa / min for 5 min ) . these two insulin infusion rates were chosen to evaluate liver and adipose tissue insulin sensitivity ( low - dose insulin infusion to submaximally suppress hepatic glucose production and lipolysis of adipose tissue triglycerides ) and skeletal muscle insulin sensitivity ( high - dose insulin infusion to adequately stimulate muscle glucose uptake ) . euglycemia was maintained at a blood glucose concentration of 5.5 mmol / l by variable rate infusion of 20% dextrose enriched to 2.5% with [ 6,6-h2]glucose . the infusion rates of [ 6,6-h2]glucose , [ 1-c]palmitate , and [ 1,1,2,3,3-h5]glycerol were reduced by 50% during stage 1 , and the infusion rate of [ 6,6-h2]glucose was reduced to 25% during stage 2 to account for changes in endogenous glucose production and lipolysis . blood samples to determine plasma leptin and insulin concentrations and substrate ( glucose , palmitate , and glycerol ) tracer - to - tracee ratios ( ttrs ) were obtained before beginning the tracer infusion and every 10 min during the final 30 min of the basal period and stages 1 and 2 of the clamp . plasma insulin and leptin concentrations were measured by using radioimmunoassay ( linco research , st . plasma glucose , palmitate , and glycerol ttrs were determined by using gas chromatography - mass spectroscopy . substrate rate of appearance ( ra ) in plasma was calculated by dividing the substrate tracer infusion rate by the average plasma substrate ttr . endogenous glucose production rate during the clamp procedure was calculated by subtracting the exogenous glucose infusion rate from glucose ra ; glucose rd was calculated as the sum of endogenous glucose ra and the rate of infused glucose . hepatic insulin sensitivity was assessed by using the hepatic insulin sensitivity index , calculated as the product of basal endogenous glucose ra and plasma insulin concentration ( 13 ) . subjects were admitted to the clinical research unit ( cru ) in the afternoon before the clamp procedure . total body mass , fat mass , and fat - free mass were determined by using dual - energy x - ray absorptiometry ( hologic qdr 4500 , waltham , ma ) . at 0600 h the next morning , after an overnight fast , one catheter was inserted into a forearm vein for infusion and a second catheter was inserted into a hand vein , which was heated to 55c by using a thermostatically controlled box , to obtain arterialized blood samples . at 0700 h , a primed , continuous infusion of [ 6,6-h2]glucose was started and maintained until the end of the study , 10.5 h later . at 0900 h , a continuous infusion of [ 1-c]palmitate and a primed , continuous infusion of [ 1,1,2,3,3-h5]glycerol were started and maintained for 5 h ( until the end of stage 1 of the clamp ) . at 1030 h , a two - stage ( 3.5 h each ) euglycemic - hyperinsulinemic clamp was started . during stage 1 ( 3.5 to 7.0 h ) , insulin was infused at a rate of 20 mu / m body surface area ( bsa)/min ( initiated by 80 mu / m bsa / min for 5 min and then 40 mu / m bsa / min for 5 min ) . during stage 2 ( 7.0 to 10.5 h ) , insulin was infused at a rate of 50 mu / m bsa / min ( initiated by 200 mu / m bsa / min for 5 min and then 100 mu / m bsa / min for 5 min ) . these two insulin infusion rates were chosen to evaluate liver and adipose tissue insulin sensitivity ( low - dose insulin infusion to submaximally suppress hepatic glucose production and lipolysis of adipose tissue triglycerides ) and skeletal muscle insulin sensitivity ( high - dose insulin infusion to adequately stimulate muscle glucose uptake ) . euglycemia was maintained at a blood glucose concentration of 5.5 mmol / l by variable rate infusion of 20% dextrose enriched to 2.5% with [ 6,6-h2]glucose . the infusion rates of [ 6,6-h2]glucose , [ 1-c]palmitate , and [ 1,1,2,3,3-h5]glycerol were reduced by 50% during stage 1 , and the infusion rate of [ 6,6-h2]glucose was reduced to 25% during stage 2 to account for changes in endogenous glucose production and lipolysis . blood samples to determine plasma leptin and insulin concentrations and substrate ( glucose , palmitate , and glycerol ) tracer - to - tracee ratios ( ttrs ) were obtained before beginning the tracer infusion and every 10 min during the final 30 min of the basal period and stages 1 and 2 of the clamp . plasma insulin and leptin concentrations were measured by using radioimmunoassay ( linco research , st . plasma glucose , palmitate , and glycerol ttrs were determined by using gas chromatography - mass spectroscopy . substrate rate of appearance ( ra ) in plasma was calculated by dividing the substrate tracer infusion rate by the average plasma substrate ttr . endogenous glucose production rate during the clamp procedure was calculated by subtracting the exogenous glucose infusion rate from glucose ra ; glucose rd was calculated as the sum of endogenous glucose ra and the rate of infused glucose . hepatic insulin sensitivity was assessed by using the hepatic insulin sensitivity index , calculated as the product of basal endogenous glucose ra and plasma insulin concentration ( 13 ) . after completion of the clamp procedure , subjects were randomized to treatment with subcutaneous low - dose r - met hu leptin ( 15-mg bid ) , high - dose r - met hu leptin ( 40-mg bid ) , or placebo ( saline , 2-ml bid ) for 14 days , given by a research nurse during twice - daily home visits . the low dose was intended to provide a modest ( threefold ) increase in plasma leptin concentrations , whereas the high dose was intended to provide a pharmacological exposure ( 200-fold above baseline ) . subjects were instructed to maintain their usual dietary and physical activity habits and were weighed daily by the research nurse . an increase in dietary intake was encouraged in any subject who demonstrated a trend toward a decrease in body weight . on day 14 of treatment , subjects were readmitted to the cru , where the body composition analyses and the clamp procedure were repeated . anova with repeated measures was used to compare between ( placebo vs. low - dose and high - dose leptin ) and within ( before vs. after treatment ) group differences . a p value of 0.05 was considered statistically significant . based on our own data evaluating the reproducibility of the effect of insulin on glucose and fatty acid kinetics during a euglycemic - hyperinsulinemic clamp in obese , insulin - resistant subjects ( 14 ) , we estimated that six subjects in each group would allow us to detect a 25 , 30 , and 35% between - group difference in insulin - mediated effects of glucose ra , palmitate ra , and glucose rd , respectively , at the 0.05 -level and a -value of 0.20 ( i.e. , 80% power ) . baseline body weight and body composition were similar between groups , and body weight and body composition did not change in any group after 2 weeks of treatment ( table 1 ) . plasma leptin remained unchanged in the placebo group and increased by threefold and 150-fold , respectively , in the low - dose and high - dose leptin treatment groups ( table 1 ) . body composition and plasma leptin concentrations before and after placebo and leptin treatment values are means se . * value significantly different from corresponding value before , p 0.05 . there were no differences between groups in basal plasma insulin concentrations , the hepatic insulin sensitivity index , and basal substrate kinetics before and after placebo or leptin treatment ( table 2 ) . plasma insulin concentrations and substrate kinetics during the clamp procedure were also not different between groups before and after placebo or either dose of leptin treatment ( table 2 ) . plasma insulin concentration increased from 17 mu / l during basal conditions to 35 mu / l during stage 1 and to 85 mu / l during stage 2 both before and after 14 days of therapy . insulin - mediated suppression of endogenous glucose ra ( 60 and 80% decrease during stages 1 and 2 , respectively ) and insulin - mediated increase in glucose rd ( 75% increase during stage 2 ) was not different between groups before and after placebo or either dose of leptin treatment . palmitate and glycerol ra decreased by 50 and 35% , respectively , during stage 1 of the clamp procedure and were not different between groups before and after placebo or either dose of leptin treatment . plasma insulin concentrations and metabolic kinetics during basal conditions and during the hyperinsulinemic - euglycemic clamp before and after placebo and leptin treatment values are means se . basal , stage 1 , and stage 2 refer to the hyperinsulinemic - euglycemic clamp as described in the text . baseline body weight and body composition were similar between groups , and body weight and body composition did not change in any group after 2 weeks of treatment ( table 1 ) . plasma leptin remained unchanged in the placebo group and increased by threefold and 150-fold , respectively , in the low - dose and high - dose leptin treatment groups ( table 1 ) . body composition and plasma leptin concentrations before and after placebo and leptin treatment values are means se . * value significantly different from corresponding value before , p 0.05 . there were no differences between groups in basal plasma insulin concentrations , the hepatic insulin sensitivity index , and basal substrate kinetics before and after placebo or leptin treatment ( table 2 ) . plasma insulin concentrations and substrate kinetics during the clamp procedure were also not different between groups before and after placebo or either dose of leptin treatment ( table 2 ) . plasma insulin concentration increased from 17 mu / l during basal conditions to 35 mu / l during stage 1 and to 85 mu / l during stage 2 both before and after 14 days of therapy . insulin - mediated suppression of endogenous glucose ra ( 60 and 80% decrease during stages 1 and 2 , respectively ) and insulin - mediated increase in glucose rd ( 75% increase during stage 2 ) was not different between groups before and after placebo or either dose of leptin treatment . palmitate and glycerol ra decreased by 50 and 35% , respectively , during stage 1 of the clamp procedure and were not different between groups before and after placebo or either dose of leptin treatment . plasma insulin concentrations and metabolic kinetics during basal conditions and during the hyperinsulinemic - euglycemic clamp before and after placebo and leptin treatment values are means se . basal , stage 1 , and stage 2 refer to the hyperinsulinemic - euglycemic clamp as described in the text . leptin is an important regulator of insulin action ; both leptin deficiency and leptin resistance are associated with insulin - resistant glucose metabolism ( 4,12 ) . furthermore , leptin replacement improves insulin sensitivity in subjects with leptin deficiency ( 58 ) . part of the beneficial effect of leptin replacement therapy could be a result of reductions in body weight in both leptin - deficient rodents and rodents with high - fat diet however , leptin therapy increases insulin sensitivity in the absence of a significant decrease in body weight or causes a greater reduction in blood glucose concentration after leptin - induced weight loss than after weight loss induced by pair - feeding alone in wild - type and leptin - deficient rodent models ( 1,2,16 ) and improves insulin sensitivity in leptin - deficient subjects , even in the absence of significant changes in body weight ( 7,8,17 ) . in contrast , our data demonstrate that increasing leptin availability above normal plasma concentrations by treatment with r - met hu leptin does not have weight - loss independent , clinically important effects on insulin sensitivity in obese people with type 2 diabetes . these results are consistent with data from previous leptin weight loss trials , which found that 812 weeks of leptin therapy and a low - calorie diet did not cause a greater change in plasma glucose or insulin concentrations than placebo therapy and a low - calorie diet ( 18,19 ) . the reason(s ) for the discrepancy in results from our study and those conducted in leptin - deficient subjects and rodent obesity models is unclear . we studied only subjects with newly diagnosed , and presumably more reversible , type 2 diabetes and used sensitive measures to evaluate insulin sensitivity in vivo to increase our ability to detect an effect of leptin therapy on insulin action . furthermore , we gave low and high doses of r - met hu leptin to our subjects to ensure adequate plasma leptin concentrations were achieved and to evaluate potential dose - dependent effects . it is unlikely that the 2-week intervention in our study was not long enough to affect insulin action , because leptin administration alters glucose metabolism within several hours in rodents ( 11 ) and the beneficial metabolic effects of leptin replacement therapy in subjects with leptin deficiency occur within 1 week of treatment ( 20 ) . in addition , current treatment strategies in obese insulin - resistant subjects , such as weight loss and pharmacotherapy , demonstrate improved insulin sensitivity can occur within days ( 13,21,22 ) . therefore , our data demonstrate that leptin treatment has different metabolic effects in subjects with leptin deficiency ( who have almost no body fat ) than in obese subjects ( who have high plasma leptin concentrations and large amounts of body fat ) . however , we can not exclude the possibility that leptin treatment would affect insulin action in overweight or obese subjects who have a lower bmi than our cohort . in addition , our results demonstrate that leptin does not worsen insulin sensitivity , which has been suggested because of data obtained from studies conducted in obese people and isolated adipocytes . at any given bmi , increased plasma leptin concentration is associated with greater insulin resistance ( 9,11 ) , and withdrawal of chronic leptin therapy in leptin - deficient subjects improves insulin sensitivity ( 23 ) . furthermore , large doses of leptin decrease insulin signaling and metabolic actions of insulin in isolated rat adipocytes ( 24,25 ) . in our study although our study was conducted in a small number of subjects , it is unlikely we missed an important therapeutic effect of leptin as a result of inadequate statistical power , because there was not even a trend in leptin - induced changes in substrate kinetics with either dose of leptin therapy compared with placebo . in contrast , current treatment strategies for insulin resistance , such as weight loss and pharmacotherapy , improve insulin - mediated glucose uptake by 25% or more ( 13,26,27 ) . in summary , we found that short - term treatment with either low - dose or high - dose r - met hu leptin did not improve liver , skeletal muscle , or adipose tissue insulin sensitivity in weight stable , obese subjects with type 2 diabetes . the absence of a therapeutic effect of leptin in our study , within the context of the observed beneficial effects of leptin replacement therapy in subjects with leptin deficiency ( 4,7,8,12 ) , suggests that a small amount of leptin is important for normal insulin action , but increasing leptin availability above normal plasma concentrations does not have weight loss

objectiveleptin therapy improves insulin sensitivity in people with leptin deficiency , but it is not known whether it improves insulin action in people who are not leptin deficient . the purpose of the current study was to determine whether leptin treatment has weight loss independent effects on insulin action in obese subjects with type 2 diabetes.research design and methodswe conducted a randomized , placebo - controlled trial in obese subjects ( bmi : 35.4 0.6 kg / m2 ; mean se ) with newly diagnosed type 2 diabetes . subjects were randomized to treatment with placebo ( saline ) , low - dose ( 30 mg / day ) , or high - dose ( 80 mg / day ) recombinant methionyl human ( r - met hu ) leptin for 14 days . multiorgan insulin sensitivity before and after treatment was evaluated by using the hyperinsulinemic - euglycemic clamp procedure in conjunction with stable isotopically labeled tracer infusions to measure glucose , glycerol , and fatty acid kinetics.resultslow-dose and high - dose leptin treatment resulted in a threefold ( p < 0.01 ) and 150-fold ( p < 0.001 ) increase in basal plasma leptin concentrations , respectively . however , neither low - dose nor high - dose therapy had an effect on insulin - mediated suppression of glucose , glycerol , or palmitate rates of appearance into plasma compared with placebo . in addition , leptin treatment did not increase insulin - mediated stimulation of glucose disposal compared with placebo ( 14.3 3.1 , 18.4 3.6 , 16.7 2.4 vs. 17.5 2.5 , 20.7 3.0 , 19.1 3.3 mol / kg body wt / min before vs. after treatment in the placebo , low - dose , and high - dose leptin groups , respectively).conclusionsr - met hu leptin does not have weight loss independent , clinically important effects on insulin sensitivity in obese people with type 2 diabetes .

RESEARCH DESIGN AND METHODS Subjects. Experimental protocol None Intervention. Statistical analyses and sample size considerations. RESULTS Body composition and plasma leptin. Substrate kinetics and insulin sensitivity. DISCUSSION

there is no diagnostic difficulty in these cases . sometimes , the cyst lining may ulcerate focally or completely without any trace of the lining squamous epithelium , eliciting a foreign body giant cell reaction . in such cases , the nature of the lesion is evident only by the presence of extruded keratin flakes surrounded by inflammatory cells and foreign body giant cells producing the keratin granuloma . a 56-year - old male patient presented with a swelling in the right gluteal region of 5 years duration with recent increase in size . the specimen was excised and sent for histopathological examination . clinical photograph of the lesion was not available because the clinical diagnosis was the common sebaceous cyst , and no special entity was expected by the clinicians . grossly , the specimen was a cystic swelling of 3 cm diameter filled with whitish - creamy material with specks of brown to black color in the wall . microscopy showed a cyst wall with complete ulceration of the lining epithelium . instead of the usual squamous lining , corresponding to the thickened area of the wall , there was a circumscribed nodule of histiocytes , foreign body giant cells and cholesterol clefts . the epidermal cyst had ruptured into the dermis forming the typical keratin granuloma [ figure 1 ] . an interesting finding was the presence of a dense collection of golden - brown pigment extra and intracellularly in the histiocytes [ figure 2 ] and multinucleate giant cells [ figure 3 ] . ulcerated epidermal cyst wall with keratin granuloma in the deep dermis ( h and e , scanner view 40 ) brown pigment seen intra / extracellularly ( h and e , 100 ) higher power view of the pigment in multinucleate giant cells ( h and e , 400 ) suspecting a fungal infection and assuming the pigment to be hemosiderin , special stains were requested gomori methenamine silver ( gms ) to look for fungus and perl 's prussian blue stain for hemosiderin . no fungus was seen in the gms stain , but the brown pigment had taken up a black color , indicating that the pigment is melanin [ figure 5 ] . perls prussian blue for hemosiderin is negative ( 100 ) the pigment stained black in gomori methenamine silver stain ( 100 ) further confirmation of the melanin pigment was performed by a masson fontana stain , in which the pigment took black color [ figure 6 ] and a bleaching reaction in which the pigment completely disappeared [ figure 7 ] . masson fontana stain showing positivity for melanin - stained black ( 400 ) bleaching reaction the brown pigment has completely disappeared , confirming the pigment as melanin ( 400 ) epidermal cyst , also known as infundibular cyst , a common benign cystic lesion of the skin is seen in the mid / lower dermis or subcutaneous tissue . rarely , multiple infundibular cysts are seen as a manifestation of gardner 's syndrome or the basal cell nevus syndrome . these cysts may become infected and rupture into the dermis , resulting in a heavy inflammatory cell infiltrate in the adjacent dermis . in the absence of infection , when an epidermal cyst ruptures and the contents of the cyst are released into the dermis , a considerable foreign body reaction with numerous multinucleated giant cells results , forming a keratin granuloma . the presence of keratin material surrounded by the reactive inflammatory cells is the only sign of a previously existed epidermal cyst . in our case only ulceration , rupture and foreign body giant cell reaction with typical keratin granuloma in the deep dermis were seen with the unusual finding of dense collection of melanin pigment in the macrophages and giant cells . observed that a large amount of pigment accumulation within epidermal cysts occurs after cyst rupture , which was seen in our case also . have also reported a case of ruptured epidermal cyst with exuberant melanophage proliferation and melanin pigment deposition . in sections stained with hematoxylin eosin , melanocytes and melanin pigmentation of keratinocytes can be seen only rarely in epidermal cysts of whites , but frequently in epidermal cysts of blacks . the present case was also from a dark - skinned patient and with massive collection of melanin in the dermis . the literature review showed a report of multiple pigmented epidermal cysts in the face of a white patient , which was effectively treated by laser therapy . studied 125 epidermal cysts from indian patients to look for the presence of melanin , and found that 63% of the epidermal cysts ( 79 of 125 biopsies ) showed presence of melanin pigment or melanocytes to a variable extent . extensive accumulation of melanin pigment is seen in only 10 of these 79 biopsies ( 12.6% ) . in our institution , we had 210 epidermal cysts in the year 2009 and 155 cases in 2010 . none of these cases showed extensive collection of melanin pigment or proliferation of melanocytes in hematoxylin and eosin ( h and e ) sections . this is the first case of pigmented epidermal cyst with massive melanin deposition that was seen in our institution . pigmented epidermal cyst should be differentiated from the entity pigmented follicular cyst , in which prominent rete ridge pattern of epidermal lining and several terminal - sized pigmented hair shafts containing abundant melanin pigment are seen within the cyst cavity . in our case , reported a case with increased number of melanocytes in the basal layer , large globular cells of melanocytic origin in the spinous layer and hair - germ - like aggregates of basaloid cells and melanocytes . because there was complete ulceration of the lining epithelium in our case , melanocytic proliferation in the basal layer or spinous layer extensive search of the literature showed only a few articles published about pigmented epidermal cyst with massive melanin deposition in the dermis . the rarity may be because the pigment visible in h and e sections may be either ignored or interpreted as hemosiderin and no further methods might have been employed to demonstrate the nature of the pigment . hence , this case is being reported to remind the pathologists about the academic importance of diagnosing this entity as such and not simply as epidermal cyst ignoring the pigment .

epidermal cyst is a very common benign cystic lesion of the skin . it is usual to find ulceration of the lining epithelium , rupture of the cyst wall with chronic inflammation and foreign body giant cell reaction . but , it is very rare to see an epidermal cyst with marked accumulation of melanin pigment . only a few cases of pigmented epidermal cyst with dense collection of melanin pigment have been published in the literature . here , we are reporting a case of ruptured epidermal cyst with keratin granuloma formation and showing dense collection of melanin pigment .

INTRODUCTION CASE REPORT DISCUSSION

there are several studies dealing with sexual behavior , people 's attitude towards hiv / aids and hiv testing[14 ] . in zimbabwe , one study found that the prevalence of hiv among monogamous women was 21.8% . these factors are the husbands having children with other women , more than five years age difference within couples , the woman 's age , and the likelihood of the woman discussing monogamy with her husband in the next three months as well as economic disparities between the sexes . in zambia there is a sizable group of hiv positive neurology patients who suffer from myelopathy , neuropathy / radiculopathy , cerebrovascular diseases and infectious diseases . a multi - country study including thailand , south africa , tanzania and zimbabwe , which used a sample of 14,818 in 48 communities , found that frequent discussions about hiv significantly influenced prior hiv testing . a study in vietnam , dealing with hiv risk behavior and the determinants among people living with hiv / aids , found that 82% of the participants were sexually active . some 20% of the participants reported having sex with multiple partners , and only one third of the participants consistently used a condom . jamaica manifests some of these problems relating to hiv / aids , hiv testing , and determinants of risky behaviors seen in other developing countries . sexual risk taking behavior among working class women were influenced by being in a relationship with a physically violent intimate partner , attitudes towards children as resources , having multiple sexual partners , being economically vulnerable , the perception about men being providers and the vetoing power of males on women 's reproductive health matters . choices and the lack of economic dependency are accounting for increasing hiv / aids / stds in developing countries . the risk of contracting hiv also affects the incarcerated where the rate of hiv infection among prisoners is higher than the incidence in the general population . a majority of university students stated that they had no avoidance intentions towards their relatives and friends living with hiv / aids . however , less than one - half of the students were sympathetic to gay and lesbians living with hiv / aids who were not their relatives or friends . it is important to understand the cultural factors at the societal , family and individual levels that influence hiv risk among jamaican youth . in 2004 , from a survey of jamaican youth , males reported greater condom use ( 67.9% ) during their last sexual intercourse with their most recent and regular partner compared to 57% in 1996 . there was no change in the use of condoms among adolescent females for the same period . the youth perceived that they were at a greater risk for hiv infection in 2004 compared 1996 . despite the aforementioned fact , there are still many youths who are inconsistently using a condom , indicating the divide between knowledge and practice . public health effort to improve the case management of sexually transmitted diseases now includes the private sector . private medical practitioners are now receiving continuing medical education to address the hiv / aids epidemic and youths reproductive health matters . despite the increased risk of contracting hiv owing to inconsistent and improper condom usage[811 ] , many jamaicans do not want to know their hiv status . a person knowing his or her hiv status allows the person to get the appropriate medical treatment if they are positive and prevent the spread of the virus through safe sexual practices . douglas opined that the major cause of mortality among women of 15 - 44 years in the caribbean is aids , and that 1 in every 50 caribbean national was infected with hiv / aids , suggesting that there are some premature mortality arising owing to not being cognizant of one 's hiv status . sexual relation is primarily the medium through which most people contract hiv / aids . in 2007/2008 , wilks et al . conducted a study of some 2,848 jamaicans between the ages of 15 - 74 years ; they found that 3.4% of females reported having been infected with a sti in the studied period compared to 1.3% of males . although the prevalence of males having had sti is greater than that of females , which is concurred by wilks et al study ( males , 18.1% ; females , 11.0% ) , with the context that more males ( having multiple partners in last 12 months , 41% ) are promiscuous than females ( 8.4% ) , there is a pending public health problem that is underlying the females allowing males to have vetoing power over their reproductive health issues and having male partners inconsistently using condoms . an extensive research of the literature found no study which has examined the attitude of non - hiv testers towards future hiv testing as well as their current attitude and sexual behavior . within this context , the objectives of this paper are to ( 1 ) investigate the sexual behavior of non - hiv testers in jamaicans , and ( 2 ) their attitudes towards future hiv testing . the current study extracted a sample of 1,192 participants , from a nationally representative survey , who indicated that they had never had an hiv test done up to 2004 . the hiv / aids / std national kabp survey comprised 1,800 participants 15 - 49 years of age who resided in jamaica at the time of the survey ( may - august , 2004 ) . the data was collected by hope enterprises limited on the behalf of the ministry of health . each of the 14 parishes in the country is stratified into electoral constituencies , with each constituency stratified into three areas - rural areas , parish capitals ( urban areas ) and main towns ( semi - urban areas ) . the areas which comprised a constituency were then stratified into primary sampling units ( psus ) or electoral enumeration districts ( eds ) . a random sample of each psu 23 eds in the urban areas , 25 eds in the semi - urban areas , and 24 eds in the rural areas . twenty - five households were systematically chosen from each ed , and cluster sampling was carried out with all the people living in the household of the designated ages interviewed for the survey . the interviewers were trained for a 5-day period , of which 2 days were devoted to field practices . interviewers were assigned to a team comprising two females , two males and a supervisor . the participants were informed of their right to confidentiality and their right to stop the interview at any time . no names , addresses or other personal information was collected from the participants to ensure anonymity . the instrument used in the survey utilized indicator measures and definitions consistent with unaids and the usaid priority prevention indicator . data were entered , stored and retrieved using spss for windows , version 16.0 spss inc ; chicago , il , usa ) . multivariate logistic regressions were fitted using one outcome measure : self - reported confirmed positive hiv test results . where collinearity existed ( r > 0.7 ) , variables were entered independently into the model to determine those that should be retained during the final model construction . a p - value < 0.05 ( two - tailed ) was used to establish statistical significance . the current study extracted a sample of 1,192 participants , from a nationally representative survey , who indicated that they had never had an hiv test done up to 2004 . the hiv / aids / std national kabp survey comprised 1,800 participants 15 - 49 years of age who resided in jamaica at the time of the survey ( may - august , 2004 ) . the data was collected by hope enterprises limited on the behalf of the ministry of health . each of the 14 parishes in the country is stratified into electoral constituencies , with each constituency stratified into three areas - rural areas , parish capitals ( urban areas ) and main towns ( semi - urban areas ) . the areas which comprised a constituency were then stratified into primary sampling units ( psus ) or electoral enumeration districts ( eds ) . a random sample of each psu 23 eds in the urban areas , 25 eds in the semi - urban areas , and 24 eds in the rural areas . twenty - five households were systematically chosen from each ed , and cluster sampling was carried out with all the people living in the household of the designated ages interviewed for the survey . the interviewers were trained for a 5-day period , of which 2 days were devoted to field practices . interviewers were assigned to a team comprising two females , two males and a supervisor . the participants were informed of their right to confidentiality and their right to stop the interview at any time . no names , addresses or other personal information was collected from the participants to ensure anonymity . the instrument used in the survey utilized indicator measures and definitions consistent with unaids and the usaid priority prevention indicator . data were entered , stored and retrieved using spss for windows , version 16.0 spss inc ; chicago , il , usa ) . multivariate logistic regressions were fitted using one outcome measure : self - reported confirmed positive hiv test results . where collinearity existed ( r > 0.7 ) , variables were entered independently into the model to determine those that should be retained during the final model construction . a p - value < 0.05 ( two - tailed ) was used to establish statistical significance . the current study extracted a sample of 1,192 participants , from a nationally representative survey , who indicated that they had never had an hiv test done up to 2004 . the hiv / aids / std national kabp survey comprised 1,800 participants 15 - 49 years of age who resided in jamaica at the time of the survey ( may - august , 2004 ) . the data was collected by hope enterprises limited on the behalf of the ministry of health . each of the 14 parishes in the country is stratified into electoral constituencies , with each constituency stratified into three areas - rural areas , parish capitals ( urban areas ) and main towns ( semi - urban areas ) . the areas which comprised a constituency were then stratified into primary sampling units ( psus ) or electoral enumeration districts ( eds ) . a random sample of each psu 23 eds in the urban areas , 25 eds in the semi - urban areas , and 24 eds in the rural areas . twenty - five households were systematically chosen from each ed , and cluster sampling was carried out with all the people living in the household of the designated ages interviewed for the survey . the interviewers were trained for a 5-day period , of which 2 days were devoted to field practices . interviewers were assigned to a team comprising two females , two males and a supervisor . the participants were informed of their right to confidentiality and their right to stop the interview at any time . no names , addresses or other personal information was collected from the participants to ensure anonymity . the instrument used in the survey utilized indicator measures and definitions consistent with unaids and the usaid priority prevention indicator . data were entered , stored and retrieved using spss for windows , version 16.0 spss inc ; chicago , il , usa ) . multivariate logistic regressions were fitted using one outcome measure : self - reported confirmed positive hiv test results . where collinearity existed ( r > 0.7 ) , variables were entered independently into the model to determine those that should be retained during the final model construction . a p - value < 0.05 ( two - tailed ) was used to establish statistical significance . table 1 presents the demographic characteristics of sample . of the sample ( n=1192 participants ) , 54.4% was males , 42.9% were employed , and the majority had secondary level education . sociodemographic characteristics of sample , n= 1192 of those who had never done a hiv test ( n=1192 ) , 78.0% indicated that they would be willing to do this in the future ; 16.3% have sti ; 55.2% are currently and actively practicing a religion ; 14.9% are laborers ; 38.8% are household helpers and office attendants ; 31.1% are security guards , hairdressers , taxi operators , machine operators and cosmetologist , 3.6% teachers , police officers , nurses and technicians ; 6.8% clerks , 2.2% manager , assistant managers and heads of small businesses , 0.2% business executives , and 0.4% professionals ( doctors , lawyers , architects , etc . ) . when the participants were asked what is the chance of [ you ] catching hiv ? 57.7% indicated none , 30.2% said little , 6.3% reported moderate and 5.8% remarked a good chance . however , 20.5% had sex with at least two partners in the last 4 weeks . only 53.4% used a condom the first time with their current partners , and 36.1% indicated that their current partner has other partner(s ) . fewer than 70% of the sample indicated that their next most recent partner had other sexual partner(s ) . a statistical difference was found between the mean number of sexual partners males had in the last 12 months ( 2.6 , sd = 4.0 ) and females ( existed between the mean age of sexual debut for males ( 14.2 years , sd = 3.2 years ) and that of females ( 16.5 years , sd = 2.8 years ) t - test = -11.443 , p < 0.0001 . table 2 shows information on the reasons the participants gave for not using a condom by current , next recent and next most recent partner . the findings reveal that between 10% and 15% of the participants do not like using condoms with current , next recent or next most recent sexual partner(s ) , and between 3 to 5% of the participants stated that their partner objected to using a condom . reason for not using a condom by current , next recent and next most recent partner table 3 provides condom usage ( ever ) and precautions taken to avoid pregnancy by current , next recent and next most recent partner . the percentage of participants who are using a condom with their current partner(s ) is less ( 68% ) compared to the next recent partner ( 89% ) and the next most recent partner ( 91% ) . condom usage ( ever ) and precautions taken to avoid pregnancy by current , next recent and next most recent partner different reasons were given for the moderate - to - good chance of contracting the hiv virus . the reasons were ; have different partners , 13.6% ; spouse has many partners , 4.1% ; do not use a condom , 7.6% ; had blood transfusion , 1.3% ; never too careful , 14.5% ; and condom can burst , 8.0% . when the participants were asked why they had not done a hiv test , 59.7% indicated that they do not want to know their status , 15.4% said they are not sexually active , 10.0% reported that they know that they do not have hiv , 5.0% reported that they are not interested , 0.5% remarked that they always practice safe sexual relations . eight percent of the participants stated that they were forced to have sexual relations , 7.0% indicated that they had forced someone to have sexual intercourse and 1.7% remarked that physical force was used in the process . significantly more males ( 12.5% ) had forced someone compared to 2.5% of females ( = 32.946 , p < 0.0001 ) . however , 35.9% of females indicated that physical force was used in the process of forced sexual relations compared to 6.7% of males ( = 17.485 , p < 0.0001 ) . furthermore , no statistical association existed between were you forced to have sexual intercourse? based on the gender of the participants males , 9.3% and females , 10.1% ( = 0.190 , p = 0.669 ) . in addition , significantly more people with sti were forced to have sexual relations ( 15.5% ) compared to those who were not forced ( 7.0% ) = 12.894 , p < 0.0001 . table 4 examines particular demographic characteristics of the participants based on gender . some demographic characteristic by gender of participants the results of the multivariate logistic regression model , shown in table 5 , were significant ( model = 32.526 , p = 0.027 ) . table 5 indicates that 20.3% of the variances accounted for by the independent variables used in the regression analyses : age of first sexual debut , age at last birthday , having had sti , sex with commercial worker , union status , and protecting self from the possibility of contracting the hiv virus . the model had statistically significant predictive power ( model = 32.526 , p = 0.027 ; hosmer and lemeshow goodness of fit ( = 3.202 , p = 0.921 ) , and correctly classified 82.5% of the sample ( table 5 ) . logistic regression analyses : variables of willing to do hiv test in the future , n = 950 no statistical existed between would you be willing to do a hiv test in the future? and whether the individual was forced to have sexual intercourse = 0.444 , p = 0.801 : yes to be willing , 9.3% ; no to be willing , 11.0% ; and do nt know , 9.4% . this study provides invaluable insights into those who have done a hiv test and their sexual behavior and attitude towards future hiv testing . from the findings , clearly , people are linking a low risk of contracting the hiv virus with having one sexual partner . based on the perception of respondents in this sample , they are omitting the close association between inconsistent condom usage , not knowing the hiv status of current and past partners , and having unprotected sexual intercourse in a heterosexual way with high risk of contracting the hiv virus . other issues which emerged from the current work are ( 1 ) the low probability of those who have had sex with commercial sex workers willingness to consider doing a hiv test in the future , ( 2 ) the inverse association between having had a sti and the likeliness of wanting to do a hiv test in the future , and ( 3 ) negative association between age of respondents and wanting to do a hiv test in the future . within the context that 16.3% of the sample ; having had stis , 58% indicated a low risk of contracting the hiv virus , 6.2% having had sexual relations with a commercial sex worker , 50.3% consistently using a condom , and 60% not wanting to know their hiv status , and notion that hiv / aids being a homosexual disease , there is a need to execute immediate public health interventions to address these findings . more programs promoting safe sexual practices are definitely needed because between 10 % and 15 % of the participants did not use a condom during sexual intercourse with their current , next recent and next most recent partner(s ) . what is worse is that ; 3 - 5% of the participants stated that their partner objected to them using a condom . moreover , 32% of the participants do not wear a condom with their current partner compared to the 11% who do not wear a condom with their next recent partner . some 16.3% of the participants have stis . despite these risks , only 49.1% of the participants gave several reasons ( participant or spouse having multiple partners , condom failure , negligence , blood transfusion , not wearing a condom ) for a moderate to good chance of contracting hiv . more than one- half of the participants gave no reasons for a good to moderate chance of contracting hiv . it is not surprising then , that 59.7% of the participants did not want to know their hiv status , which creates a problem in the reduction of hiv infection since more than 10% reported that they did not use a condom and 36.1% reported that their current partner has other partners . another explanation which accounts for high risk of contracting stis and hiv / aids , despite the current respondents perception of their seemingly low risk , is women 's acquiescence to the vetoing power of men in reproductive health decisions . a research conducted by who showed that some women opined that reproductive health decisions are made by their partner because he looks after me , suggesting a rationale for inconsistent condom use . significantly more men compared to women declared that they had coerced someone to have sex . similarly , a greater proportion of women ( 35.9% ) stated that physical force was used in coerced sexual intercourse compared to men ( 6.7% ) . participants who experienced coerced sexual relations are less likely to use a condom because of the powerful influence of the situational context in which they find themselves . the data supports this assumption because a significant majority of the participants who were forced to have sex had stis compared to those who were not forced . within the context that hiv is more easily passed from infected males to females than in the reverse and it is predicted that proportionately more cases will occur in females than in males in jamaica in the future , and females being coerced to have sexual intercourse . if this abusive practice is occurring without the use of condoms , it will be accounting for greater risk of hiv infection in the future . an important issue which must be addressed in public health intervention is financial inadequacies of females which account for them relishing their reproductive health choices , often carte blanche to males . jamaica is a middle - income developing country with the prevalence of poverty rate being 9.9% in 2007 ( urban poverty rate , 6.2% ; semi - urban poverty rate , 4.0% ; rural poverty rate , 15.3% ; prevalence of poverty among males , 7.2% ; prevalence of poverty among females , 8.0% ) . economic deprivation of females more than males can also be explained by the unemployment rates , and earnings . the unemployment rate for females was almost 2.3 times more than that of males ( 6.2 ) , indicating that if this economic inequity continues , females will relinquish their reproductive health choices to males as they are providing the economic livelihood . in 2004 , the economic and social survey of jamaica , publication showed that on an average the earnings of males ( mean wage = $ 2.4 million ) was 2 times more than that of females ( $ 1.7 million ) ; and that 76 per cent of senior positions were held by males although 54 per cent of executive and managerial positions were held by females . poverty and unemployment , therefore , are explanations of why people become engaged in risky activities as well as take short - term health risks ; in order to earning a living as they seek a change for the better . it is this belief in socio - economic development that spurs women to relinquish their reproductive health choices to men as they perceive men , as having the economic resources to make an improvement in their current status , even if it is temporary . who stated that , on a yearly basis , there are approximately 340 million new cases of stis and 5 million new cases of hiv infection . clearly , inconsistent condom usage can account for an increase in sti / hiv , indicating that the attitude of non - hiv testers in this sample is reinforcing the pending public health problems that need to be addressed with some degree of urgency . a study by henry - lee found that reasons given by jamaican women for inconsistent contraceptive use or even discontinuation were ( 1 ) cost of contraception , and ( 2 ) long waiting queues at clinics ( 3 ) . she contended that scheduling of appointments could reduce the long waiting time at the clinics . while henry - lee is correct about using appointments to reduce waiting at clinics , but the vetoing supremacy of males in the reproductive health decisions of females could account for their non - return to the clinics . due to the economic disparity , poverty , low education , low income , and inconsistent condom usage , many people do not want to know their hiv status yet they believe that they are at a low risk of contracting the virus . furthermore , significantly more females than males who have never had a hiv test indicated that they are not at risk of contracting the hiv virus , yet statistically more males than females consistently use a condom . clearly , there is a denial among jamaicans of their probability of contracting the hiv virus as people still perceive that the virus is a homosexual disease . a study by dunkle et al . showed that more people that have had heterosexual intercourse had contracted the hiv virus in zambia and rwanda than those who are engaged homosexual relationships . 's work provide some understanding of the rationale behind jamaicans reluctance to have a hiv test done even though they may be in heterosexual relations , as the predominant cultural belief is that it is a homosexual disease . one researcher noted that among jamaican males , there is a substantial peer group pressure to engage in sex in order to avert the stigma of homosexuality , and sexual initiation may occur as early as the age of 8 years indicating the dominance of the culture in causing or reverting to particular and even potentially deleterious actions . the irony here is that caribbean men are socialized to be promiscuous and indulge in premarital sexual relations[2528 ] , which is evident in the male - to - female hiv prevalence ratio ( 1.3:1)[2932 ] , but if they are infected with hiv as a direct consequence of their sexual behavior , they will be scoffed at for having being homosexual . the public health dilemma of hiv infection is that we have been estimating a probability of prevalence among jamaicans[2931 ] . the jamaican ministry of health estimated that 65% of those persons who are infected with hiv are unaware of their status , and this reiterates the rationale for public health intervention programs . due to the sobering reality that people do not want to know their hiv status owing to the stigmatization associated with the virus . currently the nation is not seeing the economic impact of hiv / aids , which may justify why non - hiv testers were not researched on their sexual behavior and attitude towards hiv testing . research findings are in on non - hiv testers , and these should be integrated with public health intervention so as to effectively address some of the present challenges of non - hiv testing in the jamaica and in the wider developing nations . world bank revealed that over 95% of persons living with hiv / aids were in low and middle income countries . of this figure over 20 million of these persons have died from aids in 2002 . this hiv / aids epidemic has reduced life expectancy by more than ten years in many countries . gebre postulated that the caribbean countries have the highest incidence rates of hiv / aids in the americas and the second highest prevalence rates in the world among adults aged 15 44 years . statistics have showed that between 270,000 to 780,000 adults are living with hiv in the region , which is about 2.3% . almost 9453 cases of aids were reported in jamaica and of this amount 1100 of these cases were identified in 2004 . this is an estimated 1.6% of the adult population that has been infected with hiv . according to who , 60% of global mortality is caused by chronic illness , suggesting that hiv / aids will substantially influence the economic and social development of developing countries in the future and that we can no longer allow risky health practices to continue unabated . in 2006 , the reasons persons have given for non - hiv testing were ( 1 ) they do not want to hear their status ( 11% ) , ( 2 ) 1.3% perceived seronegative status , ( 3 ) 1.0% stated that they were not sexually active and ( 4 ) had no partner , 0.7% said that they were not interested in doing an hiv test . based on the findings of the current study the prevalence of jamaicans not wanting to know their hiv status has increased exponentially , which speaks to the rise of inconsistent condom usage , increase in stis , increase in premarital sexual relations and promiscuity . those are the rationales which should vehemently dictate the urgency of hiv / aids registry in seeking to thoroughly understand this threat and how to effectively tailor intervention that the spread of stis and hiv / aids can be reduced . the time has come for the government to require hiv and aids to be reportable by name , and a national hiv / aids registry be kept in order to monitor the epidemic in jamaica . while the researchers recognize the stigmatization surrounding the disease , the issue of privacy , discrimination , and understand that this may result in the continuation of the low willingness of people to become tested for hiv ; the findings are far reaching and can not be allowed to continue into the indefinite future .

background : hiv / aids is a problem in developing countries including jamaica . there are several studies dealing with hiv / aids in jamaica but given the increasing rate of the infection , ongoing studies are necessary.aims:this study examines the sexual behavior and attitude of non - hiv testers in jamaica in order to provide research evidence that will direct public health policies and interventions.materials and methods : this study extracts a sample of 1,192 participants who indicated not having done a hiv test from 1,800 respondents from a 2004 hiv / aids / std national kabp survey . a detailed questionnaire was developed and used to collect data from people ages 15 - 49 years old.results:some 20.3 % of the variances , which is self reported positive hiv test results , are a function of relationship status , using protection against hiv , having sex with a commercial sex worker , having stis , the age at which the participant first had sex and age at last birthday . these findings hold across gender , occupational status and education . the majority ( 87.9% ) of the participants said they had little or no chance of getting hiv , and 59.7% did not want to know their hiv status . however , 46.6% did not wear a condom the first time they had sex with current partner , 32% do not wear a condom with their current partner and 16.3% reported having had stis.conclusion:the existing hiv prevention programs need to be expanded and fortified to target young jamaicans , particularly those who do not engage in safe sexual practices .

Introduction Patients and Methods None Sample Data sources Statistical analyses Results Discussion Conclusion

intrusion of the gas into the bowel wall is due to mucosal or immune compromise . acute massive gastric dilatation with intragastric pressure > 30 cm h2o has been shown to decrease intramural blood flow , leading to necrosis . traditionally , pneumatosis of the intestine has been considered an ominous sign suggesting bowel ischemia with risk of infarction . however , many case reports have been published on pneumatosis suggesting that pneumatosis is a radiographic finding representing a spectrum of conditions ranging from benign disease to abdominal sepsis and death . various causes of pneumatosis in patients of various ages are inflammatory bowel disease , bowel distention , diverticulitis , chronic obstructive pulmonary disease , connective tissue disease , aids , steroids , chemotherapy , pyloric stenosis , gastric outlet obstruction , duodenal stenosis , duodenal ulcers or tumors , etc . [ 3 , 4 ] . gastric pneumatosis can be a life - threatening gangrenous gastric pneumatosis ( emphysematous gastritis ) or benign non - gangrenous gastric pneumatosis ( gastric emphysema ) . the finding of gastric pneumatosis can signify a multitude of possibilities both in terms of causes and consequences . generally , gangrenous gastric pneumatosis requires emergency surgery , whereas non - gangrenous gastric pneumatosis may be managed by non - operative decompression . however , many recent case reports have concluded that the significance of pneumatosis depends upon the underlying clinical condition and physical findings . there are no case reports in the literature suggesting utilization of laparoscopy to determine full thickness necrosis in patients with radiological diagnosis of pneumatosis and esophagogastroduodenoscopy showing mucosal necrosis . however , we report a case of gastric pneumatosis which emphasizes the importance of diagnostic laparoscopy to rule out full thickness necrosis in patients with gastric pneumatosis , which can ultimately avoid major surgery such as gastrectomy . a vigorous 84-year - old man presented with recurrent adenocarcinoma and a solitary hepatic metastasis several years after laparoscopic right hemicolectomy . a wedge excision of a 1.8 cm lesion in segment 7 of the liver was performed after resecting en bloc a 3.5 cm mass at the ileo - colonic anastomosis that invaded the retroperitoneum and the serosa of the third portion of the duodenum . after recovering from adynamic ileus , the patient was sent home on postoperative day 9 to return soon after with nausea and severe vomiting . on arrival , he had normal vital signs except for tachycardia that resolved after rehydration . an abdominal x - ray was performed and showed an atypical gas pattern involving the stomach . there were also signs of gastric pneumatosis and portal venous gas . computed tomography of the abdomen with oral and intravenous contrast 1 ) which showed gastric distention and pneumatosis with portal venous gas . esophagogastroduodenoscopy confirmed the gastric mucosal findings ( fig . it showed areas of mucosal color changes in patchy distribution , along with edematous and friable areas suggesting necrosis . gastric pneumatosis with portal venous air and endoscopic concern for gastric ischemia prompted urgent diagnostic laparoscopy . this demonstrated completely normal - appearing gastric serosa with no signs suggesting full thickness infarction or distal obstruction ( fig . the patient was treated conservatively with intravenous antibiotics covering aerobic and anaerobic bacteria , nasogastric decompression and parenteral nutrition . he recovered well with this conservative approach and was discharged home in a stable condition . on follow - up , he was able to tolerate a regular diet . in the 1940s and 1950s , there were few reports of emphysematous gastritis with a mortality rate of 6080% [ 6 , 7 ] . various terminologies have been used to describe air in the wall of the stomach , including cystic pneumatosis , interstitial gastric emphysema and emphysematous gastritis . gastric pneumatosis is a generalized term that includes any cause of air in the wall of the stomach . since then , multiple case reports of gastric pneumatosis have been published with varied outcomes , ranging from conservative management to operative interventions ultimately leading to total gastrectomy [ 9 , 10 , 11 , 12 , 13 , 14 ] . gastric pneumatosis is a radiographic finding that represents a spectrum of conditions ranging from benign disease to abdominal sepsis and death . four theories have been proposed : ( 1 ) bacterial intramucosal gas is generated by gas - forming bacilli ; ( 2 ) mechanical increased intraluminal pressure during endoscopy or obstruction ; ( 3 ) mucosal damage air enters through disrupted mucosa due to gastric ulcer ; ( 4 ) pulmonary disease - alveolar air dissects down through the mediastinum within the gastric wall [ 2 , 15 ] . in our patient , the most likely reason for pneumatosis was mechanical due to retching and severe vomiting in the absence of duodenal obstruction which was confirmed by upper gastrointestinal gastrografin . gastric pneumatosis with portal venous gas is generally considered an ominous sign prompting emergent operative intervention . however , the outcome depends on multiple factors . management should be guided by the proposed mechanism and clinical signs as opposed to radiographic and endoscopic findings alone . therefore , diagnostic laparoscopy to confirm or refute full thickness ischemic necrosis and conservative management can be considered in some patients , recognizing the possibility of a benign process . diagnostic laparoscopy should be considered as an essential tool to confirm or refute full thickness ischemic necrosis in patients with gastric pneumatosis so that extremely morbid procedures such as total gastrectomy can be avoided .

gastric pneumatosis is a radiographic finding that represents a spectrum of conditions ranging from benign disease to abdominal sepsis and death . along with portal venous gas , it is generally considered an ominous sign prompting emergent operative intervention . we report a rare case showing that diagnostic laparoscopy can be used to confirm or refute full thickness ischemic necrosis and that conservative management can be considered in some patients , recognizing the possibility of a benign process .

Introduction Case Report Discussion Conclusion Disclosure Statement

treatment of the residual alveolar defect in patients with cleft lip and palate often requires bone grafting . an important goal for this treatment , apart from restoring the missing alveolar bone in the cleft area , is to obtain favorable periodontal conditions for the teeth adjacent to the defect . attempts to move the teeth into the edentulous spaces often result in significant periodontal problems . the first report about the alveolar bone grafting aiming to obtain the continuity of the alveolar process was published by lexer in 1908 , who used free bone grafting or pedicled flap of bone and soft tissues of the fifth digit . various sources of autogenous bone are used , but an anterior iliac crest is considered the gold standard for grafting . its resorption rate seems to be high within the 1 year after grafting . from since , multiple techniques and materials have been described to repair the alveolar bone cleft deformity . we describe a transoral mucosal to repair cleft alveolar bone with bioglass and z - plasty . a 10-year - old male , born with a unilateral left cleft lip and palate was presented to our clinic . the history indicated that there was no known familial incidence of cleft lip or palate . at the time of the initial dental evaluation , the patient show complete alveolar bone cleft on the left side of the premaxilla with nasolalvelar fistula [ figure 1a ] . the patient complained from the nasal regurgitation of both food and fluids into the nose and the disfigurement during a smile . ( b ) postoperative picture for patient with repaired cleft alveolar after 1-year first , a z - plasty was designed by donning a z - incision in the mucosal side of the upper lip forming two mucosal triangles ( 1 and 2 ) flap with small mucosal triangle ( 3 ) in between [ figure 2a ] , this small mucosal triangle ( 3 ) used to cover the floor of the nose [ figure 2b ] . in addition , the bioactive glass ( bioglass was mixed with blood [ figure 3 ] and then inserted into the pocket done after the closure of the nasal floor . finally , the two triangles ( 1 and 2 ) are suture together using a 5 - 0 vicryl by crossing each other . wound healing of the recipient sites wound complications such as bleeding , infection , dehiscence , and neurologic disturbance were recorded . furthermore , the evaluation of bone graft quantities was performed according to the bergland scale ( oslo grading system ) . with this scale , the obtained bone level of the interdental septum is compared with the normal side , and it was type 1 in this case after 1-year [ figure 1b ] . ( a ) z - plasty mucosal triangle ( 1 and 2 ) flap with small mucosal triangle ( 3 ) in between ( b ) the small mucosal triangle ( 3 ) used to cover the floor of the nose ( a ) bioactive glass ( bioglass ) maples . alloplastic grafts have been used successfully in the repair of artificial alveolar clefts in animals . according to the literature , autogenic grafts are mostly used ; however , alloplastic graft material started to take place in repair alveolar clefts in human beings . four major categories of surface - active biomaterials have been developed ; dense hydroxylapatite ceramics , bioactive glasses , bioactive glass - ceramics , and bioactive composites . bioactive glass is a commercially available family of bioactive glasses , composed of silicone dioxide ( sio2 ) , sodium dioxide ( na2o ) , calcium oxide ( cao ) and phosphorous pentoxide ( p2o5 ) in specific proportions . bioglass or 45s5 are bioactive glass which is composed of 45% sio , 24.5% cao , 24.5% na2o and 6% p2o5 . bioactive glass such as ( e.g. , 45s5 or bioglass ) bind to soft tissues and bone , while bioactive glass - ceramics ( e.g. , 5s4.3 or ceravital ) bind only to bone , both designed to engender surface reactions that lead to osseointegration , in this case i used the bioactive glass ( bioglass ) . johnson et al . reported that the bioactive glass material in animals was surrounded with osteoid and new bone . alloplastic graft materials that provide simply a scaffolding effect to give support to vascular ingrowth and later calcification are known as osteoconductive . osteoinductive materials , on the other hand , are those that contain morphogens , substances that initiate the development of tissues and organ systems by stimulating undifferentiated cells to convert phenotypically . suggest that one should graft as much bone as possible to obtain adequate facial appearance . their study examined 18 patients with complete unilateral cleft lip and palate using computed tomography 20 years after secondary cancellous bone graft from the iliac crest . they concluded that , even though all dental gaps were closed , and patients were functionally intact , the amount of alveolar bone in the cleft side was less than that of the noncleft side . feichtinger et al . also showed that the absence of adjacent teeth to the cleft site leads to mean the bone volume loss of 95% . hence , the augmentation of unilateral alveolar cleft could be archived perfect by using this technique and the bioactive glass material , which gave a result of bergland 's scale type 1 after 1-year of follow - up . transoral mucosal repair of cleft alveolar bone by z - plasty using bioactive glass demonstrated an alternative easy and simple approach for augmentation of unilateral alveolar cleft patients . hence , we recommend more patients to undergo this new technique and a longer time for follow - up .

multiple literature addresses cleft alveolar bone , with little consensus on the preferred surgical technique . hence , we described a transoral approach for repair of cleft alveolar bone with z - plasty and using bioactive glass . study design : case presentation and surgical technique description . in conclusion , transoral mucosal repair cleft alveolar bone z - plasty using bioactive glass demonstrated an alternative easy and simple approach for augmentation of unilateral alveolar cleft patients .

I C D C Financial support and sponsorship Conflicts of interest

dry eye disease ( ded ) , also known as keratoconjunctivitis sicca , is a complex condition that affects the ocular surface . alterations in the quality and/or quantity of the tear film result in an instable tear film . patients with ded report ocular discomfort and irritation in conjunction with photophobia and blurred / fluctuating vision.1 multiple etiologies may contribute to the various manifestations of ded , with inflammation generally playing a central role in the pathophysiology of the condition.1,2 classification systems have been developed to categorize the range of subtypes of ded , primarily emphasizing the causative factors of the disease as well as the nature of the affected components of the lacrimal functional unit.1,35 the primary subtypes of ded recognized by the classification system presented in the international dry eye workshop ( 2007 ) include aqueous - deficient dry eye and evaporative dry eye . the category of aqueous - deficient dry eye is divided into two main subtypes of sjgren s syndrome ( ss ) and non - sjgren s syndrome ded.1 ss is a systemic autoimmune condition that affects the exocrine glands . ss is a progressive condition that often affects the salivary glands , resulting in symptoms of dry mouth , and the lacrimal glands , causing a form of ded . the exact triggers or causative factors involved in the development of ss are unknown ; however , as a progressive autoimmune condition , chronic stimulation of the immune system likely plays a central role in the pathophysiology.6,7 the diagnosis of a patient with ss often occurs years after the initial onset of symptoms , such as dry mouth and the ocular irritation / discomfort associated with ded , with the average time frame for diagnosis ranging from 3.9 to 10.4 years.8,9 in addition , the detection of autoantibody biomarkers via serological testing is generally required for a confirmatory diagnosis of ss.10 traditionally , the markers for ss have consisted of anti - ss - a ( ro ) or ss - b ( la ) antibodies for primary ss , elevated antinuclear antibodies ( ana ) , or the presence of rheumatoid factor ( rf ) , indicating a diagnosis of ss secondary to another autoimmune condition . recently , a set of novel autoantibodies has been identified that appears to be associated with the early stages of ss . detection of autoantibodies to salivary protein-1 ( sp-1 ) , parotid secretory protein 1 ( psp-1 ) , and carbonic anhydrase vi ( ca-6 ) offers the opportunity to identify the development of an autoimmune condition that may have been missed by screening for only the traditional ss biomarkers.11,12 the diagnosis and treatment of patients with ded can be challenging due to the complex etiology of the condition and a frequent lack of correlation between the signs and symptoms of ded . early identification of ded patients with ss aids in obtaining a confirmatory diagnosis and management of patients with directed treatment in a timely manner . the purpose of the retrospective pilot study was to characterize the biomarkers associated with ss identified in the serological samples of patients with recalcitrant ded ; additionally , the modalities utilized in the treatment of ded were evaluated for subsets of patients with and without serum markers associated with ss . this retrospective clinical pilot study was conducted at a single - center , multispecialty ophthalmology practice . all assessments and patient queries were conducted at the clinical site by certified investigators and technicians . study documents , including the patient data collection file , were submitted to the sterling institutional review board ( atlanta , ga , usa ) for review ; the investigator was granted an exemption from full review , including approval of a waiver of authorization for the use / disclosure of protected health information based on the nature of the study as a retrospective chart review , and a minimal perceived risk to patients as no identifying information is included or presented . the patient profiles presented include data from 48 sequential patients tested for biomarkers associated with ss using the sj serological diagnostic panel ( bausch & lomb , inc . , bridgewater , nj , usa ) with test services and results interpretation provided by immco diagnostics , inc . inclusion criteria for the study were based on a demonstration of recalcitrant ded and the ability to provide a blood sample for serological testing . patients were included in the study who did not demonstrate the expected improvement in their signs and symptoms of ded with traditional treatment modalities . patients reporting dry mouth concurrent with dry eye symptoms were also included in the study population . exclusion criteria for the study centered on the ability to obtain serological test results to quantify the levels of the biomarkers associated with ss . patients were excluded from the study population based on a lack of serological test results ( test results were not available or patients refused to have the test performed ) . patient data were collected from the medical records based on a retrospective chart review . demographic information ( age , sex , and ethnicity ) , sj test results , and concurrent use of the following dry eye treatment modalities were collected from the chart review : artificial tear solutions , artificial tear ointments , microwavable hot mask therapy ( one or more times per day ) , cyclosporine ophthalmic emulsion 0.05% ( restasis , allergan , inc . , irvine , ca , usa ) , intense pulsed - light treatments , and oral omega-3 fatty acid ( re - esterified triglyceride form ) nutritional supplements ( physician recommended nutriceuticals , plymouth meeting , pa , usa ) . blood samples for serological analysis were collected at the study center through a finger prick with a provided lancet in the serological diagnostic panel kit . blood samples were collected on the sample card designed for use with the diagnostic panel and submitted to immco diagnostics for analysis of the serological biomarkers . a study report was returned to the study site for each patient that included measurements for each individual biomarker ( psp-1 , sp-1 , ca-6 , ss - a , ss - b , rf , and ana ) and an interpretation of the results based on the relative levels for each analyte . samples were determined to be positive or negative for each biomarker based on a predetermined threshold titer range , which were noted on the sample report . no calculations were conducted with respect to the sample size of the study population due to the exploratory nature of this pilot study . descriptive statistics were prepared for the patient demographic information of age ( mean , median , minimum , and maximum ) , sex ( % ) , and ethnicity ( % ) . patient subgroups were determined based on the results of the serological testing ; patients testing positive for biomarkers associated with ss , patients testing negative for biomarkers associated with ss , and patients for whom an insufficient quantity of blood sample ( quantity not sufficient [ qns ] ) was indicated on the diagnostic panel study report . additionally , the individual serological markers ( anti - ss - a , anti - ss - b , ana , rf , sp-1 , psp-1 , and ca-6 ) were evaluated ( % of patients who tested positive for each serological marker or marker group ) for the patient subgroup that tested positive for ss biomarkers . additionally , usage of dry eye treatment modalities was analyzed ( % ) for all patients in the study population and for each patient subgroup . six patients were excluded from the study based on a lack of serological test results due to either the patient s refusal to provide a sample for analysis or that the serological test results were not available for evaluation of the patient s immunological biomarkers . a total of 48 patients met the inclusion criteria for the study and were evaluated in this retrospective study analysis . the demographic information collected from the patients is listed in table 1 for the overall study population and the patient subgroups based on the results of the serological testing . a numerically higher percentage of female patients was observed in the subgroup that tested positive for serological biomarkers associated with ss ( 91% vs 83% in the overall study population ; table 1 ) . based on the results of the serological testing for biomarkers of ss , 23% ( eleven out of 48 ) of patients tested positive for biomarkers associated with ss , 62% ( 30 out of 48 ) of patients tested negative for biomarkers associated with ss , and the results for 15% ( seven out of 48 ) of patients were returned designated qns , and were thus unable to be evaluated . the frequency of detection for specific biomarkers is portrayed in figure 1 . novel biomarkers ( sp-1 , psp-1 , and ca-6 ) associated with the early development of ss were detected in 91% ( ten out of eleven ) of the patients who tested positive for ss biomarkers , whereas 27% ( three out of eleven ) of patients tested positive for the traditional ss markers , ss - a and/or ss - b . ana and rf were each detected in 18% ( two out of eleven ) of the patients testing positive for biomarkers associated with ss . treatment options that were utilized by patients in the overall study population , patients testing positive for ss biomarkers , and patients testing negative for ss biomarkers are displayed in figure 2a c , respectively ( treatment modalities utilized by the seven patients for which a qns result was returned in the diagnostic study report are not included in the summary statistics presented in figure 2 ) . in the overall study population , nutritional supplements and artificial tear solutions were reported as the most frequently utilized treatment modalities , followed by hot mask therapy and topical cyclosporine ( figure 2a ) . in patients who tested positive for ss biomarkers oral nutritional supplements , topical cyclosporine , and artificial tear unguents were observed to have higher frequency of usage as compared to the patients who tested negative for serological biomarkers or the overall study population ( figure 2b ) . a higher percentage of patients who tested negative for ss biomarkers reported use of hot mask therapy as compared to the overall population and ss positive patients while the lowest frequency of topical cyclosporine usage was recorded in this subgroup ( figure 2c ) . a higher number of patient samples were returned as qns during the early phase of utilizing the serological diagnostic test ( february july 2014 ) versus the later stage of patient sample collection ( after july 2014 ) . six out of seven patients whose serological samples were returned as qns were submitted in the initial portion of the study . the primary purpose of this study was to characterize the serological biomarkers associated with ss identified in patients with recalcitrant ded . the serological profile of the patients who were determined to be positive for ss biomarkers indicated that nearly all of this subset of patients expressed autoantibodies for the recently identified biomarkers associated with the early development of ss : sp-1 , psp-1 , and ca-6 . these novel biomarkers ( sp-1 , psp-1 , and ca-6 ) associated with the early development of ss were detected in 91% ( ten out of eleven ) of the patients who tested positive for ss biomarkers , whereas 27% ( three out of eleven ) of patients tested positive for the traditional ss biomarkers , ss - a and/or ss - b . therefore , testing patients with recalcitrant ded with these new biomarkers may aid in the earlier diagnosis of ss . the novel biomarkers were also detected in the serological samples of the three patients who tested positive for the classical ss markers ( ss - a and/or ss - b ) , indicating that the autoantibodies to sp-1 , psp-1 , and ca-6 apparently continue to be expressed beyond the early stages of ss . failure to achieve improvement in the clinical signs of dry eye and reported symptoms by the subjects , despite conventional therapy , were the primary criteria used to select patients for serological testing and inclusion in this pilot study . although a more selective set of inclusion and exclusion criteria may be necessary for a multicenter study with a larger patient population , the investigator determined that the reported inclusion criteria were sufficient to warrant serological testing based on clinical experience . despite the relatively small size of the patient population in this single - center study , 23% of the patients tested positive for biomarkers associated with ss a majority of the overall patient population was female ( 83% ) , and the preponderance of female patients was increased in the subgroup of patients who tested positive for ss ( 91% ) . the high ratio of female to male patients identified with ss biomarkers in the present study agrees with the historically reported epidemiological ratio of 9:1 female to male ratio;13 however , the high starting percentage of female subjects in the present study population limits the ability to draw conclusions based on sex distribution of patients testing positive for ss biomarkers . the assessment of the patient profile of treatment modalities for ded revealed that the use of omega-3 nutritional supplements , artificial tears , and hot mask therapy were the most commonly utilized therapies in the overall study population ( figure 2a ) . interestingly , topical cyclosporine ( 0.05% ) was recorded as therapy used by 73% of the subgroup of patients who tested positive for ss biomarkers , as compared to only 27% of patients who tested negative based on serological markers . in contrast , a higher percentage of patients with a negative biomarker test result reported the use of hot mask therapy ( 77% ) in comparison to only 27% of patients who tested positive for ss biomarkers . the inverse profile of these treatment modalities may offer insight into patients who were more likely to present with a more prominent aqueous - deficient component of ded as compared to meibomian gland dysfunction . the limited size of the study population in this pilot investigation limits the ability to draw broad conclusions based on the profile of treatment modalities for the subgroups identified in this study ; additional clinical evaluation would be required to confirm this hypothesis . samples from a total of seven patients were returned as qns regarding the serological analysis . the high rate of samples returned as qns during the early phase of the study ( february july , 2014 ) is attributed to the general inexperience of the clinic staff in obtaining the blood samples to be sent for analysis . once the clinic staff had gained experience in collecting the samples , only one qns result was returned in the latter half of the study period . the relatively high frequency of qns results in the early phase highlights the necessity for consistent staff training and strict adherence to the sample collection procedure . recent modifications in the design of the lancets used to collect the blood samples for analysis and the option to order sample collection via venipuncture for serological analysis from a centralized laboratory may also serve to reduce the frequency of qns results of the diagnostic panel testing . the conduct of this retrospective pilot study at a single - center is a limitation of this study . expansion of the number of study centers and total patient population would enhance the ability to extrapolate the study findings , particularly in regard to the characteristic profile of patients with and without the biomarkers associated with ss . despite the heightened awareness of ss by clinicians and the availability of a centralized serological diagnostic testing , the time lag for the diagnosis of ss remains at several years following the onset of patient symptoms.8,9 as an autoimmune disease , ss is considered a relatively rare condition . in the present study population , 23% of the patients tested positive for serological biomarkers associated with ss , highlighting the potential that ss may be more common than expected in our patients with recalcitrant ded . additionally , the observation in this preliminary data set that ten out of eleven patients who tested positive had antibodies against the novel biomarkers emphasizes the importance of expanding the scope of general testing for ss to evaluate patients for these early immunological indicators in order to avoid misdiagnosis . follow - up evaluation of patients identified as positive for the serological biomarkers associated with ss is an important part of management of their condition . additional measures include referral to a rheumatologist and/or oral medicine specialist for evaluation and salivary gland biopsy , as warranted , to confirm the diagnosis of ss . earlier recognition and diagnosis of the signs and symptoms of ss allow for a confirmatory diagnosis and appropriate management of this systemic , progressive condition .

purposethe purpose was to characterize the biomarkers associated with sjgren s syndrome ( ss ) identified in the serological samples of patients with recalcitrant dry eye disease ; additionally , the modalities utilized in the treatment of dry eye disease were evaluated for subsets of patients with and without ss.patients and methodsdata for this retrospective , single - center , pilot study were based on a chart review of 48 sequential patients with recalcitrant dry eye who were evaluated for ss via serological analysis . data presented include the presence of the autoantibodies identified through the serological biomarker analysis and identification of the concurrent dry eye treatment modalities.resultseleven out of 48 patients ( 23% ) tested positive for biomarkers associated with ss . autoantibodies for salivary protein-1 , parotid secretory protein 1 , and carbonic anhydrase vi , markers associated with the early development of ss , were detected in 91% ( ten out of eleven ) of the patients who tested positive for ss , whereas 27% ( three out of eleven ) of patients tested positive for the traditional ss markers , ss - a and/or ss - b . common treatment modalities utilized in ss patients included omega-3 supplements ( 82% ) , topical cyclosporine ( 74% ) , and artificial tear solutions ( 64% ) , as compared to omega-3 supplements ( 80% ) , hot - mask therapy ( 77% ) , and artificial tear solutions ( 77% ) , in ss - negative patients.conclusionevaluation for salivary protein-1 , parotid secretory protein 1 , and carbonic anhydrase vi biomarkers allows for identification of a subset of patients with biomarkers associated with ss that may not be identified through the traditional assessments ( ss - a / ss - b ) . earlier recognition of ss biomarkers allows for a confirmatory diagnosis and appropriate management of this systemic , progressive condition .

Introduction Materials and methods Results Discussion Conclusion

although cataract surgery is highly effective and relatively safe , owing to the enormous numbers , even uncommon surgical complications could be potentially harmful for many patients . endophthalmitis is one of the most serious complications of cataract surgery , affecting around 0.1% of the cases , and often resulting in severe visual impairment . this complication often occurs sporadically , and in such situations , the common source of infection may be due to the conjunctival flora of the patient . the major pathogens are coagulase - negative staphylococci ( 70% ) , staphylococcus aureus ( 10% ) , streptococci ( 9% ) , other gram - positive cocci , including enterococci and mixed bacteria ( 5% ) , and gram - negative bacilli ( 6% ) . the fact that gram - positive bacteria cause > 95% of the cases reflect the usual pathogenesis , i.e. contamination of the aqueous humor with skin bacteria flora during surgery . however , unusual germs causing the infection are sometimes isolated and should be suspected in cases with a non - typical evolution . a 65 year - old female patient was urgently admitted to a hospital emergency room 48 hours after a cataract surgery , referring to red eye and vision decrease in the operated eye . visual acuity on the initial exam was hands movement in the right eye and 20/40 in the left eye . ophthalmological examinations showed conjunctival injection in the right eye , hypopyon , 3 + cells in anterior chamber and severe vitreitis with no fundus view . she was hospitalized and 23 g pars plana vitrectomy was immediately performed and a vitreous biopsy was taken for culture . additionally , she was treated with topical ocular applications of fortified tobramycin ( 15 mg / ml ) and ceftazidime ( 50 mg / ml ) every hour and with intravitreal injections of vancomycin ( 1 mg/0.1 ml ) and ceftazidime ( 2 mg/0.1 ml ) after the vitrectomy and 2 and 4 days after operation . intravenous antibiotics ( 1 g of vancomycin ) were also administered twice a day , 500 mg ceftazidime/12 hours , as well as the administration of systemic corticosteroid after 24 hours ( oral prednisone 1 mg / kg / day ) . because of the bad evolution after 36 hours , systemic treatment was then empirically changed to linezolid 600 mg and moxifloxacin 400 mg , twice a day . vitreous was cultured in blood agar , chocolate agar ( incubated 48h in microaerophilic conditions ) and thioglycollate broth ; and was isolated in an all media pure culture of gram - positive cocci , forming white colonies and catalase positive , which was identified by the microbiology laboratory as rothia mucilaginosa through matrix - assisted laser desorption / ionization - time of flight ( maldi - tof , bruker daltonics , bruker corporation , billerica , ma , usa ) . antimicrobial sensitivity test was done by the kirby - bauer method , being susceptible to all antibiotics tested ( penicillins , cephalosporins , fluoroquinolones , erythromycin , clindamycin , and tetracycline ) . although this organism was susceptible to the empirical antibiotics used , a bad clinical response was observed with increasing hypopyon . during the next days , pain , conjunctival injection , hypopyon and anterior chamber reaction worsened and a large vitreous abscess was observed . r. mucilaginosa , formerly called stomatococcus mucilaginosus , is part of the normal flora of the upper respiratory tract and oral cavity . it was reclassified into a new genus belonging to the family micrococcaceae in 2000 , based on 16s rrna sequencing . gram staining reveals non - spore - forming , encapsulated gram - positive cocci that can appear in pairs , tetrads , or irregular clusters . it is a facultative anaerobic bacterium , which grows well on most nonselective media and in standard blood culture systems . on sheep blood and chocolate agar , the bacterium forms clear to gray / white , non - hemolytic , mucoid or sticky colonies , which adhere to the agar surface . it can be difficult to distinguish it from coagulase - negative staphylococci , micrococci , and streptococci based on the catalase test result . its inability to grow in 6.5% sodium chloride and its ability to hydrolyze gelatin and esculin distinguish it from species of staphylococcus , micrococcus , and enterococcus genera . identification from automatic methods should correlate with phenotypic identification ; otherwise , genetic sequencing may be required to identify this organism . recently , infections in immunocompetent hosts have been described in various organ systems , including patients with pneumonia , bacteremia , and septic arthritis . it has been reported in two cases of eye infection , a postoperative endophthalmitis in a 91-year - old male and a keratitis in vitamin a deficiency . the known risk factors for this infection are immunosuppression , parenteral drugs , alcoholism , diabetes , neoplastic and valvular disease , but our patient did not present any of these . none of the reported patients with ocular infections caused by r mucilaginosa had these risk factors , so it is possible that in - ocular infections could be irrelevant . in this sense , the source of the infection in our case remains unknown . as mentioned , r. mucilaginosa is part of the normal flora of the upper respiratory tract and oral cavity but has not been isolated in normal conjunctival flora . in our opinion self - contamination of the patient could be the origin of the infection , but contamination of eye drops used in the perioperative period with r. mucilaginosa is also a possibility . unfortunately , a search of r. mucilaginosa in the eye drops was not performed when the patient was admitted in the hospital . it would have been of interest to demonstrate the origin of the contamination . however , it was reported that r. mucilaginosa is able to colonize a foreign body as a vascular catheter . the organism s ability to produce a biofilm , similar to other gram - positive bacteria , is believed to be a key pathogenic mechanism . the physical protective layer provided by the biofilm presumably facilitates adhesion of the organisms to devices and renders them relatively refractory to medical therapy . to our knowledge , this is the second case report of r. mucilaginosa endophthalmitis after a cataract surgery and the first after phacoemulsification . it is increasingly recognized as an emerging opportunistic pathogen associated with eye infections and it may be difficult to identify . physicians should be aware of this organism when treating nonresponding patients infected with gram - positive bacteria in ocular infections .

we aimed at reporting the first case of rapidly progressive acute postoperative endophthalmitis after phacoemulsification cataract surgery in an immunocompetent patient caused by rothia mucilaginosa . an immunocompetent patient manifested endophthalmitis signs 48 hours after an uncomplicated cataract surgery by phacoemulsification . a bacteria of the family micrococcaceae was cultured in the vitreous biopsy , namely r. mucilaginosa . the patient did not show a favorable clinical response after vitrectomy and systemic , intravitreal , and topical fortified antibiotics . the patient s eye was very painful , and consequently , it deemed necessary to perform an evisceration . r. mucilaginosa may be an aggressive etiologic agent for postoperative endophthalmitis . although the isolated r. mucilaginosa was susceptible to empirical treatment , it was impossible to control the infection with standard treatment , probably due to its ability to create a biofilm around the intraocular lens .

Introduction Case Report Discussion Conclusions

bronchogenic carcinoma is the most common malignant lung tumor and accounts for 95% of the lung malignancies . it is divided mainly into two groups : non - small cell carcinoma ( which includes squamous cell carcinoma , adenocarcinoma , and large cell carcinoma ) and small cell carcinoma . one of the rare tumor of lung is pulmonary carcinosarcoma , which is a subgroup of pulmonary sarcomatoid carcinomas . in 2004 , who classified sarcomatoid carcinomas into pleomorphic carcinoma , pulmonary carcinosarcoma contains mixture of malignant epithelial and sarcomatous mesenchymal elements such as cartilage , bone or skeletal muscle . it occurs in elderly age mostly in 6th and 7 decade and is four times more common in males . prognosis of sarcomatoid carcinomas is worse than the bronchogenic carcinoma due to their rapid growth , invasive nature and early metastatic spread and , hence , need for their early diagnosis andtimely treatment . we report a case of elderly male patient who was found to have malignant mass in right lower lobe along with pulmonary metastases and was diagnosed as a case of carcinosarcoma in this report . a 90-years - old male patient came to our hospital with chief complaints of cough with expectoration and breathlessness for one month . chest radiograph was done and showed a large lobulated right lower lobe opacity silhouetting the right dome of diaphragm . provisional diagnosis of malignant lung mass with pulmonary metastases was made [ figure 1 ] . chest radiograph shows large mass in right lower zone silhouetting the dome , associated with multiple rounded radio - opacities in bilateral lung fields s / o lower zone malignant mass with pulmonary metastasis . also noted is fibrocavitatory lesion in right apical region computed tomography ( ct ) scan of chest was done with intravenous contrast for further evaluation on siemens somatom perspective , 128 slice . ct scan showed a large cavitating heterogeneous mass with lobulated margins measuring approximately 8.7 8.2 7.4 cm in the lower lobe of right lung [ figure 2 ] . inferiorly the mass was invading the diaphragm and infiltrating directly into the liver involving segment vii of right lobe of liver [ figure 4 ] and [ figure 5a and b ] . postero - laterally , the mass was invading the pleura [ figure 2 ] and chest wall with associated pleural thickening and subtle rib erosion , which suggested the mass to be malignant . in addition , multiple bulky lobulated hypodense masses were seen in both lung fields suggesting pulmonary metastases , one of them in right para - vertebral region was invading the mediastinum . foci of calcification were seen within the larger metastatic masses [ figure 6a and b ] and [ figure 5a and b ] . axial ct scan shows a large heterogeneous hypodense lobulated peripheral lung mass showing few calcific foci and few small areas of cavitation . mass is invading the adjacent pleura lung window shows a large mass with lobulated margins , surrounding mild ground glass haze with small cavitating areas mass is highly invasive showing infiltration of right dome with direct transdiaphragmatic infiltration into the posterior segment of right lobe of liver ( a - b ) coronal and sagittal reformatted images show transdiaphragmatic invasion of mass into liver with few calcific foci . multiple pulmonary metastatic lesions are seen with few calcific foci seen in larger ones . also noted is an old cavity in right apical region- old tubercular infection ( a and b ) axial ct scan shows bulky hypodense deposits in right paraspinal region and right lower lobe with smaller ones in posterior subpleural region bilaterally . note presence of small calcific foci within the larger metastatic lesions sequelae to old pulmonary koch 's were also seen in form of thick walled cavity and few fibrotic lesions in upper lobes and right middle lobe with foci of calcification within them . on basis of chest ct findings : highly invasive bulky lobulated hypodense mass infiltrating the diaphragm and liver inferiorly and chest wall laterally along with lobulated metastases in both lung fields and coarse foci of calcification within the primary tumor and metastases.- possibility of some sarcomatous component within the primary lung tumor along with metastases was considered . these features are unusual to bronchogenic carcinoma , which usually shows spiculated ill - defined margins and does not usually infiltrate into the liver directly . amorphous foci of calcification may be seen within the primary bronchogenic carcinoma but is not seen in its metastases . ultrasound - guided fine needle aspiration cytology ( fnac ) of the primary tumor was done . it showed extensive necrosis and scattered squamous cells having hyperchromatic , pleomorphic nuclei along with few tadpole cells and anucleate squamous cells [ figure 7 ] and [ figure 8 ] . ( hemotoxylin and eosin ) stain of fnac of primary lung tumor shows cluster of malignant squamous epithelial cells showing hyperchromatic , pleomorphic and irregular nuclei and bluish cytoplasm few tadpole cells are also seen patient was referred to another hospital for treatment of malignancy . fine - needle percutaneous biopsy of the primary tumor was done there under ultrasound guidance . few neoplastic osteoids and ossification was seen in addition to necrosis and malignant squamous cells . so final diagnosis of carcinosarcoma was made consisting of mainly squamous cell carcinoma and component of osteosarcoma . foci of calcification seen in the primary tumor and metastasis on chest ct was metaplastic osteosarcomatous component . pulmonary carcinosarcoma , a subtype of sarcomatoid carcinomas of lung is a rare lung tumor consisting of admixture of malignant epithelial and mesenchymal elements . there is strong association of this tumor with cigarette smoking , as was seen in our case . carcinosarcomas can present either as central endo - bronchial type or peripheral intra - parenchymal type . central endobronchial tumors have a better prognosis than the peripherally invasive tumors because of earlier development of symptoms due to bronchial obstruction and hence earlier treatment . it occurs more commonly in elderly males ( male : female ratio is 4:1 ) . these tumors are usually bulky ( > 5 cm ) and have a high tendency to invade the adjacent structures like pleura , chest wall , diaphragm or mediastinum depending upon the location . large low attenuation areas are found on ct that corresponds to regions of necrosis and myxoid degeneration . most common carcinomatous component is squamous cell carcinoma ( 69% ) as was seen in our case followed by adenocarcinoma ( 20% ) or large cell carcinoma ( 11% ) . sarcomatous component is mostly chondrosarcoma or osteosarcoma followed by rhabdomyosarcoma and rarely fibrosarcoma or mixture of these . calcification may be observed within the primary tumor as well as metastases on ct scan , which likely represents osteosarcomatous or chondrosarcomatous component of the tumor . prognosis is poor and median survival rate at six months is 27% and depends upon the size of sarcomatous component . once the tumor is disseminated , most of the patients die within few weeks to months from the progressive disease . we conclude that whenever we see a bulky lobulated and highly invasive lung tumor with likely invasion of adjacent structures on ct scan along with bulky metastases and amorphous /coarse foci of calcification within the primary tumor as well as metastases , possibility of some sarcomatous component should be considered .

pulmonary carcinosarcoma is a rare lung tumor , which contains both malignant carcinomatous and heterotopic sarcomatous components . there are only few case reports on the imaging diagnosis of this rare tumor . herein , we present the radiological findings of this rare tumor , which was suspected on computed tomography ( ct ) scan due to atypical ct findings of malignant lung mass ( not usually seen in bronchogenic carcinoma ) and was finally confirmed histologically .

INTRODUCTION CASE REPORT DISCUSSION CONCLUSION

the institute of medicine ( iom ) has defined patient safety as the prevention of harm to patients . patient safety includes risk assessment , identification , management of patient related risk , reporting and analysis of incidents and the capacity to learn from and follow - up incidents and implement solutions to minimize the risk of the reoccurrence of the incident . international studies have revealed that the rate of medical errors was 5 - 80/100,000 consultations in primary health - care ( phc ) settings , with prescription errors of 11% and adverse event rate at 3.7/100,000 clinic visits . medical record based study from general practice in holland revealed a prevalence of 2.2% safety incidents in 1000 patient records . in saudi arabia , patient safety and medical errors have become an important national issue discussed by the media in the past decade . however , only a few studies have been conducted on this subject . of these studies only one discussed the knowledge and attitude of undergraduate medical students , two others discussed patient safety culture in hospital , and one reported the status of medical liability claims in saudi arabia . another study explored the preference and perception of the public on the disclosure of medical errors . a study in a phc setting in riyadh city by khoja et al . , found that there were about 19% errors in prescriptions issued by doctors working at primary health - care centers ( phcc ) . one recent study was conducted at an academic center to assess the attitude of nurses toward patient safety . this scarcity of studies indicates that more studies on patient safety are required and medical errors in the provision of health - care , particularly the knowledge and attitude toward these issues have to be explored . the objective of this study was to assess the attitude of phc physicians in aseer region , saudi arabia toward patient safety . this cross - sectional study was conducted among physicians working at phcc in aseer region , saudi arabia , in august 2011 . a self - administered questionnaire consisting of three parts was used to achieve the objective of this study . the first part of the questionnaire dealt with the socio - demographics , the academic and work profile of participants . this questionnaire was developed and validated by carruthers et al . for use among students and tutors in uk this questionnaire was found to have reliability ranges between 0.64 and 0.82 for the nine factors namely ; 1-patient safety training received , 2-errors reporting confidence , 3-working hours as error cause , 4-error inevitability , 5-professional incompetence as error cause , 6-disclosure responsibility , 7-team functioning , 8-patient involvement in reducing errors , 9- and importance of patient safety in curriculum . items were assessed on a likert scale of seven points ( 7 = strongly agree , 1 = strongly disagree ) . attitude to patient safety was classified as positive , neutral or negative depending on scores given for each item : a score of less than four was negative attitude / disagree , four was neutral and a score of five or more indicated a positive attitude / agree . for questions ( 11 - 17 ) and question , ( 25 ) attitude was considered positive / agree if the score was less than 4 , neutral if the score was four and negative / disagree if it was five or greater . the last part of the questionnaire developed by the investigator consisted of three questions ; 1-receiving training on the phcc doctors who had attended family medicine essentials course in aseer region and agreed to participate in this study were invited to complete the questionnaire under the direct supervision of the investigator , who explained the purpose and the importance of this study . permission from the authors of the apsq - iii was obtained to use the questionnaire through e - mail contact . data of the questionnaire were coded and then entered into personal computer provided with statistical package for the social sciences spss version 15 for statistical analysis . appropriate statistical tests ( chi - square , anova ) were used accordingly and p values were considered significant if > 0.05 . the total number of participants in this study was 228 doctors out of 365 ( 65% ) physicians working at phcc in aseer region at the time of the study , giving a response rate of 65% . the mean age was 39 years ; 77% were males ; saudi doctors represented 18% and 80% had no post mbbs qualifications . the average experience of work at phcc was 8.3 years and the average number of patients seen daily was 44 patients . less than one - third ( 30% ) had attended a course on patient safety and only 52% defined medical error correctly . characteristics of physicians at phcc , aseer region 2011 ( n=228 ) statistical analysis showed that 20% of saudi doctors , 42% of arab doctors and 20% of non - arab doctors had attended a course on patient safety ( = 9 , p = 0.01 ) . those who had worked at a phcc for more than 5 years had attended such courses compared to those who had worked for > 5 years ( = 12 , p = 0.006 ) . the older doctors tended to attend courses related to patient safety more than young doctors ( = 8 , p = 0.02 ) . saudi doctors and those who had worked at a phcc for less than five years defined medical error correctly compared to the non - saudi ( = 20 , p = 0.01 ) and those who had worked at a phcc for more than 5 years ( = 22.5 , p = 0.03 ) . table 2 depicts the mean scores of questions assessing attitudes toward patient safety in nine areas . attitude of phc physicians toward patient safety , aseer region , ksa , 2011 the best score was given for reducing medical errors ( 6.2 points ) , followed by role of training and learning on patient safety ( 6 and 5.9 points ) . however , participants were not satisfied with undergraduate training on patient safety ( 4.8 points ) . confidence in reporting medical errors and reporting the errors of other people scored 4.8 points and the confidence to be open with supervisor about an error had made scored 4.6 points . regarding the relationship between working hours and occurrence of medical errors , it was thought that shorter shifts would reduce medical errors ( 5.4 points ) , not taking break would increase the risk of medical errors ( 5.7 points ) while the number of hours a doctor worked would increase the likelihood of medical error ( 5.4 points ) . concerning the inevitability of errors , it was agreed that even the most experienced and competent doctors make errors ( 5.7 points ) , on the other hand , participants disagreed with the statement that most medical errors resulted from the carelessness of nurses ( 5.9 points ) or careless doctors ( 4.9 points ) . reporting all medical errors was considered necessary even if they caused no harm to patients ( 3.9 points ) while teaching teamwork skills would reduce medical errors ( 6.2 points ) . patients have an important role in preventing medical errors and their involvement was considered important in reducing the risk of medical errors ( 3 and 3.9 points respectively ) . teaching students about patient safety was considered a priority in the training of medical students ( 5.6 points ) . learning about patient safety before graduating would help doctors to become more effective ( 6.2 points ) . table 3 shows association between socio - demographic variables and attitude toward patient safety . statistical analysis revealed that females scored higher points than males in many questions ( numbers 1 , 2 , 14 , 19 , 23 , 24 ) ( p = 0.00 - 0.04 ) . association between some demographic variables and mean score for some items in apsq - iii using analysis of variance ( anova ) showed that non - arab doctors scored higher points than arab and saudi doctors for the first eight questions and question numbers 12 and 18 ( p values between 0.00 and 0.03 ) , those above 45 years , scored the highest points compared to those between 36 and 45 years and less than 36 years old in nine questions with different p values ranging from 0.00 to 0.04 for questions numbers ( 1 , 2 , 3 , 6 , 10 , 12 , 14 , 20 , 21 ) . those doctors who had worked for more than 20 years were found to have a positive attitude compared with those who had worked between 11 - 20 years , 5 - 10 years and less than five years for nine questions ( 2 , 3 , 6 , 9 , 10 , 14 , 19 , 20 , 21 ) with p values ranging between 0.00 and 0.02 . regarding the association between qualification and attitude toward patient safety , it was found that those with high qualifications had a positive attitude ( highest points ) toward patient safety as observed in question numbers ( 7 , 8 , 15 , 17 , 21 , 24 ) with p values between 0.001 and 0.04 . doctors who had training on patient safety was found to have positive atitude ( items number 2 , 6 , 13 , 23 ) [ table 4 ] . impact of training on some aspects of attitude of phcc doctors toward patient safety , aseer region , 2011 most of phcc doctors in aseer region showed a positive attitude toward patient safety . however , 70% had not attended any training course on patient safety , which was reflected in their knowledge , which was 52% and 61% were able to define medical error and knew the main causes of medical errors . such important findings indicate that there is urgent need for such interventions as conducting continuous professional development programs on medical errors and patient safety for phc physicians . in spite of the importance of patient safety in the medical school curriculum , ( 2010 ) , mentioned that more than two - thirds of medical students agreed that the teaching of patient safety in medical schools and the continuous training of health staff was necessary . another study from hong kong ( 2009 ) showed that 90% of medical students strongly agreed that patient safety was an important topic for teaching in medical school . those findings should alert us on the issue of the curricula of medical colleges in ksa and the importance of making reporting medical errors is important step in improving the quality of health - care including patient safety . in other words , about one third of the participants were comfortable with reporting medical errors . these low scores which reflected the negative attitude of some physicians toward reporting adverse events could be due to the lack of a system for reporting medical errors . also , a culture of safety in health settings , as reported by alahmadi is yet to be developed . the confidence of participants in talking about their errors with their supervisor if harm is caused to the patient was given an average score of 5.6 and majority of participants ( 78% ) said they would do so . this rate is in agreement with the findings of a study conducted of gps in denmark ( 2006 ) , which revealed that more than 75% of gps would report adverse event that occurs . in the few phcc with a high number of patients attending and work overload , participants showed that a shorter shift for the doctors and taking breaks would reduce medical errors . the area of errors inevitability scored 3.2 - 5.9 points regarding competency and experience of doctors . these findings are comparable to those reported from uk . as reported by hammami et al . ( 2009 ) the disclosure of medical errors poses a big dilemma in health settings in saudi arabia . in this study , doctor had the responsibility of disclosing errors to patients only if they resulted in harm to the patient , while 40% disagreed and 16% were neutral . on the other hand , most participants ( 79% ) agreed to report all medical errors while 12% disagreed . a study of medical trainees ( 2006 ) found that 80% would disclose adverse events to patients . these diversity of attitude could be explained by the lack of training and experience of phcs in dealing with medical errors . in addition , there is a lack of established culture of reporting adverse events in phc settings in saudi arabia . most participants ( 91% ) agreed that the teaching of teamwork would reduce medical errors . these high scores confirm the vital role played by the teaching of teamwork and leadership in patient safety mentioned by firth - cozens . patients play an essential role in their health and safety by active involvement in decision making and self - management . phcc physicians showed average to high agreement ( 84% ) in this regard ( 4.9 and 5.9 points ) compared to 5.2 points in uk study , but less than what was reported from saudi arabia ( 44.7% ) . the last theme of the study was about the importance of patient safety in the curriculum . a total of 90% of the participants with a high average score of 5.6 out of seven points agreed that teaching students about patient safety is a priority in medical training while 84% with the highest score of all items in the questionnaire agreed that learning about patient safety before graduation from medical schools would produce more effective doctors , these important findings are in agreement with results of two studies conducted among medical students in the qassim region and hong kong , which emphasized the priority of putting patient safety in the undergraduate curriculum of medical colleges . in this study , attitudes toward patient safety were affected to some degree by some demographic and scientific background such as gender , age group , nationality , qualification and work experience at phcc . female doctors had a more positive attitude toward patient safety than male doctors , which was difficult to explain . the doctors with more experience in phcc had a more positive attitude toward patient safety than those with a short experience . it is known that as doctors got older and more experienced , their attitude to their profession changed for the better . it was found that doctors with high qualifications had a positive attitude toward patient safety . such a finding could be either because they had taken some courses or their training had included some sessions on patient safety and medical errors . the impact of training on patient safety and attitude was not always positive as only a few items ( items no 2 , 6 , 13 , 23 ) were positively affected by training . these associations should be interpreted carefully as less than one - third of the participants had had a course on patient safety . this study showed that phc physicians in aseer region had a positive attitude toward patient safety . undergraduate education on patient safety considered a priority to make future doctors effective was inadequate . teamwork approach and patient involvement were given a priority in the prevention of medical errors and the improvement of patient safety . phc physicians in aseer region should have training sessions on patient safety and medical errors . further studies are needed to explore the knowledge and behaviors of phcc physicians on patient safety and medical errors . phc physicians in aseer region should have training sessions on patient safety and medical errors . further studies are needed to explore the knowledge and behaviors of phcc physicians on patient safety and medical errors .

objective : the objective of this study was to assess the attitude of physicians at primary health - care centers ( phcc ) in aseer region toward patient safety.materials and methods : this study was conducted among working primary health - care physicians in aseer region , saudi arabia , in august 2011 . a self - administered questionnaire consisting of three parts was used ; the first part was on the socio - demographic , academic and about the work profile of the participants . the attitude consisting of 26 questions was assessed on a likert scale of 7 points using attitude to patients safety questionnaire - iii items and the last part concerned training on patient safety , definition and factors that contribute to medical errors . data of the questionnaire were entered and analyzed by statistical package for the social sciences ( spss ) version 15.results:the total number of participants was 228 doctors who represent about 65% of the physicians at phcc , one - third of whom had attended a course on patient safety and only 52% of whom defined medical error correctly . the best score was given for the reduction of medical errors ( 6.2 points ) , followed by role of training and learning on patient safety ( 6 and 5.9 points ) , but undergraduate training on patient safety was given the least score . confidence to report medical errors scored 4.6 points as did reporting the errors of other people and 5.6 points for being open with the supervisor about an error made . participants agreed that even the most experienced and competent doctors make errors ( 5.9 points ) , on the other hand , they disagreed that most medical errors resulted from nurses carelessness ( 3.9 points ) or doctors carelessness ( 4 points).conclusion : this study showed that phcc physicians in aseer region had a positive attitude toward patient safety . most of them need training on patient safety . undergraduate education on patient safety which was considered a priority for making future doctors work effective was inadequate .

INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION Recommendations

data uncertainty widely exists in realistic problems due to their random nature , measurement errors , or other reasons . as a result , decision making inherently involves consideration of such uncertainties since the solution of an optimization problem often exhibits high sensitivity to data perturbations , and ignoring the uncertainty could lead to suboptimal or even infeasible solutions . in past decades , developing optimization methods and tools to facilitate decision making under uncertainty has become one of the most important topics in both the operations research community and also the process systems engineering community . robust optimization belongs to an important methodology for dealing with optimization problems with data uncertainty . this type of method enforces the constraint satisfaction for all possible realizations of uncertain parameters inside a predefined uncertainty set . comparing it to other methodologies that deal with uncertainty , one major motivation of robust optimization is that in many applications the data set is an appropriate notion of parameter uncertainty , especially for those cases that the parameter uncertainty is not stochastic , or for instances where no distributional information is available . one of the earliest papers on robust counterpart optimization is the work of soyster , who considered simple perturbations in the data and aimed to find a reformulation of the original linear programming problem such that the resulting solution would be feasible under all possible perturbations . the approach admits the highest protection and is the most conservative one since it ensures feasibility against all potential realizations . thus , it is highly desirable to provide a mechanism to allow for the trade - off between robustness and performance . the work by ben - tal and nemirovski , el - ghaoui et al . , and bertsimas and sim investigated the framework of robust counterpart optimization by introducing different types of uncertainty sets . ben - tal and nemirovski proposed the ellipsoidal set based robust counterpart formulation . el - ghaoui and lebret introduced a robust optimization approach for least - squares problems with uncertain data . bertsimas and sim studied robust linear programming with coefficient uncertainty using an uncertainty set with budgets . in this robust counterpart optimization formulation , a budget parameter ( which takes a value between zero and the number of uncertain coefficient parameters in the constraints and is not necessarily integer ) is introduced to control the degree of conservatism of the solution . developed the theory of the robust optimization framework for general mixed - integer linear programming problems and considered both bounded uncertainty and several known probability distributions . the robust optimization framework was later extended by verderame and floudas and they studied both continuous ( general , bounded , uniform , normal ) and discrete ( general , binomial , poisson ) uncertainty distributions and applied the framework to operational planning problems . the work was further compared with the conditional value - at - risk based method in verderame and floudas . in the first two parts of this paper series , we systematically studied the set induced robust counterpart optimization technique for linear and mixed integer linear optimization problems . different uncertainty sets were extensively studied , including those studied in literature and novel ones were introduced in this work . the relationship between different representative uncertainty sets was discussed , and their corresponding robust counterpart formulations for both linear optimization ( lp ) and mixed integer linear optimization ( milp ) problems were derived . probabilistic guarantees on constraint satisfaction for the robust solution of those different uncertainty set induced robust counterpart optimization models were derived , for both bounded and unbounded uncertainty , with and without detail probability distribution information . a key element in applying the robust optimization framework is the selection of the type and the size of the uncertainty set , which is strongly related to the desired reliability of the solution ( i.e. , the probability of constraint satisfaction ) . it is known that chance constraint / probabilistic constraint is the most direct way to enforce the reliability of the solution of an optimization problem , where the reliability is expressed as a minimum requirement on the probability of satisfying constraints . for example , even evaluating the distribution of a sum of uniformly distributed independent random variables is very difficult . when the program has structural properties that allow for an equivalent deterministic formulation , a chance constrained problem can be converted to a deterministic problem and can be solved directly . however , if the model does not admit sufficient structure that can be exploited , an approximation method has to be used . the various approximation methods can be divided into sampling based methods and analytical approximation based methods . first , sampling based methods are designed based on the assumption that it is possible to draw observations from the distribution of the uncertainty . sampling based methods fall broadly into two categories : scenario approximation and sample average approximation . for scenario approximation , it draws a finite number of samples from a given distribution , and enforces all sampled constraints to hold . sample average approximation refers to replacing the distribution with another easy - to - use distribution , typically the empirical distribution determined from a sample drawn from the original distribution . while solving the approximation problem represents one aspect of complexity , the size of the sample required to guarantee the quality of the approximation is another important limitation . second , analytical approximation methods are based on either robust optimization or well - known probability inequalities . since the type and the size of the uncertainty set is determined based on an initial assumption on the constraint satisfaction and the a priori probability bound formulation , robust optimization provides a safe approximation of probabilistic constraint . in contrast to sampling based approximation , robust optimization based approximation is a promising deterministic alternative for certain classes of chance constrained problems . in addition , other forms of deterministic analytical approximation use probability inequalities , such as the markov inequality , chebyshev s inequality , bernstein s inequality , hoeffding s inequality , etc . although robust optimization has been used widely in different areas to achieve solution robustness / reliability , the quality of the solution is often ignored . in other words , while the desired solution feasibility ( i.e. , desired probability of constraint satisfaction ) is met , how far is the solution from optimality ? in this work , we will first illustrate the above issues and then propose an iterative strategy for improving the robust solution . in the proposed method , the tight a posteriori probability bounds are used to improve the robust solution within an iterative framework . compared to the single - step classical robust optimization method , the quality of the robust solution can be improved . on the other hand , compared to the pure a posteriori probability bound based methods , the proposed method has the advantage that it does not require the global optimization of nonconvex problem . we first present the problem of optimization with probabilistic guarantee on constraint satisfaction , that is , probabilistically constrained problem , and then introduce the traditional robust optimization based approximation framework . next , the a posteriori probability bound based approximation framework is presented in section 3 . , we present a novel iterative framework which combines the advantage of the previous two different methods . the proposed method and the traditional methods are studied through production planning and process scheduling problems in section 5 , and the paper is concluded in section 6 . consider the following linear optimization problem1where x and xj ( j = 1 , ... , n ) can be either continuous or integer variables , the left - hand - side ( lhs ) constraint coefficients ij are subject to uncertainty , and ji represents the index subset that contains the variable indices whose corresponding coefficients are subject to uncertainty . j ji with aij being the nominal value and ij being a constant perturbation amplitude ( ij > 0 ) , { ij}jji are random variables which are subject to uncertainty . with the above definition , the ith constraint in problem 1 can be rewritten as the follows:2 in many practical applications , enforcing constraint satisfaction for all possible values of the uncertain parameters ( i.e. , worst - case scenario ) can be too costly or even impossible . probabilistic constraint ( also called chance constraint ) provides a compromise to avoid this situation and ensures that the constraints are satisfied under certain given probability . a probabilistic version of the above constraint is written as follows so that a probabilistic guarantee on constraint satisfaction is applied:3or an upper bound on the probability of constraint violation is applied4where ( 0 < for instance , = 0.05 means that the constraint must be satisfied with a probability larger than 0.95 or the probability of constraint violation must be less than 0.05 . while joint probabilistic constraints are alternative for modeling solution reliability , individual probabilistic constraints are investigated in this paper . consider the following linear optimization problemand assume that the lhs constraint coefficients of the constraints are uncertain and subject to uncertainty with 11 = 10 + 11 , 12 = 20 + 212 , 21 = 6 + 0.621 , and 22 = 8 + 0.822 and 11 , 12 , 21 , 22 are independent uncertain parameters uniformly distributed in [ 1,1 ] . for the above problem , if we set the allowed violation probability for each of the constraint as 0.05 , then the probabilistic constrained version of the problem is in set induced robust optimization , the uncertain data is assumed to be varying in a given uncertainty set and the aim is to choose the best solution among those immunized against data uncertainty . for constraint 2 , the set induced robust optimization method aims to find solutions that remain feasible for any in the given uncertainty set u so as to immunize against infeasibility , that is5the corresponding robust optimization problem is6for different uncertainty sets , the robust counterpart formulation is distinct . furthermore , under specific probability distribution assumption , the probabilistic guarantee on the constraint satisfaction can be quantified using the size of the uncertainty set . in our previous work , we have systematically derived the robust counterpart formulations under different uncertainty sets and also derived their probability bounds on constraint violation . for example , if the uncertainty set is given by a box7where is the size of the box , then the robust optimization counterpart constraint is8if the uncertain parameters are subject to independent bounded symmetric distribution , then the following a priori probability bound is valid9a priori probability bound means that if the size of the box set is , then the solution of the robust optimization problem will ensure that the probability of constraint violation is less than or equal to the following bound:10 in the literature , the traditional way of applying robust optimization to solve the probabilistically constrained problem is as follows . first , the reliability level in the probabilistic constraint is set , and the type of the robust optimization model ( i.e. , uncertainty set ) is selected by the distribution of the uncertainty . next , the size of the uncertainty set is evaluated based on the a priori probability bounds . for example , assuming that the box type uncertainty set is selected for applying robust optimization , the size of the uncertainty set can be determined by the following problem11using the size parameter value determined from the above problem , the robust counterpart optimization problem can be solved and the solution ensures that the constraint is satisfied with the desired probability 1.12as a summary , the traditional framework of applying robust optimization to address the probabilistic guarantee on constraint satisfaction is shown in figure 1 . the robust optimization problem provides a safe and conservative approximation of the probabilistically constrained problem . notice that the minimum possible value for is used to find the best possible solution within this framework . in the following , we illustrate the approximation of the probabilistic constrained problem using the robust counterpart optimization through the motivating example . uncertainty set induced robust counterpart optimization model is applied to solve the motivating example problem . the robust counterpart optimization problem under the interval + ellipsoidal uncertainty set is as followswhere 1 and 2 are parameters determining the size of the interval + ellipsoidal uncertainty set . using the probability bound on constraint violation for this type of robust counterpart optimization model ( under the assumption that the uncertainty is bounded and symmetric which is satisfied in this example)we obtained the smallest possible value : 1 = 2 = 2.4477 . the above robust optimization problem is convex and can be efficiently solved using convex nonlinear optimization solvers . with this value , the robust counterpart optimization problem can be solved , the optimal objective value is obj * = 90.9091 and the robust solution is x = ( 7.2727 , 2.7273 ) . this solution ensures that the constraints are satisfied with the desired probability 0.95 on constraint satisfaction . once a robust solution is obtained , the probability of constraint violation can also be quantified by a posteriori probability bound . in our previous paper if the probabilistic distribution information on the uncertain parameters is known , then the following relationship holds:13 in the above derivation , is an arbitrary positive number . as studied in our previous work , with the above probability inequality , we can evaluate the a posteriori probability bound on constraint violation as follows once we have a set of solution x , ( i.e. , we have xj as the solution):14notice that in the above equation , a minimization with respect to ( i.e. , only one variable ) is performed to find the tightest probability bound . for the traditional robust counterpart optimization based framework , the adjustable parameter defining the size of the uncertainty set is initially selected based on the a priori probability bound which is a function of the adjustable parameter . however , usually , the resulting solution could be too conservative , since the actual probability of constraint violation is much smaller than the bound . for example , with the robust solution x = ( 7.2727 , 2.7273 ) obtained for the motivating example , the probability of constraint violation can be evaluated using the above a posteriori probability bound 14 , and the following upper bounds on constraint violation for the two constraints can be calculated : which are far less than the desired violation probability 0.05 . this implies that the obtained robust solution is conservative and there is room for improvement . while the a posteriori probability bound can be used to check the probability of constraint satisfaction with a given solution , it can also be used in another way to formulate a safe approximation of the probabilistic constraint . using inequality 13 , the following safe approximation of 4 is obtained:15because for any feasible solution satisfying 15 , it also satisfies the constraint 4 . constraint 15 can be further rewritten as16and finally the following safe approximation of the probabilistic constrained problem is obtained:17note that while for any fixed value of i > 0 , 15 is an approximation of the original probabilistic constrained problem , here i is set as a decision variables in problem 17 so as to find the tightest possible approximation and to seek the best possible solution . the a posteriori probability bound based framework addressing the probabilistic constraint can be represented using figure 2 . it is seen that the approximation optimization problem is constructed based on the selected a posteriori bound , in comparison to the selection of uncertainty set in the traditional robust optimization framework . , then the explicit formulation of above approximation problem depends on the moment generating function e[e ] . for several known distributions , their probability density functions and moment generating functions are listed in table 1 , which can be substituted into 17 and the resulting problem can be solved as a deterministic optimization problem . following the derivation in this section , the probability inequality based safe approximation of the probabilistic constrained problem of the motivating example is obtained : since the random variable 11 is subject to uniform distribution in [ 1 , 1 ] , we haveevaluate the expectation terms in the similar way and finally the following problem is obtained : the above problem is a nonconvex optimization problem , which can be solved through a deterministic global optimization approach . we solve the above problem through global optimization solver antigone 1.1 in gams 24.2.2 ( with relative optimality gap tolerance optcr = 0 and resource limit reslim = 10000 ) and obtain the following solution after 10 000 s ( with a relative gap 0.11% to the upper bound 92.33):comparing the above solution with the solution from the traditional robust optimization framework , it is observed that while both solutions ensure the desired probability on constraint satisfaction , the a posteriori probability bound based method generates a solution which is better than the classical method . comparing the previous two methods , the following observations can be made:(1)in terms of the information needed , the a posteriori probability bound based approximation method needs the exact probability distribution function while the robust optimization method only needs partial information . for instance , in the studied robust optimization formulations , the assumptions on uncertainty are only bounded and symmetric so that a probabilistic guarantee is valid.(2)in terms of the solution complexity , the probability inequality based approximation problem can be nonconvex and global optimization is necessary ( i.e. , higher computation complexity ) . the robust optimization based approximation leads to convex problem which can be solved very efficiently.(3)in terms of the quality of the solution , the a posteriori probability bound based approximation method leads to less conservative solution because it is tighter than the a priori probability bound as illustrated in previous work . in terms of the information needed , the a posteriori probability bound based approximation method needs the exact probability distribution function while the robust optimization method only needs partial information . for instance , in the studied robust optimization formulations , the assumptions on uncertainty are only bounded and symmetric so that a probabilistic guarantee is valid . in terms of the solution complexity , the probability inequality based approximation problem can be nonconvex and global optimization is necessary ( i.e. , higher computation complexity ) . the robust optimization based approximation leads to convex problem which can be solved very efficiently . in terms of the quality of the solution , the a posteriori probability bound based approximation method leads to less conservative solution because it is tighter than the a priori probability bound as illustrated in previous work . the aforementioned observations show that there is a trade - off between the two different types of approximations . to fully take advantage of both of them , an iterative solution framework , which is also the major contribution of this paper , is proposed to combine the use of the traditional robust optimization approximation and the a posteriori probability bound . the objective is to improve the quality of robust solution while still ensure the probabilistic guarantee of the robust solution . at the same time , the computational complexity is decreased comparing to the a posteriori probability bound based approximation method . the proposed solution framework is shown in figure 3 , which can be detailed as follows . initially , the type of the uncertainty set ( i.e. , the robust optimization formulation ) is selected . based on the desired degree of constraint satisfaction , the smallest possible size of the uncertainty set is determined using the specific a priori probability bounds ( e.g.,exp(/2 ) , exp(/2|ji| ) , etc . ) of that type of robust formulation . with the determined size of the uncertainty set , the robust counterpart optimization problem is solved and the robust solution is obtained . then , an upper bound on the constraint violation is evaluated using the derived solution and the a posteriori probability bound . if the gap between them is larger than a certain predefined tolerance , the size of the uncertainty set is adjusted and the robust optimization problem is solved again . the size adjusting is the important step in the algorithm . based on the fact that the a posteriori probability upper bound is monotonically decreasing function of the set size , this adjustment can be made heuristically : if the probability upper bound exceeds the desired level , the set size should be decreased ; if the bound is below the desired level , the set size should be increased . the above procedure is repeated until the gap between the desired degree of constraint violation and the computed upper bound on constraint violation is less than the tolerance . finally , the solution from the last round robust optimization step is reported . a pseudo code of the iterative algorithm is given as follows : notice that in the proposed iterative framework , the problem of evaluating the a posteriori probability bound using the right - hand side ( rhs ) of 14 is an optimization problem . since any feasible solution of the rhs of 14 will be a valid a posteriori upper bound on the probability of constraint violation , furthermore , since x is a known solution and taken fixed value , the rhs of 14 is a single variable optimization problem , which can be solved relatively efficiently . proposed iterative solution framework to improve the quality of robust solution . applying the proposed framework and using the interval + ellipsoidal uncertainty set based robust counterpart optimization formulation , we obtain the following results for the motivating example as shown in table 2 for different iterations ( i.e. , k stands for iteration ) : the detailed solution procedure is explained as follows : step 1 : initialize 1satisfy = 2.4477 , 1violate = 0 , 2satisfy = 2.4477 , 2violate = 0 using the a priori bound . set tolerance parameter = 0.01.step 2 : set 1 = 1satisfy = 2.477 , 2 = 2satisfy = 2.477.iteration 1step 3 : solve the robust optimization problem and obtain solution obj * = 90.91 , x1 = 7.2727 , x2 = 2.7273.step 4 : compute the a posteriori probability bound from solution p1 = 2.51 10 , p2 = 3.46 10.step 5 : since the probability violation upper bound is less than 0.05 for both constraints , there is room to contract the uncertainty set and improve the solution . so decrease the size of both uncertainty sets based on the value in iteration 1 : 1 = 2 = ( 0 + 2.4477)/2 = 1.2238.iteration 2step 3 : solve the robust model and obtain obj * = 91.807 , x1 = 7.2745 , x2 = 2.8009.step 4 : compute the a posteriori probability bound p1 = 0.0305 , p2 = 0.0205.step 5 : since the probability violation upper bound is still less than 0.05 for both constraints , it is necessary to further decrease the size of both uncertainty sets based on the size value in iteration 1 1 = 2 = ( 0 + 1.2238)/2 = 0.6119.iteration 3step 3 : solve the robust model and get obj * = 95.6954 , x1 = 7.6045 , x2 = 2.9049.step 4 : compute the a posteriori probability bound p1 = 0.5486 , p2 = 0.5426.step 5 : since the probability violation upper bound becomes larger than 0.05 for both constraints , this means the uncertainty set should be enlarged to satisfy chance constraint . so we adjust the uncertainty sets based on smallest size leading to constraint satisfaction so far ( 1.2238 in iteration 2 ) and the size that leads to violation ( 0.6119 in iteration 3 ) : 1 = 2 = ( 0.6119 + 1.2238)/2 = 0.9179.iteration 4step 3 : solve the robust model and obtain obj * = 93.685 , x1 = 7.422 , x2 = 2.859.step 4 : compute the a posteriori probability bound p1 = 0.222 , p2 = 0.205.step 5 : since the probability violation is still larger than 0.05 for both constraints by using size 0.9179 , we need to further enlarge the uncertainty set to satisfy chance constraint . we adjust the parameter toward the smallest size leading to constraint satisfaction ( 1.2238 in iteration 2 ) : 1 = 2 = ( 0.9179 + 1.2238)/2 = 1.0709.iteration 5step 3 : solve the robust model and obtain obj * = 92.712 , x1 = 7.335 , x2 = 2.836.step 4 : compute the a posteriori probability bound p1 = 0.105 , p2 = 0.086.step 5 : since the probability violation is still larger than 0.05 for both constraints in the previous iteration , we need to further enlarge the uncertainty sets : 1 = 2 = ( 1.0709 + 1.2238)/2 = 1.1474.iteration 6step 3 : solve the robust model and obtain obj * = 92.236 , x1 = 7.293 , x2 = 2.824.step 4 : compute the a posteriori probability p1 = 0.062 , p2 = 0.045.step 5 : since the probability violation is larger than 0.05 for the first constraints and the second constraint is satisfied , we keep 2 unchanged and further increase 1 as 1 = ( 1.1474 + 1.2238)/2 = 1.1856.iteration 7step 3 : solve the robust model and obtain obj * = 92.153 , x1 = 7.354 , x2 = 2.777.step 4 : compute the a posteriori probability p1 = 0.045 , p2 = 0.045 ; both are less than 0.05 and the gap is smaller than = 0.01 , so the iteration stops.step 6 : return the final solution obj * = 92.153 , x1 = 7.354 , x2 = 2.777 . step 1 : initialize 1satisfy = 2.4477 , 1violate = 0 , 2satisfy = 2.4477 , 2violate = 0 using the a priori bound . step 2 : set 1 = 1satisfy = 2.477 , 2 = 2satisfy = 2.477 . step 3 : solve the robust optimization problem and obtain solution obj * = 90.91 , x1 = 7.2727 , x2 = 2.7273 . step 4 : compute the a posteriori probability bound from solution p1 = 2.51 10 , p2 = 3.46 10 . step 5 : since the probability violation upper bound is less than 0.05 for both constraints , there is room to contract the uncertainty set and improve the solution . so decrease the size of both uncertainty sets based on the value in iteration 1 : 1 = 2 = ( 0 + 2.4477)/2 = 1.2238 . step 3 : solve the robust model and obtain obj * = 91.807 , x1 = 7.2745 , x2 = 2.8009 . step 4 : compute the a posteriori probability bound p1 = 0.0305 , p2 = 0.0205 . step 5 : since the probability violation upper bound is still less than 0.05 for both constraints , it is necessary to further decrease the size of both uncertainty sets based on the size value in iteration 1 1 = 2 = ( 0 + 1.2238)/2 = 0.6119 . step 3 : solve the robust model and get obj * = 95.6954 , x1 = 7.6045 , x2 = 2.9049 . step 4 : compute the a posteriori probability bound p1 = 0.5486 , p2 = 0.5426 . step 5 : since the probability violation upper bound becomes larger than 0.05 for both constraints , this means the uncertainty set should be enlarged to satisfy chance constraint . so we adjust the uncertainty sets based on smallest size leading to constraint satisfaction so far ( 1.2238 in iteration 2 ) and the size that leads to violation ( 0.6119 in iteration 3 ) : 1 = 2 = ( 0.6119 + 1.2238)/2 = 0.9179 . step 3 : solve the robust model and obtain obj * = 93.685 , x1 = 7.422 , x2 = 2.859 . step 4 : compute the a posteriori probability bound p1 = 0.222 , p2 = 0.205 . step 5 : since the probability violation is still larger than 0.05 for both constraints by using size 0.9179 , we need to further enlarge the uncertainty set to satisfy chance constraint . we adjust the parameter toward the smallest size leading to constraint satisfaction ( 1.2238 in iteration 2 ) : 1 = 2 = ( 0.9179 + 1.2238)/2 = 1.0709 . step 3 : solve the robust model and obtain obj * = 92.712 , x1 = 7.335 , x2 = 2.836 . step 4 : compute the a posteriori probability bound p1 = 0.105 , p2 = 0.086 . step 5 : since the probability violation is still larger than 0.05 for both constraints in the previous iteration , we need to further enlarge the uncertainty sets : 1 = 2 = ( 1.0709 + 1.2238)/2 = 1.1474 . step 3 : solve the robust model and obtain obj * = 92.236 , x1 = 7.293 , x2 = 2.824 . step 4 : compute the a posteriori probability p1 = 0.062 , p2 = 0.045 . step 5 : since the probability violation is larger than 0.05 for the first constraints and the second constraint is satisfied , we keep 2 unchanged and further increase 1 as 1 = ( 1.1474 + 1.2238)/2 = 1.1856 . step 3 : solve the robust model and obtain obj * = 92.153 , x1 = 7.354 , x2 = 2.777 . step 4 : compute the a posteriori probability p1 = 0.045 , p2 = 0.045 ; both are less than 0.05 and the gap is smaller than = 0.01 , so the iteration stops . step 6 : return the final solution obj * = 92.153 , x1 = 7.354 , x2 = 2.777 . the solution procedure of the above iterative method is also illustrated in figure 4 , which shows how the constraint satisfaction probability converges to the desired level and how the quality of the robust solution is eventually improved comparing to the traditional framework . iterative solution procedure for the motivating example : ( upper ) a posteriori probability bound ; ( lower ) optimal objective value of robust solution . finally , solutions from three different methods for the motivating example are summarized in table 3 . the columns traditional , a posteriori , and iterative represent the traditional robust optimization framework , the a posteriori probability bound based method , and the iterative method , respectively . the row obj * and probviolationposterioriub represent the optimal objective value of the robust optimization problem and the a posteriori probability bound based on the robust solution obtained . the percentage numbers represent the gaps between the traditional / iterative method s solutions and the a posteriori method s solution . comparing the traditional robust optimization based approximation framework and the proposed iterative method , we observed that it improves the quality of the solution while still ensures the degree of constraint satisfaction . the percentage gap to the a posteriori solution 92.2292 has been decreased from 1.43% to 0.08% as shown table 3 . comparing the pure a posteriori probability bound based approximation method and the proposed iterative method , we observed that its computational complexity is decreased , since only a set of convex robust optimization problem is solved . in addition , when the gap tolerance is defined small enough , the solution of the proposed method will be close to the solution of the probability inequality based method . in this section , we apply the different methods to solve a production planning problem and a process scheduling problem to compare their performances and illustrate the effectiveness of the proposed iterative method . all the optimization problems are solved on a unix workstation with 3.40 ghz intel core i7 - 2600 cpu and 8 gb memory . the related global optimization problems are solved via antigone 1.1 and the ( mixed integer ) linear optimization problems are solved using cplex 12.0 in gams 24.2.2 . this example was introduced by li et al . , which addresses the problem of planning the production , storage and marketing of a product for a company . it is assumed that the company needs to make a production plan for the coming year , divided into six periods of 2 months each , to maximize the sales with a given cost budget . the production cost includes the cost of raw material , labor , machine time , etc . , and the cost fluctuates from period to period . the product manufactured during a period can be sold in the same period , or stored and sold later on . operations begin in period 1 with an initial stock of 500 tons of the product in storage , and the company would like to end up with the same amount of the product in storage at the end of period 6 . this problem can be formulated as a linear optimization problem as follows:18a18b18c18d18e18f18gin this example , it is assumed that the production costs cj are subject to independent uncertainty distributions . the uncertainty is normalized using 50% of the nominal value cj as the base perturbation amplitude . then the original constraint 18b can be rewritten aswhere j are independent random variables . to ensure the reliability of the solution , the minimum probability for constraint 18b to be satisfied is set as 0.85 ( i.e. , the upper bound on the probability of constraint violation is set to 0.15 ) , then the probabilistic constrained version for this constraint isin the sequel , this example is studied under different assumptions on the uncertainty distributions . in this case , it is assumed that the production costs are subject to uniform uncertainty , that is , j are random variables that uniformly distributes in [ 1 , 1 ] . the traditional robust optimization method is applied first to solve the probabilistically constrained optimization problem . using the interval + ellipsoidal type uncertainty set , the following robust counterpart optimization constraints can be formulatedthe robust optimization problem is obtained by replacing the original deterministic constraint 18b with the above constraints . using the a priori probability bound for the interval + ellipsoidal set induced robust optimization model , the size of the uncertainty set is computed as = 1.9479 . then the robust optimization problem is solved and the corresponding objective is 2 356 977 . the corresponding robust planning solution is shown in figure 5 . the a posteriori probability bound based method is applied next and the following constraint is applied to replace the original constraint 18b : for the a posteriori probability bound based method , the objective value is 2 550 538 . it is seen that this solution is better ( higher sales ) than that of the classical robust optimization method . finally , the iterative framework is applied to solve the problem , the solution procedure is shown in table 5 and the robust solution is shown in figure 7 . in the above solution procedure , the parameter is adjusted as follows : 2 = 0.51 , 3 = 0.5(1 + 2 ) , 4 = 0.5(2 + 3 ) . notice that although the parameter value is decreased in the fourth step , it is still a conservative solution ( 0.0714 < 0.15 ) , so we do not adjust the parameter using 5 = 0.5(3 + 4 ) , but rather 5 = 0.5(2 + 4 ) . it is seen from table 6 that while all the solutions satisfy the probabilistic requirement on constraint satisfaction , the solution of the a posteriori probability bound method ( with relative optimality gap tolerance optcr = 0 and resource limit reslim = 10 000 s ) is the best although global optimization is necessary . if we compare the classical robust optimization method and the iterative robust optimization method and consider the difference between their solution and the best solution ( i.e. , the a posteriori bound based solution ) , the gap has been decreased from 193 561 to 13 679 ( a percentage of 92.93% ) , and the solution has been greatly improved through the iterative framework . comparing the solution of the a posteriori probability bound based method ( figure 6 ) and the iterative method ( figure 7 ) , it is seen that the difference between the solutions is very small . note that global optimization is not needed in the iterative framework and only five convex robust counterpart optimization problems are solved . in this case , it is assumed that the random variables j are subject to symmetric triangular distribution with support on [ 1,1 ] . notice that this type of distribution is bounded and symmetric , so we can still apply the a priori probability bounds to determine the size of the uncertainty set and then apply the traditional robust optimization framework . under the interval + ellipsoidal set induced robust optimization model , the solution will be the same as the previous uniform distribution since the a priori probability bound does not depend on the distribution . on the other hand , the solution of the other two methods will change since they depend on the distribution of the uncertainty . specifically , while the a posteriori probability bound based method is applied , the solution is 2 626 457 after a 10 000 s resource limit reaches in gams ( with a relative gap of 7.52% to the upper bound 2 840 000 ) . when the iterative framework is applied , the final solution is 2 619 188 and the solution procedure is shown in table 7 . the results of three different methods are compared in table 8 . while the traditional framework leads to an 11.4% difference to the a posteriori method solution , the iterative method s solution only has 0.28% difference . this shows that the iterative framework significantly improve the quality of the robust solution while the reliability of the solution is satisfied . in the above studies , it is assumed that the uncertainty distribution is independent , bounded and symmetric , such that we can apply the traditional robust optimization framework based on only the a priori probability bounds . however , when the uncertainty distribution does not fall into this characteristic , there is no basis for determining the size of the uncertainty set . consequently , the traditional robust optimization framework can not be directly applied ( i.e. , if we still use the a priori bound to determine the size , the solution will not ensure the probabilistic guarantee ) . on the other hand , with the proposed iterative framework next , we study two cases where the distribution does not satisfy the bounded or symmetric condition . in this case , we assume the random variables are subject to exponential distribution with rate parameter = 1 . notice that this distribution is unbounded , so we apply the ellipsoidal type uncertainty set induced robust optimization model rather than interval + ellipsoidal type in this study . with the a posteriori probability bound based method , the final solution is 1 818 269 . with the iterative solution framework , the solution is 1 803 310 and the corresponding a posteriori probability upper bound of constraint violation is 0.1474 . notice that we do not list the a priori bound value here , since it is not applicable for the asymmetric distribution in this case . it is assumed that each j is subject to normal distribution n(0,0.5 ) in this case . although for the case of the normal distribution , it is not necessary to apply an approximation scheme to solve the probabilistically constrained problem since analytical deterministic equivalent problem can be formulated and solved , we study the robust optimization approximation based method here to compare the solution quality . type uncertainty set is also used in this case to deal with the unbounded distribution . the a posteriori method lead to solution of 2 569 004 , and the iterative method leads to 2 563 734 , with the constraint violation probability less than 0.1394 as shown in table 10 . in this example , a process scheduling problem is studied . this example involves the scheduling of a batch chemical process related to the production of two chemical products using three raw materials . the mixed integer linear optimization model for the scheduling problem is formulated as follows , and the readers are directed to the paper for the detailed mixed integer linear optimization formulation and problem data19a19b19c19d19e19f19g19h19i19j19k19l19m19nin this example , we consider the demand uncertainty only and the following constraints are affected : we assume independent uncertainty distributions on the product demand parameters rs and assign a base perturbation of 20% of the nominal demand data ( rp1 = 50 , rp2 = 75 ) : rs = rs(1 + 0.2s ) . we set the expected minimum probability level on constraint satisfaction to 0.5 ( i.e. , set the upper bound on constraint violation to 0.5 ) . then the probabilistic constrained version isseveral different type of uncertainty distributions are considered to study the proposed method . specifically , we assume a uniform distribution on the product demand parameters rs ( i.e. , s is uniformly distributed in [ 1,1 ] ) . based on the traditional robust optimization method , the robust counterpart optimization constraint is formulated as follows : which is equivalent for box , ellipsoidal , and polyhedral type of uncertainty sets since the number of uncertain parameter in each demand constraint is 1 . from the desired bound on constraint violation 0.5 , we get the uncertainty set size value 1.1774 . since the uncertainty is bounded , a set with size value 1 will cover the whole uncertainty space . the a posteriori probability bound based method is applied next and the following constraint is applied : the resulting nonconvex mixed integer nonlinear optimization problem is solved using antigone 1.1 ( with tolerance parameter optcr = 0.01 and reslim = 10 000 ) and the objective is 1070.04 ( with relative optimality gap 1.82% to the upper bound 1089.47 after 10 000 s ) . since set size value 1 makes the robust optimization problem infeasible , in the iterative framework , we start from 0.5 for the parameter s to make the problem feasible . notice that the adjustments of the parameter values are based on the change of the a posteriori bounds . for example , we realize that for the first demand constraint is 0.6646 in the 2nd iteration and 0.3397 in the third iteration . to move the bound close to less than 0.5 , we set the new parameter value p1 in the fourth iteration as ( 0.5 + 0.75)/2 = 0.625 , and the resulting solution lead to a new probability bound 0.507 . notice in the sixth step , there is no change on the objective solution , so the solution procedure is stopped . the final optimal objective value is 1064.29 , and the corresponding schedule is shown in figure 9 . this solution has only 0.54% difference to the a posteriori solution as shown in table 12 . schedule obtained from the iterative method . comparing the solution from all the three different methods shown in table 12 , the following observations can be made . first , the traditional robust solution framework is conservative and even leads to an infeasible problem . however , the iterative framework successfully addresses the same problem and finds feasible solution . the reason is that the iterative framework utilizes not only the a priori probability bound but also the a posteriori probability bound , thus avoids the conservative solution . second , comparing the a posteriori probability bound based method and the iterative method , it is seen that the optimal solutions are very close . however , with the iterative framework , we obtain a solution with almost same quality but far less computational efforts . this further validates the effectiveness of the proposed iterative method . for this scheduling example , two unbounded distributions are also studied . the traditional framework is not applicable in this situation because the a priori probability bound is based on the bounded distribution assumption . box , ellipsoidal , and polyhedral type of uncertainty set lead to same robust optimization formulations here . the results of the different methods are summarized in table 13 , and the solution procedure of iterative method is given in table 14 . the iterative method s solution has only 0.6% difference to the a posteriori solution in this case . the results are given in table 15 , and the solution procedure of iterative method is summarized in table 16 . the a posteriori solution 1074.22 has a relative gap of 1.33% to the upper bound 1088.75 after 10 000 s. iterative method s solution has only 0.1% difference when compared to the a posteriori solution . from the above results , it is observed that while the traditional method is not applicable to the unbounded distribution cases , the iterative method applies the robust optimization approximation and uses the a posteriori bound to check the solution reliability . the solutions of iterative method are consistently very close to the a posteriori bound based method in terms of the optimal objective values while the computational complexity is greatly reduced since only convex robust optimization problem is solved in several iterations . the traditional robust optimization framework can be used to approximate probabilistic constraints and provide safe solution . when a detailed probability distribution on uncertainty is available , the a posteriori probability bound based method leads to less conservative approximation , but the trade - off is that the resulting nonconvex problem needs to be solved via a deterministic global optimization approach . a novel solution framework combining the robust optimization approximation and the a posteriori probability bound evaluation is proposed to improve the solution quality of traditional robust optimization framework without significant computation effort . the effectiveness of the proposed method has been illustrated through a motivating example , as well as planning and scheduling problems . furthermore , while the traditional robust optimization method requires information on certain probability distribution on the uncertainty such that the a priori probability bound is valid , the proposed iterative framework extends the application to general distributions since we can always use the a posteriori probability bound to ensure the constraint is satisfied within desired probability . finally , it is worth mentioning that the probability bounds used in this work are derived based on the assumption of independence on the uncertain parameters . one of the future research directions will be to investigate the correlation between uncertain parameters .

in this paper , we study the solution quality of robust optimization problems when they are used to approximate probabilistic constraints and propose a novel method to improve the quality . two solution frameworks are first compared : ( 1 ) the traditional robust optimization framework which only uses the a priori probability bounds and ( 3 ) the approximation framework which uses the a posteriori probability bound . we illustrate that the traditional robust optimization method is computationally efficient but its solution is in general conservative . on the other hand , the a posteriori probability bound based method provides less conservative solution but it is computationally more difficult because a nonconvex optimization problem is solved . based on the comparative study of the two methods , we propose a novel iterative solution framework which combines the advantage of the a priori bound and the a posteriori probability bound . the proposed method can improve the solution quality of traditional robust optimization framework without significantly increasing the computational effort . the effectiveness of the proposed method is illustrated through numerical examples and applications in planning and scheduling problems .

Introduction Framework for Robust Optimization A Posteriori Probability Bound Based Solution Method Iterative Solution Strategy Case Studies Conclusion

based on the type of acrylic resin used , the specimens were divided into three groups with 10 specimens each . they were group 1 - conventional denture base resins , group 2 - high impact denture base resins , and group 3 - glass reinforced denture base resins . each group was subjected to flexural strength evaluation . to make the mold space for the specimens , three stainless steel cuboidal dies were milled measuring 65.5 mm 10.5 mm 3.5 mm in length , breadth , and thickness , respectively , [ figure 1 ] . the metal dies were flasked using type ii dental plaster to complete the flasking procedure . the plaster was allowed to set for an hour , and parts of the flask were separated [ figure 2 ] . the stainless steel dies were retrieved to create the mold space for the acrylic specimen . the separating medium ( cold mold seal ) was applied to the mold space created and is allowed to set for 20 min . three different pmma were evaluated , namely milled stainless steel die three stainless steel die were placed in a single flask group 1 - conventional denture base resins ( dental products of india heat cure resins ) [ figure 3 ] group 2 - high impact denture base resins ( trevalon hi ) [ figure 3 ] group 3 - glass reinforced denture base resins ( acrylic composite ) [ figure 3 ] . group 1 - conventional denture base resins ( dental products of india heat cure resins ) [ figure 3 ] group 2 - high impact denture base resins ( trevalon hi ) [ figure 3 ] group 3 - glass reinforced denture base resins ( acrylic composite ) [ figure 3 ] . ten acrylic specimens taken from each group for testing flexural strength the polymer and monomer were proportioned as suggested by the manufacturer in a ratio of 3:1 by volume . the acrylic resin was packed into the mold , after trial closure the flask was tightened to their final position . the specimens were subjected to curing cycle starting from room temperature to reach 74c in 30 min and held at this temperature for approximately 2 h and then terminal boiling point was done at 100c for 1 h. after bench cooling , the acrylic specimens were retrieved , trimmed , finished , and polished to the required dimension measuring 65 mm 10 mm 3 mm in length , breadth , and thickness , respectively ( according to international organization for standardization [ iso ] standardization 1567 ) . the exclusion criteria for the samples were specimens with smaller dimensions , internal porosity , external porosity , worn out edges , and surface defects . all the thirty specimens were immersed in distilled water for 28 days at room temperature to simulate the oral conditions . the specimens were prepared by marking three lines a , b , and c. the first line a was drawn at a distance of 10 mm from the border of the specimen . the second line b was marked at 45 mm away from line a. these two lines a and b correspond to the location of supporting arm in the universal testing machine . a midline between these lines a and b was marked as line c , and it is the location the striker of the testing machine would come and contact with the specimen [ figure 4 ] . as the universal testing machine plunges into the specimen , the specimen would fracture at a particular load [ figure 5 ] . this maximum load before fracture ( f ) is given in newtons in the display of the testing machine . the flexural strength of the given sample in megapascals was computed from the maximum load by using the formula s = 3fl/2bd2 jigs of supporting arm mounted at 45 mm apart fracture occurring at the center of the acrylic specimen s flexural strengthf maximum load ( force ) before fracturel length of the support arm ( 45 mm)b width of the specimend thickness of the specimen . s flexural strength f maximum load ( force ) before fracture l length of the support arm ( 45 mm ) b width of the specimen d thickness of the specimen . the mean value of the flexural strength of all the groups was computed and then statistically analyzed using one - way analysis of variance ( anova ) , using spss software version 21.0 ( ibm corporation , armonk , ny , usa ) . to make the mold space for the specimens , three stainless steel cuboidal dies were milled measuring 65.5 mm 10.5 mm 3.5 mm in length , breadth , and thickness , respectively , [ figure 1 ] . the metal dies were flasked using type ii dental plaster to complete the flasking procedure . the plaster was allowed to set for an hour , and parts of the flask were separated [ figure 2 ] . the stainless steel dies were retrieved to create the mold space for the acrylic specimen . the separating medium ( cold mold seal ) was applied to the mold space created and is allowed to set for 20 min . three different pmma were evaluated , namely milled stainless steel die three stainless steel die were placed in a single flask group 1 - conventional denture base resins ( dental products of india heat cure resins ) [ figure 3 ] group 2 - high impact denture base resins ( trevalon hi ) [ figure 3 ] group 3 - glass reinforced denture base resins ( acrylic composite ) [ figure 3 ] . group 1 - conventional denture base resins ( dental products of india heat cure resins ) [ figure 3 ] group 2 - high impact denture base resins ( trevalon hi ) [ figure 3 ] group 3 - glass reinforced denture base resins ( acrylic composite ) [ figure 3 ] . ten acrylic specimens taken from each group for testing flexural strength the polymer and monomer were proportioned as suggested by the manufacturer in a ratio of 3:1 by volume . the acrylic resin was packed into the mold , after trial closure the flask was tightened to their final position . the specimens were subjected to curing cycle starting from room temperature to reach 74c in 30 min and held at this temperature for approximately 2 h and then terminal boiling point was done at 100c for 1 h. after bench cooling , the acrylic specimens were retrieved , trimmed , finished , and polished to the required dimension measuring 65 mm 10 mm 3 mm in length , breadth , and thickness , respectively ( according to international organization for standardization [ iso ] standardization 1567 ) . the exclusion criteria for the samples were specimens with smaller dimensions , internal porosity , external porosity , worn out edges , and surface defects . all the thirty specimens were immersed in distilled water for 28 days at room temperature to simulate the oral conditions . the specimens were prepared by marking three lines a , b , and c. the first line a was drawn at a distance of 10 mm from the border of the specimen . the second line b was marked at 45 mm away from line a. these two lines a and b correspond to the location of supporting arm in the universal testing machine . a midline between these lines a and b was marked as line c , and it is the location the striker of the testing machine would come and contact with the specimen [ figure 4 ] . as the universal testing machine plunges into the specimen , the specimen would fracture at a particular load [ figure 5 ] . this maximum load before fracture ( f ) is given in newtons in the display of the testing machine . the flexural strength of the given sample in megapascals was computed from the maximum load by using the formula s = 3fl/2bd2 jigs of supporting arm mounted at 45 mm apart fracture occurring at the center of the acrylic specimen s flexural strengthf maximum load ( force ) before fracturel length of the support arm ( 45 mm)b width of the specimend thickness of the specimen . s flexural strength f maximum load ( force ) before fracture l length of the support arm ( 45 mm ) b width of the specimen d thickness of the specimen . the mean value of the flexural strength of all the groups was computed and then statistically analyzed using one - way analysis of variance ( anova ) , using spss software version 21.0 ( ibm corporation , armonk , ny , usa ) . one - way anova was used to determine statistical differences among the flexural strength of three groups . data were analyzed by spss software , and the results were obtained [ tables 13 ] . the mean flexural strength of conventional pmma ( group 1 ) was 101.18 mpa , for high impact pmma ( group 2 ) it was 121.50 mpa , and for e - glass reinforced pmma ( group 3 ) it was 144.45 mpa . specimens reinforced with glass fibers showed the highest flexural strength values , followed by a specimen reinforced with butadiene styrene and followed by conventional unreinforced acrylic resin . the results of the statistical analysis for flexural strength were shown in the bar diagram [ statistical figure 1 ] . results of flexural strength between three denture base resins measured in terms of megapascals descriptive analysis of data on flexural strength and impact strength anova on flexural strength graphical representation of data on flexural strength the mean transverse strengths of all test groups have statistically significant differences ( p < 0.005 ) . the analysis of data revealed a significant difference between group 1 and group 2 , and 3 ( p < 0.005 ) . scheffe s post - hoc test was done to substantiate this [ table 4 ] . the prime and most frequent site of fracture in the upper denture is in the medial line . during chewing , flexural strength is a measure to know the resistance of the polymer to flexural deformation . therefore , in this study , flexural were evaluated for the above three denture base resins . over the years , there have been various modifications attempted to improve the mechanical properties of pmma . the modifications include chemical modification of pmma , through the incorporation of butadiene styrene to produce graft copolymer ( high impact denture base resins ) and mechanical reinforcement through the inclusion of various fibers ( fiber reinforced denture base resins ) . therefore , all the three denture base resins ( conventional resins , high impact , and glass reinforced ) were included in this study . artificial aging such as underwater storage in thermally controlled condition was simulated in this study . different authors use different time periods of underwater storage , but the important influence of water on the flexural strength occurs during the first 4 weeks of immersion causing decrease of the flexural strength values . hence , a 28 days immersion in distilled water at 37c was used in this study . regarding the fiber reinforcement in denture base resins , it has long been hypothesized that the addition of synthetic fibers to the monomer - polymer mixture may strengthen the resultant acrylic resin . different authors used various types of fibers such as carbon , aramid , glass , polyethylene fibers . the result of the present study , showed that silanized ( e)-glass fibers could considerably enhance the flexural strength of dental polymers , which could be due to the proper impregnation of fibers with resin polymer . the other factors that are related to the strength of the fiber composite are the type of fibers , percentage of fibers in polymer matrix , the orientation of the fibers , fiber form ( chopped , continuous , unidirectional , bidirectional ) , and the adhesion of the fibers to polymer . the use of carbon and aramid fibers produced an important clinical problem , namely poor esthetics and difficulties in polishing . other advantages include its white translucent appearance , negligible water sorption , and excellent polishing characteristics . glass fiber reinforcement has been the most clinically successful ; glass fibers are also esthetically suitable and are reported to have improved flexural strength , flexural modulus , fatigue strength , and impact strength of resin . therefore out of all the fibers , self - reinforced glass fibers were selected for this study . in the present study , the flexural strength of unreinforced and reinforced acrylic resin specimens specimens reinforced with glass fibers showed the highest flexural strength values , followed by specimens reinforced with butadiene styrene and followed by conventional unreinforced acrylic resin . the reason behind this would be the presence of glass fibers , which may prevent the propagation of a crack when the stresses are applied . hence , glass fibers are strongly recommended in patients with heavy occlusal load or when fracture strength of denture base resin is of great concern . this finding is in agreement with results of previous studies . for better understanding , the fracture and deformation mechanisms future researches about the effects of the residual monomer content and the viscoelastic properties on the fracture process and microstructure of acrylic resins could be performed . within the limitation of the current study , the following conclusions were drawn the flexural strength values of heat polymerized pmma were considerably enhanced by reinforcementspolymethyl methacrylate reinforced with glass fibers showed the highest flexural strength values this was followed by pmma reinforced with butadiene styrene , and the least strength was observed in the conventional denture base resins . the flexural strength values of heat polymerized pmma were considerably enhanced by reinforcements polymethyl methacrylate reinforced with glass fibers showed the highest flexural strength values this was followed by pmma reinforced with butadiene styrene , and the least strength was observed in the conventional denture base resins .

aim : the aim of this study was to evaluate whether the flexural strength of a commercially available , heat polymerized acrylic denture base material could be improved using reinforcements.materials and methods : a total of 30 specimens ( 65 mm 10 mm 3 mm ) were fabricated ; the specimens were divided into three groups with 10 specimens each . they were group 1 - conventional denture base resins , group 2 - high impact denture base resins , and group 3 - glass reinforced denture base resins . the specimens were loaded until failure on a three - point bending test machine . an one - way analysis of variance was used to determine statistical differences among the flexural strength of three groups . data were analyzed by spss software version 21.0 ( ibm corporation , armonk , ny , usa ) and the results were obtained.results:the flexural strength values showed statistically significant differences among experimental groups ( p < 0.005).conclusion : within the limitations of the study polymethyl methacrylate ( pmma ) reinforced with glass fibers showed the highest flexural strength values this was followed by pmma reinforced with butadiene styrene , and the least strength was observed in the conventional denture base resins .

Materials and Methods Making of acrylic specimen Evaluation of flexural strength Results Discussion Conclusion Financial support and sponsorship Conflict of Interest

although not a diagnostic symptom of autism spectrum disorder ( asd ) , deficits in face processing represent a model domain in which to understand some of the core behavioral and neural features of autism . for example , many components of face processing ( e.g. , identity recognition , expression recognition ) are developing at the very time that behavioral symptoms of autism are emerging and changing developmentally ( infancy through young adulthood ) , allowing researchers to track aberrant developmental trajectories , and thus identify vulnerable developmental periods . in addition , many of the individual neural regions comprising the broadly distributed circuitry that subserves face recognition abilities ( gobbini and haxby , 2007 ) are located within anatomical regions that show pathological structural growth patterns during infancy , toddlerhood , and adolescence in autism . these regions include the temporal and frontal lobes as well as the amygdala ( schumann et al . , 2010 ) , suggesting that they may be particularly vulnerable throughout the developmental course of the disorder . finally , given that faces are the pre - eminent social stimulus from which we extract multiple kinds of social information that guide behavior , they provide a useful index of atypical neural organization of social - information processing across a spectrum of social emotional disorders ( e.g. , evans et al . , 2008 ; kucharska - pietura et al . , therefore , understanding the profile of atypical neural activation during face processing in autism , particularly during vulnerable developmental periods , is a fruitful approach to studying a core feature of autism ; that is , disruption of the social brain and social information processing more generally . the central goal of the current project was to evaluate the nature and extent of disruption in the social brain during face processing in autism , particularly during adolescence . we focus specifically on adolescence ( i.e. , the second decade of life ) as this is a developmental period of emerging vulnerability for individuals with autism in terms of face processing behavior ( o'hearn et al . , 2010 ) and neural circuitry ( dalton et al . , 2005 ; 2004 ) . also , an estimated one - third of children with autism experience deterioration in functioning during adolescence , which is associated with concomitant neurological complications ( gillberg and steffenburg , 1987 ; kanne et al . , 2011 ) , a substantial increase in social withdrawal ( anderson et al . , 2011 ) , and a potential heightened risk for developing comorbid depression and anxiety ( brereton et al . , 2006 ; kuusikko et al . , , we include a particular focus on the functional profile of activation within the fusiform face area ( ffa ; kanwisher et al . , 1997 ) of the temporal lobe and the amygdala , two critical regions supporting multiple aspects of face processing ( i.e. , identity recognition , affective processing , trait attribution ) . our focus on atypical activation within the ffa and amygdala in autism stems from contradictions within the existing literature that have made it difficult to ascertain a profile of atypical functional activation and organization among these regions even in adulthood autism . importantly , while the amygdala is central for processing affective information about faces , it is only one of several other critical regions that make up the extended face network ( gobbini and haxby , 2007 ) . surprisingly , little is known about the neural profile of these extended regions in autism , which might be especially disrupted given the known social and affective impairments in autism . the ffa in the fusiform gyrus ( fg ) together with a lateral region in the inferior occipital cortex [ occipital face area , 2000 ] and the posterior superior temporal sulcus ( sts ; hoffman and haxby , 2000 ) comprise the core regions in the broadly distributed neural circuitry supporting face processing ( gobbini and haxby , 2007 ; haxby et al . , 2000 ) . although these core regions are strongly implicated in supporting the visuoperceptual and cognitive analysis of faces , they also receive strong inputs from the extended regions , which are implicated in the more social and emotional aspects of face processing ( said et al . , 2010 , 2011 ) . the extended face processing regions include the amygdala , insula , and medial prefrontal cortex , regions in the anterior paracingulate cortex , and the anterior temporal lobe ( gobbini and haxby , 2007 ) . these extended regions process more changeable aspects of faces , such as facial expressions and associating person knowledge with faces , including personal traits , attitudes , mental states , and intentions . the overwhelming majority of studies investigating the neural basis of face processing in autism have focused on understanding whether face - related activation in the ffa and the amygdala is atypical . many studies report hypo - activation in the ffa in individuals with autism during unfamiliar face processing ( dalton et al . , 2005 ; domes et al . , 2013 ; grelotti et al . , 2005 ; humphreys et al . , 2008 ; kleinhans et al . , 2011 ; sato et al . , 2012 ; schultz et al . , 2000 ; wang et al . , for example , we previously reported that during passive viewing of movies of faces , hypo - activation is evident in the ffa as well as other core ( i.e. , perceptual ) regions of the face - processing network in adults ( humphreys et al . , 2008 ) and adolescents ( scherf et al . , 2010 ) with high - functioning autism ( hfa ) . however , there are several studies that fail to find atypical activation within the fusiform gyrus ( bird et al . , 2006 ; , 2004 , 2007 ; kleinshans et al . , 2008 ) in autism . for example , who used a passive viewing task of static face photographs but asked participants to fixate a red fixation cross positioned on the bridge of the nose of the face images , failed to find differences in face - related activation in the fg of adults with autism ( hadjikhani et al . , 2007 ) . it would seem that encouraging participants with autism to fixate the face improves signal in the ffa ; however , a similar a study of adults with autism using the same procedure reported face - related hypo - activation in the fg ( humphreys et al . one important difference between these two studies is that the participants in the studies varied in the magnitude of their symptom severity with the participants in the study by hadjikhani and colleagues consisting of almost an equal distribution of autism , and asperger's / pdd participants whereas the study by humphrey and colleagues only included participants with autism . a review of this literature suggests that the pattern of mixed findings of face - related activation in the fusiform gyrus is not likely to be related to differences in task demands ( e.g. , passive viewing versus face matching ) or the specific contrast used to define the face activation ( e.g. , affective faces versus neutral faces , faces versus objects , faces versus shapes ) . patterns of both hypo- and comparable face - related activation in the ffa have been observed under the full range of these conditions . the pattern of mixed findings is also not likely to be related to the familiarity of the face stimuli since findings of both hypo- and comparable face - related activation have been observed when the face stimuli are familiar to participants ( hypo - active , dalton et al . , 2004 ; pierce and redcay , 2008 ) . instead , the studies appear to differ in terms of the relative severity of the autism participants . specifically , all the studies reporting comparable face - related activation in people with autism , particularly in the ffa , have included a large proportion of participants with asperger 's syndrome and pdd - nos , who are less severely impacted symptomatically than those with an autism diagnosis . in contrast , the studies reporting hypo - activation in the ffa have largely included participants with a diagnosis of autism who are more severely affected by the disorder . based on these findings , we suggest that the discrepancies in the existing literature , particularly with respect to face - related activation in the fusiform gyrus , may actually reflect a systematic relation between the magnitude of activation and the severity of autism symptoms and/or variation in face recognition behavior . understanding the potential relation between symptom severity , face recognition behavior , and ffa activation in response to faces may provide a critical step in reconciling the notable discrepancies about the development of the social brain in autism . findings about atypical amygdala activation during face processing in autism are equally discrepant . given the social impairments of autism and the reported difficulties in processing emotional expressions ( adolphs et al . , 2001 ; dawson et al . , 2005 ) , amygdala activation is likely to be atypical , particularly in response to affective faces . however , the nature of this atypicality is controversial and the existing results conflict , with many reporting hypo - activation ( ashwin et al . , 2007 ; , 2008 ; corbett et al . , 2009 ; critchley et al . , 2000 ; grelotti et al . , 2005 ; hadjikhani et al . , 2007 ; pierce et al . , 2001 ) , some reporting hyper - activation ( dalton et al . , 2005 ; monk et al . , 2010 ; swartz et al . , 2013 ; tottenham et al . , 2014 ; weng et al . , 2011 ) , and still others reporting comparable activation ( pierce et al . , 2004 ; wang et al . , our review of this literature suggests that , instead of symptom severity , the discrepancy in findings about amygdala activation in autism may be related to methodological differences in the way neural activation is defined , particularly with respect to the comparison baseline condition . for example , studies reporting amygdala hyper - activation in autism generally contrast affective faces ( e.g. , sad , happy ) with fixation ( e.g. , dalton et al . , 2005 ; tottenham et al . , 2014 ; , hyper - activation compared to controls could result from either higher magnitude responses to the faces and/or lower responses to the fixation , which could both contribute to a larger difference score ( i.e. , hyper - activation ) across these two conditions . in contrast , studies reporting amygdala hypo - activation in autism have employed a variety of contrasts in which affective or neural faces are compared with other visual objects , shapes , or scrambled images ( e.g. , bookheimer et al . , 2008 ; corbett et al . , 2009 ; in this case , the reduced responsivity of the amygdala in autism compared to controls could result from either lower magnitude responses to faces and/or higher magnitude responses to the other visual categories , resulting in a lower difference score ( i.e. , hypo - activation ) across these two conditions . given this pattern of findings , it is difficult to assess whether aberrant activation in the amygdala in autism is largely indicative of atypical processing of faces specifically ( as might be concluded from the work contrasting faces with fixation baseline ) , or whether there is a broader atypicality in amygdala function that affects the processing of a wide array of visual objects ( as might be concluded by the work contrasting faces with more complex comparison images ) . careful investigation of the profile of amygdala activation in response to faces ( both affective and neutral ) as well as to a wide range of other visual stimuli will help address this question . in this study , we aimed to identify disruptions in neural activation through the core and extended regions supporting face processing ( and social - information processing more generally ) in adolescents with autism and to explore individual differences as reflected in the relationship between variations in behavior and/or symptom severity and face - related activation within these regions . we studied high functioning adolescents ( hfa ) with autism ( ages 1017 years ) and age - matched typically developing ( td ) adolescents . we measured brain activation using fmri while participants performed a recognition task with both affective and neutral faces as well as a range of other visual stimuli , including common objects , houses , and scrambled images . this enabled us to map and compare face - related activation in both core and extended face processing regions across the groups to determine the extent to which atypical activation exists in the full network of regions . we also interrogated the profile of amygdala activation across the entire range of stimuli in order to evaluate the claim that faces , and not other visual objects , specifically elicit atypical activation in the amygdala . we assayed the behavioral profile of face recognition abilities for upright and inverted faces outside the scanner . the face inversion effect ( i.e. , more accurate recognition for upright compared to inverted faces , yin , 1969 ) is a hallmark of typical face perception and the magnitude of the face inversion effect has been used as a measure of individual difference in face processing studies previously ( russell et al . , 2009 ) . finally , we correlated the magnitude of face - related activation throughout the brain , and separately within our a priori regions of interest , with autism symptom severity , levels of adaptive social functioning , and face recognition behavior . because of our sensitivity to the developmental course of the disorder and age as a proxy measure of that continuum , we also included age as an independent factor in all the regression analyses between neural activation and behavior / symptom severity measures . the participants included 20 male hfa adolescents ( range 1017 years ) and 12 age - matched td adolescents ( range 1117 years ) . the mean age did not differ across groups , f(1,30 ) = 0.07 , the mean iq was in the average range for both groups ( see table 1 for demographic and iq information , as determined using the wechsler abbreviated scale of intelligence ) . the td group had higher verbal iq scores , f(1,30 ) = 5.3 , p < .025 , which contributed to slightly higher full scale iqs , f(1,30 ) = 3.6 , p = .07 . the diagnosis of autism was established using the autism diagnostic interview - revised ( adi - r ) ( lord et al . , 1994 ) , the autism diagnostic observation schedule - g ( ados ) ( lord et al . , 2001 ) , and expert clinical diagnosis ( minshew , 1996 ) . the hfa adolescents were medically healthy ; had no identifiable genetic , metabolic , or infectious etiology for their disorder ; and , were free of birth or traumatic brain injury , seizures , attention deficit disorder , and depression . td participants were included if they were medically healthy , free of regular medication usage , and had good peer relationships as determined by parent , self - report , and staff observations during the screening procedures . td participants were excluded if they or their first - degree relatives had a history of autism , neurological or psychiatric illness , acquired brain injury , learning disabilities , developmental delay , school problems , substance abuse , or medical disorders with central nervous system implications . a single episode of depression in a parent during a stressful episode was not considered grounds for exclusion providing no other family members reported depressive episodes . both hfa and td adolescents were recruited to be part of a longitudinal study investigating the effects of visuoperceptual training . in this ongoing study , pairs of participants with autism are yoked to a single td participant with each triad of participants systematically matched on age , sex , and fsiq . this explains the relatively smaller sample size of the td compared to hfa adolescents described in this project . the data reported here are from the pre - training assessment . written informed consent was obtained from participants ' guardians , and written assent from the participants themselves , using procedures approved by the internal review boards of the university of pittsburgh and carnegie mellon university . parents completed two scales of social functioning about their adolescent , the social responsiveness scale ( srs : constantino et al . , 2003 ) and the vineland adaptive behavioral scales , second edition ( vabs - ii : sparrow et al . , 2005 ) . the srs is a questionnaire that measures the severity of autism spectrum symptoms as they occur in natural social settings ; higher scores reflect more severe symptoms . the vbas - ii is a standardized caregiver interview that measures communication , social , daily living and motor skills ; higher skills reflect more adaptive functioning . the cfmt ( duchaine and nakayama , 2006 ) was used to measure face recognition behavior outside the scanner . this task has been used previously with td children and with adolescents with autism ( o'hearn et al . , 2010 ) . as in our previous work ( scherf et al . , 2008 ) , participants always performed the upright version first to maximize the possibility that participants with autism would initially approach the task in an ecologically valid way prior to having to confront the less naturally occurring inverted faces . all participants were placed in a mock mr scanner for approximately 20 min and practiced versions of the tasks that were administered in the full scan . high - resolution structural images and functional images were then acquired in a single session . participants were scanned using a siemens 3 t verio mri scanner , equipped with a 32-channel adult head coil , at carnegie mellon . anatomical images were acquired using a 3d - mprage pulse sequence with 176 t1-weighted ac - pc aligned sagittal slices ( tr / te / ti = 1700 , 2.48 , 900 ms ; voxel size = 1 mm , fov = 256 256 , ipat = 2 ) . pc aligned slices , covering most of the brain and all the occipital and temporal lobes ( tr / te = 2000 , 25 ms , fov = 192 , matrix 64 64 , flip angle = 79 , voxel size = 3 mm , this task was designed to elicit activation in response to several visual categories and to actively engage recognition behavior . functional images were acquired across two runs of a 1-back localizer task , which included blocks of neutral faces , fearful faces , common objects , vehicles , houses , novel objects ( i.e. , greebles : gauthier and tarr , 1997 ) , and scrambled images ( fig . 1 ) . scrambled images were created in adobe photoshop by scrambling pixels in the images of the common objects . each run lasted a total of 9 min and 12 s and began with a 20-s block of fixation and a 12-s block of patterns . thereafter , blocks of stimuli were presented in a randomized order followed by intervening blocks of fixation ( 6 s ) . within a block , 12 stimuli were each presented for 800 ms , followed by a 200 ms fixation . participants were required to indicate , by button press , when they detected a repeated image . there were two repeats in each of the stimulus blocks , the position of which was counterbalanced across blocks . in each run , there were four blocks of each stimulus category such that in the final analysis when the two runs were combined , each participant observed 8 blocks of each stimulus category . the neuroimaging data were analyzed using brain voyager qx v2.3 ( brain innovation , masstricht , the netherlands ) . preprocessing of functional images included 3d - motion correction , slice scan time correction , filtering low frequencies , and re - sampling the voxels to 1 mm . runs in which participants exhibited spikes in motion of more than 2.9 mm in any of the six motion directions on any image were excluded from the analyses . a single run was excluded for each of two hfa and one td participant . the average motion ( between each time point ) in each group on both runs of the task was less than 1 mm in all six dimensions and did not differ between groups ( p > .10 ) . for each participant , the time series images for each brain volume were analyzed for category differences in a fixed - factor glm . each category was defined as a separate predictor and modeled with a box - car function adjusted for the delay in hemodynamic response . following the recommendations of weiner and grill - spector ( 2012 ) , the functional data were not spatially smoothed . the time series images were then spatially normalized into talairach space , which is common practice in autism neuroimaging research , particularly in the study of adolescents and adults when brain volumes are comparable to those of td adolescents and adults ( redcay and courchesne , 2005 ) . although participants viewed multiple visual categories in the localizer task , here we focus on differences in the topography of face- , common - object , and house - related activation with respect to activation elicited by scrambled images . functional rois were defined for each individual subject for the region of interest analyses . for each participant , the time series images were submitted to a fixed - effects glm in which category was a fixed factor . as in our previous work , we defined the measures of category - selectivity with respect to all other categories ( scherf et al . , 2007 , 2010 , 2012 ) . note that these definitions are extremely conservative in that they identify many fewer voxels as compared to a contrast that would define each visual category against a fixation ( or scrambled image ) baseline . critically , these contrasts identify non - overlapping sets of voxels in all participants , indicating that they identify the most selective of voxels for each visual category . for example , face selectivity was defined with the following balanced contrast : { [ 3 * ( neutral faces ) + 3 * ( fearful faces ) ] [ 2 * ( common objects ) + 2 * ( houses ) + 2 * ( scrambled images)]}.1 similarly , object selectivity was defined as { [ 4 * ( common objects ) ] [ ( houses ) + ( neutral faces ) + ( fearful faces ) + ( scrambled images ) ] } ; and house selectivity as { [ 4 * ( houses ) ] [ ( common objects ) + ( neutral faces ) + ( fearful faces ) + ( scrambled images)]}. the resulting individual maps were corrected for false positive activation using the false discovery rate procedure ( genovese et al . , 2002 ) with a q < .01 , which is appropriate for identifying individual - level regions of interest ( roi ) . the right and left ffa were defined as the most anterior cluster of contiguous significant voxels in the fusiform gyrus generated from each participant 's face - activation map . unfortunately , the amygdalae were not definable as functional rois consistently across the individual participant face - activation maps . as a result , given our a priori hypotheses about group differences in activation in the amygdala , we defined right and left hemisphere amygdala rois by creating a 6 mm sphere around functionally defined talairach coordinates from previous work ( blasi et al . , the left amygdala roi was centered at ( 19 , 5 , 17 ) and the right centered at ( 22 , 1 , 17 ) . within each of these rois , we conducted an roi - based glm on the time series data for each individual participant to generate the resulting beta weights for each visual category . the beta weights were submitted to repeated - measures anovas with the factors of visual category ( 5 ) and group ( 2 ) separately for the right and left rois . estimates of face - selectivity were also determined for each roi by computing a balanced difference score in the beta weights ( e.g. , faces objects ) . in addition , the ffa rois were quantified in terms of the size ( number of significantly active voxels ) . category selectivity was determined separately for each group ( hfa , td ) by submitting the time - series images from each participant within the group to a random - effects glm with category as a fixed factor and participant as a random factor . the contrasts used to define face- , object- , and house - related activation at the group level were the same as those for the individual level rois ( e.g. , faces vs houses , objects , scrambled ) . however , given the addition of between - subjects variance in these maps , we used a monte carlo simulation to correct the group maps for multiple comparisons ( p < .05 ) separately for the td ( 16 contiguous voxels at a t - value 2.7 ) and hfa ( 12 contiguous voxels at a t - value 2.5 ) participants , given the different number of participants in the two groups . to compare group differences in category - selectivity , the full set of time series data from all participants was submitted to a mixed - model anova including group and category as fixed factors and subject as a random factor.2 we specifically evaluated group category interactions in each voxel in a whole brain analysis based on the contrasts of interest . for example , to compare group differences in face - selective activation , we coded the following interaction : td ( faces > other ) > hfa ( faces > other ) . to correct the resulting interaction maps for false positive activations , we used a monte carlo simulation ( p < .05 required a minimum of 33 contiguous voxels at a t - value 2.0 ) . to examine associations between patterns of brain activation and participant characteristics , we evaluated correlations between cfmt accuracy , raw srs scores , and vbas - social scores with the individually defined roi metrics ( e.g. , magnitude of activation , size of roi ) as well as in whole brain analyses . the various roi metrics were submitted to separate step - wise regressions with age as the first factor and the relevant measure of interest ( e.g. , raw srs score ) as the second factor . this enabled us to determine whether age and the relevant measure of interest independently accounted for variation in each of these roi metrics . whole brain ancovas were computed in the hfa individuals to identify voxels in which there was significant co - variance between category - selective activation and age , raw srs scores , vbas - social scores , and cfmt accuracy . these analyses generated separate whole - brain correlational maps that were thresholded at a corrected r - value of p < .01 using a monte carlo simulation to determine the number of contiguous voxels ( 8 with r > .56 ) . rois that survived this threshold were defined . to illustrate the nature of the relation between the scores and activation in each of these rois , we generated beta weights for all visual categories ( faces , objects , houses , scrambled images ) by computing a separate glm within each roi for each participant . using these beta weights , a difference score was computed that reflected the original balanced category - selective contrast ( e.g. , faces > other ) , which was then plotted against the specific measure of interest . as described for the roi - based correlations , we submitted these difference scores to a step - wise regression with age as the first factor and the relevant measure of interest as the second factor in order to determine the independent contributions of these factors to variation in the profile of face selectivity in each region . the srs and vabs - ii social scores for the two groups are plotted in fig . 2 . for both measures , there was unequal variance across the groups ( p < .005 ) . hfa adolescents had significantly higher srs scores , t(26.8 ) = 13.4 , p < .001 , indicating more severe autism - like symptoms , as well as significantly lower vabs - ii social scores , t(20.6 ) = 6.2 , p < .001 , reflecting lower adaptive functioning than the td participants . separate regressions of age on the srs and vabs - ii scores failed to reveal age - related changes in these measures in either group . the hfa adolescents were less accurate and failed to show an inversion effect in the cfmt ( fig . a repeated - measures anova including the within - subject factor of orientation and the between - subject factor of group , revealed a main effect of group , f(1,29 ) = 6.1 , p < .025 , indicating that the hfa adolescents ( m = 41.6% ) performed worse than the td adolescents ( m = 48.0% ) across both the upright and inverted versions of the task . the low performance in both groups is still above the chance rate of 33% and is comparable to the performance reported of similarly aged td and asd participants on this same task ( o'hearn et al . , 2010).3 there was also a main effect of orientation , f(1,29 ) = 4.7 , p < .05 ( upright m = 46.9% ; inverted m = 42.5% ) , but this was qualified by an orientation group interaction , f(1,29 ) = 6.2 , p < .025 . paired - samples t - tests conducted separately for each group revealed an orientation effect ( i.e. , upright > inverted ) in the td group , t(11 ) = 2.5 , p < .05 , but not in the hfa group , t(18 ) = 0.3 , p = ns . separate regressions of age on the upright cfmt scores failed to reveal age - related changes in this measure in either group . as evident from fig . 3b , there were no group differences in accuracy or reaction time ( rt ) when participants performed the 1-back memory task in the scanner . a repeated - measures anova with visual category as the within - subjects factor and group as the between - subjects factor revealed neither a main effect of group , f(1,30 ) = 0.6 , p = ns , nor a group category interaction , f(1,30 ) = .985 , p = ns . there was , however , a main effect of visual category , f(1,30 ) = 8.1 , p < .005 , with reduced accuracy for neutral faces compared to fearful faces , common objects , and houses ( all bonferroni corrected p < .01 ) , but not scrambled images ( p = ns ) . there were no significant effects in the analysis of the rt data . therefore , group differences in the bold response to these different categories of visual objects can not be attributed to performance differences in the 1-back memory task during scanning . 4a b shows the category - selective activation for each group for faces ( red ) , places ( green ) and common objects ( blue ) . td adolescents exhibited extensive activation in both core ( i.e. , right ffa , bilateral occipital face area ( ofa ) , right sts ) and extended ( i.e. , bilateral amygdala , pcc , and vmpfc ) regions ( table 2 ) . although hfa adolescents exhibited some activation in a subset of the core face - processing regions ( i.e. , bilateral ffa ) , they did not exhibit face - related activation in the ofa or sts core regions , or in the anterior temporal lobe , pcc , or vmpfc ( table 2 ) . statistical comparison of the hfa and td face - related group maps revealed significant hypo - activation in multiple core regions in the hfa adolescents , including the bilateral ofa , right sts , and right ( but not left ) ffa , as well as in extended regions , including the right atl , pcc and vmpfc ( fig . in addition , there were several other regions that were hypoactive in the hfa adolescents during face processing , including parietal , medial temporal , as well as prefrontal regions ( table 3 ) . both the hfa and td groups exhibited extensive and comparable activation of the ventral visual processing stream bilaterally during common object processing ( see figs . 4b and 5b ) except that the hfa adolescents exhibited stronger object - related activation bilaterally in the precuneus than the td adolescents ( fig . additional comparisons using a more lenient contrast for determining object - related activation ( objects versus scrambled images ) revealed no group differences in object - related activation . however , the hfa adolescents exhibited weaker activation in the left ppa than td adolescents during house blocks ( fig . a one - tailed independent - samples t - test failed to reveal a significant difference between groups in the number of voxels within the individually defined right ffa roi , t(30 ) = 0.7 , p = ns . however , the groups tended to differ with respect to the magnitude of face selectivity of the activation in these individually defined rffa rois , t(30 ) = 1.5 , p = .07 , one - tailed . this finding replicates our previous findings in a new sample of hfa and td adolescents ( scherf et al . , 2010 ) . the magnitude of activation ( i.e. , beta weights from roi - based glms ) for each visual category for each group is illustrated separately for the right and left amygdala rois in fig . , there was no main effect of group , f(1 , 30 ) = 0.0 , p = ns ; however , there was a main effect of visual category , f(4 , 120 ) = 11.7 , p < .000 , as well as a group category interaction , f(4 , 120 ) = 4.1 , p < .005 . bonferroni corrected post - hoc tests revealed that , for both groups , fearful faces elicited greater activation than houses and scrambled images ( p < .001 ) and tended to elicit more activation than neutral faces ( p = .051 ) . additionally , neutral faces and objects also elicited more activation in the right amygdala than scrambled images ( p < .05 ) . separate repeated - measures anovas within each group revealed that there were main effects of visual category in both the td , f(4 , 44 ) = 13.4 , p < .001 , and the hfa , f(4 , 76 ) = 3.9 , p < .01 , adolescents . however , the bonferroni corrected post - hoc tests revealed that the groups differed with respect to the categories that elicited the strongest activation in the right amygdala . specifically , in the td adolescents , fearful and neutral faces as well as objects elicited stronger activation than scrambled images ( p < .05 ) ; however , these categories were not different from one another . in contrast , in the hfa adolescents , the only significant difference was between fearful faces and houses ( p < .05 ) ; none of the visual categories was different from the scrambled images as was evident in the td adolescents . interestingly , the negative response to scrambled images among the td adolescents in the right amygdala was 12 times larger ( m = .201 ) than the modestly positive activation to scrambled images ( m = .016 ) in the hfa adolescents . independent samples t - tests comparing activation on each visual category between groups confirmed that the scrambled images condition was the only one for which the hfa and td adolescents differed , t(30 ) = 2.0 , p < .05 . this pattern of differences in response to the baseline condition is highly relevant for considering discrepancies in the current literature about the hyper- or hypo - active signal in the amygdala in autism . in the left amygdala , there was no main effect of group , f(1 , 30 ) = 1.7 , p = ns , and no interaction between group and visual category , f(4 , 120 ) = 1.9 , p = ns . however , there was a main effect of visual category , f(4 , 120 ) = 4.5 , p < .005 . across both groups , fearful faces elicited stronger activation than scrambled images ( p < .001 ) , and tended to elicit stronger activation than neutral faces ( p = .081 ) , but no other categories were different from one another . independent samples t - tests failed to reveal group differences in the magnitude of activation to any of the visual categories in the left amygdala . there were no regions in either the hfa or td adolescents in which there was a significant correlation between age and face- , object- , or house - related activation . the whole - brain correlation analyses between srs score and face - related activation in the hfa adolescents revealed that the right ffa was negatively correlated with srs scores ( fig . 7a ) : participants with higher srs scores had consistently lower magnitude face - related activation in the right ffa . the stepwise regression including the predictors of age and raw srs score on the beta weight difference scores generated for each participant in this roi was significant , f(2 , 17 ) = 9.4 , p < .005 , r = .53 ; however , only raw srs score was a significant independent predictor of face - related activation within this roi ( p < . the locus of the right ffa identified in this analysis ( 31 , 42 , 14 ) overlapped with the same right ffa region that was identified in the group level contrasts of face - related activation ( 37 , 47 , 20 ) during face processing . similarly , the magnitude of the face - related activation in the individually defined right ffa was significantly negatively related to the raw srs score among the hfa participants ( fig . the stepwise regression including the predictors of age and raw srs score on the beta weight difference scores generated for each participant in their individually defined right ffa was significant , f(2 , 15 ) = 6.0 , p < .025 , r = .44 ; however , only raw srs score was a significant independent predictor of face - related activation within this roi ( p < participants with higher srs scores had lower face - selective activation in their individually defined right ffa roi . however , the size of these individually defined rois was not related to srs scores , f(1 , 18 ) = 2.2 , p = ns , r = .11 , nor was the age of the participants ( p = ns ) . in contrast , the level of adaptive function in the hfa group was not significantly related to the level of face - related activation anywhere in the brain . there were no regions in the td adolescents in which either srs or vineland scores correlated with face - related activation . the whole - brain correlational analyses between cfmt performance and face - related activation among the hfa adolescents revealed that activation in the right atl ( 31 , 3 , 23 ) was positively correlated with performance . importantly , the stepwise regression analyses of the beta weights extracted from the individual participant glms in this roi with the predictors of age and upright face recognition accuracy was significant , f(2,17 ) = 13.4 , p < .001 , r = .61 . however , only upright cfmt performance was an independent predictor of face - related activation in this roi ( p < .001 ) , age was not ( p = ns ) . the stepwise regression with age and inverted face accuracy was not significant , f(2,16 ) = 1.0 , p = ns , r = .12 ( fig . there were no regions in which face - related activation was related to performance on the cfmt in the td adolescents . the central goals of this investigation were to evaluate face - related activation in adolescents with hfa in both core and extended regions of the broader face - processing network , with particular focus on the fusiform gyrus and the amygdala , and to explore a potential relation between the magnitude of this face - related activation and autism symptom severity , levels of adaptive social functioning , and variations in behavioral face recognition performance . using a classic task of unfamiliar face recognition , we replicated and extended previous findings that adolescents with autism are impaired in upright face recognition abilities compared to age- and iq - matched td adolescents . in addition , to our knowledge , we are the first to use the cfmt to evaluate the magnitude of the face inversion effect ( fie : yin , 1969 ) in adolescents with autism . the fie is often taken as a marker of typical face perception ; however , findings of the presence and magnitude of an fie in autism are mixed . a recent review suggests that people with asd do not demonstrate qualitative differences in the fie ( weigelt et al . , 2012 ) . here , we report that adolescents with autism do not exhibit an fie when tested with the cfmt , which is in contrast to our own previous findings ( scherf et al . , 2008 ) . we suggest that these findings can be explained by the relative difficulty of the cfmt . this is a much harder task than has been used to test the fie in the vast majority of previous studies . there is empirical evidence of a developmental progression in performance on the upright version of the task that continues into early adulthood in td individuals , but this progression plateaus in hfa individuals in adolescence ( o'hearn et al . , 2010 ) . the td adolescents outperformed the hfa adolescents in the upright condition of this task , but the groups were indistinguishable in their performance on the inverted condition . therefore , we suggest that the fie may only be observable in autism under conditions when upright face recognition is optimized . using a paradigm that was designed to elicit activation in both core ( i.e. , visuoperceptual and cognitive ) and extended ( i.e. , motivational and affective ) regions of the face processing system , we determined that hfa adolescents exhibit hypo - activation in the majority , but not all , regions compared to td controls . specifically , although hfa adolescents , as a group , exhibited face - related activation in the pre - eminent ffa in both hemispheres ; activation in the right , but not the left , ffa was significantly hypo - active compared to the td adolescents ( fig . also , face - related activation in the right and left ofa and in the right posterior sts were hypoactive in the hfa group as well . hfa adolescents exhibited comparable activation to tds bilaterally in the loc and hyper - activation in the precuneus during object - recognition , and comparable activation in the ppa during house - recognition . these findings were evident even though the sample size of hfa adolescents was nearly twice the size of the td adolescents . it is important to note that the smaller td sample size compared to the hfa group size is not ideal , but does not likely challenge the pattern of results reported here given that the group comparison is at most risk for type ii error ( false negative ) . in spite of the fact that we have a smaller number of control participants , we still had enough power to observe strong group differences in favor of the controls . in other words , there is more power and consistency in the face - related activation of 12 td controls than among 20 hfa adolescents . this is due , in part , to the powerful signal - to - noise ratio that is generated from the blocked fmri design and the fact that we collected two independent runs of the experiment from each participant to boost signal even more . these findings largely replicate our own and other previous findings in adolescents with autism ( dalton et al . , 2005 ; grelotti et al . , 2005 ; pierce and redcay , 2008 ; scherf et al . , 2010 ) with one exception . here , we find that hfa adolescents exhibited strong , consistent face - related activation in the left ffa that was not present among the td adolescents ( table 2 ) . our finding that adolescents with hfa recruit the left ffa during face recognition task is particularly useful for understanding that some parts of the face - processing network are preserved and even highly functional in autism . one possible explanation for the left ffa activation in the hfa adolescents relates to findings of hemispheric asymmetries in the kinds of information encoded by the fusiform gyri . there is growing consensus that the right fusiform is more specialized for holistic processing , while the left fusiform is more implicated for part - based processing ( meng et al . thus , the reliance on the left ffa during face processing in the hfa adolescents may reflect the use of a more part - based representation to process face identity . this interpretation is consistent with findings that individuals with autism have biased visuoperceptual systems that emphasize feature - based processing of local details in visual scenes ( behrmann et al . , with respect to extended regions , as a group , the hfa adolescents only activated the left amygdala . they did not exhibit activation in the right amygdala , pcc , anterior sts , right or left atl , or vmpfc . in contrast , td adolescents exhibited activation in the right amygdala , pcc , and vmpfc . note that the td adolescents did not show group level activation in the atl in either hemisphere , suggesting that these regions may continue to develop through adolescence . however , when pitted against each other directly , the hfa adolescents exhibited hypo - activation in the right atl , the right anterior sts , and the pcc , as well as in several other regions compared to the td adolescents ( table 3 ) . there were no regions in which the hfa adolescents exhibited greater activation than the td adolescents during face processing . importantly , there were no group differences in the profile of activation of the left amygdala for any of the stimulus categories . both groups exhibited the strongest magnitude response to fearful faces and a negative response to scrambled images . in contrast , in the right amygdala , there were differences between the groups in the profile of activation , but these differences were not specific to faces . in the right hemisphere , the only reliably different response in amygdala activation was to scrambled images . the td adolescents exhibited a strong negative response to scrambled images , whereas there was no such negative response in the hfa adolescents . there were no other group differences in response to either fearful or neutral faces , houses , or common objects . these findings show how a contrast between fearful or neutral faces and scrambled images would lead to a conclusion that hfa adolescents exhibited hypo - activation in the right amygdala , as has been reported in previous studies that used scrambled images as a contrast to face stimuli ( ashwin et al . , 2007 ; hadjikhani et al . , 2007 ; kleinshans et al . , 2008 ) however , a contrast between fearful and neutral faces or between fearful or neutral faces and objects would lead to a conclusion of comparable amygdala activation across the groups , which is consistent with findings from one previous study ( weng et al . , , there was no contrast that reflected hyper - activation to faces in the amygdala among hfa adolescents . this finding stands in contrast with several previous findings of relative hyper - activation in the amygdala during affective face processing in autism . there are multiple potential explanations for the absence of hyper - activation of the amygdala during face processing . importantly , many of our autism participants have been in several previous research studies , including those employing functional neuroimaging . as a result , most of our hfa adolescents were experienced and especially comfortable being in the fmri scanner , which may have significantly reduced anxiety and thus amygdala activation . we suggest that this is an important consideration for other studies reporting hyper - activation in the amygdala in individuals with autism ; it may reflect more generalized anxiety about the scanner environment compared to typically developing individuals . alternatively , one might suggest that our participants were avoiding looking at the eye region of the faces , thereby reducing amygdala activation . a recent study reported hyper - activation in amygdala responses from an autism group viewing neutral faces , particularly when they were directed to look at the eye region of the face ( swartz et al . , 2013 ) . this prediction would be consistent with the hypothesis that there is decreased motivation to attend to ( i.e. , look at ) social stimuli , like faces ( dawson et al . 2002 ) , which leads to hypo - activation in the fusiform gyrus ( dalton et al . , 2005 ) . together , these findings might suggest that the adolescents in our sample were not looking at the eye region of the faces to the same extent as were the td adolescents and that this aversion to the eye region led to the hypo - activation throughout the core and extended regions of the face processing network . we did not collect eye - tracking data , which limits our ability to investigate this possibility . however , both groups performed comparably on the 1-back recognition task for faces , and all other visual objects , in the scanner . this suggests that the adolescents with autism attended to the faces sufficiently to support near ceiling performance on the recognition task while the hypo - active bold signal was being acquired . also , it should be noted that the relation between purported atypicalities in the locus of fixations during face processing and cortical activation patterns in children with autism is controversial ( see boraston and blakemore , 2007 ) . for example , one study of young adolescents with autism found no differences from td controls in fixation patterns when observing facial expressions , despite finding impressive differences in the patterns of neural activation under these same conditions ( dapretto et al . , 2006 ) . also , at least one study in adults with autism found similar patterns of face - related hypoactivation in the fg when participants were required to fixate a central dot overlaid on the center of each stimulus and under free viewing conditions ( humphreys et al . , 2008 ) . we also report novel evidence that the magnitude of hypo - activation in the right ffa among the hfa adolescents is selectively related to the severity of autism symptoms . specifically , individuals with more severe autism symptoms ( i.e. , higher srs scores ) exhibited less face - related activation in the right ffa and no other region . in other words , there was a negative relation between the magnitude of srs scores and face - activation . the illustrative plot of the beta weights from these analyses suggest that the most severely affected adolescents with autism exhibited more object- than face - related activation in the right fusiform gyrus . this finding is consistent with the notion that individuals with autism may treat faces more like common objects with respect to the visuoperceptual strategy that they employ for recognition ( mottron et al . , 2006 ) . it is also consistent with several other studies , which report that typical face - processing regions are actually object - selective in autism ( humphreys et al . , 2008 ; scherf et al . , 2010 ; schultz et al . , 2000 ) . importantly , there were no regions in which object- or house - related activation correlated with symptom severity or levels of social functioning . these highly selective results suggest that the right ffa is particularly vulnerable in autism and that activation in this region may be related to the success with which individuals with autism interact with the social world . although these findings do not indicate a causal direction of the effect ( i.e. , impaired ffa activation leads to social deficits or vice versa ) , there may be a bidirectional influence between face - processing and symptom severity and/or social functioning in autism . the individual differences approach that we employed in this work to understand brain - behavior correspondences in autism may help reconcile discrepancies in the literature concerning hypo - activation in the ffa and suggest that studies failing to report such hypo - activation are likely to have a sample of individuals with less severe symptoms . we did not find a similar relation between face - related activation in the ffa ( or any other region ) in the td adolescents and either their autism - like behaviors or their levels of adaptive functioning . this null result may be related to the limited range of individual differences on these measures among the td adolescents and the small number of participants . it is possible that td adolescents with higher numbers of autism - like traits ( as measured by the autism quotient ; baron - cohen et al . , 2001 ) might show a similar relation between face - related activation and the severity of these traits . this kind of finding would help determine whether the relation between the neural profile of activation for faces and autism symptoms / traits is specifically vulnerable in and characteristic of autism or whether it reflects a broader relation between social information processing of human faces and levels of social functioning in the population more broadly . in spite of the association between face - activation and symptom severity in the right ffa , we did not find a relation between variation in face - recognition behavioral performance and the magnitude of face - selective activation in the fusiform gyrus among the hfa adolescents . this null result is consistent with recent findings of adults with asd ( jiang et al . , these same authors also reported that , using a novel analysis of voxelwise correlations and an fmri - adaptation paradigm to probe the sparseness of face - related representations within the ffa , adults with autism who exhibit particularly poor face recognition skills have less sparse ( and therefore less selective ) neural representations for faces in the ffa ( jiang et al . , in other words , the whole - brain correlational analysis using a category - selective definition of face - related activation ( as determined by the faces other visual categories contrast ) may not have been sensitive enough to detect the brain however , in spite of this limitation within the ffa , we did find a brain behavior relation in the right atl , a region implicated in supporting face individuation ( kriegeskorte et al . , specifically , hfa adolescents who scored higher on the upright version of the cfmt outside the scanner exhibited stronger face - related activation in the right atl during the face - recognition task in the scanner . this finding suggests that there may be substantial heterogeneity in activation patterns that might be used to predict and/or identify which individuals could benefit the most from targeted cognitive remediation ( e.g. , face training ) . given that the atl is also associated with linking biographic information about faces to perceptual representations ( haxby et al . , 2000 ) , hfa adolescents who showed stronger activation in this region might benefit from strategies such as linking names to faces or encoding a semantic detail about the face ( e.g. , looks like my teacher ) . we did not observe a similar relation between face - recognition behavior and face - related activation in the right atl ( or any other region ) among the td adolescents . the small number of td participants ( n = 12 ) likely underpowered the whole - brain correlational analyses of individual differences in this group.4 at the same time , reports of brain behavior correlations within the face - processing system are actually quite limited , with some reporting positive correlations between the volume of the right ffa with recognition behavior ( golarai et al . , 2007 , 2010 ) and others reporting positive correlations between the magnitude of behavioral and neural responses to face inversion within the right ffa ( alyward et al . , 2005 ; passarotti et al . , 2007 ) in samples that combine adolescents and adults . these correlations could be driven by developmental changes in both face recognition behavior and neural organization within the ffa and/or by individual differences in these characteristics across the age range . future work investigating the developmental emergence of these brain behavior relations separate from individual differences in these relations among typically developing individuals will be critical for interpreting our findings of individual differences among hfa adolescents . in conclusion , our findings identify the right ffa as a particularly vulnerable node in the broadly distributed face - processing network in autism , particularly during adolescence when this region is maturing among typically developing adolescents . importantly , we show that it is not the only atypical node , indicating that the extent of impairment in the functional organization of neural regions supporting face processing in autism is much broader than previously reported . interestingly , conclusions about the relative hyper- or hypo - activation of the amygdala depended on the nature of the contrast that was used to define the activation . we suggest that our findings reflect a systematic relation between the magnitude of neural dysfunction , severity of autism symptoms , and variation in face recognition behavior , which provides new insight about reconciling discrepancies in the existing literature . by elucidating brain behavior relations that underlie one of the most prominent social deficits in autism , this research helps resolve discrepancies in the literature concerning hypo - activation of the social brain in autism , and points to a specific vulnerability in the development of the fusiform gyrus .

despite the impressive literature describing atypical neural activation in visuoperceptual face processing regions in autism , almost nothing is known about whether these perturbations extend to more affective regions in the circuitry and whether they bear any relationship to symptom severity or atypical behavior . using fmri , we compared face- , object- , and house - related activation in adolescent males with high - functioning autism ( hfa ) and typically developing ( td ) matched controls . hfa adolescents exhibited hypo - activation throughout the core visuoperceptual regions , particularly in the right hemisphere , as well as in some of the affective / motivational face - processing regions , including the posterior cingulate cortex and right anterior temporal lobe . conclusions about the relative hyper- or hypo - activation of the amygdala depended on the nature of the contrast that was used to define the activation . individual differences in symptom severity predicted the magnitude of face activation , particularly in the right fusiform gyrus . also , among the hfa adolescents , face recognition performance predicted the magnitude of face activation in the right anterior temporal lobe , a region that supports face individuation in td adults . our findings reveal a systematic relation between the magnitude of neural dysfunction , severity of autism symptoms , and variation in face recognition behavior in adolescents with autism . in so doing , we uncover brain behavior relations that underlie one of the most prominent social deficits in autism and help resolve discrepancies in the literature .

Introduction Materials and methods Results Discussion Conclusion

children with craniosynostosis develop an abnormal head shape due to the premature closure of one or more cranial sutures . this congenital malformation occurs in one in 2100 to 2500 births . in up to 20% of these cases it is part of a syndrome , such as apert , crouzon , muenke and saethre - chotzen , caused by mutations in the fgfr1 , 2 and 3 and twist1 gene . different brain abnormalities are reported in patients with syndromic craniosynostosis including non - progressive ventriculomegaly , callosal agenesis or thinning , agenesis of the septum pellucidum , paucity of the antero - mesial temporal white matter , medial temporal lobe dysgenesis , pyramidal hypoplasia , venous malformations and chiari i malformations [ 3 , 4 , 7 , 8 , 14 , 15 , 19 ] . in patients with syndromic craniosynostosis the origin of the abnormalities can either be intrinsic to the genetic defect or develop secondary to the craniosynostosis and associated hydrocephalus and increased intracranial pressure ( icp ) . a mismatch between intracranial volume versus brain and ventricle volume however , in spite of the craniosynostosis the intracranial volumes are reported to be normal in patients with craniosynostosis or even enlarged in apert and crouzon syndrome [ 6 , 13 , 16 ] . they found that patients with crouzon syndrome had a similar brain volume compared to normal controls . this contradicts the assumption that a mismatch between intracranial and brain volume is the cause of raised icp . to improve our understanding of the development of raised icp , knowledge of brain and ventricular volume in this population patients diagnosed with syndromic or complex craniosynostosis based on genetic testing and treated at the dutch craniofacial center were invited to undergo mri . craniosynostosis was defined as complex if two or more sutures were closed and no mutation was found . the mri were performed on a 1.5-t mr scanner ( ge healthcare , mr signa excite hd ) between january 2004 and january 2011 . brain and ventricular volumes were calculated from the transversal 3d t1 weighted mr images with the use of brainlab. this is a post - processing programme developed for neuronavigation . if the automatic contour was questionable , it was manually edited . after outlining the brain or ventricle volume slice by slice brain volume was compared to that in normal controls at the age of 1 , 4 , 8 and 12 years , reported in literature [ 10 , 12 , 17 ] . total ventricle volume could not be compared to that of normal controls because of the lack of normative data in the literature . a multivariate analysis was performed to look for potential predictors of brain and ventricular volume ; age , gender , syndrome , chiari i malformation and vault expansion . if patients had more than one mri , only the first was used in the analysis , and patients with a ventriculoperitoneal shunt were excluded from the analysis . the intraclass correlation coefficient was calculated to compare the within - rater and between - rater reliabilities of the volume measurements . this study was approved by the medical ethical committee of the erasmus university ( mec2005 - 273 ) . between february 2004 and january 2011 , 103 patients were invited to receive an mri of whom 19 refused to participate . the 84 patient who received an mri had a mean age of 8.1 years ( range 0.318.3 years ) . of the 84 patients , 13 had apert syndrome , 31 crouzon syndrome , 15 muenke syndrome , 10 saethre - chotzen syndrome and 15 complex craniosynostosis . a vault expansion was performed in 66 patients prior to the mri , at a mean age of 1.1 years . a chiari i malformation was found in 12 ( 14% ) patients , one ( 8% ) patient with apert syndrome , 10 ( 32% ) with crouzon syndrome and one ( 7% ) with muenke syndrome . the mean brain volumes at 1 , 4 , 8 and 12 years of patients with craniosynostosis and normal controls are shown in table 1 . age had a significant influence on brain volume ( p < 0.001 ) but not on ventricular volume . the brain volume increased significantly in the first 5 years ( p = 0.004 ) after which it stabilized . patients with apert syndrome ( p = 0.004 ) had a significantly larger ventricular volume compared to all other patients . 0.001 ) had a significantly larger ventricular volume compared to patients without a chiari i malformation . unexpectedly , crouzon syndrome as such was not significantly associated with ventricular volume , although most patients ( 10 out of 12 ) with a chiari i were diagnosed with crouzon syndrome . patients with crouzon syndrome and a chiari i malformation were significantly older compared to crouzon patients without a chiari i malformation , the mean age being 10.1 versus 8.0 years ( p = 0.018 ) . furthermore , they had a larger ventricle volume ( p = 0.019 ) and were less likely to have had a vault expansion ( p = 0.049 ) . the syndrome - specific relation between age and total ventricular and brain volume is shown in figs . 1 and 2.table 1mean brain volume of patients with syndromic craniosynostosis and of normal controlscraniosynostosisnormal controls [ 10 , 12 , 17]p - value1 year n429 age0.90 ( 0.43)1.06 ( 0.03)0.048 brain volume924.25 ( 254.62)855.54 ( 12.43)0.1184 years n826 age3.95 ( 0.60)3.96 ( 0.52)0.960 brain volume1280.88 ( 162.05)1210.62 ( 109.20)0.1668 years n1620 age8.41 ( 0.83)8.60 ( 0.70)0.461 brain volume1403.44 ( 156.87)1391.42 ( 23.54)0.88312 years n1620 age11.92 ( 0.60)12.10 ( 0.60)0.396 brain volume1464.50 ( 148.01)1439.17 ( 23.54)0.455fig . 1syndrome - specific relation between age and ventricular volumefig . 2syndrome - specific relation between age and brain volume mean brain volume of patients with syndromic craniosynostosis and of normal controls syndrome - specific relation between age and ventricular volume syndrome - specific relation between age and brain volume in this study we compared the total brain volume of patients with complex or syndromic craniosynostosis to that of normal controls from the literature . we found that the total brain volume in patients with complex or syndromic craniosynostosis is similar to that in normal controls and that ventricular volume was significantly related to apert syndrome and the presence of a chiari i malformation . the majority of patients with syndromic and complex craniosynostosis have a normal or even enlarged intracranial volume , before as well as after vault expansion [ 6 , 11 , 13 , 16 ] . the finding that brain volume is normal suggests that the compensatory skull growth is sufficient , to allow normal brain growth . the excess of cerebrospinal fluid we observed may be the driving force behind this compensatory growth of the skull . therefore , in these patients , raised icp is more likely to result from raised csf pressure than from a mismatch between intracranial and brain volume . in most patients this raised csf pressure will have a communicating character with papilledema as the only sign . chiari i malformation is primarily seen in patients with crouzon syndrome . in our population 32% of the patients with crouzon syndrome had a chiari i malformation , compared to 73% perviously reported by cinnali et al . . this difference can perhaps be explained by the fact that they performed an mri in case of clinical signs , while we performed mri as part of a prospective study and in most cases without a clinical indication . the diagnosis of crouzon syndrome itself was not associated with an enlarged ventricular volume when it was corrected for chiari i malformation . this means that chiari i malformations have a stronger relation with ventricular volume than crouzon syndrome by itself . with the lack of consecutive data , we are not able to tell whether chiari i malformation precedes or follows the enlarged ventricular volume . enlarged ventricular volume could be the consequence of reduced csf outflow due to chiari i but could also be the cause of downward pressure on the cerebellum due to raised icp . chiari i malformations and raised icp are both prevalent in crouzon syndrome . in apert syndrome larger ventricles are not related to chiari i malformation , as only 2 to 8% of the patients with apert syndrome have a chiari i malformation . despite the larger ventricular volume , this could be due to their significantly larger intracranial volume before and after vault expansion [ 6 , 16 ] . in apert syndrome extra compensatory growth of the skull is facilitated by the enlarged anterior fontanelle that stays open for a relatively long period , preventing the development of increased icp . for the first time we show that patients with syndromic and complex craniosynostosis have a normal total brain volume . therefore , it is unlikely that a mismatch between intracranial and brain volume is the main cause of raised icp . furthermore , we found enlarged ventricular volume to occur particularly in patients with apert syndrome and patients with a chiari i malformation . patients with crouzon syndrome are especially at risk for chiari i , but those without a chiari i have normal ventricular volumes . we advice screening of all patients with apert and crouzon syndrome for the development of enlarged ventricle volume and the presence a chiari i malformation .

purposebrain abnormalities in patients with syndromic craniosynostosis can either be a direct result of the genetic defect or develop secondary to compression due to craniosynostosis , raised icp or hydrocephalus . today it is unknown whether children with syndromic craniosynostosis have normal brain volumes . the purpose of this study was to evaluate brain and ventricular volume measurements in patients with syndromic and complex craniosynostosis . this knowledge will improve our understanding of brain development and the origin of raised intracranial pressure in syndromic craniosynostosis.methodsbrain and ventricular volumes were calculated from mri scans of patients with craniosynostosis , 0.3 to 18.3 years of age . brain volume was compared to age matched controls from the literature . all patient charts were reviewed to look for possible predictors of brain and ventricular volume.resultstotal brain volume in syndromic craniosynostosis equals that of normal controls , in the age range of 1 to 12 years . brain growth occurred particularly in the first 5 years of age , after which it stabilized . within the studied population , ventricular volume was significantly larger in apert syndrome compared to all other syndromes and in patients with a chiari i malformation.conclusionspatients with syndromic craniosynostosis have a normal total brain volume compared to normal controls . increased ventricular volume is associated with apert syndrome and chiari i malformations , which is most commonly found in crouzon syndrome . we advice screening of all patients with apert and crouzon syndrome for the development of enlarged ventricle volume and the presence of a chiari i malformation .

Introduction Materials and methods Results Discussion Conclusion

three hospitals providing inpatient clinical care were identified in the western kenyan highlands ( figure 1 ) . these hospitals were selected because malaria epidemics had been reported within the last 5 years where they were located , and complete clinical records , spanning more than 10 years , were available for review . the three hospitals were st joseph s catholic mission hospital at kilgoris in trans mara district ( latitude 1.068 s , longitude 34.958 e ; altitude 1,683 m ) ; tabaka catholic mission hospital ( latitude 0.751 s , longitude 34.663 e ; altitude 1,684 m ) in gucha district ; and kisii district hospital ( latitude 0.684 s , longitude 34.770 e ; altitude 1,815 m ) in kisii central district . each facility is located above 1,600 m , an altitude above that defined as characterizing highland / epidemic - prone malaria ( 1820 ) , although such limits have been challenged ( 5 ) the average altitudinal limits of the wider area shown in figure 1 range from 1,600 to 2,200 m. monthly temperature and rainfall data were extracted for january 1980 to december 1995 from an interpolated global climate surface at 0.5 x 0.5 spatial resolution ( 41,42 ) , using georeferencing details from tabaka catholic mission hospital . the synoptic year ( 1980 - 1995 ) shows a remarkably stable mean monthly temperature of approximately 20c ( figure 2a ) , with peak rainfall ( approximately 200 mm ) occurring in the months of april and may ( figure 2b ) , usually referred to as the long rains . meteorologic time - series for study hospitals . ( a ) minimum ( bottom ) , mean ( middle ) , and maximum ( top ) monthly temperatures ( c ) ; ( b ) average total monthly rainfall ( mm ) . hospital admission registers for every ward at each facility were located and sequentially reviewed to identify patient age , date , and cause of admission . month- and age - tallied cases of clinical malaria were compiled for each complete year . criteria used to select malaria cases were based on whether malaria was made as a primary , co - primary , or coincidental diagnosis by the admitting physician . not all diagnoses were microscopically confirmed , and discharge diagnoses may have been different from those defined on admission , following further clinical and laboratory investigations . nevertheless , patients at each facility were treated for malaria during the initial 24 hours of admission and represent the monthly clinical commitment to malaria case management at each hospital . such data are used routinely to define epidemics by local health authorities and serve as the basis for increasing demands for resources . in these analyses we consider only the pediatric malaria admissions ( patients < 15 years of age ) , who constituted approximately two thirds of the patients at each facility ( kilgoris , 14,079 adults and 30,793 children ; kisii , 44,043 adults and 84,648 children ; and tabaka , 23,692 adults and 55,871 children during the study period ) . the rationale is that children are more likely to give an accurate picture of local malaria transmission than adults , as they are less likely to have functional immunity or to have traveled and acquired the disease elsewhere . cumulative monthly cases were also computed for each year to show the overall annual burden and acute , seasonal rises in malaria admissions . the years of exceptional malaria cases were defined simply as the 2 years of highest cases during the surveillance period . we assumed a minimum set of requirements for resource - constrained , district - level health services in kenya : access to a computer , limited knowledge of a spreadsheet application , and availability of at least 5 years of admission records from a health facility . for this reason , we focused on a subset of those techniques advocated by who for application to malaria surveillance in resource - constrained environments ( 24 ) . epidemic alerts can be based on simple incidence thresholds only , as is common with meningococcal meningitis at the district level in sub - saharan africa ( 4346 ) ; when a threshold is exceeded , an alert is triggered . the value of the threshold is usually determined from expert opinion informed by an examination of retrospective case data over wide geographic areas . this technique is not applicable to a single facility where accurate population denominator data ( necessary to calculate incidence ) are often not available and therefore not considered further . many epidemic surveillance techniques aim to identify points in a disease time - series outside the 95% confidence intervals of a normal distribution determined from the history of cases at that location . a method proposed by cullen ( 47 ) uses the previous 5 years of data ( in which epidemic years are arbitrarily excluded ) to construct an admissions profile for an average year . the alert threshold for each month is then determined as the mean plus 2 times the standard deviation ( strictly , the arithmetic mean plus 1.96 times the standard deviation should capture 95% of cases in normally distributed data ) . this technique was successfully applied to cases of plasmodium vivax malaria in northern thailand during the 1980s ( 47 ) . it has also been used for surveillance of p. falciparum malaria in the madagascan highlands ( 49 ) . who has advocated the use of a conceptually similar method that triggers an alert when current cases exceed the upper 3rd quartile or the upper normal limit determined from 5 years of retrospective monthly case data ( 50 ) . for 5 years of observations , quartile 0 is the minimum , quartile 1 the second lowest , quartile 2 the median , quartile 3 the second highest , and quartile 4 the maximum value of the series for any given month . this method has been implemented to detect highland malaria epidemics in ethiopia ( 22 ) . the centers for disease control and prevention has developed a further cumulative sum ( c - sum ) method for detecting epidemics . it is based on the construction of an average or base year , determined by calculating the expected number of cases using the average for that month ( and the previous and following month ) during the past 5 years ( n=15 ) ( 29,51,52 ) . for example , the expected number of cases for march 2000 would be derived from the average of february , march , and april admissions from 1995 to 1999 , inclusive . a ratio of present to past cases is then usually presented as a current to past history graph ( 53 ) , with values greater than one representing disease increases . who , cullen , and c - sum methods were tested on the series of pediatric malaria admissions data to evaluate their usefulness in the identification of epidemics , defined as the 2 years of highest numbers of cases . we modified the c - sum technique to provide 95% confidence intervals for the expected cases so that it could be evaluated against the other techniques . for each method , the expected cases in a given month were defined by the previous 5 years of data and sequentially updated for each new observation year in the series . epidemic years were not excluded from the base years , as no objective criteria have been offered to define years that are epidemic and excluding these years would increase the likelihood of detecting epidemics . a skewness statistic that measures the degree of asymmetry in a distribution around the mean ( microsoft excel 2000 , seattle , wa ) was also applied to the data to test assumptions of normality in the admissions data . positive or negative values indicate an asymmetric tail extending towards more positive or more negative values , respectively . the cullen and c - sum techniques were then repeated by using log10 transformed childhood admissions data to investigate potential problems with the techniques that assume normally distributed data . confidence intervals were determined for the cullen and c - sum techniques on untransformed and log10 normalized admissions data by using the mean + ( 2x standard deviation ) as well as the mean + ( t value at 0.05 confidence interval x standard error ) , as is recommended for small sample sizes ( 48 ) . three hospitals providing inpatient clinical care were identified in the western kenyan highlands ( figure 1 ) . these hospitals were selected because malaria epidemics had been reported within the last 5 years where they were located , and complete clinical records , spanning more than 10 years , were available for review . the three hospitals were st joseph s catholic mission hospital at kilgoris in trans mara district ( latitude 1.068 s , longitude 34.958 e ; altitude 1,683 m ) ; tabaka catholic mission hospital ( latitude 0.751 s , longitude 34.663 e ; altitude 1,684 m ) in gucha district ; and kisii district hospital ( latitude 0.684 s , longitude 34.770 e ; altitude 1,815 m ) in kisii central district . each facility is located above 1,600 m , an altitude above that defined as characterizing highland / epidemic - prone malaria ( 1820 ) , although such limits have been challenged ( 5 ) the average altitudinal limits of the wider area shown in figure 1 range from 1,600 to 2,200 m. monthly temperature and rainfall data were extracted for january 1980 to december 1995 from an interpolated global climate surface at 0.5 x 0.5 spatial resolution ( 41,42 ) , using georeferencing details from tabaka catholic mission hospital . the synoptic year ( 1980 - 1995 ) shows a remarkably stable mean monthly temperature of approximately 20c ( figure 2a ) , with peak rainfall ( approximately 200 mm ) occurring in the months of april and may ( figure 2b ) , usually referred to as the long rains . meteorologic time - series for study hospitals . ( a ) minimum ( bottom ) , mean ( middle ) , and maximum ( top ) monthly temperatures ( c ) ; ( b ) average total monthly rainfall ( mm ) . hospital admission registers for every ward at each facility were located and sequentially reviewed to identify patient age , date , and cause of admission . month- and age - tallied cases of clinical malaria were compiled for each complete year . criteria used to select malaria cases were based on whether malaria was made as a primary , co - primary , or coincidental diagnosis by the admitting physician . not all diagnoses were microscopically confirmed , and discharge diagnoses may have been different from those defined on admission , following further clinical and laboratory investigations . nevertheless , patients at each facility were treated for malaria during the initial 24 hours of admission and represent the monthly clinical commitment to malaria case management at each hospital . such data are used routinely to define epidemics by local health authorities and serve as the basis for increasing demands for resources . in these analyses we consider only the pediatric malaria admissions ( patients < 15 years of age ) , who constituted approximately two thirds of the patients at each facility ( kilgoris , 14,079 adults and 30,793 children ; kisii , 44,043 adults and 84,648 children ; and tabaka , 23,692 adults and 55,871 children during the study period ) . the rationale is that children are more likely to give an accurate picture of local malaria transmission than adults , as they are less likely to have functional immunity or to have traveled and acquired the disease elsewhere . cumulative monthly cases were also computed for each year to show the overall annual burden and acute , seasonal rises in malaria admissions . the years of exceptional malaria cases were defined simply as the 2 years of highest cases during the surveillance period . we assumed a minimum set of requirements for resource - constrained , district - level health services in kenya : access to a computer , limited knowledge of a spreadsheet application , and availability of at least 5 years of admission records from a health facility . for this reason , we focused on a subset of those techniques advocated by who for application to malaria surveillance in resource - constrained environments ( 24 ) . epidemic alerts can be based on simple incidence thresholds only , as is common with meningococcal meningitis at the district level in sub - saharan africa ( 4346 ) ; when a threshold is exceeded , an alert is triggered . the value of the threshold is usually determined from expert opinion informed by an examination of retrospective case data over wide geographic areas . this technique is not applicable to a single facility where accurate population denominator data ( necessary to calculate incidence ) are often not available and therefore not considered further . many epidemic surveillance techniques aim to identify points in a disease time - series outside the 95% confidence intervals of a normal distribution determined from the history of cases at that location . a method proposed by cullen ( 47 ) uses the previous 5 years of data ( in which epidemic years are arbitrarily excluded ) to construct an admissions profile for an average year . the alert threshold for each month is then determined as the mean plus 2 times the standard deviation ( strictly , the arithmetic mean plus 1.96 times the standard deviation should capture 95% of cases in normally distributed data ) . this technique was successfully applied to cases of plasmodium vivax malaria in northern thailand during the 1980s ( 47 ) . it has also been used for surveillance of p. falciparum malaria in the madagascan highlands ( 49 ) . who has advocated the use of a conceptually similar method that triggers an alert when current cases exceed the upper 3rd quartile or the upper normal limit determined from 5 years of retrospective monthly case data ( 50 ) . for 5 years of observations , quartile 0 is the minimum , quartile 1 the second lowest , quartile 2 the median , quartile 3 the second highest , and quartile 4 the maximum value of the series for any given month . this method has been implemented to detect highland malaria epidemics in ethiopia ( 22 ) . the centers for disease control and prevention has developed a further cumulative sum ( c - sum ) method for detecting epidemics . it is based on the construction of an average or base year , determined by calculating the expected number of cases using the average for that month ( and the previous and following month ) during the past 5 years ( n=15 ) ( 29,51,52 ) . for example , the expected number of cases for march 2000 would be derived from the average of february , march , and april admissions from 1995 to 1999 , inclusive . a ratio of present to past cases is then usually presented as a current to past history graph ( 53 ) , with values greater than one representing disease increases . who , cullen , and c - sum methods were tested on the series of pediatric malaria admissions data to evaluate their usefulness in the identification of epidemics , defined as the 2 years of highest numbers of cases . we modified the c - sum technique to provide 95% confidence intervals for the expected cases so that it could be evaluated against the other techniques . for each method , the expected cases in a given month were defined by the previous 5 years of data and sequentially updated for each new observation year in the series . epidemic years were not excluded from the base years , as no objective criteria have been offered to define years that are epidemic and excluding these years would increase the likelihood of detecting epidemics . a skewness statistic that measures the degree of asymmetry in a distribution around the mean ( microsoft excel 2000 , seattle , wa ) was also applied to the data to test assumptions of normality in the admissions data . positive or negative values indicate an asymmetric tail extending towards more positive or more negative values , respectively . the cullen and c - sum techniques were then repeated by using log10 transformed childhood admissions data to investigate potential problems with the techniques that assume normally distributed data . confidence intervals were determined for the cullen and c - sum techniques on untransformed and log10 normalized admissions data by using the mean + ( 2x standard deviation ) as well as the mean + ( t value at 0.05 confidence interval x standard error ) , as is recommended for small sample sizes ( 48 ) . figure 3a - c shows pediatric admissions for the three study hospitals during the surveillance period . the graphs of cumulative cases ( figure 4a - c ) show a distinct seasonality in admissions ; the sharpest rise in case numbers occurred in june and july , immediately after the long rains in april and may ( figure 2b ) . the 2 years of highest case numbers were 1994 and 1998 for kilgoris , 1996 and 1997 for kisii , and 1997 and 1996 for tabaka . in these so - called epidemic years , cases were often above normal in all months . cumulative child admissions ( < 15 years ) in kilgoris ( a ) , kisii ( b ) , and tabaka ( c ) . all years for which data were available are shown , 1980 - 1999 , 1987 - 2000 , and 1981 - 2000 time periods for kilgoris , kisii and tabaka , respectively . red lines show epidemic years , defined as the 2 years of highest total admissions . for kilgoris these exceptional years are 1994 and 1998 , for kisii they are 1996 and 1997 , and for tabaka they are 1997 and 1996 . the child admissions data at each site were positively skewed with values of 2.88 , 1.96 , and 1.78 ( skewness statistic = 0 for normal data series ) for kilgoris , kisii , and tabaka , respectively ( table 1 ) . log10 transformations of these data reduced the positive skew , thus helping normalize each series to values of -0.13 , 0.34 , and -0.08 for kilgoris , kisii , and tabaka , respectively . who methods concluded that 41.7% , 31.5% , and 42.8% of months in the surveillance period were epidemic for kilgoris , kisii , and tabaka , respectively ( table 2 ; figure 3a - c ) . the cullen method showed fewer than half of these months to be epidemic , 14.4% , 10.2% , and 12.8% , respectively . the c - sum method indicated fewer still at 9.4% , 5.6% , and 10.6 % , respectively . log10 transforming the child admissions data further reduced the proportion of months detected as epidemic . adjusting the confidence intervals for small sample sizes had the opposite effect ( table 2 ) . the who method and cullen and c - sum techniques using the kirkwood confidence intervals predicted approximately one third of all months during the surveillance period as epidemic ( average 31.7% , range 14.8% to 42.8 % ) ( table 2 ; figure 3a - c ) . strict statistical evaluation between the remaining techniques is difficult because of the problem of retrospectively determining what months were true epidemics ; thus such evaluation was simply on the criteria of identifying the 2 years of highest cases ( figure 4 ) . all techniques identified these 2 epidemic years in kilgoris , but only the untransformed cullen method with standard confidence intervals detected both epidemic years in kisii and tabaka as well . reports of epidemics in the highlands of western kenya increased in frequency in the early 1990s ( 10,12,54,55 ) ; as a consequence , detection and control of epidemics became a priority for the recently launched national malaria strategic plan ( 23 ) . this initiative forms part of a broader international effort to develop surveillance and warning systems for epidemic detection in africa as part of the who roll back malaria initiative ( 24,56 ) . the definition of epidemics continues to confuse many public health practitioners specializing in common diseases such as malaria . epidemics are more often defined in response to political necessity rather than by examining empirical data . little critical examination of long - term clinical data against proposed methods for epidemic interpretation in nominally epidemic - prone areas of sub - saharan africa has occurred . to address this , we examined time series of pediatric malaria admission data during the late 1980s and 1990s from three hospitals located in districts of the western highlands of kenya identified by the ministry of health as prone to epidemics . application of three primary epidemic detection methods indicated alert signals in most years of the test period with or without modifications . rather than representing an inadequacy in the methods , this reflected the restricted utility of these approaches in areas of acutely seasonal malaria case burdens , characterized by a large degree of between - year variability in the timing of seasonal onset and a gradual increasing trend in admissions . clearly , having such frequent epidemic alert signals makes the usefulness of such techniques in this particular area of the western kenyan highlands questionable . a further characteristic of this area is between - year variability in malaria incidence . during the 1990s , at least two important and dramatic seasonal rises in malaria occurred at each of the three hospitals ( figure 4 ) . sharp rises occurred during the months of february , and more commonly april or may ( with the onset of the rains [ figure 2b ] ) . plotting monthly cumulative cases provided a more informative tool than traditional time - series plots to show seasonal deviations from previous years and simultaneously represented overall annual malaria cases . for the two exceptional years at each of the hospitals , the most sensitive of the epidemic detection methods shown in figure 3 was the nontransformed cullen technique that used standard confidence intervals . this technique , however , would also have given rise to a substantial number of false alarms during the observation period . applying the statistical techniques we have outlined highlights several methodologic issues that deserve comment , particularly for the cullen and c - sum techniques , and should be considered by those advocating further application of these tools to common vector - borne diseases . first , mosquito - borne diseases that are sensitive to climate and hence are often seasonal , can show a skewed non - normal distribution in time . methods that depend on arithmetic means and standard deviations ( with their assumptions of data normality ) to define alerts may require data transformation . simple log10 transforms achieved data normalization and decreased the sensitivity of the techniques at all three facilities in this study . second , each technique recommends using 5 years of retrospective admissions data so that standard deviations and hence alert thresholds for an average month are based on only five samples . a more appropriate formula for calculating the standard deviation in such situations has been proposed ( 48 ) , although applying such modifications to these health facilities made the epidemic detection techniques substantially more sensitive . third , when cases are increasing over the duration of the study , it is important to take a 5-year moving average to adjust the magnitude of the base year accordingly . testing for the sensitivity of these techniques to the duration of moving average used was beyond the scope of this research but requires future investigation . for example , how many months detected as epidemic are needed in any year to prompt that year s exclusion from the moving average , and after exclusion , what data are used to define the confidence intervals for alerts ? this exercise demonstrates that many factors need to be more fully considered before widely advocating such techniques . our analyses used records of severe and complicated malaria admissions to tertiary - level health facilities , where diagnosis is often supported by microscopy . we have not applied the epidemiologic surveillance tools to patients with mild , ambulatory cases of malaria treated as outpatients . these latter data may provide a more robust tool for early detection , but they are also subject to imprecise clinical case definitions , where diagnosis is almost always made presumptively without microscopy . improvements in the provision of microscopy in the diagnosis of outpatient malaria may facilitate improvements of these surveillance tools . epidemic malaria was precisely described by macdonald as an acute exacerbation of disease out of proportion to the normal to which the community is subject.epidemics are common only in zones of unstable malaria , where very slight modification in any of the transmission factors may completely upset equilibrium and where the restraining influence of immunity may be negligible or absent , and they therefore show a very marked geographic distribution ( 57,58 ) . the term epidemic is applied more liberally today for malaria in the kenyan highlands ; it is essentially used for any occurrence of cases in excess of normal . much of the confusion around defining epidemics spatially or temporally relates to knowing what is ( or should be ) expected routinely . this can relate to climate - driven variation , seasonality , interepidemic periods resulting from population dynamics , or long - term trends ( 39 ) . these factors can all operate simultaneously and are not epidemics , although they may have substantial public health implications . deviations from any of these expected variations are true epidemics if they result from a disturbance of the normal epidemiologic equilibrium ( 50 ) . such considerations are crucially important in the determination of the normal situation against which epidemics are measured . the highlands of western kenya is an area where so - called malaria epidemics have been increasingly reported . considerable international efforts are also being made to develop and promote early warning and improved case - detection systems for epidemic - prone areas ( 24,56,59 ) . these results indicate that the simple epidemic detection techniques recommended to date require substantial refinement before they can be considered operationally robust , since they lack the required sensitivity in detecting aberrant case burdens . the further question as to whether these techniques are appropriate for facilities that have pronounced and acutely seasonal transmission of malaria is still open . the dual goals of technique development and a more comprehensive description of the local malaria epidemiology in this region are the subjects of ongoing research . a related article in this issue outlines the implications of these data for interpreting the epidemiology of p. falciparum malaria in this highland region of western kenya ( 60 ) .

epidemic detection algorithms are being increasingly recommended for malaria surveillance in sub - saharan africa . we present the results of applying three simple epidemic detection techniques to routinely collected longitudinal pediatric malaria admissions data from three health facilities in the highlands of western kenya in the late 1980s and 1990s . the algorithms tested were chosen because they could be feasibly implemented at the health facility level in sub - saharan africa . assumptions of these techniques about the normal distribution of admissions data and the confidence intervals used to define normal years were also investigated . all techniques identified two epidemic years in one of the sites . the untransformed cullen method with standard confidence intervals detected the two epidemic years in the remaining two sites but also triggered many false alarms . the performance of these methods is discussed and comments made about their appropriateness for the highlands of western kenya

Methods Study Area Clinical Data Epidemic Detection Techniques Statistical Analysis Results Discussion

metastases to organs such as the liver , bones or central nervous system appear to be a frequent complication of lung cancer , whereas metastases to the suprarenal glands are found less frequently . metastases of lung cancer to the spleen are a great rarity and they are described sporadically . an adrenal gland tumor detected incidentally during imaging tests is described as an incidentaloma [ 37 ] . primary cysts acquiring enormous proportions and hemangiomas are classified as benign tumors [ 9 , 10 ] . metastatic lesions and inflammatory pseudotumors may also be seen , but only very rarely and usually as casuistry [ 11 , 12 ] . splenic lesions may be observed in the course of malignant lymphoma [ 13 , 14 ] . lesions characteristic of sclerosing angiomatoid nodular transformation ( sant ) have also been described . in most cases , the typical characteristics of splenic tumors are established on the basis of histopathological findings , which are obtained by the surgical removal of the tumor or by post - mortem examination [ 8 , 10 , 1618 ] . this work presents a case of simultaneous dissemination of lung cancer to the adrenal gland and to the spleen . a female patient ( age 74 ) was sent from a hospital in zawiercie for further investigations and management of a left lung tumor lesion discovered during x - ray examination . chest surgeons had rejected her from an invasive therapy . however , bronchoscopy was performed and revealed no evidence of pathological bronchial lesions . in this situation the patient was sent to our hospital for the purpose of making the histopathological diagnosis ( history no . 16735/877/09 ) . computed tomography ( ct ) scan showed chest infiltration situated peripherally in the left lung . after establishing the distance , place and depth of the puncture by using ct ( fig . 1 ) , the parietal tumor was visualized by ultrasound and a biopsy was performed . we performed an ultrasound - guided ( free hand technique ) fine - needle biopsy of the lesion using a hitachi eus 515 sonographic machine ( fig . computed tomography imaging scan establishing the distance , place and depth of the puncture ultrasound imaging scan wall located left lung tumor biopsy needle the ultrasound examination of the abdomen revealed a pathological mass in the spleen and in the left adrenal gland ( fig . we also performed in local anesthesia an ultrasound - guided ( free hand technique ) fine - needle biopsy of these lesions . in our case small cell lung cancer was detected in the percutaneous biopsy of the left lung . the same type of cancer as in the left lung was observed in both the adrenal gland and in the spleen ( metastases of small cell cancer ) . imaging methods available to us showed no evidence of cancer metastases in other organs . in the existing literature , we found only a few cases of lung cancer metastases to the spleen [ 2 , 17 , 18 ] . simultaneous metastases of lung cancer to the adrenal gland and the spleen have never been described . the case presented above shows that the metastatic lesion can sometimes be an accessible place to collect tissue for diagnosing the cancer pattern of the primary cancer site . the case is exceptional because the spleen is an organ where lung cancer metastases are not frequently found , while metastases to the adrenal gland alone are common . the case is also unique because the adrenal gland and the spleen are organs where finding concurrent metastases of lung cancer is very rare .

metastases of lung cancer to such organs as the liver , bones or to the central nervous system appear to be a frequent complication of this disease . at the same time , metastases to the adrenal gland are found less frequently . metastases of lung cancer to the spleen are a great rarity and they are described sporadically . our report presents a unique case of left lung cancer with simultaneous metastases to the adrenal gland and to the spleen . all the presented lesions were diagnosed by ultrasound guided biopsy and confirmed by histopathological examination.the patient received combined chemoradiotherapy . she was closely monitored over an 18-month observation period following treatment . no new metastases were reported .

Introduction Material and methods Results and discussion None

alzheimer 's disease ( ad ) is a neurodegenerative disorder in the elderly characterized by progressive dementia [ 1 , 2 ] . the disorder probably begins many years before the first clinical symptoms are evident [ 3 , 4 ] . recent studies have demonstrated that during the presymptomatic phase , neuronal degeneration occurs even without the presence of clinical symptoms . these make preclinical discrimination between people who will and will not ultimately develop ad critical for early treatment of the disease . neuropathological hallmarks of ad include macroscopic change as reduced brain weight with cortical atrophy and ventricular enlargements primarily due to neuronal loss in the temporal and parietal structures . at the microscopic level , it can be found neurofibrillary tangles ( intracellular aggregations of tau protein filaments ) and amyloid plaques ( extracellular aggregates of amyloid beta - peptides ) that are particularly concentrated in the hippocampus , entorhinal cortex , and postcentral parietal neocortex . recent advances in molecular genetics have allowed identifying individuals carrying defective genes predisposed to develop ad . when the disease penetrance is high , the examination of apparently asymptomatic subjects carriers of defective genes allows early evaluation of different physiopathology 's processes . up to now , three genes have been unequivocally related to familial forms of ad , the presenilin-1 ( ps-1 ) gene , accounting for 1550% of the cases , the amyloid precursor protein ( app ) , and the presenilin-2 ( ps-2 ) mutation which have been identified in less than 1% . in a genetic analysis , the findings , of mutations that produce an autosomal dominant form of ad , in a patient with dementia or in a family carrying dominant autosomic form of the disease , allow a diagnosis with nearly 100% of certainty . while the cause(s ) of the most common ad the late - onset form is unknown , mutations in presenilin-1 cause many cases of familial the early onset ad . in a community based at antioquia , colombia , there is a well - documented form of early onset familial ad related with glutamic acid to alanina mutation at the codn 280 of chromosome 14 , in the presenilin-1 gene . clinically , this phenotype can not be distinguished from sporadic ad except for the early aged onset . a clinical diagnosis accuracy of approximately 85% of detection rate is commonly achieved , by a procedure of exclusion after structural or functional imaging tests including quantitative electroencephalography , laboratory , and psychometric test . annual conversion rate , from normality to dementia of ad type , it ranges between 0.2% and 4% whereas , from mild cognitive impairment to ad , is between 6% and 25% . it is an open issue with important clinical implication whether or not mild cognitive impairment is essentially prodromal stage of ad [ 1416 ] . in the aging brain including the ad ones during pre - clinical conditions plastic compensatory remodeling guarantees functional maintenance , so that the neuronal and synaptic death can occur in the absence of symptoms for an unknown period of time that might last years or even decades . this mechanism of cognitive or brain reserve motivates the use of instrumental markers of ad in association with standard assessment of cognitive functions . few studies have assessed eeg measures over the course of dementia progression . in our sample , there are groups of asymptomatic carrier which are going to develop the ad with 100% of accuracy because his mutation in the presenilin-1 gene provides autosomal dominance inheritance with virtually 100% of penetrance . systematic studies of this familial ad have enabled the identification of subjects that carry the mutation but without clinical signs or symptoms . this condition allows finding individuals at preclinical stages of ad , permitting the early electrophysiological evaluation with the quantitative eeg measure . multiple techniques have been used to evaluate the pathophysiological processes underlying early stages of ad . among them , the quantitative analysis of digital electroencephalogram ( eeg ) has been introduced as a nonexpensive , noninvasive , and objective tool for evaluating dementia . longitudinal analyses of brain eeg rhythms have provided objective evidences of disease progression from mci to ad [ 17 , 18 ] . previous studies using the eeg in demented patients have reported high sensitivity in detecting a diffuse organic damage , but low specificity in determining an etiologic diagnosis . in the last years , several research groups have started investigating the potentiality of electroencephalogram for diagnosis ad . however , in our knowledge , there are no previous research using eeg signal for the diagnosis of ad subjects to ensure 100% who develop the disease in the future will develop the disease . many researches have shown that mild cognitive impairment and ad cause eeg signals to slow down and mild cognitive impairment and ad are associated with increase of power in slow frequencies ( delta- and theta - band ) and a decrease of power in fast frequencies ( alpha- and beta - band ) . nevertheless , increased gamma - band power has been reported in mild cognitive impairment and ad patients compared to healthy age matched control subjects . the most often electroencephalographic findings in ad patients are ( a ) severe slowing of background activity with an increased power in slow eeg activity [ 2126 ] and ( b ) a concomitant decrease of the power at fast ( alpha and beta ) eeg frequency ranges [ 21 , 27 , 28 ] . many researches also hypothesize that the earliest modifications of the eeg occur in the beta- and theta - bands , while changes in alpha and delta bandwidths appear later in the time course of the disease [ 2 , 29 ] . several studies have shown a close relationship between an increase in the slow frequency and the degree of cognitive impairment in these patients [ 26 , 3135 ] . the quantitative eeg has been also used to evaluate the treatment response with cholinesterase inhibitors and the dementia follow - up [ 28 , 37 ] . when compared to healthy normal elderly subjects , ad patients evidence high power for delta and theta and low power for posterior alpha ( 812 hz ) and/or beta ( 1330 hz ) frequencies [ 2 , 7 , 3842 ] . some of these eeg changes could differentiate dementia diagnosis , as the strong decline of posterior slow frequency alpha sources that occurs specifically for mild ad group when compared to vascular dementia and normal elderly groups . eeg abnormalities were associated with altered regional blood flow / metabolism and impaired global function as evaluated by minimental state examination ( mmse ) [ 2 , 33 , 43 ] . nevertheless , in the earliest stages of ad , electroencephalographic patterns have not been completely characterized . ambiguous results have been reported ; nobili et al . found no eeg alteration or worsening in 50% of early ad cases in a one - year follow - up study . other authors described a delay in the peak of the dominant frequency [ 33 , 44 ] . normally , the classical frequency band analysis has a poor resolution and may overlook slight , but important , changes in the spectra [ 33 , 45 ] . of note , early stages of ad ( even preclinical ) are typically associated with slowing down resting occipital alpha rhythms , namely , a decrease of the individual alpha frequency ( iaf ) peak in power density . the iaf peak , defined as the frequency associated with the strongest eeg power at the extended alpha range , should be always taken into account in eeg studies in ad subjects , since power changes in theta and alpha bands might be dependent phenomena . furthermore , the conventional partition of eeg power into many conventional frequency bands allows the comparison of the results with those of most of the field studies but may prevent the separation of independent eeg rhythms or sources . the aim of the present study is to evaluate the hypothesis that quantitative eeg analysis is able to detect early functional changes in preclinical stages of familial ad in acr and clinically normal subjects . we believe that this research may help to identify an electroencephalographic pattern that could distinguish which genetically predisposed subjects will develop more rapidly the disease which in turn may in the long term improve the reliability of eeg as a diagnostic tool for ad . three groups of subjects were selected from five extended families affected by early onset ad due to an e280a presenilin-1 mutation . these were as follows.a probable ad group : eighteen patients with the e280a presenilin-1 mutation are diagnosed as probable ad , but still with mild symptoms of the disease according to the minimental state examination ( mmse : 1523 , and the functional assessment stages ( fast < 5).an acr ( asymptomatic carriers ) group there are twenty - one subjects , with the mutation but without any clinical symptoms of dementia ( mmse : > 23 and fast = 1).a normal group there are eighteen healthy subjects without history of neurological or mental disease , not carrying the mutation ( mmse : > 23 and fast = 1 ) . the subjects of this group were selected from the families in which there are any members with e280a presenilin-1 mutation with probable ad or acr . a probable ad group : eighteen patients with the e280a presenilin-1 mutation are diagnosed as probable ad , but still with mild symptoms of the disease according to the minimental state examination ( mmse : 1523 , and the functional assessment stages ( fast < 5 ) . an acr ( asymptomatic carriers ) group there are twenty - one subjects , with the mutation but without any clinical symptoms of dementia ( mmse : > 23 and fast = 1 ) . a normal group there are eighteen healthy subjects without history of neurological or mental disease , not carrying the mutation ( mmse : > 23 and fast = 1 ) . the subjects of this group were selected from the families in which there are any members with e280a presenilin-1 mutation with probable ad or acr . the exclusion criteria were severe physical illness , psychiatric or neurological disorders associated with potential cognitive dysfunction , and other dementia conditions ( fronto - temporal dementia , dementia associated with parkinsonism , lewy body disease , pure vascular or prion dementia , etc . ) . subjects with alcohol / drugs abuse , regular use of neuroleptics , antidepressants with anticholinergic action were also excluded . informed consent for participation was obtained from all subjects according to a general protocol approved by the human subjects committee of university of antioquia , medellin , colombia . the presence of signs or symptoms of ad was assessed using the criteria outlined by the national institute of neurological and communicative disorders and the alzheimer 's disease and related disorders association ( nincds - adrda ) and the dsm - iv criteria . table 1 shows the mean values of demographic and clinical characteristic of the probable ad , acr and normal groups as well as the results of a one - way anova for each of the variables . the short - circuited left and right mastoid served as reference for all 19 channels . eegs were recorded with the fenix system ( neuronic s.a ) , they were amplified with a gain of 512 , a filtering band pass of 0.530 hz , and a sampling rate of 200 hz . a 60 hz notch filter was also used . the eegs were visually inspected offline . for quantitative analysis , 24 artefact - free epochs of 2.56 seconds duration were selected . broad band spectral parameters ( absolute power , relative power , and mean frequency ) were calculated in four electroencephalographic classic bands : delta ( 0.53.5 hz ) , theta ( 3.57.5 hz ) , alpha ( 7.512.5 hz ) , and beta ( 12.519.1 hz ) while narrowband frequency model was computed with a frequency resolution of 0.39 hz from 0.78 to 19.14 hz [ 5153 ] . a logarithmic transformation was applied to the spectral estimates to obtain an approximate gaussian distribution . the spectral power at each electrode was normalized to the spectral power averaged across all frequencies ( 0.519 hz ) and electrodes . the individual alpha frequency ( iaf ) , as an anchor frequency , was selected according to literature . the iaf is defined as the frequency associated with the stronger eeg power at the extended alpha range . the frequencies bands were adjusted individually for each subject , by using iaf as the cut - off point between the lower and upper alpha band . in order to reveal difference between groups the following steps were carried . ( i ) the z standardized statistic was calculated for all spectral parameters ( 1)z = x(age , mmse)(age , mmse ) , where and are the mean and standard deviation ( sd ) estimated in the normal group . the x value is the observation of each subject . as eeg power in the theta and alpha frequency range has been described that is related to cognition and memory [ 47 , 54 ] . this was also supported by our results where we found a significance difference between mean values in each group in these covariates ( table 1 ) . the mean and standard deviation values were computed using the regression functions obtained from the normal group using the covariates aged and mmse with crossvalidation technique ( leave one out ) to compare a single individual to a population of normal individuals . in order to identify the measures that are deviant from normal and the magnitude of deviation , the z score was computed for all variables based on his / her respective age , mmse matched mean , and sd in the normal group . for example , parameters from left - right homologous derivations tend to be symmetrical , the deviation of z vector from normal group . this was carried out by consideration of the correlations between its components by means of mahalanobis distance ( d ) . the formal definition is ( 2)d2=zt1z , where z is the vector according to step ( i ) and is the inverse covariance matrix of the z vector . following standard math notation z is the transpose of vector z. the application of d for broad band frequency has been considered by john et al . in 1987 . the d is useful to combine deviations from the normal pattern in different spectral feature enhancing slight deviation at different frequency bands . this combination is in agreement with usual practice in mapping studies which involves the subjective analysis of deviation from the norm in multiple spectral maps . the d distance was calculated by selecting different parameters of the z vector as follows : taking in account all the parameters ( here called global d ) .considering all frequencies in a fixed region ( five regions were considered : frontal ( f3 , f4 , f7 , f8 , fz ) , central ( c3 , c4 , cz ) , temporal ( t3 , t4 , t5 , t6 ) , parietal ( p3 , p4 , pz ) , and occipital regions ( o1 , o2 ) . ( this was referred to as regional d ) , considering all regions in a fixed frequency interval . firstly , the classical broad band model was considered ( delta , theta , alpha , and beta bands ) , and in a second place , two news bands were defined slow and fast band . the slow band contained frequencies from 0.5 to 7.5 hz and the fast band frequencies from 7.5 to 19.14 hz ( here refered to as frequency d ) . considering all frequencies in a fixed region ( five regions were considered : frontal ( f3 , f4 , f7 , f8 , fz ) , central ( c3 , c4 , cz ) , temporal ( t3 , t4 , t5 , t6 ) , parietal ( p3 , p4 , pz ) , and occipital regions ( o1 , o2 ) . ( this was referred to as regional d ) , considering all regions in a fixed frequency interval . firstly , the classical broad band model was considered ( delta , theta , alpha , and beta bands ) , and in a second place , two news bands were defined slow and fast band . the slow band contained frequencies from 0.5 to 7.5 hz and the fast band frequencies from 7.5 to 19.14 hz ( here refered to as frequency d ) . ( iii ) to evaluate the diagnostic efficiency of d , receiving operate curves ( roc ) models were used . the p value observed under null hypothesis was corrected by bonferroni with adjusted of 0.0010 . figure 1 shows superimposed averaged log spectral power for each group of the narrow band model . in temporal regions , the probable ad group shows a higher increase of the power in the theta band compared to the acr and the normal groups . on the other hand , the probable ad group showed a decrease of the alpha power with respect to the other two groups . the sd of the spectra for all frequencies and derivations in the groups were normal group 0.55 , acr group 0.87 , and probable ad 1.02 . the difference between these log spectral value should be demonstrated statistically , as a significant difference between the accuracy of classified using the individually distance to normal group ( according to step ( ii ) in section statistical analysis ) . the d was computed using the z log spectra of the narrow band model . the histogram of the d ( figure 2 ) shows that the maximal distance ( in decrease order of magnitude ) was reached in the probable ad followed by the acr group and the normal group . the discriminative accuracy of d was quantitative measured q by means of the roc area . the areas were estimated between normal and acr groups ( area roc = 0.90 ) , normal probable ad groups ( area roc = 0.98 ) , and acr - probable ad groups ( area roc = 0.92 ) . that means that it is possible to separate acr and probable ad groups from the normal group , but also that the diagnostic performance is higher to separate acr and probable ad groups . in order to determinate the accuracy according to the regions and frequency bands , d was calculated for selected component of the z log spectra in five different regions : frontal ( f3 , f4 , f7 , f8 , fz ) , central ( c3 , c4 , cz ) , temporal ( t3 , t4 , t5 , t6 ) , parietal ( p3 , p4 , pz ) , and occipital ( o1 , o2 ) . roc areas were obtained for five regions ( see table 2 ) . the maximal difference among the normal group and the other groups ( in decrease order of magnitude ) was localized in temporal , frontal , central , occipital and parietal regions . in the other hand , the best diagnostic performance between acr and probable ad groups was obtained at the temporal region ( area roc = 0.94 ) . the d applied to the broad - band model considered ( delta , theta , alpha , and beta bands ) . the roc areas were also obtained for each band of the broadband model ( see table 2 ) . the discrimination index using d showed that beta band was more accurate to discriminate between normal and acr groups ( area roc = 0.89 ) and between probable ad and normal groups ( area roc = 0.99 ) than the rest of the classical bands . the accuracy of classification in the fast band was higher than that in the slow frequency bands . in decrease order of magnitude , the best indexes were among normal probable ad groups ( area roc = 0.98 ) , acr probable ad groups ( area roc = 0.91 ) , and normal acr groups ( area roc = 0.89 ) ( see table 2 ) . the goal of the present study was to determine the possible impact of spectral eeg analysis to detect early functional changes in preclinical stages of familial ad . the main finding of the present research was the presence of beta - bands alteration in acr groups in the absence of clinical sign . fast frequency bands change mainly in gamma - frequencies , in people with clinical sign of mild cognitive impairment . other researches also found a significant decrease of eeg power in the 1418 hz and 1822 hz in ad patients . the researcher suggested that a reduction of beta power is not only due to ageing , but may reflect an alteration of ad especially in the early stage , this change in the eeg could be found in another dementia , but generally the subjects have clinical sign of cognitive disorders . however , the findings of difference in beta band activity in acr when compare with normal group suggest the possibility that this disturbance in the cholinergic system begins before cognitive impairment appears . this finding could imply that beta band could be affected before the alteration in the gamma described in subjects with mild cognitive impairment . it is possible that early modifications in beta band as found in this study are not sufficient to produce clinical cognitive impairment but could be an early sign to develop the disorder in the future . the modification in delta and alpha bands observed in this study was similar to that previous reports in subjects with ad [ 5961 ] . the importance of high frequency for cognitive process has been recently stressed in several studies [ 6264 ] . reported that loss of beta band power is an independent predictor of unfavorable prognosis in ad . stam et al . suggested that loss of beta band power may also be important for early diagnosis of ad . besides a corticocortical uncoupling progression , a decrease of synaptic coupling is likely to contribute selectively to reducing eeg coherence for faster rhythms , as observed in healthy humans by transient use of a cholinergic synaptic blocker like scopolamine . animal models suggest that acetylcholine reduction produces a decrease of high frequency eeg coupling and an increase of slow frequency coupling . significant drop in eeg synchronization in faster rhythms has also been correlated with decreased mmse scores in mci and ad patients . our findings suggest a decrease of functional connectivity in beta band in acr . this may be explained by a loss of intracortical connections , which are essential for interactions between brain regions . our research showed changes in the eeg spectral parameters in frontal and temporal region likely the most early and sensible than other regions . a related study further supports the role of eeg as a noninvasive tool used in the early identification of dementia demonstrated the earliest subcortical and cortical changes , associated with neural decline according to baar and gntekin , the left fronto parietal connections are highly affected by ad pathology primarily occurring within the parietal regions during the early stages of the disease . in summary , our results suggest that the modification in the qeeg of subjects with genetic predisposition to develop ad is characterized by change in beta frequency band and modification in fronto - temporal regions of the spectral parameters before clinical sign of cognitive impairment appears in subjects with e280a presenilin-1 mutation but this finding needs to be finding with increase of the number of subjects with this mutation and corroborates in another genetic form of dementia . these results should be carefully handled because it has been reported that families with ps-1 mutation present a more severe clinic syndrome than families with ps-2 mutation or another genetic form of dementia . nevertheless , these results open a possibility to recognize an electroencephalographic pattern that could distinguish which genetically predisposed subjects will develop more rapidly the disease and perhaps which subjects with mild cognitive impairment or sporadic ad form could suffer a quick deterioration in their cognitive function through functional disturbances that are indirectly present in the eeg activity . another important issue is to quantify the severity of the disease using spectral eeg analysis to provide patients , acr subjects , and their families with a more reliable prediction of the disease 's course . an appropriate clinical treatment , even at early stages , begins with several actions by means of cognitive rehabilitation and planning for necessary social resources . however , many crucial issues will need to be addressed before using eeg in the clinical practice as a tool for diagnosing ad . this research field offers sufficient opportunities for exciting and clinically relevant research and opens the possibilities to increase the number of subjects with this or another genetic form of dementia .

to evaluate the hypothesis that quantitative eeg ( qeeg ) analysis is susceptible to detect early functional changes in familial alzheimer 's disease ( ad ) preclinical stages . three groups of subjects were selected from five extended families with hereditary ad : a probable ad group ( 18 subjects ) , an asymptomatic carrier ( acr ) group ( 21 subjects ) , with the mutation but without any clinical symptoms of dementia , and a normal group of 18 healthy subjects . in order to reveal significant differences in the spectral parameter , the mahalanobis distance ( d 2 ) was calculated between groups . to evaluate the diagnostic efficiency of this statistic d 2 , the roc models were used . the roc curve was summarized by accuracy index and standard deviation . the d 2 using the parameters of the energy in the fast frequency bands shows accurate discrimination between normal and acr groups ( area roc = 0.89 ) and between ad probable and acr groups ( area roc = 0.91 ) . this is more significant in temporal regions . theses parameters could be affected before the onset of the disease , even when cognitive disturbance is not clinically evident . spectral eeg parameter could be firstly used to evaluate subjects with e280a presenilin-1 mutation without impairment in cognitive function .

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

teaching - learning is a dynamic process and depending on the subject it can be accomplished by various ways and means . problem - based learning ( pbl ) is a student - centered approach in which students learn through analyzing and solving problems . the goals of pbl are to help the students develop flexible knowledge , effective problem - solving skills , self - directed learning , effective collaboration skills , and intrinsic motivation . pbl is a style of active learning and represents a paradigm shift from traditional teaching and learning philosophy , which is more often lecture - based . working in groups , students identify what they already know , what they need to know , and how and where to access new information that may lead to resolution of the problem . the role of the tutor is to facilitate learning by supporting , guiding , and monitoring the learning process . problem - based learning addresses the need to promote lifelong learning through the process of inquiry and constructivist learning . pbl can be considered a constructivist approach to instruction , emphasizing collaborative and self - directed learning and being supported by flexible teacher scaffolding . yew and schmidt , schmidt et al . , and hung elaborate on the cognitive constructivist process of pbl . this central role places high demands on the quality of problems which in turn affects the quality of the small group process and students educational achievements . in general , problems are composed of a relatively neutral description of phenomena or events that appear to be related in some way and require further explanation . a problem can be of different types : explanation problem , discussion problem , strategy problem , study problem , application problem , and multi - level problem . most of these are based on clinical scenarios and are effective in triggering the generation of learning objectives covering many areas of clinical pharmacology . however , there are topics which are not addressed because they are not within the domain of a typical clinical scenario . it is important to sensitize students to issues such as the prerequisites for clinical evaluation of new drugs in humans , phases and types of clinical trials , ethical and legal aspects of testing drugs in humans , adverse drug reactions , postmarketing surveillance , drug safety and drug - related injuries . with these learning objectives in mind , life - cycle of a drug was introduced in the undergraduate medical curriculum during the preclerkship phase in the last unit ( unit ix ) : medicine , science and technology . it is important for medical students to be aware of what is involved in terms of effort , time , resources , and professionalism in the development of a new drug . patient safety is a relatively new discipline and introducing any new material into an existing curriculum is always challenging . unfortunately , this increase in use has also brought with it an increase in hazards , error and adverse events associated with medication use . several instructional strategies have been proposed to teach medical students about medication safety , and a combination of approaches is likely to be most effective . the various options include pbl , interactive lectures , small group discussions , practical workshops , tutorials , project work including tasks to be undertaken in the clinical environment and at the bedside , online learning packages , reading and case analysis . several organizations such as world health organization ( who ) , association of american medical colleges , and general medical council have endorsed increased teaching of patient safety and quality improvement during medical school . despite policy consensus , few medical schools have implemented curricula addressing these topics , and there is little evidence on the best method to teach them . perhaps the vast majority of literature on drug safety reports investigations of postgraduate teaching of these topics . a recent systematic review of patient safety curricula in medical schools demonstrated that most teaching about patient safety occurred during the 3 year , varied in length and was taught by clinicians , ethicists , and medical education experts . current teaching does not meet the goals established by the who and includes no controlled assessment of a standardized curriculum . a recent study examined students preferences on timing , setting , and pedagogical methods for teaching these topics . to address all these concerns related to new drug development in a pbl setting , we thought of using a real - life event to generate relevant learning objectives . the story of rofecoxib ( vioxx ) , a nonsteroidal anti - inflammatory drug with selective inhibition of cycloxygenase-2 appeared very appealing as it had all the elements and issues discussed earlier . we used it as a platform , developed it into a problem for students to trigger the necessary learning objectives and satisfy the requirements of self - directed learning in a pbl curriculum during the preclerkship phase of the medical program . in this paper , we describe our experience on these educational issues : problem development , implementation , and evaluation . the arabian gulf university ( agu ) in the kingdom of bahrain is a regional leader in pbl . the pbl curriculum of the college of medicine and medical sciences ( cmms ) is a 6-year program leading to the md degree and is divided into three phases , as shown in the curriculum map [ table 1 ] : phase i ( premedical ) , phase ii ( preclerkship ) and phase iii ( clerkship ) . a comprehensive description of the medical curriculum of cmms , agu has been reported . medical curriculum map of the agu phase ii has 9 units , of which units iii viii are system - based while units i , ii , and ix are more conceptual and integrative . in each unit , problems are discussed in small tutorial groups to generate learning objectives in session 1 . during the week , students undertake self - study supplemented with structured educational activities such as resource sessions , laboratory sessions , professional clinical skills training , community health activity , and interaction with faculty members . in session 2 , the group assembles again , and students present and discuss what they have learned . in the beginning of the next week , students review the problem and provide feedback about the previous week 's problem . at the end of each unit development of a problem starts with expected learning outcomes and learning objectives within the broader framework of the unit and the entire curriculum . we retrieved the literature available on the internet and gathered information about vioxx , its development , trials , marketing , adverse effects , withdrawal from the market , and the legal and ethical ramifications . keeping in mind the expected learning outcomes and objectives [ table 2 ] , the information was organized into a problem : life - cycle of a drug consisting of a series of triggers to encompass the different stages and steps involved in the process of new drug development [ table 3 ] . the intended learning needs of life - cycle of a drug problem the triggers of life - cycle of a drug in session 1 , students discussed the problem in small groups facilitated by a faculty tutor . any difficult terms were clarified , and discussion focused on the events in the triggers , revisiting previously acquired knowledge , and more importantly , what new knowledge was required to fully understand the contents of a trigger . they came up with learning objectives as generated by each trigger [ table 2 ] . after completing the discussion in session 1 , students devoted themselves to self - study about the learning objectives , acquiring new knowledge . this was supplemented with a mini problem on fen - phen ( an appetite suppressant withdrawn from the market in the 90s due to concerns about its safety ) , mcqs , interaction with resource people and attending structured resource sessions , and a workshop on medication errors and patient safety . in session 2 , each tutorial group gathered again and students shared the knowledge acquired about various learning objectives with each other . in the following week , the problem was reviewed , and queries were addressed . each tutorial group of students , along with respective tutors , provided feedback about different aspects of the problem and the educational activities of the week as a whole . problem evaluation feedback was provided by each group of students in discussion with the tutor by completing a questionnaire with close - ended and open - ended items [ table 4 ] . the close - ended items were scored from 1 to 5 ( 1 = disagree , 5 = strongly agree ) . each year , based on the previous year 's feedback , the problem is modified . the arabian gulf university ( agu ) in the kingdom of bahrain is a regional leader in pbl . the pbl curriculum of the college of medicine and medical sciences ( cmms ) is a 6-year program leading to the md degree and is divided into three phases , as shown in the curriculum map [ table 1 ] : phase i ( premedical ) , phase ii ( preclerkship ) and phase iii ( clerkship ) . a comprehensive description of the medical curriculum of cmms , agu has been reported . medical curriculum map of the agu phase ii has 9 units , of which units iii viii are system - based while units i , ii , and ix are more conceptual and integrative . in each unit , problems are discussed in small tutorial groups to generate learning objectives in session 1 . during the week , students undertake self - study supplemented with structured educational activities such as resource sessions , laboratory sessions , professional clinical skills training , community health activity , and interaction with faculty members . in session 2 , the group assembles again , and students present and discuss what they have learned . in the beginning of the next week , students review the problem and provide feedback about the previous week 's problem . at the end of each unit development of a problem starts with expected learning outcomes and learning objectives within the broader framework of the unit and the entire curriculum . we retrieved the literature available on the internet and gathered information about vioxx , its development , trials , marketing , adverse effects , withdrawal from the market , and the legal and ethical ramifications . keeping in mind the expected learning outcomes and objectives [ table 2 ] , the information was organized into a problem : life - cycle of a drug consisting of a series of triggers to encompass the different stages and steps involved in the process of new drug development [ table 3 ] . the intended learning needs of life - cycle of a drug problem the triggers of life - cycle of a drug in session 1 , students discussed the problem in small groups facilitated by a faculty tutor . any difficult terms were clarified , and discussion focused on the events in the triggers , revisiting previously acquired knowledge , and more importantly , what new knowledge was required to fully understand the contents of a trigger . they came up with learning objectives as generated by each trigger [ table 2 ] . after completing the discussion in session 1 , students devoted themselves to self - study about the learning objectives , acquiring new knowledge . this was supplemented with a mini problem on fen - phen ( an appetite suppressant withdrawn from the market in the 90s due to concerns about its safety ) , mcqs , interaction with resource people and attending structured resource sessions , and a workshop on medication errors and patient safety . in session 2 , each tutorial group gathered again and students shared the knowledge acquired about various learning objectives with each other . in the following week , the problem was reviewed , and queries were addressed . each tutorial group of students , along with respective tutors , provided feedback about different aspects of the problem and the educational activities of the week as a whole . problem evaluation feedback was provided by each group of students in discussion with the tutor by completing a questionnaire with close - ended and open - ended items [ table 4 ] . the close - ended items were scored from 1 to 5 ( 1 = disagree , 5 = strongly agree ) . each year , based on the previous year 's feedback , the problem is modified . the arabian gulf university ( agu ) in the kingdom of bahrain is a regional leader in pbl . the pbl curriculum of the college of medicine and medical sciences ( cmms ) is a 6-year program leading to the md degree and is divided into three phases , as shown in the curriculum map [ table 1 ] : phase i ( premedical ) , phase ii ( preclerkship ) and phase iii ( clerkship ) . a comprehensive description of the medical curriculum of cmms , agu has been reported . medical curriculum map of the agu phase ii has 9 units , of which units iii viii are system - based while units i , ii , and ix are more conceptual and integrative . in each unit , problems are discussed in small tutorial groups to generate learning objectives in session 1 . during the week , students undertake self - study supplemented with structured educational activities such as resource sessions , laboratory sessions , professional clinical skills training , community health activity , and interaction with faculty members . in session 2 , the group assembles again , and students present and discuss what they have learned . in the beginning of the next week , students review the problem and provide feedback about the previous week 's problem . at the end of each unit development of a problem starts with expected learning outcomes and learning objectives within the broader framework of the unit and the entire curriculum . we retrieved the literature available on the internet and gathered information about vioxx , its development , trials , marketing , adverse effects , withdrawal from the market , and the legal and ethical ramifications . keeping in mind the expected learning outcomes and objectives [ table 2 ] , the information was organized into a problem : life - cycle of a drug consisting of a series of triggers to encompass the different stages and steps involved in the process of new drug development [ table 3 ] . the intended learning needs of life - cycle of a drug problem the triggers of life - cycle of a drug in session 1 , students discussed the problem in small groups facilitated by a faculty tutor . any difficult terms were clarified , and discussion focused on the events in the triggers , revisiting previously acquired knowledge , and more importantly , what new knowledge was required to fully understand the contents of a trigger . they came up with learning objectives as generated by each trigger [ table 2 ] . after completing the discussion in session 1 , students devoted themselves to self - study about the learning objectives , acquiring new knowledge . this was supplemented with a mini problem on fen - phen ( an appetite suppressant withdrawn from the market in the 90s due to concerns about its safety ) , mcqs , interaction with resource people and attending structured resource sessions , and a workshop on medication errors and patient safety . in session 2 , each tutorial group gathered again and students shared the knowledge acquired about various learning objectives with each other . each tutorial group of students , along with respective tutors , provided feedback about different aspects of the problem and the educational activities of the week as a whole . problem evaluation feedback was provided by each group of students in discussion with the tutor by completing a questionnaire with close - ended and open - ended items [ table 4 ] . the close - ended items were scored from 1 to 5 ( 1 = disagree , 5 = strongly agree ) . each year , based on the previous year 's feedback , the problem is modified . the problem triggers that were created to bring out these learning needs are presented in table 3 . feedback pertaining to various aspects of the problem was collected over a period of 5 years ; provided by 57 tutorial groups , each comprising 810 students . the pooled results of problem evaluation are presented in table 4 . as can be seen , overall the problem has been perceived positively . several approaches have been used in addressing this issue both from curriculum design and implementation perspectives , in traditional as well as innovative medical curricula . the speed with which new technology , including drugs , is introduced into health care treatments demonstrates the constant change in health care which , in turn , is changing the nature of the work or tasks undertaken by different health professionals . taking into account the workplace context , it is necessary to bring authenticity to the learning experience and prepare the students for the work environment they will be entering . it is important to delineate the distinction between pbl and case - based learning ( cbl ) . there are fundamental differences in terms of educational philosophy , curriculum design , and implementation between pbl and cbl . first , the problems must raise the concepts and principles relevant to the content domain . second , the problem must be real . because the students are open to explore all discussions of the problem , there is real difficulty in creating a rich problem with a consistent set of information . several elements need to be considered in problem design : the learning outcomes ( educational objectives ) , the type of problem and its format . a written problem is the most common format for trigger material ; the triggers should be relevant , interesting and provoke discussion in small groups . the curriculum committee , in consultation with different departments , identified the core learning objectives in each discipline . because of the integrated and spiral nature of pbl curriculum different departments ( epidemiology , biochemistry , family medicine , professionalism , pharmacology , and therapeutics ) worked together in contributing to the problem . core learning objectives of basic and clinical pharmacology for undergraduate medical students have been identified and listed . we noticed a gap : new drug development was not being addressed , and there was no way to introduce it through the existing problems . a new problem was developed in which students could understand the different stages and issues involved in the process of drug development . the story of vioxx appeared very appealing as it had all the elements of new drug development . it went even further : it brought out several other important issues , such as ethics of clinical research , particularly of clinical trials , patient safety , and legal implications of drug - induced harm . the process of developing new drugs generally starts with the identification or development of a new lead chemical molecule . this is brought out in trigger 1 against a backdrop of nonsteroidal anti - inflammatory drugs ( nsaids ) , their widespread use , their limitations , and the need for better drugs . students reviewed nsaids , which they had studied in previous problems , for example , in unit i ( inflammation ) , unit iii ( pulmonary embolism ) , unit v ( peptic ulcer disease ) , unit vi ( immunothrombocytopenic purpura ) and unit vii ( arthritis ) . this raised the topic of the need for new nsaids and how new drugs are discovered and developed . it raised discussion about the preclinical testing of new drugs in animals as a prerequisite for testing them in humans . success in animal testing leads to the emergence of an investigational new drug ( ind ) . consent for participation in trials , ethics , and strategies to minimize bias are also discussed . students who were better informed also raised topics from history , e.g. , unethical testing on prisoners of war or on vulnerable people . once a drug has been successfully tested in humans , it is time for new drug application ( nda ) . in trigger 4 discussion is about the prerequisites for nda , drug regulatory affairs in general , generic name and brand name , evidence - based medicine and critical appraisal of the literature . mk-966 ( rofecoxib ) was marketed as vioxx and merck actively advertised their new pharmaceutical product . unlike nonselective cox inhibitors , it caused fewer gastrointestinal adverse effects . students discussed the necessity for new drugs , various influences on the prescriber , and drug nomenclature . results from the vioxx gastrointestinal outcomes research ( vigor ) study were promising , with advantages of vioxx over naproxen , a nonselective nsaid , in terms of gastrointestinal adverse effects . but later results showed that the incidence of cardiovascular events in the vioxx group was double compared to the naproxen group . however , reasoning that vioxx appeared risky in comparison to naproxen because naproxen protected patients by acting like low - dose aspirin , the company decided to continue the study . it is a turning point , and merck could have avoided litigations associated with vioxx if only it had taken the alternative decision to discontinue the study . the vigor study was completed , and the paper was published in the new england journal of medicine . this trigger raised important issues of ethics and professionalism in research . no doubt developing new drugs other topics raised in this trigger were the use of controls in study design , type of controls , conflict of interests , and bias in data interpretation and nondisclosure . trigger 7 is about one of the first reported deaths associated with the use of vioxx and the subsequent lawsuit against merck . postmarketing surveillance and reporting ( phase iv ) is the last ( but not the least ) step in new drug development . , there was an increased risk of cardiovascular events ( myocardial infarction and stroke ) associated with vioxx . in september 2004 revisiting adverse drug reactions , there was a discussion about the possible reasons for myocardial infarction and stroke associated with vioxx . it triggered interesting discussions : publication bias , critical appraisal of the scientific literature , and public accountability and liability of the medical profession . it was also pointed out that the company did win some legal cases , because in other cases with multiple diseases and multi - drug therapy , it can be impossible to link an adverse event to one particular cause with certainty due to nonestablishment of causality . along with the main problem , mini - problems and mcqs are also given to the students for elaboration and self - assessment . mini - problems are used for elaboration or for presentation in different clinical settings or contexts . in general one approach is to evaluate whether students are able to generate the same learning goals as intended by the curriculum . the degree of congruence between the two is considered to be reflective of problem effectiveness . the problem was perceived positively with a mean overall score of 3.8 0.4 out of 5 . all close - ended items scored 3 and above except item no . 5 : problem cuts across disciplines which scored 2.9 . it seems most groups perceived the problem as mainly pharmacology / clinical pharmacology discipline - oriented . nevertheless , the problem does integrate with some other discipline , e.g. , biochemistry , evidence - based medicine , epidemiology , and professionalism . according to the open - ended comments by various groups , the problem was perceived as very interesting , informative , and an eye - opener . it indicates the effectiveness and success of this innovative approach in teaching - learning such important topics of clinical pharmacology . in a pbl curriculum , having identified the learning outcomes , problems can be developed from real - life events for teaching - learning clinical pharmacology topics for which patient - based scenarios might be inadequate . a systematic approach described in this paper can be used for the development and validation of educational material for introducing focal topics of pharmacology / clinical pharmacology integrated with other disciplines in innovative medical ( and other health profession ) curricula . the world federation of medical education and the medical council of india have recommended the introduction of innovative medical curricula . other variants like case - based learning and team - based learning are also used in some schools across the world . these being relatively novel concepts in india , we chose to publish this article to create awareness among the readers about the trends in health professions education ( including pharmacology education ) . a note of caution , however , against adopting these methods without evaluating its feasibility and validity in the indian context .

aim : this paper describes how in a problem - based learning ( pbl ) medical curriculum , having identified the learning outcomes , problems can be developed from real - life events for teaching - learning clinical pharmacology topics for which pbl cases might be inadequate . such problems can be very interesting and educational.methodology:using the story of the development and withdrawal of rofecoxib ( vioxx ) , we developed a problem for undergraduate medical students to address important issues related to clinical pharmacology and therapeutics such as new drug development , preclinical testing , clinical trials , adverse drug reactions , professionalism , and critical appraisal of literature . these topics would otherwise be difficult to address in patient - based problems.results:the evaluation of the problem based on pooled feedback from 57 tutorial groups , each comprising 810 students , collected over 5 years , supported the effectiveness of the problem.conclusion:a systematic approach described in this paper can be used for the development and validation of educational material for introducing focal topics of pharmacology / clinical pharmacology integrated with other disciplines in innovative medical ( and other health profession ) curricula .

Introduction Methodology None Setting Problem Designing Problem Implementation Problem Evaluation Results Discussion Conclusion Note:

since 2010 , the us national health interview survey ( nhis ) has included questions assessing pain persistence and pain bothersomeness . these questions were developed and piloted as part of an international initiative under the purview of the washington group on disability statistics,1 constituted by the united nations statistical commission . the goal of the washington group is to promote and coordinate international cooperation in the area of health statistics focusing on disability measures suitable for censuses and national surveys and to develop tools to collect the basic data necessary to provide information on disability that is comparable throughout the world.2 based on qualitative and mixed - method studies performed in the united states and other countries affiliated with the washington group , miller and loeb3 have proposed a coding system that combines data from the nhis questions on pain persistence and bothersomeness to create discrete categories of increasing pain severity . in a previous study,4 we not only demonstrated the concurrent validity of the coding system , with the pain categories discriminating between different levels of health status , disability , and health care use , but also identified steps that needed to be taken before the coding scheme could be fully incorporated into the armamentarium of the pain clinician or scientist . in particular , we noted that the category cut points for the coding system needed to be confirmed in quantitative analyses and in different anatomical sites of pain . such data would offer additional support as to whether the coding system proposed by miller and loeb3 provides a valid measure of an individual s current pain experience . should the miller and loeb pain categories eventually fulfill all the criteria of a well validated and clinically useful pain scale , then the brevity of the miller and loeb approach will make it an excellent choice in situations where clinician time and patient burden are limiting factors . in the current analyses , using data from the 2012 nhis , we quantitatively assessed the pain category definitions proposed by miller and loeb3 by adapting a statistical approach widely used in the pain field for grading pain intensity with functional interference.5 three goals that guided this study are 1 ) to assess whether the cut point analysis method proposed by serlin et al5 can be applied to pain severity categories defined by self - reported pain persistence and bothersomeness , using measures of health status and health care use as cut point criteria ; 2 ) to determine whether the optimal pain category definitions identified in the entire 2012 nhis adult samples would be evidenced in randomly derived subsamples of the full sample ; and 3 ) to determine if the same optimal pain category definitions would be evidenced in back pain and joint pain subpopulations . the data used in this study are from the 2012 nhis sample adult core and the nhis adult functioning and disability ( afd ) supplement.6 the nhis is an annual survey of the health of the us civilian , noninstitutionalized population conducted by the national center for health statistics , centers for disease control and prevention . this in - person survey contains four main modules : household , family , sample child , and sample adult . the first two modules collect health and sociodemographic information on each member of all families residing within a sampled household . within each family , additional information is collected from one randomly selected adult ( the sample adult ) aged 18 years or older . bilingual interviewers or interpreters were recruited to interview all respondents who preferred the use of a language other than english . the survey uses a multistage clustered sample design and oversamples black , asian , and hispanic populations . when combined with centers for disease control and prevention - derived sampling weights , this design allows accurate extrapolation of findings to the civilian , noninstitutionalized us adult population . for the 2012 interview sample , there were 42,366 households consisting of 108,131 persons in 43,345 families . the total household response rate was 77.6% . from the households interviewed , 34,525 adults completed interviews , resulting in an overall sample adult response rate of 79.7% . approximately one - quarter of sampled adults were randomly chosen to participate in the afd supplement . almost all chosen adults ( 8,781 ) completed the supplement resulting in a 98% supplement response . the 2012 nhis was approved by the national center for health statistics research ethics review board . the afd collected information on the persistence and bothersomeness of self - reported pain in the previous 3 months . respondents were first asked how often they had pain in the previous 3 months : never , some days , most days , or every day . data from this question provide estimates of the 3-month persistence ( period prevalence ) of pain . for those who had pain on at least some days , a follow - up question assessing bothersomeness was asked : thinking about the last time you had pain , how much pain did you have a little , between a little and a lot , or a lot . ninety - six percent of afd participants completed these pain questions . given this high response rate , no attempt was made to impute missing data . miller and loeb3 have suggested a coding scheme that combines persistence and bothersomeness of pain to create four discrete categories of increasingly severe pain ( figure 1 ) . this coding scheme was tested and validated using a variety of qualitative assessments such as cognitive testing and mixed methods analyses2,3,7 and has been shown to have concurrent validity.4 in order to be consistent with the original pain category definitions of miller and loeb,3 the alternative definitions studied were restricted to four discrete categories that varied in only in their distributions of pain persistence and bothersomeness . to aid in identifying a set of alternative pain category definitions , we visually explored the relationship between nine pain persistence / bothersomeness combinations and measures of health status and health care use as defined later . chronic pain often predicts the onset of psychological distress.8,9 in turn , psychological distress has been identified as one of the factors mediating pain s relationship to disability.10 the nhis measures nonspecific psychological distress over a 30-day recall period with the kessler 6 ( k6 ) scale.11 the k6 scale asks respondents about six manifestations of psychological distress : during the past 30 days , how often did you feel ( a ) so sad that nothing could cheer you up ? possible responses are all of the time , most of the time , some of the time scoring of the individual questions is based on a scale of between 0 and 4 points , according to increased frequency of the problem , yielding a total score on the scale from 0 ( no psychological distress ) to 24 ( extreme psychological distress).11 this was examined as a continuous variable . pain often results in disability days.1214 health - related bed - disability days were assessed with the survey question : during the past 12 months , about how many days did illness or injury keep you in bed more than half of the day ( include days while an overnight patient in a hospital ) ? pain is associated with increased health care use,13,15,16 including visits to emergency departments.17,18 health care use was examined with two nhis survey questions : 1 ) how many times did you visit a doctor or other health care professional during the last 2 weeks ? and 2 ) during the past 12 months , how many times have you gone to a hospital emergency room about your own health ( this includes emergency room visits that resulted in a hospital admission ) ? the 2012 nhis adult core included questions on the presence of low back pain within the previous 3 months ( yes and no ) , and on the presence of joint , aching or stiffness in the last 30 days ( yes and no ) . we studied the alternative definitions of the four pain categories using a variation of the statistical method described by serlin et al.5 each alternative definition was related simultaneously to four dependent measures the k6 score , the number of health related bed - disability days , the number of visits to a health professional , and the number of emergency room visits using multivariate analysis of variance ( manova ) . the manova yields f values , based on wilk s lambda , for the between - category effect on the dependent variables . following serlin et al,5 the pain category definition yielding the best model fit , as measured by the f value , is considered the optimal definition . as suggested by hirschfeld and zernikow,19 we quantified the variability in the f value by running manova in 100 random samples ( with replacement ) of 2,000 participants chosen from the entire sample population of 8,781 . for each alternate definition , we calculated f score means and 95% confidence intervals ( cis ) around the means . also as suggested by hirschfeld and zernikow,19 we counted the number of times that each alternate definition was identified as the optimal definition based on manova f scores generated from the random samples . in the 12 cases where f scores were tied , each definition was counted as optimal . finally , using the approach described above , the alternative pain category definitions were further explored in two disease - specific populations : 1 ) those with back pain and 2 ) those with joint pain . the data used in this study are from the 2012 nhis sample adult core and the nhis adult functioning and disability ( afd ) supplement.6 the nhis is an annual survey of the health of the us civilian , noninstitutionalized population conducted by the national center for health statistics , centers for disease control and prevention . this in - person survey contains four main modules : household , family , sample child , and sample adult . the first two modules collect health and sociodemographic information on each member of all families residing within a sampled household . within each family , additional information is collected from one randomly selected adult ( the sample adult ) aged 18 years or older . bilingual interviewers or interpreters were recruited to interview all respondents who preferred the use of a language other than english . the survey uses a multistage clustered sample design and oversamples black , asian , and hispanic populations . when combined with centers for disease control and prevention - derived sampling weights , this design allows accurate extrapolation of findings to the civilian , noninstitutionalized us adult population . for the 2012 interview sample , there were 42,366 households consisting of 108,131 persons in 43,345 families . the total household response rate was 77.6% . from the households interviewed , 34,525 adults completed interviews , resulting in an overall sample adult response rate of 79.7% . approximately one - quarter of sampled adults were randomly chosen to participate in the afd supplement . almost all chosen adults ( 8,781 ) completed the supplement resulting in a 98% supplement response . the 2012 nhis was approved by the national center for health statistics research ethics review board . the afd collected information on the persistence and bothersomeness of self - reported pain in the previous 3 months . respondents were first asked how often they had pain in the previous 3 months : never , some days , most days , or every day . data from this question provide estimates of the 3-month persistence ( period prevalence ) of pain . for those who had pain on at least some days , a follow - up question assessing bothersomeness was asked : thinking about the last time you had pain , how much pain did you have a little , between a little and a lot , or a lot . miller and loeb3 have suggested a coding scheme that combines persistence and bothersomeness of pain to create four discrete categories of increasingly severe pain ( figure 1 ) . this coding scheme was tested and validated using a variety of qualitative assessments such as cognitive testing and in order to be consistent with the original pain category definitions of miller and loeb,3 the alternative definitions studied were restricted to four discrete categories that varied in only in their distributions of pain persistence and bothersomeness . to aid in identifying a set of alternative pain category definitions , we visually explored the relationship between nine pain persistence / bothersomeness combinations and measures of health status and health care use as defined later . chronic pain often predicts the onset of psychological distress.8,9 in turn , psychological distress has been identified as one of the factors mediating pain s relationship to disability.10 the nhis measures nonspecific psychological distress over a 30-day recall period with the kessler 6 ( k6 ) scale.11 the k6 scale asks respondents about six manifestations of psychological distress : during the past 30 days , how often did you feel ( a ) so sad that nothing could cheer you up ? possible responses are all of the time , most of the time , some of the time , a little of the time , and none of the time . scoring of the individual questions is based on a scale of between 0 and 4 points , according to increased frequency of the problem , yielding a total score on the scale from 0 ( no psychological distress ) to 24 ( extreme psychological distress).11 this was examined as a continuous variable . pain often results in disability days.1214 health - related bed - disability days were assessed with the survey question : during the past 12 months , about how many days did illness or injury keep you in bed more than half of the day ( include days while an overnight patient in a hospital ) ? pain is associated with increased health care use,13,15,16 including visits to emergency departments.17,18 health care use was examined with two nhis survey questions : 1 ) how many times did you visit a doctor or other health care professional during the last 2 weeks ? and 2 ) during the past 12 months , how many times have you gone to a hospital emergency room about your own health ( this includes emergency room visits that resulted in a hospital admission ) ? the 2012 nhis adult core included questions on the presence of low back pain within the previous 3 months ( yes and no ) , and on the presence of joint , aching or stiffness in the last 30 days ( yes and no ) . we studied the alternative definitions of the four pain categories using a variation of the statistical method described by serlin et al.5 each alternative definition was related simultaneously to four dependent measures the k6 score , the number of health related bed - disability days , the number of visits to a health professional , and the number of emergency room visits using multivariate analysis of variance ( manova ) . the manova yields f values , based on wilk s lambda , for the between - category effect on the dependent variables . following serlin et al,5 the pain category definition yielding the best model fit , as measured by the f value , is considered the optimal definition . as suggested by hirschfeld and zernikow,19 we quantified the variability in the f value by running manova in 100 random samples ( with replacement ) of 2,000 participants chosen from the entire sample population of 8,781 . for each alternate definition , we calculated f score means and 95% confidence intervals ( cis ) around the means . also as suggested by hirschfeld and zernikow,19 we counted the number of times that each alternate definition was identified as the optimal definition based on manova f scores generated from the random samples . in the 12 cases where f scores were tied , using the approach described above , the alternative pain category definitions were further explored in two disease - specific populations : 1 ) those with back pain and 2 ) those with joint pain . figure 2a d shows the relationships between the nine combinations of pain persistence and bothersomeness and measures of health status and health care use . those with a lot of pain either most days or every day had the highest mean k6 scores , 6.28 and 6.23 , respectively ( figure 2a ) , indicating they were in more psychological distress then individuals with other combinations of pain persistence and pain severity . conversely , those individuals with a little pain some days had the lowest mean k6 score ( 2.07 ) and least amount of psychological distress . similar patterns were seen when examining the mean number of health - related bed days ( figure 2b ) , the mean number of office visits ( figure 2c ) , and the mean number of emergency room visits ( figure 2d ) , with those with a lot of pain either most days or every day having the highest values ( mean number of bed days , 9.73 and 17.31 , respectively ; mean number of office visits , 0.69 and 0.77 , respectively ; and for mean number of er visits , 1.35 and 1.38 , respectively ) and those with a little pain some days having the lowest values ( mean number of bed days , 2.41 ; mean number of office visits , 0.24 ; and for the mean number of er visits , 0.27 ) . two other consistent patterns were seen 1 ) individuals with between a little and a lot of pain either most days or every day had the third and fourth highest mean k6 score ( 3.74 and 4.07 , respectively ) and had the third and fourth highest mean number of bed days ( 8.74 and 9.27 , respectively ) and office visits ( 0.56 and 0.69 , respectively ) and 2 ) those with a lot of pain some days had the fourth lowest value for the k6 score ( 3.29 ) and the fourth lowest mean number of bed days ( 5.5 ) and office visits ( 0.46 ) . the ranking of the other categories defined by pain persistence and pain severity varied considerably depending on the specific dependent measure . given the variability in the data , visual inspection of the graphs in figure 2a d was used to suggest alternative definitions of miller and loebs3 original pain categories ( figure 1 ; alternative definitions 110 ) . for instance , the original definition and alternative definitions # 1 and # 2 defined pain categories 1 and 2 identically but varied in how they defined pain categories 3 and 4 . conversely , alternative definitions # 8 and # 10 had pain categories 3 and 4 in common with the original definition but varied in how they defined pain categories 1 and 2 . alternative definition # 5 coded pain category 1 and pain category 4 identical with the original definition but varied in the other pain categories . alternative definitions # 3 , # 4 , # 6 , # 7 , and # 9 shared only one pain category in common with the original definition . along with the original definition table 2 shows the wilk s lambda f values calculated from the entire sample of 8,781 for each of the alternative definitions . the original definition produced the largest f value ( 185.87 ) followed consecutively by alternative definitions # 5 ( 184.17 ) , # 10 ( 180.95 ) , and # 9 ( 179.5 ) . nearly identical ordering was found when looking at the mean f value means generated from 100 random samples ( table 2 ) . however , considerable overlap was seen between the 95% confidence interval ( ci ) for the original definition and alternative definition # 5 , # 9 , and # 10 . figure 3 presents the frequencies with which each alternative definition achieved the optimal f value over the 100 random samples . interestingly , while there are only small differences seen in either the overall f values or mean f values ( table 2 ) , large differences were seen in the observed frequencies only two definitions had achieved the optimal f value > 5% of the time : the original definition ( 46% ) and alternative definition # 5 ( 41% ) . table 2 also shows the overall f values and the mean f values in those with back pain or joint pain , while figures 4 and 5 show the frequency of the optimal f values in those with back pain and joint pain , respectively . for both those with back pain ( table 2 ; figure 4 ) and those with joint pain ( table 2 ; figure 5 ) , the original definition had the highest overall f value ( back pain , 72.42 ; joint pain , 97.18 ) and the highest mean f value ( back pain , 16.44 ; joint pain , 22.71 ) . as in the overall adult sample , small differences in f values translated into large differences in the percent of time a given definition had the optimal f value . in the random samples of those with back pain ( figure 4 ) , 38% of the time the original definition was identified as having the optimal f value across the 100 samples , followed by alternative definition # 5 at 36% . in those with joint pain ( figure 5 ) , the frequencies were more divergent : 49% for the original definition and 28% for alternative definition # 5 . using a series of manova , we sought to identify the optimal definition of discrete pain categories using existing nhis questions that assessed pain persistence and severity . the results of manova on a large nationally representative sample of adults indicated that the original pain category definitions suggested by miller and loeb3 provided the best model fit versus ten alternative definitions . we confirmed this finding in 100 random samples of the total sample , as well as in two disease - specific subpopulations , those with back pain and those with joint pain . therefore , the present data provide additional support that the nhis coding system proposed by miller and loeb3 is a valid measure of an individual s current pain experience . given the large number of statistical tests employed in this analysis ( > 300 ) , it is possible that some of our observations occurred purely by chance . this multiple - test issue has been suggested as a possible weakness of the serlin approach.19 to overcome the multiple - test issue , hirschfeld and zernikow19 suggest examining the variability of the observed optimal definitions , which we have done in the present report . data based on f values for the entire sample , mean f values with 95% cis derived from the random samples , and the frequency with which optimal definitions are observed produced the same conclusion , that the coding scheme proposed by miller and loeb3 is the optimal definition . however , the data additionally show that one alternative definition ( # 5 ) also does very well across comparisons . the original definition and alternative definition # 5 code pain category 1 ( least severe pain ) and pain category 4 ( most severe pain ) identically and vary only slightly in how pain persistence and bothersomeness are combined to create pain categories 2 and 3 ; specifically , the original definition places slightly more emphasis on grouping by pain bothersomeness , while alternative definition # 5 places slightly more emphasis on grouping by pain persistence . future analyses , such as that using receiver operating characteristic curves , might help clarify the test characteristics of these two definitions . first , the categories we used were based on only pain persistence and bothersomeness , and therefore do not reflect the multidimensional nature of pain . second , potential confounders or effect modifiers , such as age , education , race , and sex were not included in this study as we were first concerned with exploring alternative cut points in the general population versus optimizing the manova for specific demographic groups . whether the miller and loeb definition remains the optimal definition in specific demographic populations will need to be assessed in future . third , the nhis did not include a well - validated scale for measuring pain - related disability . instead , we were limited to only a handful of variables measuring health status and health care use and for which continuous data were collected ( required for manova ) . as shown by zelman et al20 and fejer et al,21 the conclusions from cut point analyses vary depending on the dependent variables used . other results may have been found using the brief pain inventory , other pain - related disability measures , or more widely used generic measures of function such the sf-36 . therefore , the present results are suggestive rather than definitive and await corroboration in different samples . finally , in order to be consistent with the original pain category definitions suggested by miller and loeb,3 we have limited our analyses to alternative definitions derived to create four discrete pain categories . it is not known if fewer or greater numbers of categories would better explain the variance in manova . this study provides additional support for the coding of nhis - derived pain as proposed by miller and loeb.3 we demonstrated that the miller and loeb3 coding scheme provided optimal manova cut points for pain severity relative to health status and health care use , both in the general population and in disease - specific subpopulations . still to be explored is how well the nhis questions correlate with established validated measures of pain and whether they are sensitive enough to assess changes in pain severity over time . with further validation , the miller and loeb approach may prove to be a brief , viable option for assessing pain severity in clinical and research settings .

backgroundbased on qualitative and mixed - method approaches , miller and loeb have proposed a coding system that combines questions on pain persistence and bothersomeness to create discrete categories of increasing pain severity for use in large population - based surveys . in the current analyses , using data from the 2012 national health interview survey , we quantitatively assess the pain category definitions proposed by miller and loeb and compare this original definition to ten alternative definitions.methodsusing multivariate analysis of variance , each definition was related simultaneously to four dependent measures the kessler 6 score for measuring psychological distress , the number of health - related bed - disability days , the number of visits to a health professional , and the number of emergency room visits . following the protocol of serlin et al , the definition yielding the largest f score was considered the optimal definition.resultsthe miller and loeb definition produced the largest f value ( 185.87 ) , followed consecutively by several alternative definitions # 5 ( 184.17 ) , # 10 ( 180.95 ) , and # 9 ( 179.5 ) . a nearly identical ordering was found when looking at the mean f value generated from 100 random samples . we also examined the frequencies with which each alternative definition achieved the optimal f value over the 100 random samples . only two definitions had achieved the optimal f value > 5% of the time : the miller and loeb definition was optimal 46% of the time , while alternative definition # 5 was optimal 41% of the time . similar results were seen in subpopulations with back pain and joint pain.conclusionadditional support was provided for the miller and loeb coding of pain persistence and bothersomeness to produce discrete categories of increasing pain severity . this two - question coding scheme may prove to be a viable option for assessing pain severity in clinical settings where clinician time and patient burden are limiting factors .

Introduction Methods Population Dependent variable: assessment of pain Alternative definitions of the four pain categories Psychological distress Health-related disability Health care use Pain-related health conditions Statistical analyses Results Discussion Conclusion

new - generation sequencing technologies have substantially enhanced the development of genomic tools to assist in breeding decisions . among many techniques , genotyping technologies have enabled breeders or researchers to identify dna regions or quantitative trait loci ( qtl ) associated with a particular phenotypic trait of domesticated animals . especially , several single - nucleotide polymorphisms ( snps ) and qtl associated with holstein complex traits , including economic traits related to milk production , were investigated in the approximate 10-year wave of genome - wide association studies ( gwass ) . in the near past , researchers discovered genetic markers and created marker panels for use in performing marker - assisted selection ( mas ) . although mas provided a first genomic approach to achieve breeding goals to animal breeders , the domesticated animal genome in mas was taken lightly with narrow perspectives . mas is restricted when it comes to predicting animal capacity , as it depends on a small number of qtls that are tagged by markers associated with each trait . additionally , the gap between the proportion of phenotypic variance accounted for by the top snps that reach genome - wide significance level in a gwas and the heritability estimated from pedigree analyses remained unexplained for most complex traits of human . this was called the missing heritability problem , explanations to which have been debated in the field . recent studies using whole - genome estimation approaches demonstrated that a large proportion of heritability for complex traits of humans can be captured by all common snps on current genotyping arrays . it implies that there are a large number of variants with an effect that is too small to pass the stringent genome - wide significance level . in animal breeding , genomics selection ( gs ) has been developed to overcome restriction factors of mas ; it has covered a large number of variants containing small effects . the fundamental difference between mas and gs as important animal selection tools is that scale gs embraces a much larger number of markers than mas in predicting animal capacity . gs uses a dense set of markers from across the entire genome to use all markers containing snps with small effects . for predictions based on genomic data for humans , a previous study has already estimated the proportion of phenotypic variance . it was elucidated by the common snps all together on a genotyping array for a range of quantitative traits in a large homogenous human sample , using the whole - genome estimation and partitioning approaches . the novelty of our research lies in animal capacity prediction based on genomic data due to the fact that animal evaluation is a core content in the breeding industry . finally , to conduct our animal breeding research on polygenic inheritance , which was likely to be ubiquitous for complex holstein traits associated with milk production traits , the genomic selection technique was performed along with various different analyses . this study used 462 holsteins from korea , and they had information on three phenotypes related to milk production for parity 1 ( fig . 1 ) . three traits used in this study were milk yield , milk fat , and milk protein ; 339 of 462 holsteins in records of parity 1 had records of parity 2 . all candidates were measured for a range of quantitative traits through public surveys for livestock improvement . the genomic dnas were isolated from snivel by using the nasal collection kit and were genotyped with 54,609 snps on the illumina bovinesnp50 beadchip . we excluded the snps with a missingness rate of 0.05 , minor allele frequency ( maf ) 0.01 , and hardy - weinberg equilibrium test p - value 10 using plink . then , we removed snps on the sex chromosome and retained 41,099 autosomal snps for further analysis . we estimated the genetic relationship matrix ( grm ) between all pairs of individuals from all genotyped snps for each trait , we then estimated the variance that can be captured by all snps using the restricted maximum likelihood approach in a mixed linear model : y = xb + gg + e where y is a vector of phenotypes , b is a vector of fixed effects with its incidence matrix x , and gg is a vector of aggregate effects of all snps . var(gg ) = agg ag is the snp - derived grm , and g is the additive genetic variance . the proportion of variance explained by all snps is defined as hg = g p p is the phenotypic variance . when we estimated genetic variance using snp data , we have to consider seasonal effects ( winter , december february ; spring , march may ; summer , june august ; and autumn , september november based on birth date ) as environmental effects , because milk production traits are closely related to season . then , we performed anova test to investigate the relationship between each trait ( parity 1 and 2 ) and seasonal effect . milk fat had only a close relation to season in the anova test of both parity 1 ( p = 0.0267 ) and 2 ( p = 0.000432 ) . in addition , using the same method as above but allowing multiple genetic components to be fit simultaneously in the model , we partitioned hg into the contributions of genic ( hgg ) and intergenic ( hgi ) regions of the whole genome across all traits . the genic regions were defined as 0 kb of the 3 ' and 5 ' untranslated regions ( utrs ) . a total of 13,297 snps were located within the boundaries of 7001 protein - coding genes for this definition ( 0 kb ) . the total length of the 7001 protein - coding genes was approximately 585 mb , which covered 21.91% of the genome . this study used 462 holsteins from korea , and they had information on three phenotypes related to milk production for parity 1 ( fig . 1 ) . three traits used in this study were milk yield , milk fat , and milk protein ; 339 of 462 holsteins in records of parity 1 had records of parity 2 . all candidates were measured for a range of quantitative traits through public surveys for livestock improvement . the genomic dnas were isolated from snivel by using the nasal collection kit and were genotyped with 54,609 snps on the illumina bovinesnp50 beadchip . we excluded the snps with a missingness rate of 0.05 , minor allele frequency ( maf ) 0.01 , and hardy - weinberg equilibrium test p - value 10 using plink . then , we removed snps on the sex chromosome and retained 41,099 autosomal snps for further analysis . we estimated the genetic relationship matrix ( grm ) between all pairs of individuals from all genotyped snps for each trait , we then estimated the variance that can be captured by all snps using the restricted maximum likelihood approach in a mixed linear model : y = xb + gg + e where y is a vector of phenotypes , b is a vector of fixed effects with its incidence matrix x , and gg is a vector of aggregate effects of all snps . var(gg ) = agg ag is the snp - derived grm , and g is the additive genetic variance . the proportion of variance explained by all snps is defined as hg = g p p is the phenotypic variance . when we estimated genetic variance using snp data , we have to consider seasonal effects ( winter , december february ; spring , march may ; summer , june august ; and autumn , september november based on birth date ) as environmental effects , because milk production traits are closely related to season . then , we performed anova test to investigate the relationship between each trait ( parity 1 and 2 ) and seasonal effect . milk fat had only a close relation to season in the anova test of both parity 1 ( p = 0.0267 ) and 2 ( p = 0.000432 ) . in addition , using the same method as above but allowing multiple genetic components to be fit simultaneously in the model , we partitioned hg into the contributions of genic ( hgg ) and intergenic ( hgi ) regions of the whole genome across all traits . the genic regions were defined as 0 kb of the 3 ' and 5 ' untranslated regions ( utrs ) . a total of 13,297 snps were located within the boundaries of 7001 protein - coding genes for this definition ( 0 kb ) . the total length of the 7001 protein - coding genes was approximately 585 mb , which covered 21.91% of the genome . these data were collected from 462 cows recruited from 63 farms in south korea , genotyped at 54,609 snps on the illumina bovinesnp50 beadchip . there were 462 individuals and 41,099 autosomal snps after quality control ( methods section ) . all individuals were measured for three traits ( milk yield , milk fat , and milk protein ) , which were related to milk production . we estimated the proportion of variance explained by fitting all snps in a mixed linear model for each of the three traits . in general , there was a substantial amount of variance explained by all autosomal snps after quality control on the illumina bovinesnp50 beadchip ( g ) for three traits of the records of two parities , with a mean of 43.51% ( a range from 36.3 to 47% ) across all three traits of the records of two parities ( table 1 ) . in the records , the estimate of hg was non - zero and reached the nominal significance level ( likelihood ratio test , p = 0.05 ) . we compared the estimates of hg with the narrow - sense heritability h , estimated from pedigree analyses in the literature based on canada holstein . the value of hg was similar to h from pedigree analysis in the literature , and all common snps explained most ( average , 43.51% ; range from 36.3% to 47% ) of the narrow - sense heritability ( average , 41.38% ; range from 36.3% to 47% ) , despite the estimates of h being from different country populations ( table 1 ) . in contrast , when we performed a genome - wide association analysis in the same sample , we tried to identify genome - wide - significant ( bonfferoni correction p = 0.05 ) snps for three traits of parity 1 . three snps and one snp were significant in milk yield and fat , respectively , and there were no significant snps for milk protein ( supplementary fig . we hypothesized that there was a major reason for only a few significant large - effect snps associated with holstein traits related to milk production . first of all , many snps with small effects affected the three traits related to milk production . second , according to previous studies , there are many common variants associated with the traits in humans at a nominally significant level , but their effect sizes are too small to be genome - wide - significant . using the same method as above but allowing multiple genetic components to be fit simultaneously in the model , we then partitioned hg into the contributions of individual chromosomes for each of the three traits of two parities and plotted the estimate of variance explained by each chromosome ( hc ) against the chromosome length for each trait . in a previous study , no linear correlation between hc and chromosome length for any particular traits was observed for most traits in humans as strongly as shown in the other studies . then , the average value of estimates of hg over all of tens traits to reduce the sampling error variance , and the average estimated hc was strongly correlated with chromosome length . however , we dealt with only three traits related to milk production , and we aimed to identify a linear correlation between hc and chromosome length for each trait . the squared correlation between hc and chromosome length using trait information of parity 1 was 0.46 , 0.532 , and 0.509 for milk yield , fat , and protein , respectively ( fig . the regression slope of the proportion of the genetic variance attributed to each chromosome on the proportion of the genome represented by each chromosome was significant ( p<0.1 ) , suggesting a proportional relationship between genome length and genetic variance in holstein . consistent results were achieved from myriad genetic variants , each possessing a small effect throughout the widespread whole genome in holstein . in addition , we partitioned hg into the contributions of genic ( hgg ) and intergenic ( hgi ) regions of the whole genome ( methods section ) . we estimated the variance explained by the genic ( hcg ) and intergenic ( hci ) regions of each chromosome . the numbers of genic and intergenic snps on each chromosome are presented in supplementary table 2 . we showed that the variance explained by the genic ( intergenic ) regions on each chromosome is also proportional to the total length of the genic ( intergenic ) regions ( fig . previous studies using the whole - genome estimation approach have shown that common snps explicate a large proportion of heritability for traits in human . the reason why gwass in this field have not yet identified all common snps that contain information on the amount of variation is mainly that many peculiar effects are too small to pass the stringent genome - wide significance level . therefore , we hypothesized that inheritance of korean holstein milk production traits would consist of a lot of genetic variants with small effects . finally , we estimated and partitioned the genetic variance that met certain conditions , tagged by all common snps for three complex traits related to milk production and showing ubiquitous polygenic inheritance of korean holstein . the estimates of hg for three traits were different from 0 at the significance level ( p<0.1 ) . the estimate of hg for milk yield of parity 1 was 43.0% ( se = 10.3% ) , which was smaller than the estimate from a study in canada ( hg = 52% ) . there could be three possible reasons : ( 1 ) there is a difference in the reference population size between korea and canada . in fact , koreans import a lot of canada holstein semen , but the present reference population is smaller than canada . ( 2 ) the korean environment is different from canada . ( 3 ) we can consider only one environmental effect ( season ) due to data size in this study . the estimate for milk fat ( hg = 47.0% , se = 10.1% ) and protein ( hg = 44.3% , se = 10.2% ) in korea was larger than in canada ( fat hg = 36.9% , protein hg = 42.3% ) . it is demonstrated by the genome partitioning analysis that there was a significant linear relationship between the estimates of variance explained by individual chromosomes and chromosome length ( fig . 2 ) . if we define the genic region as 0 kb of the utrs , the correlation between variance explained and chromosome length was stronger in the intergenic regions than in the genic regions ( fig . 3 ) . a previous study showed by a number of analyses that the result in this was driven neither by the difference between the number of snps in genic regions and in intergenic regions nor by the difference in maf distribution between genic and intergenic snps . if trait - associated genetic variants are enriched in functional elements , such as introns and utrs , and diluted in exons , the relationship between the explained variance and dna length will be attenuated in the genic region , as in human . however , it could possibly be just a sampling problem . because our data size was smaller than our expectancy , some chromosomes did not reach the significance level ( p = 0.01 ) in the genome partitioning analysis . in the genome partitioning analysis for milk yield using snps in the intergenic region and milk protein using snps in the genic region , the linear regression results were also insignificant , and we could identify some outliers . moreover , when these outliers were removed , the linear regression results were significant ( e in fig . we hypothesize that some chromosomes could have a slightly larger effect than our expectation , based on the fitted line in special cases ( chromosome 17 or 4 in milk protein ) . we showed by whole - genome estimation and partitioning analyses that holstein milk production traits appeared to be highly polygenic , which means that there were a large number of genetic variants segregating in the population with a small effect widely distributed across the whole genome . all common snps expressed most of the heritability on average over all 3 traits analyzed in this study . the slight , remaining unexplained heritability could be due to causal variants , including common and rare ones that are not well tagged by snps on this illumina snp chip , or possibly because the heritability was over - estimated in the pedigree study . in conclusion , heritability consists of many variants with small effects for holstein milk production traits in korea . overall , this research will become a cornerstone for genomic selection applications in animal breeding . supplementary data , including two figures and two tables , can be found with this article online at http://www / genominfo.org / src / sm / gni-13 - 146-s001.pdf . numbers of genic and intergenic snps on each chromosome variance explained by all the genic and intergenic snps on individual chromosomes for the 3 traits all pairwise phenotypic correlation of the three traits related to milk production . manhattan plot of genome - wide association studies results for three traits of parity 1 ( milk yield , milk fat , and milk protein , respectively ) .

previous studies in holstein have shown 35% to 51.8% heritability in milk production traits , such as milk yield , fat , and protein , using pedigree data . other studies in complex human traits could be captured by common single - nucleotide polymorphisms ( snps ) , and their genetic variations , attributed to chromosomes , are in proportion to their length . using genome - wide estimation and partitioning approaches , we analyzed three quantitative holstein traits relevant to milk production in korean holstein data harvested from 462 individuals genotyped for 54,609 snps . for all three traits ( milk yield , fat , and protein ) , we estimated a nominally significant ( p = 0.1 ) proportion of variance explained by all snps on the illumina bovinesnp50 beadchip ( h2 g ) . these common snps explained approximately most of the narrow - sense heritability . longer genomic regions tended to provide more phenotypic variation information , with a correlation of 0.46~0.53 between the estimate of variance explained by individual chromosomes and their physical length . these results suggested that polygenicity was ubiquitous for holstein milk production traits . these results will expand our knowledge on recent animal breeding , such as genomic selection in holstein .

Introduction Methods Korean Holstein data Genotyped and imputed data Estimating and partitioning genetic variance using SNP data Results Discussion Supplementary materials

pulmonary embolism ( pe ) is a relatively common cardiovascular emergency , and is associated with a high mortality.1 ) in this respect , early diagnosis of pe is clinically important . because of the variable and nonspecific clinical presentations of pe , a diagnostic testing like computed tomographic pulmonary angiography ( ctpa ) is necessary to diagnose and determine the extent of disease . however , we sometimes experience a patient with a significant discrepancy between clinical findings and ctpa results . here , we describe a patient with pe , in whom ctpa did not suggest the therapeutic role of surgical embolectomy . notwithstanding , based on the clinical and echocardiographic judgment , the patient was referred to surgery , resulting in a progressive , hemodynamic improvement postoperatively . a 42-year - old man presented to the emergency department with recently aggravated severe dyspnea . the patient had been treated for crohn 's disease . he experienced acute pe 2 years ago and thereafter had been on warfarin , with a target inr of 2.0 to 3.0 . the last follow - up ctpa that had been conducted 7 weeks before revealed a decreased extent of eccentric filling defects in descending braches of both pulmonary arteries , which was improved compared to the initial exam ( fig . concomitant baseline echocardiography revealed mildly to moderately depressed right ventricular ( rv ) systolic function , mild tricuspid regurgitation ( tr ) and pulmonary hypertension with estimated systolic pulmonary arterial pressure of 62 mm hg . his blood pressure was 81/70 mm hg , pulse 89 bpm , respiration rate 40 breaths per minute , and body temperature 36.0 with oxygen saturation of 81% on room air . serum level of cardiac enzymes was within normal ranges , but bnp was elevated at 396 pg / ml , and d - dimer was also elevated at 1,940 ng / ml . initial arterial blood gas analysis showed combined metabolic acidosis and respiratory alkalosis ( ph 7.237 , pco2 12.8 mm hg , hco3 9.6 mmol / l ) and pao2 of 126 mm hg with supplemental oxygen at 10 liters / min via a partial rebreathing mask . shortly after arrival at emergency department due to high clinical suspicion of acute pe , ctpa with venous phase imaging of lower extremities was conducted . but ctpa did not demonstrate any interval change in the extent of pre - existing chronic pe ( fig . 1b ) , and ct venography also did not demonstrate a source of pulmonary embolism . there was no lung parenchymal lesion which could explain the patient 's acute respiratory failure . however , echocardiography revealed an increased rv size with severely depressed rv systolic function ( fig . moderate tr was found with maximal velocity of tr jet of 3.58 m / sec , corresponding to an estimated systolic pulmonary arterial pressure of 71 mm hg with an assumption of ra pressure of 20 mm hg ( fig . because of refractory respiratory failure unresponsive to maximum medical management , he was subsequently connected to a venovenous extracorporeal membrane oxygenation system in medical intensive care unit . although ctpa did not support the role of surgical intervention , pulmonary embolectomy under cardiopulmonary bypass was performed on the third hospital day with a high probability of hemodynamically significant acute pe , based on clinical and echocardiographic findings . in the operating room , both pulmonary arteries were extensively inspected , and a large amount of organized thrombi with fresh thrombi were found in right and left pulmonary arteries extending to the segmental artery levels . his hemodynamic parameters had progressively improved over the following 2 days , with a significant reduction in systolic pulmonary artery pressure to 40 mm hg on invasive monitoring . however , pulmonary hemorrhage and ventilator associated pneumonia developed and he finally passed away on the 43rd postoperative day . since the first introduction of multi - detector ct , ctpa has become the method of choice for the diagnosis of suspected acute pe in everyday clinical practice.2 ) in hemodynamically unstable patients with acute pe , ctpa is strongly recommended because of its high sensitivity for detecting ' fresh ' emboli in " large " pulmonary artery branches.3 ) however , there are two points that we should keep in mind in this setting . first , the predictive value of ctpa depends on the pretest probability of pe . stein et al.3 ) have reported that in patients with a high pretest probability of pe , as assessed by the wells score,4 ) a negative ctpa result had a low ( 60% ) negative predictive value for pe , highlighting that clinical finding should be considered a high priority over ctpa findings in therapeutic decision - making . second , in the setting of acute thromboembolic event on top of chronic pe , ctpa can underestimate the distribution of pulmonary emboli in the pulmonary branches . this is especially true when the emboli are linearly located along the pulmonary arterial wall or at the subsegmental artery level despite use of multi - detector ctpa.5 ) in addition , we suggest based on this case that in - situ thrombosis occurring slowly on chronic thromboemboli is difficult to detect with ctpa . as shown in fig . 3 , fresh thrombi were clearly demonstrated on operative findings , although ctpa could not suggest their presence . in this situation , clinical presentation of the patient must be discrepant from anatomic burden of pulmonary emboli . in this particular condition , echocardiography is able to guide therapeutic decision - making because it can reflect hemodynamic status of the patient , not simple anatomical thrombi burden . echocardiography is frequently performed in pe patients , because it permits reliable , qualitative , hemodynamic assessment of rv size , rv systolic function , and pulmonary arterial pressure.6)7 ) prognostic information can be obtained with the help of echocardiography.8)9 ) furthermore , in a critically ill patient with worsening symptoms who can not undergo ctpa without delay , bedside echocardiography contributes to early diagnosis and treatment of pe . kucher et al.10 ) have suggested that echocardiographic signs of rv pressure overload and dysfunction in a hemodynamically unstable patient with high pretest probability of pe may justify invasive treatment for pe . in this case , worsened hemodynamic parameters on serial echocardiography helped us to apply an invasive approach for recurrent pe . since the patient was on a venovenous extracorporeal membrane oxygenation system , intravenous thrombolysis was not considered an initial treatment option . as described above , ctpa can underestimate the burden of pulmonary thrombi , especially those that is distributed along the pulmonary arterial wall , at the subsegmental artery level , and in - situ thrombosis on top of chronic thrombi . therefore , we believe that the present case is important and instructive in that hemodynamic assessment with echocardiography should be conducted to more accurately guide therapeutic strategy in clinically suspected pe patients .

we describe a 42-year - old man presenting to the emergency department with cardiogenic shock . he had a prior history of acute pulmonary embolism ( pe ) , and had been on anticoagulation for 2 years . although computed tomographic pulmonary angiography performed at the emergency department showed no change in the extent of pe and did not support a role of surgical treatment , pulmonary embolectomy was recommended by attending physician based on clinical and echocardiographic hemodynamic findings like unstable vital sign and markedly enlarged right ventricle with severely depressed systolic function . surgery confirmed the presence of fresh thrombi . after surgery , hemodynamic status was progressively improved , but the patient died due to pneumonia and pulmonary hemorrhage .

Introduction Case Discussion

the english longitudinal study of ageing ( elsa ) is a prospective observational study of community - dwelling people aged 50 years and over in england . at baseline , in 20022003 , the sample comprised 11 391 core participants who were recruited from households that had earlier participated in the health survey for england in 1998 , 1999 and 2001 . the health survey for england is an annual health examination survey , which each year recruits a different nationally representative sample using a multistaged stratified random probability design . after the baseline , follow - up interviews took place every 2 years and health examinations every 4 years ( the first health examination was in 20042005 ) . a detailed description of the study can be found at : http://www.elsa - project.ac.uk/. the analytical sample comprised 10 305 after the exclusion of 362 participants with proxy or partial interviews , 459 participants who did not consent to the mortality linkage , 5 participants who died the same month they granted their baseline interview and 260 participants with missing values in baseline variables ( excluding body mass index ( bmi ) ) . the elsa has collected detailed information on different dimensions of wealth both at baseline and each of the follow - up interviews . we used total net non - pension household wealth , which is a summary measure of the value of financial , physical and housing wealth owned by the household ( ie , a single respondent or a responding couple along with any dependent individuals ) minus any debt . the estimation of this variable was based on 22 different wealth and debt components , which were either observed or imputed . the imputed wealth and debt components were integral part of the original elsa data set and generated by the data depositors and not the authors of this study . most of these imputations referred to incomplete values in one or two components and were based on information that the participants provided about the upper and lower boundaries within which the actual values of those components lay . a detailed description of wealth and its components can be found at : http://bit.ly/1yrrghd and http://bit.ly/1awp6iz . as our aim was to study wealth inequalities in mortality , we categorised wealth into tertiles . death registrations up to february 2013 were obtained from the office for national statistics for all consenting participants . deaths with icd10 codes c00 to c97 were classified as cancer deaths and those with icd10 codes i00 to i99 as cardiovascular deaths . paternal ( or main carer 's ) occupational class when participants were 14 years old , education , occupational class , income , smoking , physical activity , measured bmi and elevated depressive symptoms were also measured . we used repeated measurements of wealth , physical activity and depressive symptoms to generate time - varying variables . to minimise non - response bias in analyses involving time - varying variables we could not impute the missing values in smoking , because there was not enough variation in this variable over time , and consequently did not derive a time - varying smoking variable . bmi was not measured in all waves of the study , and thus we did not have the necessary data to derive a time - varying bmi variable . among the 10 305 participants with complete baseline data ( excluding bmi data ) , 5758 ( including 2401 who died after the baseline ) did not have complete data on wealth tertiles , physical activity and depressive symptoms at all four follow - up interviews . we imputed all missing values and censored the imputed data at the date of death . we also imputed missing values in the baseline bmi categories variable ( n=944 ; in our analyses bmi was the only baseline variable with missing values ) . cox proportional hazard regression analyses were performed to investigate the associations between wealth and all - cause and cause - specific mortality . survival time was measured as the amount of time that had elapsed from the date of baseline interview to the first of either the date of death or censoring ( ie , february 2013 ) . significant interactions by age , but not sex , were identified using the likelihood ratio test . for this reason , all analyses were stratified into two age groups : 5064 and 65 years or older . we used the cut point of 65 years because that was the national state pension age for men and the mean age of our sample . the use of this cut point resulted in two age groups of similar size with an adequate number of cases per group . the cox regression models were initially adjusted for age , sex and marital status , then , in addition , for smoking and physical activity , and finally for bmi and elevated depressive symptoms . we repeated the survival analysis using repeated measurements of wealth , physical activity and depressive symptoms . we also examined whether the association between wealth and all - cause mortality persisted after adjustment for other socioeconomic position measures . further , we examined whether wealth mediated the associations between either childhood or adult socioeconomic position measures and all - cause mortality . we confirmed that the proportionality assumption was met using survival plots and the schoenfeld residuals test . to investigate the potential influence of reverse causality , that is , baseline chronic diseases and subsequent proximity to death leading to a decrease in wealth , we repeated our analyses after excluding people with major chronic conditions ( see web table 1 ) . to explore whether the use of any imputed wealth data biased our findings , we repeated our analyses after excluding participants with imputed values in any of the 22 baseline wealth and debt components that were generated by the elsa data depositors ( see web table 2 ) . the english longitudinal study of ageing ( elsa ) is a prospective observational study of community - dwelling people aged 50 years and over in england . at baseline , in 20022003 , the sample comprised 11 391 core participants who were recruited from households that had earlier participated in the health survey for england in 1998 , 1999 and 2001 . the health survey for england is an annual health examination survey , which each year recruits a different nationally representative sample using a multistaged stratified random probability design . after the baseline , follow - up interviews took place every 2 years and health examinations every 4 years ( the first health examination was in 20042005 ) . a detailed description of the study can be found at : http://www.elsa - project.ac.uk/. the analytical sample comprised 10 305 after the exclusion of 362 participants with proxy or partial interviews , 459 participants who did not consent to the mortality linkage , 5 participants who died the same month they granted their baseline interview and 260 participants with missing values in baseline variables ( excluding body mass index ( bmi ) ) . the elsa has collected detailed information on different dimensions of wealth both at baseline and each of the follow - up interviews . we used total net non - pension household wealth , which is a summary measure of the value of financial , physical and housing wealth owned by the household ( ie , a single respondent or a responding couple along with any dependent individuals ) minus any debt . the estimation of this variable was based on 22 different wealth and debt components , which were either observed or imputed . the imputed wealth and debt components were integral part of the original elsa data set and generated by the data depositors and not the authors of this study . most of these imputations referred to incomplete values in one or two components and were based on information that the participants provided about the upper and lower boundaries within which the actual values of those components lay . a detailed description of wealth and its components can be found at : http://bit.ly/1yrrghd and http://bit.ly/1awp6iz . as our aim was to study wealth inequalities in mortality , we categorised wealth into tertiles . death registrations up to february 2013 were obtained from the office for national statistics for all consenting participants . deaths with icd10 codes c00 to c97 were classified as cancer deaths and those with icd10 codes i00 to i99 as cardiovascular deaths . paternal ( or main carer 's ) occupational class when participants were 14 years old , education , occupational class , income , smoking , physical activity , measured bmi and elevated depressive symptoms were also measured . we used repeated measurements of wealth , physical activity and depressive symptoms to generate time - varying variables . to minimise non - response bias in analyses involving time - varying variables we could not impute the missing values in smoking , because there was not enough variation in this variable over time , and consequently did not derive a time - varying smoking variable . bmi was not measured in all waves of the study , and thus we did not have the necessary data to derive a time - varying bmi variable . among the 10 305 participants with complete baseline data ( excluding bmi data ) , 5758 ( including 2401 who died after the baseline ) did not have complete data on wealth tertiles , physical activity and depressive symptoms at all four follow - up interviews . we imputed all missing values and censored the imputed data at the date of death . we also imputed missing values in the baseline bmi categories variable ( n=944 ; in our analyses bmi was the only baseline variable with missing values ) . cox proportional hazard regression analyses were performed to investigate the associations between wealth and all - cause and cause - specific mortality . survival time was measured as the amount of time that had elapsed from the date of baseline interview to the first of either the date of death or censoring ( ie , february 2013 ) . significant interactions by age , but not sex , were identified using the likelihood ratio test . for this reason , all analyses were stratified into two age groups : 5064 and 65 years or older . we used the cut point of 65 years because that was the national state pension age for men and the mean age of our sample . the use of this cut point resulted in two age groups of similar size with an adequate number of cases per group . the cox regression models were initially adjusted for age , sex and marital status , then , in addition , for smoking and physical activity , and finally for bmi and elevated depressive symptoms . we repeated the survival analysis using repeated measurements of wealth , physical activity and depressive symptoms . we also examined whether the association between wealth and all - cause mortality persisted after adjustment for other socioeconomic position measures . further , we examined whether wealth mediated the associations between either childhood or adult socioeconomic position measures and all - cause mortality . we confirmed that the proportionality assumption was met using survival plots and the schoenfeld residuals test . to investigate the potential influence of reverse causality , that is , baseline chronic diseases and subsequent proximity to death leading to a decrease in wealth , we repeated our analyses after excluding people with major chronic conditions ( see web table 1 ) . to explore whether the use of any imputed wealth data biased our findings , we repeated our analyses after excluding participants with imputed values in any of the 22 baseline wealth and debt components that were generated by the elsa data depositors ( see web table 2 ) . the lower the wealth of participants the more likely they were to be older , female , non - married , smokers , physically inactive , depressed , obese and of lower socioeconomic position ( table 1 ) . response association between wealth and all - cause and cause - specific mortality that was stronger among younger participants ( table 2 ) . the adjustment for risk factors only partially explained these associations , except for the association between wealth and cancer mortality , which was fully explained ( table 2 ) . the adjustment for other socioeconomic position measures also did not affect the association between wealth and all - cause mortality ( compare model 2 , right - hand side panel ( household wealth tertiles ) , table 3 to model 1 , table 2 ) . in contrast , the associations between paternal occupational class at age 14 years , education , occupational class , and income and all - cause mortality were considerably attenuated after adjustment for wealth ( see left - hand side panel ( socioeconomic position indicator ) , table 3 ) . the adjustment for time - varying physical activity and depressive symptoms and the use of time - varying wealth did not much change the results ; except for a decrease in the strength of the association between and cardiovascular mortality among those aged 5064 years that was brought about mostly by the use of time - varying wealth ( compare table 4 to table 2 ) . the baseline characteristics of 10 305 women and men aged 50 years and over by household wealth * p values were calculated using , kruskal - wallis and analysis of variance tests for categorical , ordinal and continuous covariates , respectively . defined as 4 symptoms on the eight - item center for epidemiological studies - depression scale . the other category included 73 participants with missing values and was not used in the calculation of the p value . the association between household wealth and all - cause and cause - specific mortality by age * adjusted for age , sex and baseline marital status . as model 2 , plus adjustment for baseline elevated depressive symptoms and baseline body mass index . the association between different socioeconomic position indicators and all - cause mortality by age * adjusted for age , sex and baseline marital status . for clarity reasons , results for the small never worked category are not presented . for clarity reasons , results for the small other category are not presented . the association between time - varying household wealth and all - cause and cause - specific mortality by age * adjusted for age , sex and baseline marital status . as model 1 , plus adjustment for baseline smoking and time - varying physical activity . as model 2 , plus adjustment for time - varying elevated depressive symptoms and baseline body mass index . in a national sample of people 50 years or older , we found pronounced gradients in all - cause and cause - specific mortality by total household wealth . adjustment for covariates neither fully explained the associations , except for cancer mortality , nor affected their dose response pattern . additional adjustment for time - varying covariates and the use of time - varying wealth did not change the associations to a great extent . the only exception was a decrease in the strength of the association between wealth and cardiovascular mortality among participants aged 5064 years . wealth was found to be a more powerful predictor of mortality than any other socioeconomic position indicator used in our analyses and explained most of the associations between other socioeconomic position indicators and all - cause mortality in both younger and older participants . our study is one of the first to thoroughly study wealth inequalities in mortality and the role of wealth in the associations between commonly used socioeconomic position indicators such as education and occupational class and mortality over a long follow - up . the use of a national sample of community dwellers and rich longitudinal data from a well - established survey are strengths of our study . the 10-year follow - up allowed for the investigation of the longer term effect of wealth on the risk of mortality . the quality measurement of wealth minimised the possibility of measurement bias , while the examination of age differences widened the scope of our work . the use of a selected sample of people who survived at least to the age of 50 years to be included in our sample is a limitation that affects the applicability of our findings to younger generations . although our study had a household response rate at baseline of 70% and was inclusive of the vast majority of people who had participated at baseline , we can not rule out the possibility of non - response bias . nevertheless , the consistency between the main analyses findings and those of supplementary analyses that excluded participants with major chronic diseases or imputed wealth values suggests that it is unlikely non - response bias to considerably influence our findings . finally , despite retaining in the sample participants who moved to care homes and other institutions after the baseline , the exclusion of people living in institutions at baseline makes our findings less relevant to institutionalised older people . few , mostly us , studies have studied wealth inequalities in mortality . notwithstanding methodological differences , their findings largely concur with ours and highlight wealth as an important predictor of mortality.2128 significant wealth inequalities were observed in both participants aged 5064 years and those aged 65 years or older , but they were much more pronounced in the former age group . the decline in the relative strength of socioeconomic3 16 and more specifically wealth21 23 25 inequalities in people aged 65 years or older compared with younger adults is a consistent finding of the literature and accords with the declining strength of the associations between mortality and most risk factors with age , which is likely , in part , to be an effect of survivor bias . nevertheless , the importance of wealth inequalities for people aged 65 years and older remains as they translate into great differences in the absolute risk of dying . the great difference in the relative risk of premature death between the top and bottom tertile of wealth among people aged 5064 years likely indicates the detrimental effect of the accumulation of disadvantage over the life course and identifies a population at risk . that the majority of participants who did not own their home were classified in the bottom tertile of the wealth , our findings support earlier findings suggesting that the inverse association between home ownership and mortality risk should be interpreted as part of wealth inequalities in mortality.29 wealth inequalities in cardiovascular mortality were pronounced and persisting . the magnitude and persistence of wealth inequalities in cardiovascular mortality probably indicates a systematic life course effect socioeconomic position on cardiovascular health that might be related to early life programming and mediated by subsequent epigenetic and physiological modifications . wealth inequalities in cancer mortality were less pronounced and mostly a result of the higher prevalence of unhealthy behaviours among people in the bottom tertile of the wealth distribution . the persisting wealth gradient in mortality from other causes , including respiratory causes , may at least partly reflect the impact of social disadvantage and inadequate housing conditions on respiratory health . in this study , a set of behavioural and psychosocial risk factors failed to completely account for the observed mortality gradient . a recent study of wealth inequalities in a nationally representative us sample aged 51 years and older reported similar findings.23 to the extent that our work is comparable with studies of occupational cohorts that did not use total wealth,18 19 our findings contradict findings suggesting a full explanation of socioeconomic inequalities after adjustment for time - varying unhealthy behaviours18 and lend support to those suggesting a partial attenuation.19 we found that adjustment for time - varying covariates explained only a small additional part of the association between wealth and mortality compared with the conventional analysis that did not use time - varying covariates . the use of time - varying predictor also did not bring about considerable changes , except for the partial attenuation of the association between wealth and cardiovascular mortality among those aged 5064 years , an indication that fluctuations in wealth at older ages likely are less important for survival than baseline wealth . the adjustment for paternal occupational class at age 14 years , education , occupational class and household income these findings indicate that wealth is an appropriate socioeconomic position indicator to use when studying socioeconomic inequalities in health in older adults . previous findings concur with our findings indicating that wealth remained associated with mortality after adjustment for other socioeconomic position measures21 22 24 2628 with any discrepancy stemming from methodological differences such as the use of data from administrative sources.27 in agreement with our findings , studies also reported full or partial attenuation of the associations between either education or income and mortality after adjustment for wealth,27 30 except for one instance.30 the full attenuation of the associations between paternal occupational class , education and occupational class and mortality after adjustment for wealth in our data suggests that wealth accumulation is one of the main pathways through which childhood and adult socioeconomic positions are associated with later life risk of mortality . wealth appears to be more strongly associated with the risk of death than any other socioeconomic position indicator at older ages . future research should propose and test causal models of the most pertinent pathways linking socioeconomic position indicators from different stages of the life course to mortality at older ages in an attempt to improve our understanding of the association between socioeconomic position and mortality . what is already known on this subjectour understanding of social inequalities in mortality at older ages remains limited.it is unclear how best to measure socioeconomic position at older ages and which aspects of socioeconomic position are mostly relevant to mortality at older ages . wealth , an important socioeconomic position measure that may be mostly appropriate to use when studying middle - aged and older people , has rarely been used in epidemiological research.it also remains unclear whether unhealthy behaviours , depression and obesity explain the association between socioeconomic position and mortality at middle and older ages.finally , to better understand the association between socioeconomic position and mortality over the life course , it is important to establish whether the associations between childhood and early and middle adulthood socioeconomic position and mortality at older ages are explained by contemporary socioeconomic position measured using wealth . what this study addsthis study is one of the first to thoroughly examine the associations between total net non - pension household wealth and all - cause and cause - specific mortality using repeated measurements of wealth and behavioural and psychosocial factors.we found important and persisting wealth inequalities in all - cause and cause - specific mortality at older ages . they were less pronounced but still significant among those aged 65 years and older.we also found that the associations between measures of childhood and young and middle adulthood socioeconomic position and mortality were largely explained by wealth . wealth was more strongly associated with mortality than any other socioeconomic position indicator and appears to be an excellent socioeconomic position measure to use in studies of middle - aged and older people.unhealthy behaviours , elevated depressive symptoms and obesity provided only a partial explanation of the association between wealth and mortality , even when multiple measurements of these factors and wealth were used.wealth was more strongly associated with cardiovascular and other non - cancer mortality than cancer mortality . it is unclear how best to measure socioeconomic position at older ages and which aspects of socioeconomic position are mostly relevant to mortality at older ages . wealth , an important socioeconomic position measure that may be mostly appropriate to use when studying middle - aged and older people , has rarely been used in epidemiological research . it also remains unclear whether unhealthy behaviours , depression and obesity explain the association between socioeconomic position and mortality at middle and older ages . finally , to better understand the association between socioeconomic position and mortality over the life course , it is important to establish whether the associations between childhood and early and middle adulthood socioeconomic position and mortality at older ages are explained by contemporary socioeconomic position measured using wealth . this study is one of the first to thoroughly examine the associations between total net non - pension household wealth and all - cause and cause - specific mortality using repeated measurements of wealth and behavioural and psychosocial factors . we found important and persisting wealth inequalities in all - cause and cause - specific mortality at older ages . they were less pronounced but still significant among those aged 65 years and older . we also found that the associations between measures of childhood and young and middle adulthood socioeconomic position and mortality were largely explained by wealth . wealth was more strongly associated with mortality than any other socioeconomic position indicator and appears to be an excellent socioeconomic position measure to use in studies of middle - aged and older people . unhealthy behaviours , elevated depressive symptoms and obesity provided only a partial explanation of the association between wealth and mortality , even when multiple measurements of these factors and wealth were used . wealth was more strongly associated with cardiovascular and other non - cancer mortality than cancer mortality . our study is one of the first to thoroughly study wealth inequalities in mortality and the role of wealth in the associations between commonly used socioeconomic position indicators such as education and occupational class and mortality over a long follow - up . the use of a national sample of community dwellers and rich longitudinal data from a well - established survey are strengths of our study . the 10-year follow - up allowed for the investigation of the longer term effect of wealth on the risk of mortality . the quality measurement of wealth minimised the possibility of measurement bias , while the examination of age differences widened the scope of our work . the use of a selected sample of people who survived at least to the age of 50 years to be included in our sample is a limitation that affects the applicability of our findings to younger generations . although our study had a household response rate at baseline of 70% and was inclusive of the vast majority of people who had participated at baseline , we can not rule out the possibility of non - response bias . nevertheless , the consistency between the main analyses findings and those of supplementary analyses that excluded participants with major chronic diseases or imputed wealth values suggests that it is unlikely non - response bias to considerably influence our findings . finally , despite retaining in the sample participants who moved to care homes and other institutions after the baseline , the exclusion of people living in institutions at baseline makes our findings less relevant to institutionalised older people . few , mostly us , studies have studied wealth inequalities in mortality . notwithstanding methodological differences , their findings largely concur with ours and highlight wealth as an important predictor of mortality.2128 significant wealth inequalities were observed in both participants aged 5064 years and those aged 65 years or older , but they were much more pronounced in the former age group . the decline in the relative strength of socioeconomic3 16 and more specifically wealth21 23 25 inequalities in people aged 65 years or older compared with younger adults is a consistent finding of the literature and accords with the declining strength of the associations between mortality and most risk factors with age , which is likely , in part , to be an effect of survivor bias . nevertheless , the importance of wealth inequalities for people aged 65 years and older remains as they translate into great differences in the absolute risk of dying . the great difference in the relative risk of premature death between the top and bottom tertile of wealth among people aged 5064 years likely indicates the detrimental effect of the accumulation of disadvantage over the life course and identifies a population at risk . that the majority of participants who did not own their home were classified in the bottom tertile of the wealth , our findings support earlier findings suggesting that the inverse association between home ownership and mortality risk should be interpreted as part of wealth inequalities in mortality.29 wealth inequalities in cardiovascular mortality were pronounced and persisting . the magnitude and persistence of wealth inequalities in cardiovascular mortality probably indicates a systematic life course effect socioeconomic position on cardiovascular health that might be related to early life programming and mediated by subsequent epigenetic and physiological modifications . wealth inequalities in cancer mortality were less pronounced and mostly a result of the higher prevalence of unhealthy behaviours among people in the bottom tertile of the wealth distribution . the persisting wealth gradient in mortality from other causes , including respiratory causes , may at least partly reflect the impact of social disadvantage and inadequate housing conditions on respiratory health . in this study , a set of behavioural and psychosocial risk factors failed to completely account for the observed mortality gradient . a recent study of wealth inequalities in a nationally representative us sample aged 51 years and older reported similar findings.23 to the extent that our work is comparable with studies of occupational cohorts that did not use total wealth,18 19 our findings contradict findings suggesting a full explanation of socioeconomic inequalities after adjustment for time - varying unhealthy behaviours18 and lend support to those suggesting a partial attenuation.19 we found that adjustment for time - varying covariates explained only a small additional part of the association between wealth and mortality compared with the conventional analysis that did not use time - varying covariates . the use of time - varying predictor also did not bring about considerable changes , except for the partial attenuation of the association between wealth and cardiovascular mortality among those aged 5064 years , an indication that fluctuations in wealth at older ages likely are less important for survival than baseline wealth . the adjustment for paternal occupational class at age 14 years , education , occupational class and household income these findings indicate that wealth is an appropriate socioeconomic position indicator to use when studying socioeconomic inequalities in health in older adults . previous findings concur with our findings indicating that wealth remained associated with mortality after adjustment for other socioeconomic position measures21 22 24 2628 with any discrepancy stemming from methodological differences such as the use of data from administrative sources.27 in agreement with our findings , studies also reported full or partial attenuation of the associations between either education or income and mortality after adjustment for wealth,27 30 except for one instance.30 the full attenuation of the associations between paternal occupational class , education and occupational class and mortality after adjustment for wealth in our data suggests that wealth accumulation is one of the main pathways through which childhood and adult socioeconomic positions are associated with later life risk of mortality . wealth appears to be more strongly associated with the risk of death than any other socioeconomic position indicator at older ages . future research should propose and test causal models of the most pertinent pathways linking socioeconomic position indicators from different stages of the life course to mortality at older ages in an attempt to improve our understanding of the association between socioeconomic position and mortality . what is already known on this subjectour understanding of social inequalities in mortality at older ages remains limited.it is unclear how best to measure socioeconomic position at older ages and which aspects of socioeconomic position are mostly relevant to mortality at older ages . wealth , an important socioeconomic position measure that may be mostly appropriate to use when studying middle - aged and older people , has rarely been used in epidemiological research.it also remains unclear whether unhealthy behaviours , depression and obesity explain the association between socioeconomic position and mortality at middle and older ages.finally , to better understand the association between socioeconomic position and mortality over the life course , it is important to establish whether the associations between childhood and early and middle adulthood socioeconomic position and mortality at older ages are explained by contemporary socioeconomic position measured using wealth . what this study addsthis study is one of the first to thoroughly examine the associations between total net non - pension household wealth and all - cause and cause - specific mortality using repeated measurements of wealth and behavioural and psychosocial factors.we found important and persisting wealth inequalities in all - cause and cause - specific mortality at older ages . they were less pronounced but still significant among those aged 65 years and older.we also found that the associations between measures of childhood and young and middle adulthood socioeconomic position and mortality were largely explained by wealth . wealth was more strongly associated with mortality than any other socioeconomic position indicator and appears to be an excellent socioeconomic position measure to use in studies of middle - aged and older people.unhealthy behaviours , elevated depressive symptoms and obesity provided only a partial explanation of the association between wealth and mortality , even when multiple measurements of these factors and wealth were used.wealth was more strongly associated with cardiovascular and other non - cancer mortality than cancer mortality . it is unclear how best to measure socioeconomic position at older ages and which aspects of socioeconomic position are mostly relevant to mortality at older ages . wealth , an important socioeconomic position measure that may be mostly appropriate to use when studying middle - aged and older people , has rarely been used in epidemiological research . it also remains unclear whether unhealthy behaviours , depression and obesity explain the association between socioeconomic position and mortality at middle and older ages . finally , to better understand the association between socioeconomic position and mortality over the life course , it is important to establish whether the associations between childhood and early and middle adulthood socioeconomic position and mortality at older ages are explained by contemporary socioeconomic position measured using wealth . this study is one of the first to thoroughly examine the associations between total net non - pension household wealth and all - cause and cause - specific mortality using repeated measurements of wealth and behavioural and psychosocial factors . we found important and persisting wealth inequalities in all - cause and cause - specific mortality at older ages . they were less pronounced but still significant among those aged 65 years and older . we also found that the associations between measures of childhood and young and middle adulthood socioeconomic position and mortality were largely explained by wealth . wealth was more strongly associated with mortality than any other socioeconomic position indicator and appears to be an excellent socioeconomic position measure to use in studies of middle - aged and older people . unhealthy behaviours , elevated depressive symptoms and obesity provided only a partial explanation of the association between wealth and mortality , even when multiple measurements of these factors and wealth were used . wealth was more strongly associated with cardiovascular and other non - cancer mortality than cancer mortality .

backgrounddespite the importance of socioeconomic position for survival , total wealth , which is a measure of accumulation of assets over the life course , has been underinvestigated as a predictor of mortality . we investigated the association between total wealth and mortality at older ages.methodswe estimated cox proportional hazards models using a sample of 10 305 community - dwelling individuals aged 50 years from the english longitudinal study of ageing.results2401 deaths were observed over a mean follow - up of 9.4 years . among participants aged 5064 years , the fully adjusted hrs for mortality were 1.21 ( 95% ci 0.92 to 1.59 ) and 1.77 ( 1.35 to 2.33 ) for those in the intermediate and lowest wealth tertiles , respectively , compared with those in the highest wealth tertile . the respective hrs were 2.54 ( 1.27 to 5.09 ) and 3.73 ( 1.86 to 7.45 ) for cardiovascular mortality and 1.36 ( 0.76 to 2.42 ) and 2.53 ( 1.45 to 4.41 ) for other non - cancer mortality . wealth was not associated with cancer mortality in the fully adjusted model . similar but less strong associations were observed among participants aged 65 years . the use of repeated measurements of wealth and covariates brought about only minor changes , except for the association between wealth and cardiovascular mortality , which became less strong in the younger participants . wealth explained the associations between paternal occupation at age 14 years , education , occupational class , and income and mortality.conclusionsthere are persisting wealth inequalities in mortality at older ages , which only partially are explained by established risk factors . wealth appears to be more strongly associated with mortality than other socioeconomic position measures .

Methods Study population Assessment of household wealth Mortality Covariates Statistical analysis Results Discussion Strengths and weaknesses Interpretation of the findings Supplementary Material

children with spina bifida develop a wide variety of congenital and acquired orthopedic deformities including knee flexion contractures . knee flexion contractures in children with spina bifida affect children s functional activities and interfere with the ability of children to transfer and walk1 . a limited number of studies have looked at the management of knee flexion contractures in children with spina bifida . surgical intervention is the most effective choice for correcting joint contracture in children with spina bifida2 , 3 . however , with surgical intervention there is a great potential risk of complications arising from anesthesia and the varying skills of surgeons4 . serial casting is the process of successfully applying and removing corrective plaster of paris casts to increase extensibility in the soft tissues surrounding the casted joint5 . serial casting for improving range of motion in the knee joint has been used in conditions such as cerebral vascular accident ( cva ) , traumatic brain injury ( tbi ) and pediatric cerebral palsy6,7,8 . these previous studies show that serial casting can increase joint range of motion , muscle extensibility , and improve functional mobility . however , to date , no work has specifically looked at the use of serial casting in the management of children with spina bifida , and there is a lack of documented evidence on the long term benefits of serial casting apart from the evaluation at the time of the final cast removal . hence , the purpose of this case report was to describe for the first time , the use of serial casting in the management of knee joint flexion contracture in a young child with spina bifida , with follow - up to document the long term benefit of serial casting after one year . the child was 6 years old with normal cognitive abilities , and had l3l4 spina bifida level lesion with quadriceps muscle strength grade 3 + . the child was born through a normal vaginal delivery and had normal developmental milestones . at 4 years old a clinical examination revealed there was an associated history of neurological weakness , gait abnormalities and urinary and bowel incontinence . neurological examination revealed reduced sensation to pain and touch in the distribution of l3l4 nerves . the child was referred for physical therapy intervention for the first time at 4 years old with lower limb joint contractures including knee flexion contractures . physical therapy training included : manual stretching exercise and prolonged stretch through strapping on a tilt table or application of sandbag weights over the distal femur . physical therapy training was conducted in one hourly session per week by a trained physical therapist but no improvement was seen . pre - intervention testing was completed one day prior to intervention which consisted of standard goniometer measurement and was used to measure the childs knee joint ranges of motion ( rom ) before and after casting over 4 weeks . the universal goniometer ( ie . the child s parents signed an informed consent statement that had been approved by the al - hussein center ethical committee and al - hashmite university research committee board . the child s legs were immobilized in a long cast from the thigh to the ankle ( fig . the serial casting technique applied in this study was modified to be appropriate for a child with spina bifida . children with spina bifida are usually prone to decubitus ulcers and other types of skin breakdown , especially on bony prominent areas10 . careful measures were taken to avoid bed sores or other complications which may occur with casting . one of these measures included reduction of cast interval changes ; the duration was reduced to two to three days . p.o.p is cheap , strong and radio translucent but the limb can not be inspected . in order to avoid skin breakdown and friction between the cast and the child s skin one held the limb and the other immersed the 6 inches p.o.p roll in luke warm water until all air bubbles within the bandage had disappeared . then the p.o.p roll was removed from the water and squeezed gently to expel excess water . the wet roll was then applied around the limb with gentle firmness , while the assistant maintained the knee in maximum possible extension . while the plaster was wet the physiotherapist smoothed it and molded it to conform to the contours of the limb . this process was repeated on a further 8 occasions at intervals of 23 days . special care was taken to maintain gained rom while checking the skin for breakdown or sores . measurements were taken by orthotist for knee ankle foot orthosis ( kafo ) with a hip attachment . during this period all processes were performed by pediatric physiotherapists and each casting process took around 30 minutes for each limb . arrangements were made with orthotist and the child was fitted with kafo and hip support days after cast removal . following the kafo fitting , the child received intensive physiotherapy including gait training . gait training began in parallel bars for three weeks with the locked knee kafo attached to the hip and was followed by walking outside the parallel bars using a brace and reverse walker for about 2 months . knee flexion contracture was measured in degrees before applying the serial casts and immediately after cast removal . the knee flexion contracture , which was not corrected with passive stretching , improved with casting from 40 knee extension to 5. at one year follow - up , the child continue to ambulate independently for short distances ( home and school ) with an unlocked kafo and forearm crutches . this case report shows that using serial casting and follow - up with bracing may be a useful intervention for a young spina bifida child with knee flexion contracture . one possible explanation for the knee flexion contracture presented in this child , was that in addition to muscle imbalance there was delayed physical therapy intervention as treatment started after 4 years of age . the result in the current case study was consistent with those studies which have examined the effectiveness of serial casting in the management of joint contracture3 , 10 , 11 . in these studies , the improvements translated into reduced spasticity and improved rom , but whether the improvement of rom resulted in function improvement was not clear . in this case report , immediately after cast removal and at one year follow - up , the gain in rom directly translated into improvement in mobility function . however , in this case report gain in joint range was maintained at one year after cast removal . a possible explanation for the rom gain and improvement in mobility function after one year may be the arrangements which were made in advance with orthotist , and the intensive gait training and direct supervision provided by physical therapists . fear of skin breakdown in serial casting to correct joint contracture in children with sensory loss may be behind the limited use of serial casting trials in this patient group . in this case report careful measure such as padding well and changing the cast in a short period of time may have contributed substantially to the improvement in joint rom without skin breakdown or any other complications . to date , this case report is the first to report using serial casting for children with sensory loss without complications . another advantage of using the serial casting approach is that avoided surgical intervention . despite the fact that , surgical intervention is the most effective choice for correction of joint deformities , there is great potential risk of complications , arising from anesthesia and the varying skills of surgeons12 . in addition , surgical intervention requires a long waiting time for an appointment at public hospitals and if done in a private hospital it is very expensive . other recognized complications of surgical intervention of knee flexion contractures in children with spina bifida include over lengthening , infection and scarring1 . the child s parents were extremely pleased when they noticed that knee joint rom had improved without complications and at a later stage when they saw their child could stand and walk . correll and gabler13 reported that musculoskeletal deformities are one of the main factors influencing ambulation in children with spina bifida . on the other hand , children who stand or walk early in life , even if they become non - walkers later , will be more independent in their homes , have a lower incidence of medical complications , fewer fractures and greater independence compared to non - walkers14 . the outcome of this study should be treated with caution because only one case study is presented . further research is needed to determine the effectiveness of using serial casting with larger samples including children with various levels of spina bifida .

[ purpose ] the purpose of this case report is to describe for the first time , the use of serial casting in the management of knee joint flexion contracture for a young child with spina bifida . [ case description ] the child was 6 years old , and had l3l4 spina bifida level lesion with quadriceps muscle strength grade 3 + . the child had previously received weekly physiotherapy including stretching for knee flexion contracture on both lower limbs , but without improvement . [ results ] the knee flexion contracture , which was not corrected with passive stretching , improved with casting from 40 knee extension to 5 knee extension as measured by a standard goniometer over a period of 4 weeks . careful measures were taken to ensure skin integrity . at follow up after one - year , the child could ambulate independently with the help of walking aids . [ conclusion ] the outcome indicates that using serial casting and follow - up with the use of bracing may be useful for enhancing the walking ability of a young child with spina bifida with knee flexion contractures . further investigations of serial casting as well as investigation of serial casting with other interventions are warranted .

INTRODUCTION SUBJECT AND METHODS RESULTS DISCUSSION

however , only a minority of patients with such a history are actually allergic to penicillin(s ) , based on the results of allergological work - up . in real life , most physicians prefer to prescribe alternate antibiotics to these patients , unless a penicillin allergic status can be excluded [ 3 , 4 ] . since the prescription of alternate antibiotics may have undesirable consequences in terms of antibiotic susceptibility , adverse reactions , and health economics , the confirmation of penicillin allergic status would be beneficial in patients with a suspected history of penicillin allergy . in clinical practice , the confirmation of penicillin allergic status is not always feasible , which results in unnecessary avoidance of beta - lactam antibiotics in patients who are overdiagnosed . it is well recognized nowadays that the cross - reactivity rate among beta - lactams is lower than previously expected . however , the consensus recommendation of antibiotic selection in these suspected penicillin allergic patients has yet to be established [ 79 ] . allergists , physicians specialized in managing allergy and immunology disorders , are responsible for confirming penicillin allergic status , preventing overdiagnosis , and determining appropriate alternative antibiotics . in reality , the number of certified allergists in many countries may not be sufficient to manage this common problem . this task is particularly difficult in thailand , as there are only 148 certified allergists ( 28 adult allergists and 120 pediatric allergists ) , among a total of 45,124 medical doctors , or an equivalent of 0.3% registered medical doctors in the country . as a result , the selection of antibiotics in these patients is often decided by doctors of other specialties in real life before they can seek help from allergists . even among thai allergists themselves , penicillin skin test is mainly performed by using penicillin g alone and the use of penicilloyl - polylysine and minor determinant mixture is mostly limited for research purposes . the confirmation of drug allergy status has not been emphasized in medical school curriculum resulting in inadequate knowledge for non - allergists to handle this problem . the potential role of pharmacists in conducting penicillin skin testing in these patients has also been suggested but rarely implemented . it is understandable that doctors in other specialties may have limited knowledge and different perspectives about managing allergic disorders , as compared to allergists . their perspectives and fundamental knowledge on penicillin allergy might have an impact on how they manage these patients in clinical practice . the lack of allergists and standard skin test reagents for the diagnosis of penicillin allergy makes the confirmation of penicillin allergic status not always possible [ 1317 ] . types of medical practice settings and clinical practice duration may also influence the selection of antibiotics in patients with a history of penicillin allergy . the selection of antibiotic prescription in patients with different types of previous penicillin reactions could be influenced by several factors such as the availability of skin test reagents and medicolegal risk . it would be interesting to know how patients with a history of penicillin allergy are mainly managed in the real life by non - allergists where certified allergists may not always be available . the purpose of this study was to comparatively survey the management of penicillin allergy between allergists and non - allergists in thailand in patients with different patterns of penicillin - induced suspected allergic reactions . the awareness of cross - reactivity among beta - lactam antibiotics as well as the knowledge and attitudes towards penicillin allergy management was also determined . the impacts of areas of expertise , types of medical practice settings , and duration of clinical practice on penicillin allergy management were also analyzed . this study was a cross - sectional survey on the management of patients with a suspected history of penicillin allergy in thailand . after approval by the ethics and research committee of faculty of medicine at chulalongkorn university , the web - based questionnaire was created by using google docs and e - mailed to 1,000 thai physicians ( 57 allergists and 943 non - allergists in various fields ) throughout the country . the questionnaire was focused on four aspects : ( 1 ) the management of patients with a history of penicillin - induced immediate reactions in the presence and absence of penicillin skin test reagents , ( 2 ) the management of patients with a history of penicillin - induced non - immediate reactions , ( 3 ) the prescription of other beta - lactams in patients with a history of penicillin allergy , and ( 4 ) the fundamental knowledge of penicillin allergy skin testing and attitudes towards the management of patients with a history of penicillin allergy in thailand . details of the web - based questionnaire are available in supplemental appendix ain supplementary material available online at http://dx.doi.org/10.1155/2014/214183 . the online survey responses were automatically collected and subsequently analyzed using spss 17.0 for windows ( spss , chicago , il , usa ) . the actual rates of penicillin allergy in patients with a suspected history estimated by physicians were calculated by using class interval arithmetic means . chi - square test and multinomial logistic regression were used for univariate and multivariate analysis . a total of 205 completed surveys were received ( a 20.5% response rate ) from the online questionnaire . 14.1% were adult or pediatric allergists , while 26.3% , 40.5% , and 19.0% were general practitioners , internists or pediatricians ( excluding allergists ) , and other specialists , respectively . almost half ( 45.9% ) of the responders worked for an academic institute ( university hospital or research center ) and 19.0% worked in a primary care hospital , 22.9% in a general / provincial hospital , and 12.2% in the private practice sector . about 45.9% of the responders have less than 5-year experience in clinical practice , while 30.2% and 23.9% of them have 510 years and more than 10 years of practice experience , respectively ( table 1 ) . when penicillin administration was indicated , the different management between allergists and non - allergists in patients with a history of penicillin - induced immediate reaction was not statistically different if skin testing was positive ( p value = 0.06 ) but was clearly demonstrated if the penicillin skin test was negative ( p value < 0.01 ) ( figure 1(a ) ) . 79.3% of allergists and 88.1% of non - allergists would avoid penicillin or both penicillin and cephalosporins if penicillin skin testing yielded a positive result . in contrast , 75.9% of allergists would use graded challenge first if penicillin skin test was negative , while 53.4% of non - allergists would prescribe penicillin normally . a question was addressed whether the severity of the immediate reaction influenced patient management in the presence or absence of penicillin skin test reagents ( figure 1(b ) ) . the difference of decision making between allergists and non - allergists was observed in patients for whom penicillin was indicated regardless of clinical severity and who had been skin - tested with penicillin reagents ( p values < 0.01 ) . if a penicillin skin test was not available , the management of patients with a history of penicillin - induced severe immediate reaction ( anaphylaxis ) between allergists and non - allergists was still different ( p value < 0.01 ) , but no longer different ( p value = 0.12 ) in patients with a history of penicillin - induced mild immediate reaction ( urticaria and/or angioedema ) . in patients with a history of anaphylaxis , 83.584.7% of non - allergists would avoid both penicillin and cephalosporins regardless of skin testing availability . in contrast , 34.5% of allergists would perform a penicillin skin test if available , and 20.7% considered prescribing penicillin using a desensitization technique in patients requiring penicillin therapy . in patients with a history of penicillin - induced urticaria or angioedema , 82.8% of allergists would perform penicillin skin testing first , while only 25.0% of non - allergists would do so . however , if the penicillin skin test was not available , the majority of physicians ( 65.5% of allergists and 77.3% of non - allergists ) would avoid both penicillin and cephalosporins . regarding the management of patients with a history of penicillin - induced non - immediate reaction , the prescription pattern between allergists and non - allergists was significantly different in stevens - johnson syndrome ( sjs , p value = 0.03 ) , borderline different in drug reaction with eosinophilia and systemic symptoms ( dress , p value = 0.05 ) , but not statistically different in maculopapular exanthema ( mpe , p value = 0.20 ) ( figure 2 ) . only 3.4% of allergists would readminister penicillin with graded challenge or desensitization methods in patients with a history of dress , while 21.1% of non - allergists considered doing so . some of non - allergists ( 5.7% ) still considered prescribing penicillin with graded challenge or desensitization methods in patients with a history of sjs , while none of allergists would do it . in contrast , the majority of both allergists ( 65.5% ) and non - allergists ( 63.6% ) would avoid only penicillin in patients with a history of penicillin - induced mpe . when penicillin skin test reagents are not available , the decision making between allergists and non - allergists in terms of beta - lactam prescription in patients with an unconfirmed history of an immediate reaction to penicillin was different in the case of first generation and third / fourth generation cephalosporins ( p values < 0.01 ) but not with other drugs ( figure 3 ) . aminopenicillins were completely avoided by more than 60% of all thai physicians , while about half of them considered carbapenem and monobactam prescribable . first generation cephalosporins would be completely avoided by 48.3% of allergists or prescribed with precaution by 41.4% . in contrast , only 18.8% of non - allergists would completely avoid these drugs , while 58.0% of non - allergists indicated that they would prescribe them with precaution . for third and fourth generation cephalosporins , 62.1% of allergists said that they could be prescribed , while only 29.5% of non - allergists agreed so . regarding basic knowledge of penicillin skin testing for diagnosis of an immediate reaction , less than half of non - allergists ( 29.1% ) have accurate knowledge on the appropriate recommended skin test reagents ( penicilloyl - polylysine and minor determinants ) and only 5.1% of them know how to correctly interpret penicillin skin test results according to the enda recommendation ( an increase in wheal diameter greater than 3 mm read 1520 minutes after the test compared to the initial wheal size ) . it is worth noting that only 48.3% of trained allergists in thailand could properly interpret the result of penicillin skin tests as well ( figure 4 ) . the actual rate of penicillin allergy in patients with a suspected history estimated by allergists was significantly lower than that estimated by non - allergists ( 18.9% to 35.6% , resp . , p value = 0.02 ) . a difference in opinion between allergists and non - allergists was observed . the preferred approach to manage patients with a history of an immediate reaction to penicillin was significantly different between allergists and non - allergists ( p value = 0.04 ) . while the majority of allergists ( 69.0% ) favored penicillin skin tests over penicillin avoidance ( 24.1% ) , both approaches were equally elected by the non - allergist group ( 39.240.3% each ) . the reasons for prescribing an alternate drug without confirming penicillin allergy status were not statistically different between allergists and non - allergists ( p value = 0.50 ) . the easy availability of alternate antibiotics was the main reason , followed by a convincing drug allergy history . interestingly , the medicolegal problem was another main concern for thai physicians , especially among non - allergists . factors possibly influencing penicillin allergy management were analysed based on physician 's area of expertise , medical practice setting , and clinical practice duration . the results indicated that the most important factor determining the management of penicillin allergy was the area of expertise ( see appendix b ) . after multivariate analysis , penicillin allergy management by allergists was significantly different from non - allergist counterparts ( figure 5 ) . non - allergists preferred to confirm an allergic history by means of skin tests in patients with a history of penicillin - induced urticaria and anaphylaxis , much less than allergists would do ( 0.07- and 0.10-fold , resp . ) , and less likely to administer penicillin with graded challenge technique ( 0.10-fold ) in patients with a suspected history of penicillin allergy after negative skin test , compared to allergists . in contrast , non - allergists were more likely to avoid not only penicillin but also cephalosporins in patients with a history of penicillin - induced anaphylaxis and sjs than allergists would do so ( 6.62- and 5.15-fold , resp . ) . interestingly , they avoided first generation cephalosporins much less than allergists ( 0.17-fold ) in patients with a history of penicillin allergy . probably due to limited knowledge in penicillin skin test procedure and interpretation , non - allergists rather preferred to avoid penicillin ( 7.29 folds ) than to confirm allergic status ( 0.14 fold ) , as compared to their allergist counterparts . clinical practice duration also had some influence since physicians with less than 5 years of experience in practice were more in favor of penicillin allergy confirmation ( 4.65-fold ) than those practicing medicine longer than 10 years and were less likely to avoid cephalosporins in patients with a history of penicillin - induced dress ( 0.11-fold ) . although allergists are primarily responsible for the management of patients with a history of a drug allergy , in the real life , these patients are often cared for by doctors in other specialties due to the shortage of certified allergists in some areas . this study surveyed how the problem of penicillin allergy in different circumstances was managed by allergists and non - allergists , including their knowledge and attitudes towards this problem . our study shows that allergists are more willing to confirm the status of penicillin allergy and more inclined to use a desensitization procedure in patients with a history of penicillin - induced immediate reaction , as compared to non - allergists . the study also shows that the limited availability of penicillin skin tests clearly impacts their clinical judgment . if the penicillin skin testing was not available , the discrepancy of patient management between allergists and non - allergists in patients with a history of penicillin - induced urticaria or angioedema would no longer be observed , since allergists also avoided penicillin cephalosporins in these cases . interestingly , the majority of allergists were very cautious when prescribing penicillin in patients with a suspected history even after a negative penicillin skin test , while half of non - allergists would prescribe penicillin normally in a similar circumstance . different views on beta - lactam cross - reactivity between allergists and non - allergists were noted . interestingly , allergists were more reluctant to prescribe first generation cephalosporins in penicillin allergic patients while being less concerned about third / fourth generation cephalosporins , as compared to their non - allergist counterparts . current data indicates that the potential cross - reactivity with penicillin is noteworthy only in first generation cephalosporins . in terms of non - immediate reactions , more non - allergists considered avoiding cephalosporins in patients with a history of penicillin - induced sjs , as compared to allergists . no statistical difference was observed between allergists and non - allergists in the management of penicillin - induced mpe and dress . however , allergists seemed less likely to perform graded challenge or desensitize patients with previous dress . regarding knowledge and attitudes towards penicillin allergy management , it was clear that non - allergists have limited knowledge regarding penicillin skin test reagents and interpretation as compared to certified allergists . even though both allergists and non - allergists agreed that majority of patients with a history of penicillin allergy are not truly allergic , the allergists ' estimated rate of penicillin allergy in these patients was significantly lower than that of their non - allergist counterparts . surprisingly , less than half of thai allergists could correctly interpret penicillin skin test results as well . a refresher course for allergists on drug allergy testing should be organized . the appropriate modalities of allergological work - up in patients with suspected beta - lactam hypersensitivity should be finalized . the lack of penicillin metabolites ( penicilloyl - polylysine and minor determinant mixture ) could potentially be replaced by the soluble forms of the suspected beta - lactams or other beta - lactams from the same classes , along with benzylpenicillin and aminopenicillin , as skin test reagents since they are more easily available and have good predictive values in clinical practice . while penicillin skin testing was more favoured among allergists , many non - allergists still preferred penicillin avoidance . the availability of alternate drugs and the convincing drug allergy history were the main factors for prescription of alternate antibiotics instead of the confirmation of penicillin allergy status . it is worth mentioning that the medicolegal problem from harmful reactions , which are possible after a drug rechallenge , was one of the major concerns for not confirming the status of penicillin allergy particularly among non - allergists . the launch of a national standard practice guideline to manage patients with a history of penicillin allergy would be helpful to prevent medicolegal problems . health economics and outcome research regarding the confirmation of penicillin allergy in patients with a suspected history should be carefully investigated . the study demonstrates the diversity of management in patients with a history of penicillin allergy among thai physicians , although most of them knew that only a minority of these patients are truly allergic . although the duration of medical practice may also play a role , the results of our study emphasize that the area of expertise was the most important factor determining penicillin allergy management . as the different opinions between allergists and non - allergists were statistically significant in patients with a history of severe non - immediate reaction and in patients with a history of mild immediate reaction if penicillin skin test reagents are available , we recommend that these patients are managed by allergists . nevertheless , penicillin skin test reagents should be provided to practice allergists and the accurate skin test procedure emphasized . in contrast , it might be possible that non - allergists should be allowed to handle patients with a history of penicillin - induced mild non - immediate reaction ( simple mpe ) if no certified allergist is available . according to our study , the current approach between allergists and non - allergists in this patient group is already similar . in fact , graded challenge test is the recommended procedure for any physicians knowledgeable in treating adverse drug reactions to manage these patients , since the risk to develop severe reaction is small . in this regard , updated information about beta - lactam cross - reactivity and drug readministration by graded challenge technique should be provided to non - allergists to minimize patient risk . the ratio of non - allergists to allergists was 6 : 1 due to the limited number of allergists in the country . in fact , the number of allergists who replied to this survey was already one - fifth of total certified allergists in thailand . the response rate was rather low but still within a similar range to prior studies [ 3 , 9 ] . data from this study may not represent the views of thai physicians as a whole since almost half of the responders worked in academia . although medical practice setting alone did not have a significant impact on penicillin allergy management after multivariate adjustment , stratified survey in all types of medical practice could possibly be conducted to reduce response bias . the different management of penicillin allergy between allergists and non - allergists was mainly observed in patients with a history of severe non - immediate reaction and in patients with a history of an immediate reaction , particularly in the patients who have been skin - tested with penicillin reagents . the possible role of non - allergists in the management of patients with a history of penicillin - induced mild non - immediate reaction has been raised .

the purpose of this study was to compare the management of patients with a history of penicillin allergy between allergists and non - allergists in thailand . a questionnaire was distributed to thai physicians by online survey . the answers from 205 physicians were analyzed . the discrepancy of penicillin allergy management between allergists and non - allergists was clearly demonstrated in patients with a history of an immediate reaction in the presence of penicillin skin test ( p < 0.01 ) and in patients with a history of stevens - johnson syndrome ( p < 0.05 ) from penicillin . allergists are more willing to confirm penicillin allergic status , more likely to carefully administer penicillin even after negative skin test , but less concerned for the potential cross - reactivity with 3rd and 4th generation cephalosporins , compared to non - allergists . the lack of penicillin skin test reagents , the reliability of penicillin allergy history , and medicolegal problem were the main reasons for prescribing alternate antibiotics without confirmation of penicillin allergic status . in summary , the different management of penicillin allergy between allergists and non - allergists was significantly demonstrated in patients with a history of severe non - immediate reaction and in patients with a history of an immediate reaction when a penicillin skin test is available .

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

traumatic brain injury ( tbi ) is characterized by intracranial damage resulting from an external force and can be the product of physical insults such as puncture , blunt impact , or blast ( maas et al . , 2008 ) . tbi affects 1.7 million people annually , presenting a significant economic burden , and is particularly prevalent in military casualties , where an increase in explosive warfare has led to a parallel rise in tbi occurrence ( okie , 2005 ; acosta et al . tbi can be classified as mild , moderate , or severe ( lozano et al . , 2015 ) . determining the severity of tbi is commonly accomplished in humans with the glasgow coma scale ( gcs ) , a simple questionnaire used to evaluate the patient 's degree of consciousness in conjunction with medical imaging techniques ( lozano et al . , 2015 ) . associated symptoms such as headache , dizziness , fatigue , or nausea may be short - lived in the case of mild tbi , while in more severe instances cognitive symptoms may progress chronically to resemble neurodegenerative diseases such as alzheimer 's disease ( ad ) and parkinson 's disease ( pd ) ( lozano et al . , epidemiological data also suggest that sufferers of tbi are at an increased risk for developing ad and pd later in life through poorly understood mechanisms ( lozano et al . , 2015 ) . the acute phase consists of the immediate damage produced from the insult , and the chronic phase may extend for years and is defined by spreading inflammation , cell death , and neural dysfunction that is triggered by the primary injury ( werner and engelhard , 2007 ; acosta et al . the expanded distribution of pro - inflammatory molecules , reactive species , and other damaging byproducts from the primary injury site causes a progressive wave of cell damage , forming a region of dead and endangered cells called the penumbra ( zhao et al . this region poses the greatest threat of chronic symptom development , but also points to a significant opportunity for cell rescue ( zhao et al . , 2005 ; once considered to be an acute injury only , our understanding of tbi as possessing this chronic aspect has revealed the opportunity of a greater time frame for therapeutic intervention ( acosta et al . the chronic phase of tbi is marked by a multitude of complex metabolic , immune , and cellular responses which are seen primarily in tissue adjacent to the injury site , but can also spread to distal regions of the brain ( kumar and loane , 2012 ; lozano et al . , while these responses are seen universally in tbi , researchers have shown that the specific pathology and intricacies of the injury response are heterogeneous , and vary depending on the nature and location of the tbi ( pabon et al . , 2016 ) . the immediate damage that occurs during a tbi includes necrosis , excitotoxicity , and mutilation of local neurons , microglial activation , and vascular cells - none of which present a significant opportunity for intervention ( kumar and loane , 2012 ) . instead , recent research has focused on the multiple aspects of chronic tbi as a means of reducing the secondary cell death that ensues after the initial acute phase ( lozano et al . , in particular , the neuroinflammatory aspect of tbi physiopathology has been explored as a target for preventing secondary cell death and symptom progression ( lozano et al . , 2015 ) . inflammation plays a dual role in the brain after a tbi ; in the acute phase , inflammation has been shown to be neuroprotective , while aberrant inflammation throughout the chronic phase is a key factor in perpetuating cell death ( lozano et al . , 2015 ) . sequestration of the chronic inflammation has been proven to be neuroprotective ( lozano et al . , 2015 ) . the transition from protective inflammation to degenerative inflammation - as well as the large number of competing pro - inflammatory molecules ( i.e. , tumor necrosis factor ( tnf- ) and interleukin ( il-1 ) and anti - inflammatory molecules ( i.e. , il-10 and tgf- ) - make for a complex pathology ( lozano et al . , 2015 ) . the neurodegenerative inflammation and edema seen after tbi is linked to the dysfunction of the blood - brain barrier ( bbb ) that occurs after the insult ( shlosberg et al . , 2010 ; the protective effects of the bbb are contingent on the presence of uninterrupted , selective endothelial cells which moderate the entrance of blood - borne particles ( neuwelt et al . , 2011 ) . additionally , the health of these endothelial cells and the fidelity of the bbb is intimately connected to the health of supportive astrocytes . the mechanical force from the tbi compromises this fidelity , allowing a host of exogenous proteins such as albumin , fibrinogen , and thrombin , as well as peripheral immune cells , to enter the brain parenchyma ( shlosberg et al . , 2010 ; neuwelt et al . , 2011 ; lozano et al . , 2015 ) . these invading substances trigger the activation of microglia within the injured brain region , causing an immune response that persists for as long as the bbb remains compromised ( shlosberg et al . , 2010 ; initially , the microglial activation is a protective response , but the activation can become excessive and self - perpetuating over time ( lozano et al . , 2015 ) . additionally , the invading peripheral cells have been shown to release pro - inflammatory mediators such as cytokines , chemokines , prostaglandins , and free radicals , serving to progressively exacerbate the inflammatory process as well as increasing the permeability of the bbb ( lozano et al . , 2015 ) . this elevated permeability to large molecules and cells shifts the osmotic pressure within the brain , causing edema and an increase in intracranial pressure ( neuwelt et al . damage to the bbb is a continuing issue , as increased levels of molecules which damage tight junctions such as matrix metallopeptidase-9 ( mmp-9 ) and vascular endothelial growth factor ( vegf ) occur post - tbi and impede the recovery of the bbb ( guo et al . , 1989 ; the immune response is further complicated by differentiation of environment - sensitive microglia into multiple phenotypes ( lozano et al . , 2015 ) . depending on the microenvironment that the microglia is exposed to , the cell may assume an m1 , pro - inflammatory , phenotype or an m2 , anti - inflammatory phenotype that attenuates the pro - inflammatory m1 microglia ( lozano et al . , 2015 ) . possible therapeutic techniques aim to minimize harmful chronic inflammation , while not preventing acute - phase neuroprotective inflammation , by developing means of downregulating pro - inflammatory molecules , upregulating anti - inflammatory molecules , and aiding in bbb repair ( lozano et al . , 2015 ) . the only defense against the primary injury caused by a tbi is prevention with the use of safety equipment such as helmets and seatbelts ( hirschenfang et al . the window of opportunity for potential therapies lies within the period of secondary cell death which may last for days , months , or years ( iida et al . , of the different secondary death mechanisms , neuro - inflammation provides the greatest potential for intervention due to its delayed onset . glutamate excitotoxicity for example has a much narrower range for intervention as it is an immediate response and may normalize within 120 hours after insult ( matsumoto et al . , 1986 ) . based on the length of time required for enough inflammatory cells to migrate and induce secondary cell death , the onset of damaging neuroinflammmation is a slower process ( offit et al . , 1986 ) . neuroinflammation may also persist as a chronic consequence of tbi and this secondary effect is closely linked to many neurodegenerative diseases such as dementia pugilistica ( dp ) , ad , pd and other pathologies ( colmano and gross , 1971 ) . while targeting neuroinflammation is a reasonable approach to sequestering secondary cell death in tbi , it should be noted that neuroinflammation also has beneficial effects . previous studies have shown that high doses of anti - inflammatory drugs administered after tbi led to worse outcomes due to the loss of neuroprotective effects in addition to the existing harmful effects ( ziebell and morganti - kossmann , 2010 ) . several stand - alone anti - inflammatory drugs have been tested for efficacy in tbi treatment . minocycline is a tetracycline derivative known to have both neuroprotective and anti - inflammatory properties ( kumar and loane , 2012 ) . minocycline is a prime candidate for clinical trials due to its ability to cross the bbb in addition to its safety for human use as declared by the us food and drug administration ( saatman et al . , 2008 ) . this drug minimizes the release of proinflammatory cytokines and chemokines , in conjunction with other mediators of inflammation , decreases nitric oxide by directly inhibiting the overactivation and proliferation of microglia cells ( homsi et al . , 2009 ; kovesdi et al . , 2012 ) . reducing and inactivating microglial cells is key as it attributes to the decline in cytokines il-1 , il-6 , and matrix metallopeptidase 9 ( mmp-9 ) , all of which facilitate the proinflammatory response ( homsi et al . , 2009 ; ziebell and morganti - kossmann , 2010 ; the use of minocycline in animal models has illustrated a significant reduction in inflammation and tissue damage , thereby improving outcomes ( homsi et al . however , results of other studies contradict this , suggesting there is no beneficial effects of minocycline on tbi . these opposing outcomes are a predicted result of deviations in dosage and administration intervals , calling for further investigation ( homsi et al . , 2009 ; kelso et al . , 2011 ) melatonin is a hormone produced in the pineal gland and is currently being explored as a stand - alone drug due to its neuroprotective characteristics ( lozano et al . , 2015 ) . it is an enzyme that easily passes through cell membranes due to it lipophilic properties . the mechanism of action for its anti - inflammatory effects are by way of inhibiting microglial activation and reducing pro - inflammatory cytokine secretion , such as il-1 and tnf- ( wang et al . , 2013 ; ding et al . , 2014a ) the effects of melatonin as a tbi therapeutic drug vary , similar to the results of minocycline administration . lower brain edema and reduced cortical neural degeneration was displayed in successful trials , implying improvement of cognitive deficits ( ding et al . , 2014b ) . however , there was no significant cognitive enhancements in experimental models , the likely cause being attributed to dosage ( kelso et al . , 2011 ) . while melatonin is a prospective therapy to sequester secondary damage caused by inflammation , additional studies are necessary to address the long - term feasibility , safety , and efficacy of melatonin in multiple models of tbi ( hirschenfang et al . , 1968 ; statins are a well - known group of drugs used to treat high cholesterol that have additionally shown neuroprotective and anti - inflammatory effects in a mouse model of subarachnoid hemorrhage ( uekawa et al . , 2014 ) . statins inhibits the signaling pathways of toll - like receptor 4 , nuclear factor b ( nf-b ) activation , and some small g - proteins , all of which contribute to the reduction of microglia activation ( loane and faden , 2010 ; wang et al . , 2014 ) . by additionally inhibiting epidermal growth factor receptors that play a part in astrogliosis , statins also decrease astrocyte activation ( wu et al . , 2010 ) . without microglia and astrocytes contributing their proinflammatory effects , there is a reduction in the expression of proinflammatory cytokines il-1 and tnf- , as well as intracellular and intercellular adhesion molecules , and consequently an overall sequestration of the neuroinflammatory process ( loane and faden , 2010 ; wu et al . , 2010 ; uekawa et al . , 2014 ) . in tbi models accompanied by statin administration , results have displayed an increase in neuronal survival , growth , and differentiation and a lessening of functional impairment ( loane and faden , 2010 ) . minor improvements in amnesia and disorientation ( as assessed by the galveston orientation amnesia test ) were achieved after rosuvastatin was given over a 10 day period to tbi patients within a clinical trial ( tapia - perez et al . , 2008 ) . statins are an already well established class of drugs that are tolerated by patients , with occasional mild side effects that are distinct and can be monitored without difficulty . statins show promise in preclinical for other neurological deficits such as stroke , intracerebral hemorrhage , but more extensive laboratory studies in tbi models are needed to improve clinical outcomes ( hirschenfang et al . , 1968 ; lozano et al . , 2015 ) . currently , there remain safety and efficacy optimization hurdles about the use of anti - inflammatory pharmacotherapy for the treatment of tbi . the use of these drugs has demonstrated a notable reduction in the inflammatory response ; however , it is crucial to further investigate the applicability of these treatment options in a clinical setting . as previously mentioned , concerns regarding dosage and long - term outcomes warrant further investigation of these drugs prior to their clinical use . stem cells are undifferentiated cells that have the potential to regenerate damaged tissue secondary to their ability to proliferate numerously , differentiate into multiple cell lines , and provide restorative resources to surviving cells ( antonucci et al . , 2014 ; preclinical studies have demonstrated the remarkable regenerative ability of stem cells to transform into newly differentiated neurons following tbi ( antonucci et al . , 2012 ; rodrigues et al . , 2012 ; liu et al . , 2013 ; acosta et al . , 2014 ; tajiri et al . following brain injury , stem cell therapy shows notable potential in sequestering neural cell death and a prolonged inflammatory response , which results in increased recovery in both cognitive and motor function ( acosta et al . , 2014 ; de la pena et al . , 2014 ) . the hallmark of stem cells dampening the tbi - induced inflammatory response lies within their mechanism of action . mesenchymal stem cells ( mscs ) possess the ability to migrate to the site of injury and activate cellular effectors of the inflammatory response such as microglia , t lymphocytes , and neutrophils ( borlongan , 2011 ; borlongan et al . key inflammatory signals , such as tnf- and il-1 , induce mscs secrete an anti - inflammatory protein called tnf--stimulated gene / protein 6 ( tsg-6 ) ( watanabe et al . , 2013 ; zhang et al . , 2013 ) . the anti - inflammatory role of tsg-6 is carried out by disrupting the inflammatory signaling pathways of both toll - like receptors ( tlrs ) and nf-b ( watanabe et al . , 2013 ; zhang et al . , 2013 ) the inflammatory nf-b signaling cascade is characterized by the production of proinflammatory cytokines by t cells , such as interferon ( russo et al . , 2011 ) . interestingly , tsg-6 is able to modulate the activity of t cells to instead produce anti - inflammatory cytokines , such as il-4 ( russo et al . , 2011 ) . once transplanted , stem cells are faced with the struggle of surviving in the hostile environment at the site of injury ( dela pena et al . , 2014 ) . certain factors , such as granulocyte - colony stimulating factor ( g - csf ) , can be introduced alongside stem cells to promote the neuroprotection of the transplanted stem cells ( acosta et al . , 2014 ) . combining stem cells with factors such as g - csf provide a significant improvement in neurogenesis and a reduction in cell death , when compared to being administered alone ( acosta et al . , 2014 ) . g - csf , a cytokine , has the inherent capability to reduce brain edema , enable recovery of motor function , and to improve control of glutamate levels ( acosta et al . , 2014 ) . the mechanism of g - csf utilizes receptor - mediated transport to recruit endogenous stem cells from the bone marrow into the peripheral blood . these mobilized stem cells can then migrate to the site of injury , where they can synthesize and release growth factors , chemokines , and cytokines that aid in the process of brain tissue repair ( acosta et al . , 2014 ) . recent studies demonstrate that transplanted stem cells preferentially migrate to the spleen , rather than the brain ( acosta et al . , 2015a ) . the concept of increased stem cell survival in the spleen over the brain suggests that direct , targeted therapy may not always be the best treatment option ( acosta et al . , 2015a ) . indeed , studies are investigating the efficacy of systemic delivery of stem cells in tbi and other disease models , such as stroke ( acosta et al . several types of transplantable cells have been tested in the laboratory , with a few reaching clinical trials , for cell therapy in stroke , including fetal cells , nt2n cells , ctx0e3 , embryonic stem cells , neural stem / progenitor cells , umbilical cord blood , amnion , adipose , and induced pluripotent stem cells ( hara et al . , 2008 ; li et al . , 2008 ; , 2011 ; liu et al . , 2014 ; de la pena et al . , 2015 ) . primarily due to solid safety profile in other disease indications , preclinical studies and on - going clinical trials have given special attention to bone marrow and its cellular derivatives ( borlongan et al . . direct intracerebral implantation and peripheral transplantation , such as intravenous , intra - arterial , and intranasal , have documented the functional benefits of bone marrow - derived stem cells ( borlongan et al . , 2004 ; borlongan , 2011 ; borlongan et al . , 2011 ; prasad et al . , 2014 ; acosta et al . , 2015a ) . clinical trials have been initiated , and preliminary reports have demonstrated safety , although efficacy warrants additional investigations ( steinberg et al . , 2016 ) . this concept highlights the need for investigations of combination therapy ( figure 1 and table 1 ) , in order to improve the outcomes of drugs and stem transplantation in tbi and other related brain disorders . indeed , combining drugs , such as granulocyte - colony stimulating factor ( g - csf ) , erythropoietin ( epo ) , and statins may improve the functional benefits of stem cells towards brain repair in tbi and other related disorders . in this review , the outlined studies detailed treatment options for tbi that focus on either stand - alone pharmacotherapy or cell therapy . a single therapeutic approach may not be optimal in reducing inflammation and in initiating the brain repair process after tbi . it is more likely that combination therapy of anti - inflammatory drugs alongside stem cell transplantation will yield enhanced results that can be translated to the clinical setting . in addition , most preclinical studies are limited by their use of small animal models for tbi , which may not accurately mimic the clinical scenario in humans . thus , limited clinical trials of stand - alone therapies , alongside more advanced laboratory studies incorporating optimization of safety and efficacy on dosages and long - term functional assessments of combined pharmacotherapy and stem cell transplantation in large animal models , are required to allow these treatment options to become available at the bedside .

the pathologic process of chronic phase traumatic brain injury is associated with spreading inflammation , cell death , and neural dysfunction . it is thought that sequestration of inflammatory mediators can facilitate recovery and promote an environment that fosters cellular regeneration . studies have targeted post - traumatic brain injury inflammation with the use of pharmacotherapy and cell therapy . these therapeutic options are aimed at reducing the edematous and neurodegenerative inflammation that have been associated with compromising the integrity of the blood - brain barrier . although studies have yielded positive results from anti - inflammatory pharmacotherapy and cell therapy individually , emerging research has begun to target inflammation using combination therapy . the joint use of anti - inflammatory drugs alongside stem cell transplantation may provide better clinical outcomes for traumatic brain injury patients . despite the promising results in this field of research , it is important to note that most of the studies mentioned in this review have completed their studies using animal models . translation of this research into a clinical setting will require additional laboratory experiments and larger preclinical trials .

Traumatic Brain Injury and Inflammation Anti-inflammation Pharmacotherapy Stem Cell Therapy for Sequestration of Inflammation Future Directions

the study protocol was approved by the local ethics committee , and written informed consent was obtained from the parents or guardians of each patient . we studied 44 probands with pndm diagnosed before 6 months of age , who had been referred to the molecular genetics laboratory at the peninsula medical school in exeter , u.k .. mutations in kcnj11 , abcc8 , ins , and gck had been excluded . the relevant clinical information was obtained from the medical records . the single coding exon of neurod1 was pcr amplified in three overlapping fragments using specific primers for each amplicon tagged with 5 m13 tails to allow sequencing to be performed with a universal m13 primer ( primers and conditions available upon request ) . single - strand sequencing was carried out using standard methods on an abi 3730 sequencer ( applied biosystems , warrington , u.k . ) . high - density single nucleotide polymorphism ( snp ) genotyping was carried out on the affymetrix human 10k xba chip by medical solutions nottingham ( formerly geneservice ) ( nottingham , u.k . ) . house perl scripts were developed to automatically identify genomic homozygous segments , defined by at least 20 consecutive homozygous snps marking a region that exceeded 3 cm ( 13 ) . we studied 44 probands with pndm diagnosed before 6 months of age , who had been referred to the molecular genetics laboratory at the peninsula medical school in exeter , u.k .. mutations in kcnj11 , abcc8 , ins , and gck had been excluded . the single coding exon of neurod1 was pcr amplified in three overlapping fragments using specific primers for each amplicon tagged with 5 m13 tails to allow sequencing to be performed with a universal m13 primer ( primers and conditions available upon request ) . single - strand sequencing was carried out using standard methods on an abi 3730 sequencer ( applied biosystems , warrington , u.k . ) . high - density single nucleotide polymorphism ( snp ) genotyping was carried out on the affymetrix human 10k xba chip by medical solutions nottingham ( formerly geneservice ) ( nottingham , u.k . ) . processing of genomic dna was performed in accordance with the affymetrix protocol . in - house perl scripts were developed to automatically identify genomic homozygous segments , defined by at least 20 consecutive homozygous snps marking a region that exceeded 3 cm ( 13 ) . two novel homozygous mutations in neurod1 , a single base pair duplication ( c.364dupg ) and a two - base pair ct deletion ( c.427_428del ) , were identified in two unrelated probands . both mutations result in a frameshift and a premature truncation of the c terminus of the expressed protein ( p.asp122glyfs*12 and p.leu143alafs*55 , respectively ) , leading to mutated proteins completely lacking the transactivation domain ( fig . these mutations had not been previously documented and were not present in 200 alleles from healthy unrelated individuals . the abnormal protein sequence between the frameshift and the termination codon is colored in gray . a with the c.364dupg mutation , parents were known to be first cousins and , consistent with parental consanguinity , snp genotyping analysis of the proband revealed a total genomic homozygosity value of 6.0% ( 13 ) . the mutation - containing homozygous segment was the largest homozygous segment ( 46.6-mb long ) and spanned 2q31.12q36.1 delimited by the snps rs726032 to rs724149 . in contrast , in family b , the parents of the patient with the homozygous c.427_428del mutation were not known to be related and , in keeping with this , total genomic homozygosity value was very low ( 0.3% ) . however , the mutation in both parents was inherited on an extended haplotype of 10.4 mb between positions chr2q31.132.1 ( snps rs2884471rs722385 ) , suggesting that the mutation arose from a single common ancestor . extended pedigrees of the two families showing inheritance of neurod1 mutations ( family a , c.364dupg [ a ] ; family b , c427_428del [ b ] ) . genotype is shown underneath each symbol ; m and n denote mutant and wild - type alleles , respectively . directly below the genotype is the age of the individual at testing or the age at diagnosis of diabetes if diabetic , followed by the most recent treatment for diabetes . black - filled symbols denote patients with neonatal diabetes , and gray - filled symbols represent patients with later - onset diabetes . the two probands were diagnosed with permanent diabetes within the first 2 months of life and had presented with intrauterine growth retardation ( birth weights 1,490 and 2,230 g at 34 and 38 weeks of gestation , respectively ) , reflecting reduced insulin secretion in utero . they had no evidence of pancreatic exocrine dysfunction and normal pancreatic size on abdominal scanning ( see supplementary information , available in an online appendix [ http://diabetes.diabetesjournals.org/cgi/content/full/db10-0011/dc1 ] ) . in addition to diabetes , they presented with a similar pattern of neurological abnormalities including moderate - to - severe developmental delay , profound sensorineural deafness , and visual impairment due to myopia and diffuse retinal dystrophy . brain magnetic resonance imaging scans showed severe cerebellar hypoplasia with no other major intracranial abnormalities ( fig . 3 and supplementary information ) . magnetic resonance imaging of the brain in a proband from family a demonstrating the typical neuroimaging findings of neurod1-pndm . a : sagittal t1-weighted image . b : coronal t2-weighted image . there is significant cerebellar hypoplasia , particularly of cerebellar vermis inferiorly . . clinical features of the two patients with homozygous neurod1 mutations ct , computed tomography ; erg , electroretinography ; mri , magnetic resonance imaging ; sds , sd score the diabetes status , age of diagnosis , treatment , and genetic testing result of family members are shown in fig . 2 . we assessed glucose tolerance in the four parents of the two probands who were proven heterozygous carriers of the mutations . in family a ( c.364dupg mutation ) , the mother had been diagnosed with type 2 diabetes at 33 years , despite having a normal bmi , and was treated with glicazide . in contrast , the father ( also aged 33 years ) had normal fasting ( 46 mmol / l ) and postprandial ( 57 mmol / l ) blood glucose levels on several occasions . in family b ( c.427_428del mutation ) , the mother and father underwent standard oral glucose tolerance tests ( aged 33 and 37 years , respectively ) that confirmed normal glucose tolerance ( 6.2 and 4.8 mmol / l , respectively , at 2 h ) . no heterozygous family members in either family had any developmental delay or neurological features on clinical examination . two novel homozygous mutations in neurod1 , a single base pair duplication ( c.364dupg ) and a two - base pair ct deletion ( c.427_428del ) , were identified in two unrelated probands . both mutations result in a frameshift and a premature truncation of the c terminus of the expressed protein ( p.asp122glyfs*12 and p.leu143alafs*55 , respectively ) , leading to mutated proteins completely lacking the transactivation domain ( fig . these mutations had not been previously documented and were not present in 200 alleles from healthy unrelated individuals . the abnormal protein sequence between the frameshift and the termination codon is colored in gray . a with the c.364dupg mutation , parents were known to be first cousins and , consistent with parental consanguinity , snp genotyping analysis of the proband revealed a total genomic homozygosity value of 6.0% ( 13 ) . the mutation - containing homozygous segment was the largest homozygous segment ( 46.6-mb long ) and spanned 2q31.12q36.1 delimited by the snps rs726032 to rs724149 . in contrast , in family b , the parents of the patient with the homozygous c.427_428del mutation were not known to be related and , in keeping with this , total genomic homozygosity value was very low ( 0.3% ) . however , the mutation in both parents was inherited on an extended haplotype of 10.4 mb between positions chr2q31.132.1 ( snps rs2884471rs722385 ) , suggesting that the mutation arose from a single common ancestor . extended pedigrees of the two families showing inheritance of neurod1 mutations ( family a , c.364dupg [ a ] ; family b , c427_428del [ b ] ) . genotype is shown underneath each symbol ; m and n denote mutant and wild - type alleles , respectively . directly below the genotype is the age of the individual at testing or the age at diagnosis of diabetes if diabetic , followed by the most recent treatment for diabetes . black - filled symbols denote patients with neonatal diabetes , and gray - filled symbols represent patients with later - onset diabetes . the two probands were diagnosed with permanent diabetes within the first 2 months of life and had presented with intrauterine growth retardation ( birth weights 1,490 and 2,230 g at 34 and 38 weeks of gestation , respectively ) , reflecting reduced insulin secretion in utero . they had no evidence of pancreatic exocrine dysfunction and normal pancreatic size on abdominal scanning ( see supplementary information , available in an online appendix [ http://diabetes.diabetesjournals.org/cgi/content/full/db10-0011/dc1 ] ) . in addition to diabetes , they presented with a similar pattern of neurological abnormalities including moderate - to - severe developmental delay , profound sensorineural deafness , and visual impairment due to myopia and diffuse retinal dystrophy . brain magnetic resonance imaging scans showed severe cerebellar hypoplasia with no other major intracranial abnormalities ( fig . 3 and supplementary information ) . magnetic resonance imaging of the brain in a proband from family a demonstrating the typical neuroimaging findings of neurod1-pndm . a : sagittal t1-weighted image . b : coronal t2-weighted image . there is significant cerebellar hypoplasia , particularly of cerebellar vermis inferiorly . clinical features of the two patients with homozygous neurod1 mutations ct , computed tomography ; erg , electroretinography ; mri , magnetic resonance imaging ; sds , sd score . the diabetes status , age of diagnosis , treatment , and genetic testing result of family members are shown in fig . 2 . we assessed glucose tolerance in the four parents of the two probands who were proven heterozygous carriers of the mutations . in family a ( c.364dupg mutation ) , the mother had been diagnosed with type 2 diabetes at 33 years , despite having a normal bmi , and was treated with glicazide . in contrast , the father ( also aged 33 years ) had normal fasting ( 46 mmol / l ) and postprandial ( 57 mmol / l ) blood glucose levels on several occasions . in family b ( c.427_428del mutation ) , the mother and father underwent standard oral glucose tolerance tests ( aged 33 and 37 years , respectively ) that confirmed normal glucose tolerance ( 6.2 and 4.8 mmol / l , respectively , at 2 h ) . no heterozygous family members in either family had any developmental delay or neurological features on clinical examination . the patients with this novel autosomal recessive syndrome not only had early - onset permanent diabetes but also presented with developmental delay , cerebellar hypoplasia , and hearing and visual impairment . this is the 13th gene in which mutations have been described in patients with permanent neonatal diabetes . neurod1 , a tissue - specific member of the bhlh family of transcription factors , is expressed in developing pancreatic islets and in mature -cells . it forms a heterodimer with the ubiquitous bhlh transcription factor e47 that binds to specific e - box motifs on specific target genes , including ins , gck , and abcc8 , to regulate their expression ( 1416 ) . the two homozygous neurod1 mutations both introduce a frameshift that results in truncated proteins lacking the transactivation domain , which has been shown to be important for the interaction of neurod1 with its main coactivator , p300 ( 17 ) . these are likely to have no biological activity , as shown previously for a different frameshift mutation ( c.616dupc , p.his206profs*38 ) identified in a patient with neurod1-mody ( 9 ) . the two patients have a remarkably consistent phenotype ( table 1 ) , with clinical features in keeping with the known expression and biology of this transcription factor , and this provides further evidence that the homozygous mutations in neurod1 are causative . both patients have neonatal diabetes but a normal pancreas on scanning and no evidence of exocrine dysfunction . histological examination of the neurod1-deficient pancreas shows an impaired islet morphogenesis with a reduction in the number of endocrine cells especially -cells ( 8) . in addition to diabetes , our two patients presented with a similar pattern of neurological features , including developmental delay , cerebellar hypoplasia , and visual and hearing impairment . this is in keeping with the abundant expression of neurod1 in the developing and mature nervous system . interestingly , the initial neurod1-null mice that rapidly died from diabetes had no obvious anatomic and histologic abnormalities of the brain ( 8) . however , it is possible to explore the role of neurod1 in the nervous system by rescuing neurod1-null mice either by expressing a transgene encoding the mouse neurod1 gene under the insulin promoter ( 18 ) or by crossing the null mutation into a different genetic background to reduce the severity of the diabetes ( 19 ) . the rescued neurod1-null mice show a similar neuronal phenotype consisting of impaired balance , ataxic gait , circling , and swaying head movement as a result of impaired cerebellum development ( 1820 ) . furthermore , rescued neurod1-deficient mice have abnormal hearing and vision as a result of severe sensory neuronal defects in the inner ear and neural retina , respectively ( 2022 ) . the main feature seen in the mouse that was not present in our patients was epilepsy ( 19 ) . the remarkable similarity between the neurod1-deficient patients and the neurod1-deficient mice ( table 2 ) strongly supports a similar biological role of this transcription factor across species . comparison of the major features seen in neurod1-deficient mice ( refs . 8 and 1822 ) and neurod1-deficient patients with homozygous neurod1 mutations early - onset ketosis - prone diabetes failure of mature islets development striking reduction in both beta and alpha cells permanent neonatal diabetes postnatal - onset acinar cell polarity defects ( indirect effect ? ) lack of secretin- and cholecystokinin - producing cells ( remaining enteroendocrine cells normal ) > 95% decrease of granule cells impaired coordination and ataxia decrease of granule cells decreased synapses and loss of outer nuclear layer deafness and imbalance shortened cochlear duct , sensory epithelia abnormalities , and degeneration of acoustic ganglions sensorineural deafness homozygous mutations in ptf1a , which encodes another bhlh transcription factor , also cause a syndrome of neonatal diabetes and cerebellar hypoplasia / agenesis ( 23 ) . however , in this condition the pancreatic phenotype is not limited to the islets ; affected patients have pancreatic hypoplasia / aplasia . in keeping with the islets representing < 1% of the endocrine pancreas , the size of the pancreas was found to be normal in our two patients with homozygous neurod1 mutations . this suggests that shared developmental pathways are important during development in the pancreas and the cerebellum . although heterozygous loss - of - function mutations in neurod1 have previously been identified as a very rare cause of diabetes in humans ( 912 ) , diabetes was present in only one of four heterozygous mutation carrying parents . their age at the time of the study ranged from 33 to 39 years and does not exclude the possibility of developing diabetes later in life . in addition , incomplete penetrance has been described in some of the families with neurod1 diabetes ( 9 ) . homozygous mutations in other known mody genes , namely gck and ipf1 , have previously been associated with isolated pndm and isolated pancreatic agenesis , respectively ( 24,25 ) . we have shown that homozygous mutations in another mody gene are also associated with a more severe phenotype of neonatal diabetes . in conclusion , homozygous mutations in neurod1 constitute a rare novel autosomal recessive cause of neonatal diabetes with severe neurological abnormalities . this confirms the important role that neurod1 plays in the development of both the pancreas and the nervous system in humans .

objectiveneurod1 is expressed in both developing and mature -cells . studies in mice suggest that this basic helix - loop - helix transcription factor is critical in the development of endocrine cell lineage . heterozygous mutations have previously been identified as a rare cause of maturity - onset diabetes of the young ( mody ) . we aimed to explore the potential contribution of neurod1 mutations in patients with permanent neonatal diabetes.research design and methodswe sequenced the neurod1 gene in 44 unrelated patients with permanent neonatal diabetes of unknown genetic etiology.resultstwo homozygous mutations in neurod1 ( c.427 _ 428del and c.364dupg ) were identified in two patients . both mutations introduced a frameshift that would be predicted to generate a truncated protein completely lacking the activating domain . both patients had permanent diabetes diagnosed in the first 2 months of life with no evidence of exocrine pancreatic dysfunction and a morphologically normal pancreas on abdominal imaging . in addition to diabetes , they had learning difficulties , severe cerebellar hypoplasia , profound sensorineural deafness , and visual impairment due to severe myopia and retinal dystrophy.conclusionswe describe a novel clinical syndrome that results from homozygous loss of function mutations in neurod1 . it is characterized by permanent neonatal diabetes and a consistent pattern of neurological abnormalities including cerebellar hypoplasia , learning difficulties , sensorineural deafness , and visual impairment . this syndrome highlights the critical role of neurod1 in both the development of the endocrine pancreas and the central nervous system in humans .

RESEARCH DESIGN AND METHODS Study population. None Homozygosity mapping. RESULTS Molecular genetics. Clinical features. DISCUSSION Supplementary Material

the systems for controlled delivery of the medicaments in the body are causing real revolution in the medicine and pharmacy in the recent years , and all in favor of better medical treatments of the patients . using the system for the controlled and balanced release of medicaments , opposing to standard and conventional methods , constant and uniform concentration of medicament in the body is achieved throughout longer period of time . copolymer poly(d , l - lactide - co - glycolide ) is used for the controlled delivery of several classes of medicaments like anticancer agents , anti - hypertensive agents , immunomodulatory drugs , hormones , and macromolecules like nucleic acid , proteins , peptides , antibodies , dlplg nanospheres are very efficient mean of transdermal transport of medicaments in the body , for example , ascorbic acid . dlplg polymer particles allow the encapsulation of the medicament within the polymer matrix , where the principle requirement for the controlled and balanced release of the medicament in the body is the particle 's ideal spherical shape and narrow distribution of its size . the size and shape of the particles play key role in their adhesion and interaction with the cell . dynamic of the release ( pace and concentration ) depends of the morphology , that is , structure of the copolymer . the chemical structures , molecular weight , composition , as well as the synthesis conditions , are parameters which influence the final morphology of the polymer . the direct relation between these parameters and morphology is inadequately examined thus making it a topic of many researches . depending on the nature and matrix of the selected material , methods of obtaining polymer particles can be divided in general into dispersion of the polymer solution method , polymerization of the monomer method , and coacervation [ 36 ] . the plga spheres obtained with emulsion process are in range of 150200 m , 45 m , 30 m . with modified emulsion method , further modification of the process for synthesis of the particles , that is , emulsification solvent evaporation method , the obtained particles are in nanometer scale of 570970 nm and 244260 nm [ 1214 ] . the latest researches in this field indicated the possibility of producing dlplg spheres with average diameter under 100 nm . controlling the conditions of obtaining dlplg by solvent / nonsolvent method , changing the parameters like aging time , after adding nonsolvent , time and velocity of centrifugal processing , it is possible to influence on morphology ( size and shape ) and uniformity of dlplg polymer powder . dlplg powder with short aging time with nonsolvent and longest time and velocity of the centrifugal processing has smallest particles and highest uniformity . dlplg copolymer has potential to be used for transport of ascorbic acid in the body , thus considerably increasing its efficiency . ascorbic acid reduces free radicals , and in that way damages created by oxidative stress which is a root cause of , or at least associated with , many diseases are minimized . the aim of this research is obtaining the nanoparticles of copolymer poly(d , l - lactide - co - glycolide ) in which ascorbic acid is encapsulated , as well as examining the influence of the synthesis method on morphological characteristics of poly(d , l - lactide - co - glycolide ) particles with the different content of ascorbic acid . poly(d , l - lactide - co - glycolide ) ( dlplg ) was obtained from durect , lactel , adsorbable polymers international and had a lactide to glycolide ratio of 50 : 50 . time of complete resorption of this polymer is 48 weeks . molecular weight of ascorbic acid was 176.13 g / mol . copolymer powder dlplg was obtained by means of physical methods from commercial granules using solvent / nonsolvent systems ( figure 1 ) . commercial granules poly(d , l - lactide - co - glycolide ) ( 0.05 g ) were dissolved in 1.5 ml of acetone and , after approximately two hours , 2 ml of methanol was added into solvent mixture . the polymeric solution thus obtained was very slowly poured into 20 ml of aqueous pva solution ( 0.02% w / w ) while continuous stirring at 1200 rpm by a stirrer . pva is used as a stabilizer which creates negative charge of the dlplg particles , that is , it creates negative zeta potential . by creating specific zeta potential the ascorbic acid was encapsulated into the polymer matrix by means of homogenization of water and organic phases . the water solution with the variable ratio of the ascorbic acid was added to the polymer solution . this was followed by the precipitation using alcohol methanol . in the particles of dlplg copolymer , different concentration of ascorbic acid has been encapsulated with ratios 85% dlplg to 15% ascorbic acid , 70% dlplg to 30% ascorbic acid , 50% dlplg to 50% ascorbic acid , and 30% dlplg to 70% ascorbic acid . the ir measurements were performed on perkin - elmer 983 g infrared spectrophotometer , using the kbr pellet technique , in the frequency interval of 4004000 cm . the morphology of obtained particles of dlplg was examined by scanning electron microscope ( sem ) jeol jsm-646olv . the powder samples for sem analysis were coated with gold using the physical vapor deposition ( pvd ) process . samples were covered with gold ( scd 005 sputter coater ) , using 30 ma current from the distance of 50 mm during 180 seconds . the particle size and morphology were examined using the area analysis method [ 18 , 19 ] by semiautomatic image analyzer ( videoplan , kontron ) , connected with a scanning electron microscope ( sem ) . from 200 to 300 particles in the sem were measured and the following parameters were determined : area section aa , perimeter lp , maximal diameter of the particle dmax , feret x and feret y , and form factor ( fl ) ( figure 2 ) . release of the ascorbic acid from dlplg particles in vitro in physiological solution ( 0.9% sodium chloride in water ) was studied with uv spectroscopy . the uv measurements were performed on perkin - elmer lambda 35 uv - v is spectrophotometer in the frequency interval of 200400 nm . the ir spectra in figure 3 illustrate all characteristic groups for copolymer poly(d , l - lactide - co - glycolide ) . the ir spectra of dlplg show peaks at 2994 , 2946 , 2840 ( ch bend ) , 1769 ( c = o ester ) , 1460 , 1424 , 1371 ( ch3 ) , 1150 , 1069 984 ( co stretch ) , 732 509 ( ch - bend ) cm while the band on 31003600 cm belongs to the oh group of the water molecule . comparing the obtained ir spectra for dlplg and ascorbic acid ( figure 4 ) with the ir spectra charecteristics for ascorbic acid shown in the literature [ 21 , 22 ] , it is confirmed that obtained nanoparticles are composed of poly(d , l - lactide - co - glycolide ) and ascorbic acid . besides the characteristic groups for copolymer dlplg , the four oh bands of ascorbic acid could be assigned by means of infrared investigations at 3528 , 3411 , 3317 , 3217 cm . the spectra show bands that can be assigned to ch3 , ch2 , or ch groups in the ascorbic acid environment at 2720 cm and the spectra also clearly show the band corresponding to c = o groups at 2916 cm . the bands that correspond to the wave number 1754 cm belong to c = c groups , 1673 cm coc , and 1020 cm co , respectively . the morphological characteristics of the obtained dlplg particles , with and without encapsulated ascorbic acid , were examined with a scanning electron microscope . from the sem recordings of dlplg particles without ascorbic acid ( figure 5(a ) ) , it is visible that the particles have spherical shape , smooth surface , low level of agglomeration , and high level of uniformity higher than other samples . from the sem recordings of the second sample ( figure 5(b ) ) , where dlplg copolymer has encapsulated ascorbic acid in ratio dlplg / ascorbic acid 85/15% , it is visible that particles also have spherical shapes , that is , spherical shape of the initial dlplg dlplg / ascorbic acid 85/15% nanoparticles are uniform with sizes from 130 to 200 nm depending on which stereological parameters are considered ( dmax , maximum diameters , feret x , or feret y ) . the particles of the sample dlplg / ascorbic acid 70/30% ( figure 5(c ) ) also have spherical shapes , but their sizes are increased . in case of the fourth sample , dlplg / ascorbic acid 50/50% ( figure 5(d ) ) uniformity is perturbated , particles have both spherical and irregular shapes and they are much agglomerated . for the fifth sample , dlplg / ascorbic acid 30/70% ( figure 5(e ) ) , the stereological analysis is giving us the parameters which are characterizing the particle sizes ( area section dmax , and feret 's diameters ) and parameter which is characterizing the particle shape ( perimeter form factor maximum , and mean values were recorded and presented in table 1 . based on the obtained results of the stereological analysis of dlplg particles , it is visible that they are uniform , their average mean size varies from 0.15 to 0.23 m depending on the stereological parameter taken in consideration ( dmax , feret x , or feret y ) ( table 1 ) . dmax values range from 0.09 to 0.39 m with particle 's mean size 0.23 m ( figure 6 ) . figures 7 and 8 present comparative results of dlplg particles with and without ascorbic acid based on their area section and perimeter form factor . from the comparative results of the stereological analysis of the area section ( aa ) of dlplg with and without encapsulated ascorbic acid ( figure 7 ) as well as comparative results of the perimeter form factor ( fl ) , ( figure 8) we can see that dlplg particles without ascorbic acid have the smallest area section ( minimum value for aa is 0.02 mand maximum is 0.08 m ) and the highest mean value of perimeter form factor which is 0.89 . nanoparticles dlplg / ascorbic acid 85/15% have minimum dmax of 0.09 m and maximum dmax of 0.49 m , where their mean size is 0.20 m ( figure 6 ) . the mean value of the area section is 0.03 m ( figure 7 ) and of the perimeter form factor is 0.87 ( figure 8) . for particles dlplg / ascorbic acid 70/30% , minimum dmax is 0.30 m and maximum dmax is 2.59 m , where their mean size is 0.67 m ( figure 6 ) , which indicates that the uniformity is decreased and size is increased . the mean value of the area section is 0.41 m ( figure 7 ) and of the perimeter form factor is 0.77 ( figure 8) . for particles dlplg / ascorbic acid 50/50% , minimum dmax is 0.28 m and maximum dmax is 4.51 m , where their mean size is 1.60 m ( figure 6 ) . the mean size of the area section is 2.30 m ( figure 7 ) and of the perimeter form factor is 0.74 ( figure 8) . in case of dlplg / ascorbic acid 30/70% the release amount of the ascorbic acid from the polymer particles was determined periodically during the eight weeks with uv spectroscopy . a calibration curve of the ascorbic acid in physiological solution at different concentrations has been prepared using the specific absorbance peak of the ascorbic acid at 264 nm . figure 9 shows the dependence of the maximum absorption from the degradation time in cases of dlplg without ascorbic acid , dlplg / ascorbic acid 85/15% , dlplg / ascorbic acid 70/30% , and dlplg / ascorbic acid 50/50% . this absorbance is correlated with the calibration curve and amount of ascorbic acid is determined in percentages . figure 10 gives cumulative curves of the release of the ascorbic acid in percentages over the period of time of the degradation . figure 10 also shows the relative review in percentages of the ascorbic acid release in periods of up to two days , 211 , 1117 , 1724 , 2431 , 3139 , 3946 , and 4655 days . in the first 24 days of the degradation , for all samples , less than 10% of the encapsulated ascorbic acid have been released . for all dlplg / ascorbic acid samples , the overall quantities of the encapsulated ascorbic acid have been released in 8 weeks of the degradation . the particles obtained with solvent / nonsolvent physical method and technique of the centrifugal processing have potential use in transdermal systems for controlled delivery of ascorbic acid . it is possible to encapsulate ascorbic acid into dlplg particles in various concentrations thus producing particles with different morphological characteristics . the nanoparticles of dlplg / ascorbic acid with lesser ratio of ascorbic acid have higher uniformity , lower level of agglomeration , and smaller sizes . the nanoparticles of dlplg / ascorbic acid 85/15% have spherical shapes and their sizes are from 130 to 200 nm .

this paper is covering new , simplistic method of obtaining the system for controlled delivery of the ascorbic acid . copolymer poly ( d , l - lactide - co - glycolide ) ( dlplg ) nanoparticles are produced using physical method with solvent / nonsolvent systems where obtained solutions were centrifuged . the encapsulation of the ascorbic acid in the polymer matrix is performed by homogenization of water and organic phases . particles of the dlplg with the different content of ascorbic acid have different morphological characteristics , that is , variable degree of uniformity , agglomeration , sizes , and spherical shaping . mean sizes of nanoparticles , which contain dlplg / ascorbic acid in the ratio 85/150% , were between 130 to 200 nm depending on which stereological parameters are considered ( maximal diameters dmax , feret x , or feret y ) . by introducing up to 15% of ascorbic acid , the spherical shape , size , and uniformity of dlplg particles are preserved . the samples were characterized by infrared spectroscopy , scanning electron microscopy , stereological analysis , and ultraviolet spectroscopy .

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

morphine , the main component of opium , is perhaps the oldest drug known to man . pure morphine was isolated in 1803 by sertrner ( schmitz 1985 ) , and its structure was elucidated 120 years later . full systemic name of morphine is 7,8-didehydro-4,5-epoxy-17-methyl-(5 , 6)-morphinan-3,6-diol . morphine was found to be a particularly good analgesic and sedative , far more effective than crude opium . morphine was shown to exert its action through opioid receptors ( , , and ) localized in the brain ( pasternak 1993 ; reisine and bell 1993 ; harrison et al . 1998 ; kieffer 1999 ; kieffer and gaveriaux - ruff 2002 ) . however , morphine binds to the -opioid receptor with nearly two orders of magnitude greater affinity compared with the other two opioid receptors ( zadina et al . although morphine acts directly on the central nervous system ( cns ) to relieve pain , its activity on peripheral tissues is responsible for many of the secondary complications . besides its strong analgesic effect , morphine exerts a number of adverse side - effects , including addiction , tolerance , respiratory depression , immunosuppression , and constipation . the lack of equally strong painkillers is the reason that despite the drawbacks mentioned above , morphine is still the most commonly used analgesic for management of severe pain , including cancer pain ( mantyh 2006 ) . administration of morphine to cancer patients gave evidence that apart from its analgesic action , morphine can significantly alter tumor growth . in the last decade , numerous studies employing cancer cell lines and experimental animals have been performed to reveal complex mechanisms by which morphine affects tumor cells . whereas the pharmacology and function of opioids in the cns have been extensively characterized , still little is known about their effect on cancer cells . morphine was reported to increase the proliferation of endothelial and tumor cells ( simon and arbo 1986 ; moon 1988 ; ishikawa et al . on the other hand , morphine and other opioids were also found to promote tumor cell death ( maneckjee et al . 1990 ; yeager and colacchio 1991 ; page et al . 1993 ; hatzoglou et al . 1996 ; sueoka et al . 1996 , 1998 ) . several reviews have covered this important research area from different perspectives ( rasmussen et al . 2002 ; fichna and janecka 2004 ; tegeder and geisslinger 2004 ; chen et al . this review will focus on the latest findings on the influence of morphine on tumor cell proliferation , apoptosis , angiogenesis , and migration . despite extensive research , it is still not well understood whether morphine itself directly modifies the growth of tumor cells . some authors postulate that morphine can promote tumor growth and reduce the survival rate of tumor - bearing animals due to immunosuppression , since the negative effects of morphine and other opioids on the immune system are well established ( odunayo et al . multiple research data indicate that morphine can accelerate or inhibit cancer cell growth in vitro and in vivo by different mechanisms . ( 2003 ) reported that morphine inhibited tumor cell proliferation at concentrations of > 10 m . in nude mice , intermittent injections of morphine decreased the growth of tumors in a rat model of metastasizing colon cancer ( yeager and colacchio 1991 ) . on the other hand , morphine was shown to trigger stimulation of human glioblastoma t98 g cell proliferation ( lazarczyk et al . gupta et al . ( 2002 ) demonstrated that morphine , in clinically relevant doses , promoted tumor neovascularization in a human breast tumor xenograft model in mice , leading to increased tumor progression . the discrepancies in results may be due to the differences in administered doses or / and the mode of administration ( systemic versus localized ) . 2002 ) , showed that tumor suppression occurs after chronic high doses of morphine , while tumor - enhancing effects with morphine occur after a single dose or low daily doses ( zong and pollack 2000 ) . this dual concentration - dependent effect , i.e. , mitogenesis at low and growth inhibition at higher concentrations was observed both in vitro and in vivo . the lack of effects of morphine on the proliferation of colon cancer ht-29 cells ( zagon et al . 2008 ) and on breast cancer mcf-7 cells was also reported ( janecka et al . 2004 ) . recently , it was demonstrated that the -opioid receptor regulates cancer progression in animal models ( moss and rosow 2008 ; wang et al . the -opioid receptor - knockout mice were shown not to develop significant tumors when injected with lewis lung cancer cells as did the wild - type controls ( mathew et al . 2009 ) . silencing the expression of the -opioid receptor in lewis lung cancer cells inhibited lung metastasis in wild - type mice by about 75% . finally , infusion of the -opioid receptor antagonist , methylnaltrexone , markedly attenuated tumor growth in wild - type mice treated with lewis lung cancer cells by up to 90% ( mathew et al . these experimental data strongly support the hypothesis that the -opioid receptor promotes tumor growth and metastasis . possible mechanisms of morphine action mediated through the opioid receptors are demonstrated in fig . 1 and through the non - opioid pathways in fig . 2 . the summary of in vivo growth - promoting and growth - inhibiting effects of morphine is presented in table 1 . morphine binds to the -opioid receptor and a stimulates mapk / erk pathway , which results in the cell cycle progression ; b activates pi3k / akt pathway mediating anti - apoptotic effects ; c up - regulates upa expression and secretion promoting metastasis ; d transactivates vegf receptors and induces angiogenesis ; e suppresses the function of t lymphocytes , leading to immunosuppressionfig . 2possible pathways , other than through the opioid receptors , by which morphine influences cancer progression and suppressiontable 1summary of in vivo growth - promoting and growth - inhibiting effects of morphinecell line / tumor typein vivo effectsin vivo dose / timesuggested mechanismantagonist / inhibitionref.ehrlich carcinoma injected s.c . in the left thigh of balb / c micestimulation of angiogenesis0.714 mg / kg / day for 7 days ( equivalent to 50 mg per day for a 70 kg human)ustun et al . 2010colon cancer cells injected i.p . in fisher 344 ratsinhibition of tumor growth and metastasis ( significant decrease in the hepatic tumor burden)20 mg / kg / day s.c . , the day before and for 2 days after colon cancer cell inoculationenhancement of nk cell activity at the time of tumor cell injectionyeager and colacchio 1991mcf-7 , mda - mb231 breast cancer or ht-29 colon cancer cells injected s.c . in nmri - nu / nu miceinhibition of tumor growth ( mcf-7 and mda - mb231 ) no effect on ht-29 tumors1030 mg / kg / day i.p . for 3 weeks ( stepwise 10 , 20 , and 30 mg / kg / day i.p . for the first , second , and third week)inhibition of tumor growth through a p53 dependent mechanism ( up - regulation of p53-dependent p21 , bax , 1993walker 256 carcinosarcoma cells injected i.v . in sprague dawley ratincrease of the number metastases5 mg / kg single dosesuppresion of nk cellsnaloxone itself had no significant influence on the number of metastases . the effect was inhibited by preadministration of naloxonesimon and arbo 1986b16-bl6 melanoma cells injected into hindpaw in c57bl miceinhibition of tumor growth and metastasis ( decreased number of tumor nodules in the lung)5 and 10 mg / kg / day s.c . for 6 days , starting 16 days after cancer cells injectionpain reduction and blockade of pain signalssasamura et al . 2002colon 26-l5 carcinoma cells injected i.v . in balb / c miceinhibition of tumor metastasis ( reduction of the number of tumor colonies in the lung)10 mg / kg / day i.p . for 6 days , starting on day 2 after i.v . inoculation of tumor cellsinhibition of adhesion and invasion of cancer cells ( inhibition of mmp-2 and 9 production)harimaya et al . 2002mcf-7 cells injected into the mammary fat pad of nude miceincrease of tumor growth associated with increased angiogenesis ( neovascularization , increased microvessel density , higher vessel number , increased total length , and more vessel branching)0.714 mg / kg mouse / day for first 15 days and then 1.43 mg / kg mouse / day ( equivalent to 50 mg and 100 mg morphine per day , respectively , for a 70 kg human)activation of pi3k / akt and erk pathways , resulting in inhibition of apoptosis and promotion of cell cycle progressionnaloxone itself had no significant effect on angiogenesis . the anti - angiogenic effect of morphine was not inhibited by naloxonegupta et al . 2002madb-100 mammary adenocarcinoma cells injected i.v . in fisher 344 rats , 5 h after surgeryinhibition of surgery - induced increase in metastasis5 mg / kg 3 doses ( pre , post and 5 h post surgery)inhibition of postoperative pain and stress - induced immunosuppressionpage et al . 1993murine ccl-11 sarcoma cells injected into femur of the right leg in c3h / hej micemorphine did not alter tumor burden50 , 25 , 7.5 , 2.5 mg / ml implanted ( s.c . ) for 7 days following injection of ccl-11 cells into the femurking et al . 2007lewis lung carcinoma cells injected s.c . in nude micereduction of tumor cell - induced angiogenesis and tumor growtha continuous slow - release morphine pellet resulting in morphine plasma levels within 250400 ng / mlsuppression of the hypoxia - induced mitochondrial p38 mitogen - activated protein kinase ( mapk ) pathwaythe effect was abolished with naltrexonekoodie et al . 2010tc-1 cells injected s.c . into the right leg of c57bl/6j micedose - dependent increase of tumor growth10 mg / kg i.p . , twice daily from day 10 before tumor injectionimmunosuppression ( -opioid receptor mediated suppression of t lymphocytes proliferation , promotion of apoptosis of t lymphocytes through bcl-2 and bax apoptosis - related molecules)cheng et al . 2006 possible mechanisms of opioid receptor mediated influence of morphine on tumor growth . morphine binds to the -opioid receptor and a stimulates mapk / erk pathway , which results in the cell cycle progression ; b activates pi3k / akt pathway mediating anti - apoptotic effects ; c up - regulates upa expression and secretion promoting metastasis ; d transactivates vegf receptors and induces angiogenesis ; e suppresses the function of t lymphocytes , leading to immunosuppression possible pathways , other than through the opioid receptors , by which morphine influences cancer progression and suppression summary of in vivo growth - promoting and growth - inhibiting effects of morphine apoptosis is an active process of controlled cell death in the development and maintenance of tissue homeostasis . apoptosis is controlled by two main pathways , the intrinsic or the mitochondrial - mediated pathway ( green and reed 1998 ) and the extrinsic or death receptor - mediated pathway ( ashkenazi and dixit 1999 ) . mitochondrial - mediated apoptosis is controlled by bcl-2 family of proteins ( cory and adams 2002 ; cory et al . the death receptor - mediated pathway is initiated by the ligation of cell death ligands with their death receptors ( sartorius et al . caspase-3 is activated in both apoptotic pathways and plays central role in the execution phase of cell apoptosis ( ashkenazi and dixit 1999 ; hengartner 2000 ; fulda and debatin 2006 ) . apoptosis is usually deregulated in cancer cells , and this deregulation can contribute to uncontrollable proliferation and tumor growth ( hanahan and weinberg 2000 ; hengartner 2000 ; kaufmann and hengartner 2001 ) . morphine was shown to induce the apoptosis of human endothelial cells ( hsiao et al . morphine in high concentrations ( 1010 m ) was demonstrated to induce apoptotic cell death in human tumor cell lines ( kawase et al . in other studies , the pro - apoptotic effect of morphine in human tumor cell lines was observed at clinical concentration ( 10 m ) ( smith et al . for example , morphine produced a higher number of necrotic cells in the mcf-7 breast cancer cell line than in the a549 lung cancer cell line ( hatsukari et al . 2003 ) , it was recently demonstrated that chronic high - dose morphine treatment was able to cause apoptotic cell death of sh - sy5y cells in an opioid receptor - independent manner ( lin et al . a pivotal role in this process was played by c - jun n - terminal kinase ( jnk ) . activation of jnk by morphine led to reactive oxygen species ( ros ) generation and induced cytochrome c release and caspase-9/3 activation through enhancement of expression of the pro - apoptotic protein bim and reduction of expression of the anti - apoptotic protein bcl-2 . the key role of the jnk pathway in morphine - induced mitochondria - dependent apoptosis was further confirmed by the observation that decreased levels of jnk in cells transfected with specific small interfering rna resulted in resistance to the pro - apoptotic effect of morphine . it can be concluded that morphine can induce apoptosis and inhibit the growth of cancer cells by activating different signal pathways . apart from the mitochondrial pathway mentioned above , apoptosis of mcf-7 cells was shown to be mediated by a novel sigma-2 receptor and p53 and caspase - independent pathway ( crawford et al . cne2 human epithelial tumor cell line was reported to undergo apoptosis by the activation of the -opioid receptor via the phospholipase pathway ( diao et al . the growth - inhibitory or apoptosis - inducing effects of morphine might be directly associated with morphine tolerance ( wu et al . 1999 ; mao et al . 2002 ) or receptor desensitization , as assessed by a lack of morphine - stimulated gtp - ase activity at concentrations that inhibit tumor growth ( tegeder et al . drugs that prevented the development of morphine tolerance in rats also prevented cell death ( mao et al . 2002 ) . this close association between apoptosis and receptor desensitization suggests that receptor internalization may be the key event in initiating opioid - evoked cell death . on the other hand , evidence of the anti - apoptotic activity of it was shown that morphine can antagonize the pro - apoptotic activity of a well - known anti - tumor drug , doxorubicin , in neuroblastoma sh - sy5y cells ( lin et al . 2007 ) . the effect could not be reversed by naloxone , indicating a non - opioid receptor - mediated signaling pathway . further studies showed that morphine attenuated doxorubicin - induced apoptosis by the inhibition of ros generation and mitochondrial cytochrom c release , as well as by blockade of nuclear factor b ( nf-b ) transcriptional activation . nf-b is a ubiquitous nuclear transcription factor that plays a major regulatory role in apoptosis and inflammation . ( 2003 ) demonstrated that in sh - sy5y neuroblastoma cells , morphine ( 1010 m ) promoted cell survival after serum deprivation without inducing cell proliferation , and this effect was fully reversed by naloxone . it was shown that in neuronal cells , -opioid agonists do not directly induce apoptosis , but are able to activate the phosphatidylinositol 3-kinase / akt ( pi3k / akt ) signal transduction pathway , thus leading to cell survival . successful tumor growth depends on many aspects , of which the most important are proliferation of tumor cells and angiogenesis or the formation of new blood vessels which are necessary for metastasis ( carmeliet and jain 2000 ) . to date , very few studies have investigated the effect of morphine on tumor cell - induced angiogenesis . gupta et al . ( 2002 ) showed that at clinically relevant concentrations , morphine stimulated human microvascular endothelial cell proliferation and angiogenesis in vitro , and that in vivo these effects translated into enhanced tumor neovascularization in the mcf-7 breast cancer model . in addition , this group demonstrated that morphine promoted activation of vascular endothelial growth factor ( vegf ) receptor and increased metastasis and reduced survival in animal model of hormone - dependent breast cancer ( chen et al . however , the opioid receptor antagonist , naloxone , did not inhibit the pro - angiogenic activity of morphine , indicating that the effect was not mediated by the typical opioid receptors . on the other hand , singleton and moss ( 2010 ) demonstrated that -opioids ( morphine ) transactivate the vegf receptors and promote angiogenesis . the -opioid receptor antagonist methylnaltrexone blocks opioid - induced angiogenesis in cultured human endothelial cells . it was postulated that the pro - angiogenic activity of morphine is connected with the stimulation of mitogen - activated protein kinase ( mapk ) signaling pathway via g protein - coupled receptors and nitric oxide ( no ) . chronic morphine treatment increased the levels of nitric oxide synthase ( nos ) , no , and cyclooxygenase-2 ( cox-2 ) in mouse kidney ( stefano et al . 1995 ; arerangaiah et al . 1993 ) , and activated cox-2 in turn increased prostaglandin e2 production ( salvemini et al . two weeks of chronic morphine treatment of experimental mice with highly invasive sck breast cancer model stimulated cox-2 , prostaglandin e2 , and angiogenesis , which was accompanied by increased tumor weight , increased metastasis , and reduced survival . it was postulated that products of cox-2 , such as prostaglandin e2 produced in tumor cells , act on the tumor endothelium and promote angiogenesis . notably , morphine induced a much stronger expression of cox-2 in tumor cells than in endothelial cells . co - administration of cox-2 inhibitor , celecoxib , prevented morphine - induced promotion of angiogenesis and tumor growth . therefore , morphine - induced tumor growth may in part be due to the up - regulation of cox-2 and prostaglandin e2-mediated stimulation of angiogenesis . it was also shown that morphine did not have any effect on mcf-7 human breast cancer cell proliferation in vitro ( farooqui et al . 2006 ) but stimulated mcf-7 breast tumor growth by promoting angiogenesis ( gupta et al . that means that morphine by itself did not modulate tumor cell growth in culture , but due to up - regulation of cox-2 in tumor cells , stimulated angiogenesis in vivo , resulting in increased tumor growth and metastasis . it is well known that when the solid tumor grows , new tumor cells are localized further away from their vascular supply , and low oxygen tensions or hypoxia stimulate tumor cells to secrete pro - angiogenic factors ( folkman and d'amore 1996 ) . a potent pro - angiogenic factor secreted by hypoxic tumor cells within developing solid tumor is vascular endothelial growth factor ( vegf ) . this factor promotes new blood vessel formation to sustain tumor growth and initiates endothelial cell proliferation and migration ( brekken and thorpe 2001 ; ferrara 2004 ) . ( 2001 ) reported that morphine inhibited hypoxia - induced vegf secretion in rat cardiomyocytes and human umbilical vein endothelial cells and therefore attenuated the ability of hypoxic tumor cells to induce angiogenesis . most recently , the same effect was observed in a murine lewis lung carcinoma tumor model ( koodie et al . morphine was administered to mice at the clinically relevant analgesic doses by implantation of continuous slow - release pallets . morphine plasma level within 250400 ng / ml was sufficient to significantly reduce tumor cell - induced angiogenesis and tumor growth . the effect of morphine was abolished by concomitant administration of opioid antagonist , naltrexone , and also in the -opioid receptor knockout mice , supporting the involvement of classical opioid receptors in the process . the authors also demonstrated that the inhibitory effect of morphine was mediated through the suppression of the hypoxia - induced mitochondrial p38 mapk pathway . much earlier , it was shown that high concentrations of morphine ( 10 mg / ml of plasma ) inhibited angiogenesis in the chick chorioallantoic membrane assay ( pasi et al . 1991 ) . however , morphine is cytotoxic to endothelial cells at such high concentrations ( gupta et al . 2002 ) , so the observed effect could have been non - specific . in wound healing experiments in mice , systemic administration of high - dose morphine resulted in impared mobilization of endothelial progenitor cells and angiogenesis , delaying the healing process . consistent with the in vivo experiment , in cultured endothelial cells , morphine reduced capillary tube formation in a concentration - dependent manner ( lam et al . a major feature of cancer cells is their ability to migrate and settle in surrounding or distant tissues . most cancer deaths and the main cause of failure in cancer treatment is not due to growth of the primary tumor but results from its intensive spread ( metastasis ) to secondary sites ( engbring and kleinman 2003 ; widel and widel 2006 ) . the critical step of cancer dissemination is migration of cancer cells through the extracellular matrix ( ecm ) . indispensable in this process is activation of urokinase plasminogen activator system , which includes a serine proteinase , urokinase plasminogen activator ( upa ) , two inhibitors , pai-1 and pai-2 , and the membrane - linked receptor ( upar ) ( duffy and duggan 2004 ) , plasmin and matrix metalloproteinases ( mmps ) ( mignatti and rifkin 2000 ) capable of degrading the ecm . it was shown that upa , pai-1 , and upar levels are up - regulated in most types of cancers ( shapiro et al . ( 2009 ) showed that morphine caused a markedly increased secretion of upa in mcf-7 breast cancer cells , which correlated well with up - regulation of upa and upar mrna levels . naloxone reversed morphine - induced up - regulation of upa and upa mrna levels confirming the involvement of the opioid receptors in this process . ( 2008 ) who demonstrated that morphine stimulated upa secretion in ht-29 colon cancer cells . the mmps constitute a family of zinc - dependent endopeptidases , whose primary function is remodeling of components in the ecm ( engbring and kleinman 2003 ; widel and widel 2006 ; jespersen et al . , it seems that mmp-2 ( gelatinase a ) and mmp-9 ( gelatinase b ) play a crucial role in cancer invasion because of their ability to degrade type iv collagen , a major component of basement membranes ( widel and widel 2006 ) . the activity of mmps is strictly regulated at the transcription and translation levels by endogenous inhibitors , including 2-macroglobulin and tissue inhibitors of metalloproteinases ( timps ) . several studies have shown that mmp expression and activity were increased in several tumor types , especially breast and lung cancer . the up - regulated levels of mmps correlated well with tumor stage , increased invasion , and potential metastasis ( lynch and matrisian 2002 ; widel and widel 2006 ) . morphine was found to significantly reduce experimental lung metastasis and invasion of colon 26-l5 cells by inhibiting adhesion and migration of these cells to ecm ( harimaya et al . wehi 164 , activity of mmp-2 was shown to be under control of no system and was attenuated by low doses of morphine in a non - opioid receptor - related manner ( shariftabrizi et al . morphine inhibited expression and secretion of mmp-2 and -9 in time- and concentration - dependent manner , and the process was not mediated by opioid receptors but was also under the control of the no system ( gach et al . disorders in the immune system can result in disease , including inflammatory diseases and cancer . the expression of the -opioid receptor gene in neuronal cells is regulated by cytokines , released from the cells of the immune system . on the other hand , -opioid receptor is also expressed in the cells of immune system , such as lymphocytes and macrophages ( brner et al . 2007 ) . this points at the importance of interactions between opioid and immune system ( kraus 2009 ) . the fact that morphine can inhibit numerous immune cell functions and cause immunosuppression is well known ( peterson et al . 1993 ; sacerdote et al . 1997 ; eisenstein and hilburger 1998 ) . in particular , morphine decreases the activity of natural killer cells which play a major role in the rejection of tumors ( sacerdote et al . 1997 ) and increases the susceptibility of animals and humans to bacterial and viral infections ( yeager et al . 1995 ; risdahl et al . 1998 ; macfarlane et al . it was also shown that morphine inhibits production of pro - inflammatory cytokines ( e.g. , tnf , il-6 ) in monocytes ( bonnet et al . 2008 ) , modulates the t helper cell balance by inducing up - regulation of an anti - inflammatory il-4 mrna ( roy et al . 2005 ) and produces immunosuppression by inhibition of transcription of il-2 in activated lymphocytes t ( brner et al . 2009 ) . however , future research is necessary to provide clearer understanding of the cellular and molecular targets of morphine action within the immune system . for more than two decades , researchers have been trying to document the effects that morphine exerts on tumor cells . it has been recognized that morphine can affect tumor growth by either directly acting on the tumor cells or by directly acting on the endothelial cells or on the cns - mediated secretion of growth factors that may alter tumor microenvironment . however , the results obtained both in in vitro and in vivo studies are conflicting . on one hand , morphine was shown to induce tumor growth , inhibit apoptosis , promote angiogenesis and migration of tumor cells , but on the other hand , pro - apoptotic and anti - angiogenic properties of morphine were also demonstrated . performed studies were very heterogenic due to the different doses of morphine applied , and the effects may vary when the experiments were performed in vivo or on isolated cells . the dose and route of administration of morphine might be critical factors that need to be taken into consideration in clinical settings . the final answer to the question whether morphine is an inhibitor of tumor growth or whether it promotes cancer is still to be sought . the effects of morphine can also depend on a cancer type , since different cancer cells can overexpress certain enzymes which are possible morphine targets .

morphine is considered the gold standard for relieving pain and is currently one of the most effective drugs available clinically for the management of severe pain associated with cancer . in addition to its use in the treatment of pain , morphine appears to be important in the regulation of neoplastic tissue . although morphine acts directly on the central nervous system to relieve pain , its activities on peripheral tissues are responsible for many of the secondary complications . therefore , understanding the impact , other than pain control , of morphine on cancer treatment is extremely important . the effect of morphine on tumor growth is still contradictory , as both growth - promoting and growth - inhibiting effects have been observed . accumulating evidence suggests that morphine can affect proliferation and migration of tumor cells as well as angiogenesis . various signaling pathways have been suggested to be involved in these extra - analgesic effects of morphine . suppression of immune system by morphine is an additional complication . this review provides an update on the influence of morphine on the growth and migration potential of tumor cells .

Introduction Effect of morphine on tumor growth Effect of morphine on tumor apoptosis Effect of morphine on angiogenesis Effect of morphine on migration and invasiveness of tumor cells Effect of morphine on immune system Conclusions

from january 1999 to december 2000 , aft was performed on patients aged 2575 years who had type 2 diabetes at the university - affiliated diabetes center of st . patients with normal aft results were recruited ( n = 1,021 ) and received follow - up aft from january 2006 to february 2008 . patients were excluded if they were > 75 years old , if they were mentally ill or unable to undertake the test , if they had arrhythmia including atrial fibrillation , or if they had any severe illness , such as malignancy , severe infection , severe hypoglycemia , liver cirrhosis , heart failure , or alcoholism . our institutional ethics committee approved the study . at the beginning of the study , patient height , body weight , and systolic and diastolic blood pressure hypertension was defined as systolic blood pressure 140 mmhg or diastolic blood pressure 90 mmhg or as any use of antihypertensive medications . fasting and postprandial plasma glucose levels were measured using an automated enzymatic method and a1c levels were determined by high - performance liquid chromatography with a reference range of 4.46.4% . a1c levels were measured every 6 months to evaluate the status of glycemic control during the follow - up period . blood lipid concentrations for total cholesterol , triglycerides , and hdl cholesterol were measured enzymatically using an automatic analyzer ( model 736 - 40 ; hitachi , tokyo , japan ) . biochemical laboratory tests were performed at baseline and at the follow - up time points . cardiovascular aft was performed by one examiner using the ewing method , which includes tests for heart rate variability , such as the expiration - to - inspiration ( e / i ) ratio and responses to the valsalva maneuver and to a postural change from lying to standing ( 2,6,14 ) . patients were asked to fast for 12 h before aft and to avoid taking antidepressants , neuroleptics , caffeine , nicotine , or antihistamines . the e / i ratio was calculated as the mean of the longest r - r interval during expiration divided by the mean of the shortest r - r interval during inspiration . the ratio of postural change was the ratio of the longest r - r interval during beats 2040 after standing to the shortest r - r interval during beats 525 after standing . for the heart rate response to the valsalva maneuver , the ratio of the longest r - r interval to the shortest r - r interval was checked during forced exhalation into the mouthpiece of a manometer to 40 mmhg for 15 s ( 2,14 ) . an e / i ratio below the age - related reference value , a valsalva ratio < 1.2 , and a posture ratio < 1.03 were considered abnormal ( 14 ) . each of the three items was scored as 0 for normal or 1 for abnormal , to a maximum score of 3 . normal aft was defined as a score of 0 , and can was defined on the basis of at least one abnormal standard test . progression of can was defined as an increase in a follow - up aft score from the baseline value ( a score of 0 ) to a score of 13 ; such patients were designated the progressor group . nonprogressors were defined as type 2 diabetic patients whose can scores did not show any increase at the follow - up time point . the urinary albumin excretion ( uae ) rate was measured by enzyme immunoassay from a single 24-h urine collection ( 15 ) using immunoturbidimetry ( eiken , tokyo , japan ) . patients were classified into three groups : those with no diabetic nephropathy ( uae < 20 g / min ) , those with microalbuminuria ( mau ) defined as a uae of 20200 g / min , and those with overt proteinuria with uae > 200 g / min . diabetic retinopathy was assessed from retinal photographs at baseline , and the findings were reviewed by one ophthalmologist . all of the results are expressed as means sd or median ( range ) . statistical analyses were performed using spss statistical software ( spss , chicago , il ) . because of their skewed distribution , triglyceride and hdl cholesterol concentrations and microalbuminuria levels were compared after logarithmic transformation . tests were used to test the univariate relationships of categorical variables , and independent student 's t tests were used for continuous variables . we used multiple logistic regression analysis to test associations between the outcome ( progression of aft at follow - up ) and potential explanatory variables . the relationships were analyzed after adjustment for the following risk factors : age , duration of diabetes , presence of hypertension , smoking , diabetic foot ulceration , and diabetic retinopathy and nephropathy . all of the results are expressed as means sd or median ( range ) . statistical analyses were performed using spss statistical software ( spss , chicago , il ) . because of their skewed distribution , triglyceride and hdl cholesterol concentrations and microalbuminuria levels were compared after logarithmic transformation . tests were used to test the univariate relationships of categorical variables , and independent student 's t tests were used for continuous variables . we used multiple logistic regression analysis to test associations between the outcome ( progression of aft at follow - up ) and potential explanatory variables . the relationships were analyzed after adjustment for the following risk factors : age , duration of diabetes , presence of hypertension , smoking , diabetic foot ulceration , and diabetic retinopathy and nephropathy . of the 1,021 patients who had normal cardiovascular aft results , 783 ( 76.7% ) completed a follow - up aft evaluation ( fig . the total study population consisted of 392 men and 629 women with mean sd age of 50.2 10.7 years . duration of diabetes was 7.1 6.5 years , and a1c level was 8.6 2.2% . the 783 participants who were evaluated did not differ from the 248 patients who were not assessed , with respect to age ( 50.7 10.4 vs. 51.7 11.6 years , p = 0.190 ) , sex ratio ( p = 0.649 ) , presence of hypertension ( p = 0.279 ) , duration of diabetes ( 7.7 6.7 vs. 7.4 7.7 years , p = 0.390 ) , or a1c level ( 8.7 2.0% vs. 8.5 2.4% , p = 0.915 ) . during follow - up , seven patients died : four women and three men . the causes of death were sepsis in two patients , myocardial infarction in two patients , cerebral hemorrhage in one patient , and malignancy in two patients . 307 ( 39.2% ) patients had hypertension and all of them were taking antihypertensive medication . thirty - two ( 10.4% ) patients were treated for hypertension with calcium channel blockers , 6 ( 2.0% ) with -blockers , 140 ( 45.6% ) with ace inhibitors or angiotensin receptor blockers , 4 ( 1.3% ) with thiazides , and 125 ( 40.7% ) with any combination of antihypertensive agents . baseline sex ratios and bmis were similar , and there was no difference in the treatment of diabetes ( p = 0.773 ) or hypertension ( p = 0.326 ) in the progressor and nonprogressor groups . however , the patients with progression were older ( p < 0.001 ) with a longer duration of diabetes ( p < 0.001 ) , more were smokers ( p = 0.045 ) , and more had hypertension ( p < 0.001 ) . moreover , the progressor group had higher levels of fasting glucose ( p = 0.004 ) , postprandial glucose ( p = 0.010 ) , and microalbuminuria ( p = 0.002 ) than patients without progression ( table 1 ) . however , there were no differences in total cholesterol , triglyceride , or hdl cholesterol levels between the two groups . the patients with progression also showed higher frequencies of diabetic nephropathy ( p = 0.004 ) and diabetic retinopathy ( p < 0.001 ) during the observation period than the patients in the nonprogressor group ( table 1 ) . at follow - up , 270 patients ( 34.5% ) showed abnormal cardiovascular aft scores ( total score 1 , the progressor group ) . the results of the deep breathing , valsalva maneuver , and lying - to - standing tests were abnormal in 59 ( 21.9% ) , 210 ( 77.8% ) , and 159 ( 58.9% ) patients , respectively . in terms of total score , 138 ( 51.1% ) patients had a score of 1 , 106 ( 39.3% ) had a score of 2 , and 26 ( 9.6% ) had a score of 3 . the distribution of abnormal e / i ratio , valsalva maneuver , and posture ratios in patients with a total score of 1 ( n = 138 ) at follow - up was 12 ( 8.7% ) , 83 ( 60.1% ) , and 43 ( 31.2% ) patients , respectively . even among the 133 patients with newly diagnosed diabetes at baseline , 43 ( 32.3% ) showed abnormal aft results at follow - up , with total scores of 1 . these included an abnormal e / i score in 4 ( 9% ) , an abnormal valsalva ratio score in 34 ( 79% ) , and an abnormal posture score in 21 ( 49% ) ( table 2 ) . logistic regression analysis revealed that the mean value of biannually measured a1c was correlated significantly with the future development of can . compared with the patients whose mean a1c value was 7.0% , having a level of > 11.0% the conventional risk factors , such as age ( p < 0.001 ) , diabetic retinopathy ( p = 0.036 ) , and diabetic nephropathy ( p = 0.035 ) at baseline predicted the progression of can after adjustments were made for baseline age , hypertension , smoking habit , and the duration of diabetes . duration of diabetes or having a smoking habit was not associated with the progression of aft . in this study , this was a longitudinal observational study designed to investigate factors that might influence the progression of can in patients with type 2 diabetes . we demonstrate that can progressed according to glycemic control status and that it was associated with preexisting chronic diabetes complications . diabetes is an important risk factor for development of cardiovascular and cerebrovascular diseases ( 16,17 ) . dan is a serious and common complication of diabetes associated with an increased risk of cardiovascular mortality . many studies have shown consistently that there is an increased risk of cardiovascular mortality among patients with an abnormal can assessment , compared with those with normal assessment ( 18,19 ) . therefore , early detection of high - risk diabetic patients and aggressive intervention should be promising therapeutic strategies . in 2005 , a consensus statement from the american diabetes association ( ada ) recommended the battery of tests for the assessment of can including heart rate variability ( hrv ) , e / i ratio , response to the valsalva maneuver , and standing up ( 2,14 ) . hrv methods are age dependent but independent of the intrinsic heart rate ( 2,14,20 ) . until now , measuring hrv has been a standard screening method in the diagnosis of autonomic dysfunction . aft is easy to perform and is a reproducible test using a simple device at the patient 's bedside ( 21,22 ) . there have been limited observations with regard to the progression of can in patients with type 2 diabetes . poor glycemic control plays a central role in its development and progression ( 12,13 ) . moreover , strict glycemic control can slow the progression and delay the appearance of abnormal afts ( 10,23 ) . in the european diabetes ( eurodiab ) prospective complication study , the incidence of neuropathy was 23.5% , and the change in a1c value during a follow - up period of 7 years was independently associated with the incidence of neuropathy ( 11 ) . the diabetes control and complications trial ( dcct ) research group also documented that intensive therapy can slow the progression and the development of an abnormal autonomic test . however , these well - performed prospective studies were only conducted on patients with type 1 diabetes . similar to the eurodiab study , we found that 35% of our patients showed progression of can . even in the patients with newly diagnosed type 2 diabetes , 30% showed abnormal aft results after 7.5 years . we used the mean a1c value , measured every 6 months for 7.5 years , as a representative index of glycemic control status . blood glucose control , as determined by a1c levels , seemed to be improved more during follow - up in the progressor group than in the nonprogressor group . however , the mean a1c values during the observation period were significantly higher in the progressor group . we suggest that , as for type 1 diabetes , glycemic control could influence the development and progression of can in patients with type 2 diabetes . in addition , previous reports about can were designed to test only the e / i ratio or orthostatic hypotension . however , in our results , each of the three aft tests showed a different rate of progression ; a partial measurement of aft offers only limited information about can . with use of the ada criteria for the diagnosis of can in this study , the frequency of abnormal aft results were , in order , the valsalva maneuver , posture , and the e / i ratio among patients whose initial aft results were normal . we suggest that the valsalva ratio is the most sensitive parameter of the three values . interestingly , the duration of diabetes was not significantly associated with the development of can . although diabetes duration has been reported to play an important role in patients with type 1 diabetes ( 12 ) , the influence of diabetes duration seems to play a less important role in type 2 diabetes . consistent with previous studies , we found an association between can and microvascular complications ( 24,25 ) . can progression is correlated with diabetic retinopathy , diabetic nephropathy , and an increased urinary microalbumin excretion rate . this finding suggests that clinicians should pay more attention to patients with diabetic retinopathy or nephropathy , over and above the strict glycemic control required for the prevention of can first , there have been no normal values established for the e / i ratio , valsalva maneuver , and posture tests specific to normal korean subjects or to korean patients with diabetes . it remains to be clarified whether the normal values for western populations can be applied to korean subjects ; however , we performed aft with the same method by one examiner throughout . second , tests of blood pressure response to standing , blood pressure response to a sustained handgrip , and the presence of orthostatic hypotension were not checked because these tests were not available in our diabetes center from 1999 to 2000 . however , each of the three tests of cardiovascular autonomic function used had a sensitivity and specificity of > 90% ( 2 ) . moreover , the ada suggests that the three tests used here adequately fulfill their requirements because of their reliability , reproducibility , general mutual correlation , and correlation with the tests of peripheral somatic nerve function and well - established normal values . however , our ongoing follow - up studies should provide some insight into this issue . in summary , prolonged exposure to uncontrolled hyperglycemic conditions could predict future can , independent of the duration of diabetes among patients at baseline . cardiovascular aft should be monitored to pay attention to major potential cardiovascular complications even in asymptomatic patients , but especially among those for whom glycemic control is not maintained adequately .

objective we investigated whether cardiovascular autonomic dysfunction was associated with glycemic control status over time in patients with type 2 diabetes.research design and methods from 1999 to 2000 , cardiovascular autonomic nerve function testing ( aft ) was performed on patients with type 2 diabetes ( n = 1,021 ) and was followed - up in 2006 and february 2008 . tests for cardiovascular autonomic functions measured heart rate variability parameters ( expiration - to - inspiration [ e / i ] ratio , responses to the valsalva maneuver , and standing ) . aft scores were determined from the results of the each test as follows : 0 for normal and 1 for abnormal . we began with those who had a score of 0 and assessed the changes in total score along with biannual a1c levels.resultsat follow - up , the development of cardiovascular autonomic dysfunction was 34.5% ( e / i ratio 21.9% , valsalva maneuver 77.8% , and posture 58.9% ; n = 783 ) . the development of cardiovascular autonomic dysfunction was higher in older patients ( p < 0.001 ) ; in those with longer duration of diabetes ( p < 0.001 ) ; of hypertension ( p = 0.005 ) , and of diabetic retinopathy ( p < 0.001 ) ; and in those who had higher levels of microalbuminuria ( p = 0.002 ) . logistic regression analysis revealed that the development of cardiovascular autonomic dysfunction was strongly associated with the mean a1c level during the follow - up period ( mean a1c > 9.0% vs. 7.0% , odds ratio 2.984 , 95% ci 1.1777.561 ; p = 0.021).conclusions the development of cardiovascular autonomic dysfunction was independently associated with microvascular complications and glycemic control status during this 7.5-year follow - up in patients with type 2 diabetes .

RESEARCH DESIGN AND METHODS Statistical analyses RESULTS CONCLUSIONS

since the introduction of the cephalometer in 1931 , cephalometric analysis has become an important clinical tool in diagnosis , treatment planning , evaluation of growth , or treatment results and research [ 2 , 3 ] . recently , due to the affordability of digital radiographic imaging , the demand for the medical profession to completely automate analysis and diagnostic tasks has increased . in this respect , automatic cephalometric analysis is one of the main goals , to be reached in orthodontics in the near future . the main problem , in automated cephalometric analysis , is landmark detection , given that the measurement process has already been automated successfully . different approaches that involved computer vision and artificial intelligence techniques have been used to detect cephalometric landmarks [ 522 ] , but in any case accuracy was the same or worse than the one of manual identification ; for a review see leonardi et al . . none of the proposed approaches solves the problem completely , that is , locating all the landmarks requested by a complete cephalometric analysis and with accuracy suitable to clinical practice . it should be emphasized that reliability in the detection of landmarks is mandatory for any automatic approach , in order to be employed for any clinical use . as previously stated , among the possible factors that reduce reliability the loss of image quality , inherent to digital image conversion and compression in comparison with the original radiograph , has been claimed [ 3 , 23 , 24 ] . in fact , this analogue - to - digital conversion ( adc ) results in the loss and alteration of information due to the partial volume averaging ; consequently many edges are lost or distorted . to the best of our knowledge , every study on automatic landmarking has been carried out on scanned lateral cephalograms transformed into digital images , and this could explain in some way the inaccuracies of automatic location compared to the manual identification of landmarks . recently , a new hybrid approach , which is based on cellular neural networks ( cnns ) , has been proposed for automatic detection of some landmarks [ 21 , 22 ] . results of evaluation of the method 's performance on scanned cephalograms were promising ; nevertheless , for some landmarks the error in the location was often greater than the one of manual location . due to the promising results already obtained with cnns [ 21 , 22 ] , the aim of this study was to evaluate the accuracy of the ccns - based approach for the automatic location of cephalometric landmarks on direct digital cephalometric x - rays . thus the method proposed in [ 21 , 22 ] has been extended in two respects : by improving the algorithms employed to detect 7 landmarks and by developing the algorithms needed to locate 3 additional landmarks ( porion , basion , and pterygoid point ) ; of these latter , two especially difficult landmarks ( basion and pterygoid point ) that are used in the most common cephalometric analysis were located for the first time in literature . for an overall evaluation of the clinical viability of automated landmarking of this extended method , in this investigation the following null hypothesis was tested : there is no statistically significant difference in accuracy between the 10 landmarks automatically located by this approach and the true location of every landmark . forty - one lateral cephalometric radiograph files taken at the orthodontic department of policlinico , university hospital of catania , italy , were used in this study . a written informed consent to participate in the study , approved by the ethical committees of the relevant institution , was obtained from all subjects . the radiograph files were randomly selected , disregarding the quality , from the patients currently undergoing orthodontic treatment within the department . the type of occlusion and the skeletal pattern were , deliberately , not taken into consideration in the study design . exclusion criteria were the following : obvious malpositioning of the head in the cephalostat , unerupted or missing incisors , no unerupted or partially erupted teeth that would have hindered landmark identification , patients with severe cranio facial deviations , and posterior teeth not in maximum intercuspation . x - ray files collection was approved by the local research ethics committee , and informed written consent was obtained from each subject . the direct - digital cephalometric radiographs were obtained by a siemens orthophos ds ceph ( sirona dental , bensheim , germany ) . the signals are acquired at a bit depth of 12 bit ( 4096 grey levels ) , but this is subsequently reduced in the default preprocessing procedure to 8 bits ( 256 grey levels ) . the resulting image is saved as tiff file at 300 dpi ; thus the pixel size in the image was 0.085 mm and the resolution was 11.8 pixels per mm . the exposure parameters for the digital cephalographs were 73 kv , 15 ma , and 15.8 seconds . according to the manufacturer 's specifications , the machines provide focus - to - receptor distances of 1660 mm . the digital images were stored on a personal computer with intel pentium iv , 3.2 gh with 2 gb ram , 300 gb hard disk ( asustek computer incorporated ) with microsoft windows xp professional service pack 2 as operating system and were displayed on a 19-inch flat tft screen ( samsung syncmaster 913 v ) , set to an average resolution of 1280 1024-pixel , with bandwidths between 60 and 75 hz , and a dot pitch of 0.294 mm , with standard setting : 80% for contrast and 20% for brightness ) , at first to find the best estimate for each landmark and thereafter to obtain the cephalometric points automatically located . a software tool was designed and implemented in borland c++ version 5.0 produced by borland software corporation ( austin , texas , usa ) . this software tool allowed the digitization of landmarks by experienced orthodontists directly on the x - ray shown on the monitor as well as their recording on an x - y system . prior to the study the apparatus was checked for its accuracy by repeated recording of an image of known exact dimensions and by measuring known distances . the 41 cephalograms were landmarked directly on the computer monitor , by experienced orthodontists to provide the best estimate . five orthodontists with at least 6 years of clinical experience from the orthodontic department of catania university evaluated the images . an agreement was reached on the definitions of landmarks before carrying out this study , and these written definitions for each landmark were reviewed with and provided to the 5 evaluating participants ( table 1 ) . 10 landmarks ( table 1 ) were target of automatic landmark identification ; the observers were asked to identify them . complete anonymity of the 41 films was maintained with image code names and random assignment of the images to study participants . landmarks were pointed by using a mouse controlled cursor ( empty arrow ) linked to the tracing software in a dark room , the only illumination being from the pc - monitor itself . no more than 10 radiographs were traced in a single session to minimize errors due to the examiner 's fatigue . every landmark was expressed as x ( horizontal plane ) and y ( vertical plane ) coordinates with an origin fixed to a given pixel . location of every landmark or best estimate was defined as the mean of these five records from the five observers . the mean clinicians ' estimate was then used as a baseline to be compared with the cephalometric points detected by the automated system . the automatic analysis was undertaken once on each of the 41 images to detect the same 10 landmarks ( table 1 ) . the approach used for automated identification of cephalometric points is based on cellular neural networks ( cnns ) . a cnn is an analog dynamic processor , where dynamics can be described either in a continuous manner ( continuous time cnn or ct - cnn ) or in a discrete manner ( discrete time cnn or dt - cnn ) . cnns are formed by a set of processing elements , called neurons , usually but not necessarily arranged along a matrix in two or more dimensions . communication is allowed between elements inside a neighborhood , whose size is defined by the user . the dynamics of each neuron depends on the set of inputs on its state and produces one output ( there are also cnn variations with multiple outputs ) . in general , the state of a computational element is a linear combination of inputs and outputs . in this case we have linear cnn , the type used in this work . since every neuron implements the same processing function , the use of cnn is suitable for image processing algorithms . in this work we deal with a 2d matrix topology . in this framework a cell in a matrix of m n is indicated by c(i , j ) . the neighborhood of the interacting cells is defined as follows : ( 1)nr(i , j)={c(k , l):max ( |ki|,|ij|)r , 1km ; 1kn}. cnn dynamics is determined by the following equations , where x is the state , y the output , and i the input : ( 2)xi , j=xi , j+c(k , h)nr(i , j)a(i , j , k , h)ykh(t ) + c(k , h)nr(i , j)b(i , j , k , h)uk , h(t)+i , yi , j=12(|xi , j+1|+|xi , j1| ) . a compact representation of the cnn is by means of a string called gene that contains all the information needed for its simulation ; for a 5 5 neighborhood this gene is represented by 51 real numbers : the threshold i , twenty five control ( feed forward ) coefficients for the b matrix ( bi , j ) , and twenty five feedback coefficient for the a matrix . the values in the a and b matrices correspond to the synaptic weights of a neural network structure . matrices a and b are known , respectively , as feedback and control templates , and the way each cnn processes its input is determined by the values of these two templates and by the number of cycles of computation . cnns are especially suitable for image processing tasks because they allow pixel by pixel elaboration taking into account the pixel neighborhood . libraries of known templates for typical image processing operations are available [ 27 , 29 ] and the sequential application of different templates can be used to implement complicated image processing algorithms . to completely define the previous equations the boundary conditions for those cells whose typical boundary conditions are the dirichlet ( fixed ) boundary condition , in which a fixed ( zero ) constant value is assigned to the state xi , j to all cells that lie outside the input matrix , and the neuman ( zero flux ) condition , in which the state xi , j of corresponding cells perpendicular to the boundaries is constrained to be equal to each other . finally , equation ( 2 ) is fully defined if we specify the initial state xi , j(0 ) for all the cells . frequent choices for this initial state matrix are all zero or equal to the input image . various cnn models have been proposed for medical image processing . in the 128 128 cnn - um chip has been applied to process x - rays , computer tomography , and magnetic resonance image of the brain , by applying different cnn templates . discuss two classes of cnn , in particular binary cnn and multivalued cnn , and their use for image filtering and enhancement . in gacsdi et al . presents image enhancement techniques such as noise reduction and contrast enhancement with cnn and discuss the implementation on chips . giordano et al . applied cnn techniques to cephalometric analysis . by choosing appropriate templates , chosen to take into account different x - ray qualities ( in terms of brightness and contrast ) , all filtering task can be performed in a more robust and precise way . then - edge based , region - based , and knowledge based tracking algorithms are used to find the different landmarks . this technique has also been used to find landmarks in partially hidden regions , such as sella and orbitale . these studies have demonstrated that cnns are versatile enough to be used also for the detection of landmarks that are not located on edges , but on low - contrast regions with overlapping structure . with respect to other image filters , such as the one obtained by applying to the image the laplace , the prewitt , or the sobel operators , binary cnns can obtain a more precise edge detection , especially in case of a small difference between brightness of the neighbor objects . in this work , to locate the new landmarks porion , basion , and pterygoid point an adaptive approach is proposed , by which the cnn parameters are chosen adaptively based on the type of landmark to be located and on the x - ray quality , this is followed by the application of algorithms for landmark location that encode knowledge about anatomical constraints and perform an adaptive search strategy . the software that performs the automated landmarking has two main processing modules : a cnn simulator that suitably preprocesses the digital x - ray accordingly to the landmark being sought , and a module containing a set of algorithms that apply anatomical knowledge ( morphological constraints ) to locate each landmark on the preprocessed image . a first image preprocessing step is applied in order to eliminate noise , and enhance brightness and image contrast . then the method proceeds with a sequence of steps in which identification of the landmarks coordinates is done , for each point , by appropriate cnn templates followed by the application of landmark - specific search algorithms . the cnn simulator has been developed in borland c++ version 5.0 and proceeds with a sequence of steps in which the landmarks of table 1 are identified in their x and y coordinates . a detailed description of the simulator and of the interface of the landmarking tool has been previously reported [ 21 , 32 ] ; in this work we use a new version that allows the landmarking of the three new points and also has additional facilities , such as allowing the user to define an arbitrary portion of the image to be processed , an adaptative brightness improvement algorithm , a contrast enhancement function , possibility to store the cnn output as initial state or as initial input value or both , and to allow the execution of an algorithm specified as a sequence of templates . the grey level of each image pixel is normalized in the interval ( 1 , 1 ) where 1 corresponds to white and 1 to black . the interface ( figure 2 ) allows to set the template parameters , the boundary conditions ( dirichlet the default , or zero - flux ) , the initial state x(0 ) , and the input value u for each network cell . in the present work we use cnn output not necessarily in the steady state . for this investigation new algorithms have been implemented , to locate new landmarks ( porion , basion and pterygoid point ) and to improve landmark identification accuracy for the previously detected ones ( nasion , a point , b point , protuberance menti , pogonion , upper incisor edge , and lower incisor edge ) [ 21 , 22 , 33 , 34 ] . the feedback and control templates used by the software have been designed based on knowledge of cnn templates ' behavior and subsequent fine - tuning . the search algorithms are based on a laterolateral head orientation but do not require a head calibration procedure or a fixed head position . the cnns are simulated , if not otherwise stated , under the following conditions : ( 1 ) initial state : every cell has a state variable equal to zero ( xij = 0 ) ; ( 2 ) boundary condition : uij = 0 ( dirichlet contour ) . the majority of the feedback templates ( a ) used in this work are symmetrical , which ensures that operations reach a steady state , although in our approach we exploit the transient solutions provided by the cnns . for these reasons number of cycles and integration steps become important information for point identifications . if not mentioned otherwise , we consider bias = 0 ; integration steps = 0.1 ; cycle 60 . in the following we exemplify the methodology that has been used . since the incisors configuration variability is high , in this work a configuration where the up incisor is protruding with respect to the inferior one has been hypothesized . after noise removal and preprocessing , two steps are performed : the first one uses the control template reported in figure 3 , and the second one uses the control template reported in figure 4 . in both cases bias = 0.1 ; integration step = 0.2 and 60 cycles . with these templates we search for the more protruding tooth and hence find its tip . from this point we proceed for searching the tip of the second incisor that , under our hypothesis , is located downward . a simple extension will be to remove the hypothesis and find the tip in an roi going upward or downward . in order to find the points a and b we apply the template shown in figure 5 , with bias 0 and 25 cycles . the landmark search starts from the upper incisor , for which only a rough approximation of the landmark coordinates is needed , since the algorithm is robust even for error greater than 5 millimeters in up incisor location ( error never experienced by our method in the processed x - rays ) . the a point is searched going up following the bone profile and stopping when the bone profile column stops decreasing ( going to the left ) and starts increasing ( going to the right ) or when a jump in the column coordinate of the profile is found . differences in the luminosity level in the x - ray that produce a different output level in the cnn are taken into account by using a dynamic threshold to find the bone profile : a threshold of 0.99 that corresponds to a white normalized color is used to start ; if there is not a pixel whit this level in the considered row , the threshold is increased by 0.01 , until a bone pixel is found . then the coordinates of the candidate a point are checked : if the point is too much to the left compared to the up incisor or too much up or near the up incisor the search is repeated by a more aggressive saturation : in the previous template the 1 values are replaced with 2 and the result is checked against the soft tissue profile , since a greater saturation also highlights the soft tissue profile . for b point a similar procedure is used , but instead of going upward the bone profile is followed going downward from the up incisor ; also in this case the checking procedure may lead to repeat the process with a more aggressive saturation . soft tissue identification is avoided by comparing the column of the candidate with the column of the incisor : if the column of the candidate is too near or to the right to the incisor column , we have found a point in the soft tissue . the result is corrected by finding the first highlighted point in the same row but to the left ( the bone ) . soft tissue information could be used to overcome problems in finding the b point for symphysis of type b and d that present a flat or almost flat profile . in this case the soft tissue profile can guide the search in order to find the row and then from the located row search the column corresponding to the bone profile . in order to find the nasion two steps are needed : first the most anterior point in the frontal bone profile is located , and then from this point the bone profile is followed searching for the posterior point at the intersection with the nose bone profile . in order to find the most anterior point of the frontal bone after noise removal the image is binarized as shown in figure 6 . in the binarized x - ray since binarization could lead to bone removal ( as shown in figure 6 ) , the point is double checked by applying a similar search in the x - ray after applying the template in figure 7 with bias = 0 and 25 cycles . thus a search for other highlighted pixels to the left is performed , and if a point to the left with a column value greater than the value found in the binarized image is found , this is taken as the posterior point in the frontal bone . as is shown in figure 7 , the template has highlighted the frontal bone profile and the nose profile . by following this profile , we search for the anterior point and the intersection between the frontal bone profile and the nose bone profile , that is , the nasion . the found coordinates are checked : if nasion coordinates are equal or too close to the posterior point of the frontal bone , we are dealing with an x - ray with a flat bone profile . in this case the search is repeated starting from few rows downward or by a less aggressive saturation ( by replacing 3 with 2 in the previous template ) since a greater saturation tends to flatten all the bone profiles . in order to find pogonion and protuberance menti , first the jaw profile is highlighted by applying the template of figure 8 , with bias = 0 and 25 cycles , and is used as starting point for the search . the pogonion and protuberance menti are found by following the bone up in the front profile . this profile is highlighted by a vertical derivate template , such as the one reported in figure 5 , with parameters depending on the luminosity level in the region of interest ( roi ) . in order to find the landmarks basion and porion , the preprocessing step aims to find the width of the spine including the soft tissue , its axis , and a bell - shaped region of interest ( roi ) that , with high probability , will contain the basion and porion . to find this roi the template of figure 9 with bias 1.34 and 3 cycles is applied . then a highlighted zone ( values of the output < = threshold ) is searched in the first 2/3 of the x - ray height and in the left half of x - ray width . the threshold is dynamically changed from a starting point of 0.99 until a region of white pixels of suitable size is found . then a rounding of the zone is performed by cutting the tails of the bell - shaped area . the process is repeated with the same templates but with fewer cycles in order to give robustness to the algorithm and take into consideration differences in the x - rays quality . if the difference between the two procedures is too high , the second result is taken , otherwise the first one is taken since basion lies at the bottom of the region : if the zone is too narrowed the searched point can be lost the width of the spinal column including the soft tissue is found by applying the template that corresponds to a vertical derivative that sharpens vertical edge . the template , applied with bias = 0 for 25 cycles , and its result are shown in figure 10 . the landmark basion will be searched in the second half of the height of the found roi for basion and porion landmarks . its location is checked against anatomical constraints , in particular its distance from the spine and its upper bone , length of the occipital bone starting from the candidate basion , and its position with respect to the spine axis . to locate the basion we first apply the template in figure 11 with bias = 0 for 25 cycles . the value of the template could be increased until at least a point in the roi is highlighted . the cnn output is stored on the initial state of the cnn , and the template of figure 12 is applied in order to follow the occipital bone ( that must be present and have a sufficient length ) and check the basion against anatomical constraints . the template is applied with bias = 0.8 and 5 cycles . in order to find porion , starting from the same roi used to find basion , the template shown in figure 13 is applied , with bias 1.34 and 1 cycle ; then the area is binarized and the inside is searched for black rounded shape spots resembling the auditory conduct . for better separation of the spots from the walls of the area the same template is re - applied with a greater number of cycles ( 2 cycles ) , the image is binarized , the spots are located , and the union with the black spots located with the previous template is performed . for each spot , parameters such as its area , its height , its width , its shape , and the center are determined . these parameters are used to discriminate between spots that correspond to auditory conducts and spots that are noise . figure 13 shows the area binarized for the application of the template with 1 and 2 cycles . in this case if the parameters of the spots do not satisfy anatomical constraints ( i.e. , area , or width , or height ) or the located landmark is too far from the center of the spot where it lies the same template with an increased bias until the spots and the located landmark satisfy the anatomical constraints , the algorithm is able to correctly find the porion landmark even if in the x - ray both the auditory conducts are visible . in this case the landmark is computed as the middle point between the two auditory conducts . figure 14 shows the location of the automatically detected porion ( in green ) and the expert location ( in red ) in three different cases . in order to locate the pterygoid point , first a roi delimited by the right side of the basion - porion roi and by the left most side of the ocular cavity is located . the ocular cavity is highlighted by applying the feedback template of figure 12 with bias = 1.34 and 3 cycles , followed by image binarization . the next step consists in locating the pterygoid fissure ; this is done in two steps : first we locate the right wall of the fissure by the template in figure 15 , with bias = 0 and 60 cycles . the template parameters and the number of cycles are changed until the right wall of the pterygoid fissure is detected , with a search constrained by distance from the orbital cavity . the left wall is searched by applying a cnn with similar templates but different parameters , and by using a dynamic threshold to differentiate between white and black pixels and to follow it until the upper part of the pterygoid fissure is reached and hence the landmark is located . the landmark coordinates are checked against the distance from the right wall of the fissure . if the constraint is not satisfied , a recovery procedure , that depends on the distance between the landmark and the right wall of the fissure ( too low or too high ) , is implemented by changing both the templates ( more aggressive or less aggressive ) and the threshold used to follow the highlighted wall of the fissure . landmarks from the automatic system 's estimate with the best estimate ( mean clinician 's estimate ) were compared in a horizontal ( x ) and vertical ( y ) coordinate system at first in pixels . the mean errors and standard deviations from the best estimate were calculated for each landmark . mean errors in this study were defined as mean magnitude in distance between the best estimate and selected landmarks for all the 41 radiographs . the data set of point coordinates obtained from the landmarking software was screened for outliers , and for each landmark coordinate mean substitution was used for the missing data points . differences in the absolute mean errors of automatic landmarking and the best estimate were compared with a 1-way analysis of variance to test the null hypothesis that the mean errors obtained by automatic landmark detections are the same of mean value errors obtained by manual detection . if p < .05 , the test rejects the null hypothesis at the = 0.05 significance level . all statistical analyses were done with the software statistical package for social science ( spss inc , chicago , usa ) . landmarks automatically detected and best estimates obtained from 41 randomly selected digital images were available for statistical analysis . a first indication on the rate of success of the method is provided by the analysis of the outliers . the number of outliers was different according to the point sought . for 4 of the points ( pogonion , protuberance menti , upper incisor edge , and lower incisor edge ) the percentage of outliers was less than 3% ( 0 or 1 case out of 41 ) ; for 3 points ( nasion , basion , and porion ) it was less than 10% ( 3 or 4 cases out of 41 ) ; for 3 points ( a point , pterygoid point and b point ) it was between 15% and 20% ( 6 to 8 cases out of 41 ) . seven out of twenty measurements ' errors were statistically significant ( p < .05 ) . however , the magnitude of mean errors between automatic identification of each landmark and the best estimate of cephalometric points was very small ; in fact every error landmark automatically detected was found within 0.59 mm with respect to the best estimate . only 1 measurement ( a point ) had values above 0.5 millimeter on the x axis , and 1 measurement ( porion ) above 0.5 millimeter on the y axis ( table 2 ) . the total time required to automatically extract all the ten landmarks from each individual x - ray was 4 minutes and 17 seconds . our investigation was carried out on 41 randomly selected x - ray files of direct digital radiography , deliberately disregarding the quality in order to simulate clinical condition . errors between automatic identification of each landmark and the best estimate of cephalometric points were different in the horizontal ( x ) and vertical ( y ) coordinate system . some cephalometric points yield better results on the horizontal ( x ) axis ( nasion , pogonion , porion , and protuberance menti , ) ; others showed less error on the vertical ( y ) axis ( a point and pterygoid point ) . this is in line with the statement that the distribution of errors for many landmarks is systematic and follows a typical pattern ( noncircular envelope ) . in fact it has been reported that some cephalometric landmarks are more reliable in the horizontal dimension while others are more reliable in the vertical dimension . the reasons for these differences in distribution of landmark identification error are often related to the anatomical variability of the landmark location . for 6 landmarks out of 10 , it was possible to carry out a comparison with data on accuracy reported by a meta - analysis study . most of the points , automatically detected in our study , showed lower errors compared to findings obtained for each point by meta - analysis . however , even if some differences between automatic detection and best estimate were statistically significant , the mean errors were so low to be , in most instances , clinically meaningless . all in all , a better level of accuracy , with respect to our previous data [ 21 , 22 ] and findings reported in literature was obtained in this study . there could be at least two reasons for this improvement , namely , the use of a softcopy of the digital x - rays , ( therefore , no need of analogue - to - digital conversion and an increased resolution of the x - ray file ) and the use of improved algorithms that worked with the cnns technique . in fact , before the introduction of direct digital radiography or the indirect one through stored image transmission technology , digital forms of cephalometric images were obtained by indirect conversion of analogical x - rays , that is , by scanning hard copies of radiographs or using a video camera . every previous study on automatic cephalometric landmarks detection was limited to these types of conversion , which not only required an additional time consuming step but could also introduce errors that lead to distortion [ 21 , 23 , 33 ] . another issue with digital image is resolution , which has a significant impact on the outcome of automatic detection landmarks studies . the quality of a digital image is strongly dependent on the spatial resolution is , the relationship of grey level values of the pixels to the optical density of the radiograph and image display . the minimum resolution used by previous studies on automatic detection of landmarks may have contributed to the larger errors presented in their findings . the higher the resolution , the fewer landmark identification errors , automatic analysis will yield . the cost , however , is computation time and memory usage , unless there are used automatic techniques like ours that allow to the computerized systems to be implemented in a hardware chip form as reported previously , without penalizing time efficiency . it should be underlined that these problems related to image resolution are encountered mainly , if not only , when an automatic search of landmarks is performed and not when the human eye is involved , because there are available optical systems that have higher resolution than the human optical system . although our method has some limitations that actually hinder clinical application , for example , the 10 cephalometric points detected are not enough to perform a cephalometric analysis , and errors of some of them are very close but not better than manual identification , it seems to have a promising future in automatic landmark recognition . investigations on more landmarks to enable a complete automatic cephalometric analysis and on softcopy of cephalometric x - rays should be strongly encouraged as methods and techniques here presented may be of some help for a completely automated cephalometric analysis and if opportunely modified may be used one day in 3d cephalometry . an acceptable level of accuracy in automatic landmark detection was obtained in this study , due to the use of a softcopy of the digital x - rays and the use of improved algorithms that worked with the cnns technique . hypothesis tested had to be rejected for some cephalometric points , respectively , on their x - or y - axis or both . none of the differences in landmark identification error between the automatically detected and manually recorded points were greater than 0.59 mm . this indicates that any statistically significant difference between the two methods seems unlikely to be of clinical significance .

several efforts have been made to completely automate cephalometric analysis by automatic landmark search . however , accuracy obtained was worse than manual identification in every study . the analogue - to - digital conversion of x - ray has been claimed to be the main problem . therefore the aim of this investigation was to evaluate the accuracy of the cellular neural networks approach for automatic location of cephalometric landmarks on softcopy of direct digital cephalometric x - rays . forty - one , direct - digital lateral cephalometric radiographs were obtained by a siemens orthophos ds ceph and were used in this study and 10 landmarks ( n , a point , ba , po , pt , b point , pg , pm , uie , lie ) were the object of automatic landmark identification . the mean errors and standard deviations from the best estimate of cephalometric points were calculated for each landmark . differences in the mean errors of automatic and manual landmarking were compared with a 1-way analysis of variance . the analyses indicated that the differences were very small , and they were found at most within 0.59 mm . furthermore , only few of these differences were statistically significant , but differences were so small to be in most instances clinically meaningless . therefore the use of x - ray files with respect to scanned x - ray improved landmark accuracy of automatic detection . investigations on softcopy of digital cephalometric x - rays , to search more landmarks in order to enable a complete automatic cephalometric analysis , are strongly encouraged .

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

end - stage renal disease ( esrd ) is a potential outcome of chronic kidney disease ( ckd ) . the prevalence and incidence of esrd as a worldwide public health concern are increasing in developed and developing countries . according to the report published by the management center for transplantation and special disease ( mctsd ) affiliated to iran 's ministry of health in 2007 , the total number of patients with esrd undergoing renal replacement therapy ( rrt ) was 32,686 , which indicates a prevalence of 435.8 per million population ( population = 75 million people ) . usually , these patients are treated by hemodialysis , kidney transplantation , and peritoneal dialysis . these rrts were started in iran in 1964 , 1967 , and 1975 and at present kidney transplantation is the most common among them in iran ( 48.8% ) . kidney transplantation is the preferred treatment for most patients with esrd and is far more profitable than hemodialysis . the first kidney transplant in iran that was performed in shiraz , fars province , iran in 1967 was the first organ transplant among the countries that are current members of the middle east society for organ transplantation ( mesot ) . over the past three decades , the number of kidney transplants has steadily increased that is concurrent with the development of the primary health care system , reduction of mortality , population growth , increase of life expectancy , and a significant shift from communicable to noncommunicable diseases . there are three different resources for kidney transplants including living - related , living - unrelated , and deceased donors and the function of kidneys from living donors is better than from deceased individuals . in 2013 , there were 2,670 kidney transplants in iran including 1,501 living donors and 1,169 deceased donors . although kidney transplantation in iran started from living - related donors , currently the main source of kidney donations is living nonrelated donors . analyzing renal transplantation data can help to evaluate the effectiveness of transplantation activities and estimation of future costs in the health care system . several studies have been conducted to estimate the survival rate of renal transplantation in different transplantation centers of iran such as tehran , shiraz , mashhad , hamadan , but there is no any study about the survival rate in patients of one city who had undergone kidney transplantation in other centers . therefore , the aim of this study was to determine the organ survival rate after kidney transplantation during a period of 10 years ( march 2001-march 2011 ) in transplanted patients from arak , markazi province , iran . in this historical cohort study , all recipients of kidney transplantation from arak , in all transplant centers of iran who have medical records in valiasr hospital and charity for kidney patients of arak , markazi province , iran during a period of 10 years from march 2001 to march 2011 were included . in this period , 225 cases of transplantation , out of which 189 from living donors and 36 from deceased donors were performed . data collected by using checklists were completed from patients hospital records . in this study , the exact time of transplantation was considered as the initial event and the time of irreversible transplant rejection leading to a return to dialysis was defined as the end - point event . cases in which did nt experienced this event due to finishing the study , the patients who were lost to follow - up , or who died for reasons other than transplant rejection were considered as censored data . deaths due to surgical consequences immediately after surgery were excluded . there was no ethical problem in this research because the data were extracted from patient records without mentioning the name , and there was no any intervention on the patient . the research proposal was approved by the deputy of research of arak university of medical sciences with project number 1063 and the ethics committee with ethics number 92 - 160 - 14 . the studied variables were donor source ( deceased , related , unrelated ) , donor and recipient 's age , gender , recipient 's place of residency ( city , rural ) , family history of kidney transplantation , marital status ( single , married , widow ) , blood group ( a , b , ab , o ) , rh ( positive , negative ) , underlying cause of esrd ( diabetes , glomerulonephritis , hypertension , kidney poly cystic , other ) , and the number of dialysis after transplantation in 1 week . the obtained data were analyzed using statistical package for the social sciences ( spss ) 20 and stata 11 ( ibm spss statistics for windows , version 20.0 . kaplan - meier method was employed to determine the graft cumulative survival rate , log - rank test to compare survival curves in subgroups , and cox regression model to define the hazard ratio and for ruling out the intervening factors . the proportional hazards assumption for all variables was checked using the estat phtest , and the results confirmed this assumption . in this historical cohort study , all recipients of kidney transplantation from arak , in all transplant centers of iran who have medical records in valiasr hospital and charity for kidney patients of arak , markazi province , iran during a period of 10 years from march 2001 to march 2011 were included . in this period , 225 cases of transplantation , out of which 189 from living donors and 36 from deceased donors were performed . data collected by using checklists were completed from patients hospital records . in this study , the exact time of transplantation was considered as the initial event and the time of irreversible transplant rejection leading to a return to dialysis was defined as the end - point event . cases in which did nt experienced this event due to finishing the study , the patients who were lost to follow - up , or who died for reasons other than transplant rejection were considered as censored data . deaths due to surgical consequences immediately after surgery were excluded . there was no ethical problem in this research because the data were extracted from patient records without mentioning the name , and there was no any intervention on the patient . the research proposal was approved by the deputy of research of arak university of medical sciences with project number 1063 and the ethics committee with ethics number 92 - 160 - 14 . the studied variables were donor source ( deceased , related , unrelated ) , donor and recipient 's age , gender , recipient 's place of residency ( city , rural ) , family history of kidney transplantation , marital status ( single , married , widow ) , blood group ( a , b , ab , o ) , rh ( positive , negative ) , underlying cause of esrd ( diabetes , glomerulonephritis , hypertension , kidney poly cystic , other ) , and the number of dialysis after transplantation in 1 week . the obtained data were analyzed using statistical package for the social sciences ( spss ) 20 and stata 11 ( ibm spss statistics for windows , version 20.0 . kaplan - meier method was employed to determine the graft cumulative survival rate , log - rank test to compare survival curves in subgroups , and cox regression model to define the hazard ratio and for ruling out the intervening factors . the proportional hazards assumption for all variables was checked using the estat phtest , and the results confirmed this assumption . in this study , among the 225 patients followed up over 10 years , 30 cases ( 13.3% ) of irreversible transplant rejection that led to the initiation of dialysis or death was observed . 214 ( 95.11% ) out of 225 patients were followed successfully until the end of the study . the mean age of the kidney donors and recipients was 28.78 5.23 years and 36.42 14.34 years , respectively . frequency distribution of demographic and clinical variables in study the mean duration of follow - up was 55.43 42.02 months and the time at risk in the study was 12471.5 person - months . the mean of entry and exit time was 0 months and 55.68 months , respectively . as shown in figure 1 , by using the kaplan - meier method , the cumulative probability of graft survival and 95% confidence interval ( ci ) at 1 , 3 , 5 , 7 , and 10 years after transplantation were 99.10 ( 96.45 - 99.77 ) , 97.72 ( 93.89 - 99.16 ) , 94.33 ( 88.74 - 97.18 ) , 85.66 ( 77.27 - 91.13 ) , and 62.12 ( 48.45 - 73.15)% , respectively . graft survival rate in renal transplant recipients from arak , 2001 - 2011 as shown in table 2 , based on the log - rank test results , the recipients gender showed a significant relationship with the cumulative survival rate of kidney transplantation such that survival was significantly higher in female recipients compared with male recipients ( p = 0.02 ) . the recipient age was one of the affecting factors of survival so that survival in the age group of 21 - 40 years was significantly high and in the age group above 60 years was low ( p < 0.001 ) . also , in terms of the underlying cause of esrd , survival was significantly lower in diabetes and glomerulonephritis groups ( p = 0.01 ) . in the present study , kidney survival rate has not been significantly different in terms of following factors : donor 's gender ( p = 0.31 ) , donor 's age ( p = 0.69 ) , donor 's source ( p = 0.47 ) , recipient 's rh ( p = 0.14 ) , recipient 's residency ( p = 0.41 ) , recipient 's family history ( p = 0.38 ) , recipient 's marital status ( p = 0.24 ) , and recipient blood group ( p = 0.29 ) . comparison of survival rates at different time intervals including 1 year , 3 years , 5 years , 7 years , and 10 years after kidney transplantation in studied variables univariate cox regression model was used to determine the affecting quantitative variables on the kidney rejection that not entered in log - rank test . based on the results of univariate cox regression , the number of dialysis was significantly associated with cumulative probability of survival [ hazard ratios and 95% ci : 1.71 ( 1.21 - 2.40 ) ] so that for every one unit increase in the number of dialysis after transplantation , the hazard ratio of kidney transplantation rejection increased 1.71 times . the recipient 's weight was not significantly associated with cumulative probability of survival [ hazard ratios and 95% ci : 0.99 ( 0.96 - 1.01 ) ] . finally , in order to have a multivariate analysis and determination of the affecting variables on the kidney rejection , multivariate cox regression models were used . at this point , the variables that had a p value 0.2 in univariate analysis were entered into the model . results show that only the number of dialysis by controlling the effect of other variables had a significant association with the risk of kidney transplant rejection [ table 3 ] . this study showed that graft survival rates at 1 year , 3 years , 5 years , 7 years , and 10 years after transplantation in transplanted patients from arak city , markazi province , iran were 99.1 , 97.7 , 94.3 , 85.7 , and 62.1% , respectively . based on the report of iran organ procurement network , based on the results of the largest survival analysis in the country , so far 1- , 5- , 10- , and 15-year graft survival rates were 85% , 68% , 46% , and 24% , respectively . in the studies that were conducted in 2010 at the shiraz transplant center , the survival rates for these intervals from the deceased donor were 96.6% , 93.7% , 88.9% , 87.1% , and 85.8% , respectively , and from the living donor were 98.3% , 96.4% , 92.5% , 90.8% , and 89.2% , respectively ; the survival rate at intervals of 1 year , 3 years , and 5 years was less than our study but on the contrary , at intervals of 7 years and 10 years it was greater . in the study of javanrouh givi et al . in mashhad qaem hospital , 1- , 5- , and 10-year survival rates were estimated to be 98.3 , 92.5 , and 89.2% , respectively . in another study that was conducted in tehran , tehran province , iran in 2011 , the survival rates at 1 month , 1 year , and 5 years after transplantation were 93.2% , 89% and 82.5% , respectively , and the survival rate was lower than our study . in a study in taiwan the 5-year survival rate of kidney transplantation was estimated to be 84.7% that was lower than our study . saatchi et al . conducted a retrospective cohort study in hamadan province on 475 patients who had undergone kidney transplantation from 1994 to 2011 ; they found that 1- , 5- , 10- , 15- , and 18-year survival rates of transplantation were 97.1% , 92.3% , 86.2% , 77.6% , and 60.3% , respectively . kwon et al . conducted a similar study in south korea on 614 patients and reported that 1- , 5- , and 10-year survival rates of kidney transplantation patients were 92.62% , 82.37% , and 76.07% , respectively . based on the results of a single - center experience from turkey , 1-year graft survival rates were estimated to be 99.3% and 95.8% in the preemptive and nonpreemptive transplantation groups , respectively . in general , the observed differences on the one hand could be due to lack of synchronicity in these studies and changes in transplantation techniques or prescription drugs and type of immunosuppressive regimen for patients during the time while on the other hand they could be due to the nonuniform experience of transplant centers and physicians . also , this inconsistency could be attributed to the study setting , individual characteristics , and clinical status of the patients . in this study , in univariate model the cumulative probability of survival was significantly higher in female recipients compared to male recipients . this was consistent with the study of chen et al . in taiwan but was different from the study of hassanzadeh et al . the higher survival rate in women could be due to hormonal or immunological changes in them , however , this association was not significant after adjustment for the effect of other variables . a recent study has confirmed this issue and concluded that the association between graft rejection and sex was confounded by other variables . the donor 's gender showed no association with survival rate and this finding was consistent with the results of many studies while in some studies graft failure has been reported to be higher in women donors that has been attributed to lower body weight and fewer nephrons in women . in the present study , there was no significant association between the age of donors and survival rate of transplantation while the results of many studies and the literature review shows the negative effect of age on the cumulative probability survival of kidney transplantation . this negative effect attributes to the structural changes of the elderly renal parenchyma and also , inadequacy of the nephrons of elderly graft to fulfill the functional requirements of a poorly equipped recipient . however , survival in recipients aged 21 - 40 years was significantly high while in the age groups of above 60 years and below 20 years it was low , which was consistent with the results of many existing studies . the relationship between the recipient 's age and graft survival in young subjects is justifiable due to their small anatomical body parts and probability of the existence of technical problems , and in the older subject can be explained by loss of nephron function . in this study , no association was observed between the donor sources ( deceased , living - related , living - unrelated ) with the probability of graft survival while in the study of almasi - hashiani et al . , the percentage of irreversible rejections , which led to the initiation of dialysis in the cases of transplantation from deceased donors was more . also , this finding has been confirmed in other studies , which may have been caused by differences in the transmission system of donated organs or could have been due to cold ischemia time of the kidney since increasing the time of ischemia resulted in decreased graft survival . the absence of association in our study might have been due to the low sample size . in this study similar to most of the existing studies , there was no association between the underlying cause of esrd and graft survival . however , based on the study results of courtney et al . the underlying cause of esrd may influence the survival time . in the present study , dialysis time after transplantation had a significant effect on the survival of renal transplantation after adjusting the effect of other variables . based on the results of other studies , the waiting time on dialysis before transplantation was one of the largest independent risk factors for graft loss after kidney transplantation . the strength of this study was successful follow - up of survival rate of more than 95% of the patients while its major limitations were low sample size and conducting a retrospective cohort study and using medical records instead of prospective design . the lack of accurate registration of some factors such as creatinine in the patient 's records did not allow assessment of the effect of these variables . in this study , 1- , 3- , 5- , 7- , and 10-year graft survival rates were satisfactory and similar to the results of single center studies in the world . dialysis time after transplantation was a significant predictor of survival in recipients of kidney transplantation this issue should be considered during kidney transplant in order to increase the survival . mr designed and conducted the research , performed the statistical analyses , participated in all of the research , and prepared the manuscript . fs and sk designed and conducted the research , collected the data , and participated in the manuscript preparation . hs corrected the english manuscript , revised further statistical data , and participated in manuscript preparation . hj provided assistance in conducting the research , and participated in data - gathering and manuscript preparation .

background : kidney transplantation is a preferred treatment for many patients with end - stage renal disease ( esrd ) and is far more profitable than hemodialysis . analyzing renal transplantation data can help to evaluate the effectiveness of transplantation interventions . the aim of this study was to determine the organ survival rate after kidney transplantation during a period of 10 years ( march 2001-march 2011 ) among transplanted patients in arak , markazi province , iran.materials and methods : in this historical cohort study , all recipients of kidney transplantation from arak , markazi province , iran who had medical records in valiasr hospital and charity for kidney patients of arak , markazi province , iran during a period of 10 years from march 2001 to march 2011 were included . data collected by using checklists were completed from patients hospital records . kaplan - meier method was used to determine the graft cumulative survival rate , log - rank test to compare survival curves in subgroups , and cox regression model to define the hazard ratio and for ruling out the intervening factors . statistical analysis was conducted by statistical package for the social sciences ( spss ) 20 and stata 11.results:mean duration of follow - up was 55.43 42.02 months . by using the kaplan - meier method , the cumulative probability of graft survival at 1 , 3 , 5 , 7 , and 10 years was 99.1 , 97.7 , 94.3 , 85.7 , and 62.1% , respectively . the number of dialysis by controlling the effect of other variables had a significant association with the risk of graft failure [ hazard ratios and 95% confidence interval ( ci ) : 1.47 ( 1.02 - 2.13)].conclusion : this study showed that the graft survival rate was satisfactory in this community and was similar to the results of single - center studies in the world . dialysis time after transplantation was a significant predictor of survival in the recipients of kidney transplantation that should be considered .

INTRODUCTION MATERIALS AND METHODS Study design and participants Study's variables Statistical analysis RESULTS DISCUSSION CONCLUSION Financial support and sponsorship Conflicts of interest AUTHORS CONTRIBUTION

the rat genome database ( rgd , ) is the model organism database for the laboratory rat , rattus norvegicus . the primary goal of the database is to provide convenient access to high quality data about the genes and genome of the rat and to support the goals of researchers using the rat as a genetic and genomic model [ see ( 1 ) for a review of the impact of genomics in rat research ] . in pursuit of these goals we curate a variety of other relevant information such as mapping information ( genetic maps , radiation hybrid maps and most recently the genome assembly ) , quantitative trait loci ( regions of the rat genome believed to contain a gene or genes related to a particular phenotype or trait ) , rat strains used in genetic and genomic experiments and the microsatellite markers used to genotype inbred rat strains . this data is acquired from a variety of sources : the literature , other scientific databases and through bioinformatic analyses performed locally . the public website provides convenient access to this data through search tools , web page reports and more specialized bioinformatics tools that can be used to do novel analyses directly online . data curated at rgd is also available through other global genomics resources such as ncbi / entrez genes ( 2 ) and on the ensembl ( 3 ) and ucsc genome browsers ( 4 ) . in addition , our data is freely available as ftp files to enable anyone to use it in their research or analysis . a resource like rgd evolves in parallel with its community and for a model organism database , this community includes the researchers using that model organism and also the broader genomics and bioinformatics communities wishing to access and use this data . one of the most significant trends in recent years that has greatly influenced the database has been the use of biological ontologies , of which the gene ontology ( 5 ) is perhaps the most well known . by providing a set of standardized terms , definitions and relationships between the terms , ontologies are very convenient for researchers as a way to consistently annotate and categorize data . for the bioinformatics community they are emerging as a way to standardize data representations , enabling cross - organism data mining and analysis and another significant trend facing anyone using online resources and one that is particularly acute for modern biologists is the massive increase in information available ( 6 ) . the problem ranges from not finding enough data to finding too much and being unable to efficiently comprehend what is available . driven by the needs of our community we have been exploring ways to utilize the descriptive power of our ontology annotations combined with innovative visualization tools and web designs to begin to address this problem . for a number of years we have been using a variety of biological ontologies as annotation tools to organize and classify the data we curate . these include the gene ontology ( 5 ) , the mammalian phenotype ontology ( 8) , a disease ontology that we developed from mesh disease terms and a pathway ontology created at rgd . these have provided a comprehensive platform with which to annotate genes , qtl and strains and provide a snapshot of the most relevant information from the molecular level to that of the whole organism . as of september 2006 rgd has entries for 23 599 genes , 746 rat strains , 1093 rat qtl and 535 human qtl . these in turn have been annotated with ontology terms , giving rise to 92 137 go annotations , 2990 disease annotations , 1756 pathway annotations and 8242 phenotype annotations . the increasing numbers of annotations and other data has necessitated improvements to the layout of our gene report page . information has been organized into sections that provide brief summaries of the gene or qtl with links to more comprehensive information . the ontology annotation section was combined with the free text notes into a functional annotation block , providing a single location for this information . the technical details of each annotation such as evidence codes , ontology identifiers and reference links were moved to a new detail page to reduce the clutter on the main gene report . a similar approach was taken to move the lists of literature references and sequence identifiers from the main page to separate detail pages . other improvements to the gene report included providing snp and synteny tracks on the genome image for a gene and more comprehensive external database links . the major new addition to the rgd site in the past year was guided by three user - centric goals(i ) providing easy access to data related to diseases , ( ii ) allowing multiple perspectives of rgd data according to the needs of the user , and ( iii ) presenting a broader overview of data and allowing users to zoom in , filter the data and then drill down to the details as required . analysis of rat publications and trends in rat research demonstrate that much rat research is done in the context of disease - related studies . this is borne out by the types of grants that are funded and the types of searches that are undertaken on rgd . figure 1 shows a tag cloud view of a typical month 's top searches on rgd . the search keywords of interest to our users are primarily disease terms , centered on cardiovascular , autoimmune and neurological disease areas . based on this demonstrated need , we have introduced a variety of disease - centric resources to the database . top search terms from the rgd web logs ( june 1july 4 , 2006 ) . the individual search terms are shown with the number of times that term was searched shown in parentheses . a general disease portal page was released to enable one - click access to some of the most popular disease areas ( ) . for each disease this provides preconfigured links into our ontology annotation tools and the genome browser to enable users to quickly find gene lists and see genomic locations for genes and qtls . building from this , we identified a subset of diseases that were clearly of high interest to the research community and developed a two - pronged approach to providing enhanced support for research in these areas . for each disease area , two methods were utilized to identify data for inclusion . disease - related genes were identified from existing sources such as online mendelian inheritance in man ( omim ) ( 9 ) , the genetic association database ( 10 ) , genecards ( 11 ) and ncbi 's genes and disease database . in addition , genes at the mouse genome database ( mgd ) ( 12 ) annotated with related phenotypes were included . these genes were prioritized and targeted searches of rat and human literature were undertaken to provide comprehensive annotations for functional , disease , phenotype and pathway information . in a complimentary approach , focused literature searches were conducted to identify additional genes , qtls and strains related to the disease area . to facilitate translational studies human and mouse data was also included . as there is limited data on human qtl available electronically , a similar strategy was followed to identify relevant human qtl papers for inclusion in the portal . mouse and human gene orthologs are already curated as part of the normal rgd gene curation process . by following this targeted approach , all rat genes , strains , qtl related to a disease area could be added to the database , along with their functional annotations ( go , pathway , phenotype , disease ) . similarly , the human and mouse gene orthologs and human qtl were also identified to provide comparative resources for the database . to complement the dedicated curation effort , an online portal was created to provide access to this information . to date , one disease portal has been released for neurological diseases ; a second for cardiovascular diseases will be released in the autumn of 2006 . the portal combines text data with visual elements to allow the user to quickly get an overview of knowledge in a disease area while providing hyperlinks to more details as desired . a screenshot of the neurological portal is shown in figure 2 and the main elements are described below : disease category selection based on the disease ontology structure , three levels of disease specificity are provided . when the page is first opened data is presented for all neurological diseases . using the two dropdown menus a disease category and , optionally , a disease from this category can be selected to further narrow down the data displayed . a summary table lists the number of genes , qtl and strains for the three species , rat , mouse and human for the selected disease.genome view using flash gviewer this provides a graphical overview of the chromosomal locations of all genes and qtl annotated to the selected disease category . maps for rat , mouse and human genomes are provided and syntenic maps are also available . the gviewer package is written using adobe flash and allows zooming to view individual chromosomes and their features . the zoomed view also allows chromosomal regions to be selected , providing a dynamic link out to visualize the selected region in a genome browser . gviewer is freely available via the gmod project ( ) gene , qtl and strain lists the symbols for the genes , qtl and strains associated with the selected disease are shown in tabular form in the center of the page . these allow easy browsing of the data related to the disease and each symbol is a hyperlink to the appropriate object report in rgd . the strain table provides quick access to the rat models used to study the selected disease.gene ontology overview three bar charts provide an overview of the prominent gene ontology annotations available for the rat genes annotated to the selected disease . the individual go annotations for each rat gene are converted to the corresponding go slim annotation and graphed to provide a visual indication of popular go categories relevant to the selected disease . disease category selection based on the disease ontology structure , three levels of disease specificity are provided . when the page is first opened data is presented for all neurological diseases . using the two dropdown menus a disease category and , optionally , a disease from this category can be selected to further narrow down the data displayed . a summary table lists the number of genes , qtl and strains for the three species , rat , mouse and human for the selected disease . genome view using flash gviewer this provides a graphical overview of the chromosomal locations of all genes and qtl annotated to the selected disease category . maps for rat , mouse and human genomes are provided and syntenic maps are also available . the gviewer package is written using adobe flash and allows zooming to view individual chromosomes and their features . the zoomed view also allows chromosomal regions to be selected , providing a dynamic link out to visualize the selected region in a genome browser . gviewer is freely available via the gmod project ( ) gene , qtl and strain lists the symbols for the genes , qtl and strains associated with the selected disease are shown in tabular form in the center of the page . these allow easy browsing of the data related to the disease and each symbol is a hyperlink to the appropriate object report in rgd . the strain table provides quick access to the rat models used to study the selected disease . gene ontology overview three bar charts provide an overview of the prominent gene ontology annotations available for the rat genes annotated to the selected disease . the individual go annotations for each rat gene are converted to the corresponding go slim annotation and graphed to provide a visual indication of popular go categories relevant to the selected disease . screenshot of the rgd neurological disease portal showing the combined data for all neurological diseases . the various subsections of the page ( a d ) are described in more detail in the text . complementing the disease section of the portal are similar views for phenotype , biological process and pathways . these list selected phenotype ontology terms , biological process terms or pathway ontology terms for genes that have been linked to neurological or cardiovascular disease . these allow the scientist to view the disease data from alternative perspectives , to quickly ask questions like what pathways are involved in neurological disease , where are these genes on the genome , what cellular location do they typically occupy? the strain models section provides a comprehensive background on specific disease models and the strains used to study these diseases . it includes information on the experimental model , how it can be induced , the disease course , phenotypic indices and strains that are susceptible or resistant to the induction of the disease phenotypes . the disease portal approach utilizes in - depth curation and dedicated web tools to provide detailed coverage for specific disease areas . upcoming portals will cover autoimmune diseases , cancer , metabolic and nutritional diseases , renal diseases , respiratory diseases and other high priority research areas for the rat model . until these become available the general disease portal page and search tool ( ) does provide a convenient way to find genes , orthologs and qtl associated with any disease that are curated by the regular rgd literature curation effort . the first describes strategies for using rgd in support of specific research areas including comparative genomics , positional cloning and microarray with a particular focus on using the rgd tools to access data useful in these types of studies ( 13 ) . a chapter is also available in current protocols in bioinformatics which provides a more step - by - step practical guide to using rgd and its various resources ( 14 ) . as part of its role in supporting and informing the broader rat genomics community rgd hosts the rat community forum ( ) , an online mailing list with almost 900 subscribers that is regularly used to discuss practical questions pertaining to rat genetics and biology . we also produce a quarterly newsletter , the pied piper ( ) , which contains more in depth articles and announcements on current developments in rat genomics in both academia and the commercial fields . in addition to providing information to the community , rgd is also trying to solicit input from the community . a recent addition has been the genome discrepancy form ( ) , developed as a way for rgd to begin to collect and publicize known or suspected errors in the current genome assembly . we will take information submitted through this form and display it on the genome browser to publicly identify potential problem regions across the genome . ultimately these regions may be considered for further sequencing and analysis , as the rat genome assembly is refined at a future date . the importance of continuing to improve the assembly can not be overstated as the quality of annotations and sequence analyses are directly proportional to the quality of the underlying sequence . critical genomic reagents such as microarray chips are designed and annotated using the latest assemblies and the utility of these reagents are lagging behind those in human and mouse due to the difference in assembly coverage and quality . the focus of much rat research and of federally sponsored biomedical research as a whole is translational medicine ( 15 ) , the application of basic research to address problems in human health . rgd has been supporting this trend through an emphasis on comparative mapping resources and tools ( 16 ) and most recently through the disease portals . this will continue as we develop and release future disease portals and refine the portal interface . we will be introducing improved data mining tools through the use of the biomart package that will be integrated with the disease portals and other rgd tools and reports . this will greatly improve access to raw data within rgd and also allow rgd data to be more directly integrated with other external biomart resources such as ensembl 's ensmart ( 17 ) and rat proteomic and phenotypic datasets housed at the medical college of wisconsin . biomedical ontologies have been and will continue to be a core part of our data curation process with an emphasis on incorporation into tools that benefit both the novice and power user . the disease portals demonstrate this commitment , utilizing the underlying power of the disease and other ontologies through a more user - centered , visual interface . we hope to continue this trend , building on the visual nature of much biological data ( and many biologists ) by developing graphical tools to present and summarize the ever more complicated biological datasets .

the rat genome database ( rgd , ) is one of the core resources for rat genomics and recent developments have focused on providing support for disease - based research using the rat model . recognizing the importance of the rat as a disease model we have employed targeted curation strategies to curate genes , qtl and strain data for neurological and cardiovascular disease areas . this work has centered on rat but also includes data for mouse and human to create disease portals that provide a unified view of the genes , qtl and strain models for these diseases across the three species . the disease curation efforts combined with normal curation activities have served to greatly increase the content of the database , particularly for biological information , including gene ontology , disease , pathway and phenotype ontology annotations . in addition to improving the features and database content , community outreach has been expanded to demonstrate how investigators can leverage the resources at rgd to facilitate their research and to elicit suggestions and needs for future developments . we have published a number of papers that provide additional information on the ontology annotations and the tools at rgd for data mining and analysis to better enable researchers to fully utilize the database .

INTRODUCTION DATABASE CONTENTS DISEASE PORTALS OUTREACH DISCUSSION AND FUTURE DIRECTIONS

female sexual dysfunction is a serious , common , multifactorial general health problem which is usually neglected in the general population while affecting the women 's quality of life to a great extent ( 1 ) . lack of sexual health and security will result in anger , excessive rage , depression , drug abuse , lack of physical and psychological capability for parenting and child care , lack of sufficient skills for having a healthy emotional relationship , inability to flourish in the society , infanticide and even death ( 2 ) . in one study conducted in hong kong in 2007 , zhang interviewed 1510 chinese women who were between 19 and 49 years old and showed that 37.9% of the participants suffered from at least one sexual disorder ( 3 ) . one other study in egypt showed the prevalence of sexual dysfunction to be 76.9% among the studied women , with low sexual desire being the most prevalent one ( 66.4% ) . besides , as the women 's age increased , the prevalence of sexual disorders increased , as well ( 4 ) . one study on kurdish women in iran indicated that 77% of the studied participants had one sexual disorder ( 5 ) . another study in iran also revealed that 10.5% of women had never experienced orgasm ( 6 ) . in the study that safarinejad ( 2006 ) conducted on 20 - 60 year old women in iran , the prevalence of low sexual desire , lack of sexual arousal , lack of orgasm , and pain during intercourse was reported as 35% , 30% , 37% , and 26% , respectively ( 7 ) . up to now , most studies regarding the pharmacological treatments for sexual dysfunction have been conducted on males and female sexual dysfunction has been less taken into account ( 810 ) . recently , pharmacological treatments have been considered for female sexual dysfunction mainly focusing on improvement of androgen deficiency , increase of blood flow to the genital area and stimulation of the central nervous system ( 11 ) . estrogen , progesterone , and testosterone are the main effective hormones in females sexual response ( 12 ) . in addition , androgen deficiency is one of the main causes of female sexual dysfunction ( 13 ) . nonetheless , testosterone has been less welcomed due to its side - effects , including hirsutism and acne ( 9 , 14 ) . according to some researches , sildenafil citrate is effective in treatment of orgasm as well as arousal disorders and increase of females sexual experiences ( 1518 ) . sildenafil citrate is among the phosphodiesterase type 5 ( pde5 ) inhibitors which improve female sexual dysfunction through increasing blood flow to corpus cavernosum of clitoris , vagina and labia minor ( 1517 , 1925 ) . however , in rare cases , its consumption might be accompanied by complications , such as mild headache , urinary tract infection and nausea ( 14 ) . complementary and alternative medication ( cam ) is also used in treatment of sexual disorders ( 26 ) . ginseng which is a chinese traditional medication is one of the effective herbal medicines in treatment of sexual disorders ( 2728 ) . however , yohimibine has been shown to be more effective in treatment of sexual dysfunction in males as compared to females ( 2931 ) . in folk medicines , elaeagnus angustifolia ( elaeagnaceae ) is a mediterranean medicine representative of the family elaeagnaceae , commonly named oleaster , which is cultivated in north american , eurasia as far southwards as malaysia , australia , northern regions of asia to the himalayas and europe and for centuries in the desert and sub desert regions of western asia including iran ( 3235 ) . a previous study showed that e. angustifolia flower and its derivatives had antibacterial and antifungal properties . it also led to cutaneous wound healing by increasing re - epithelialization and collagen deposition ( 31 ) . e. angustifolia flower is one of the herbal medications which , according to traditional medicine , is hot and dry , aromatic , and can stimulate sexual activity especially in young girls and women ( 36 ) . it is also believed to have antioxidant effects ( 37 ) . due to its analgesic and anti - inflammatory effects , e. angustifolia extract is used in oils for external application ( 38 ) . moreover , one study showed e. angustifolia ointment to be effective in treatment of some skin disorders ( 39 ) . other studies have also confirmed that the fruit core and leaves of this plant had analgesic and anti - inflammatory effects ( 4043 ) . furthermore , one study evaluated the effect of aqueous and alcoholic extracts of e. angustifolia on intestinal smooth muscle relaxation in mice . it indicated that the presence of flavonoids in the fruit was effective in intestinal smooth muscle relaxation . therefore , the researcher suggests that this plant is a suitable candidate for treatment of muscle - skeletal disorders ( 40 ) due to its muscle relaxant effect . on the other hand , e. angustifolia has been found to contain flavonoid components and some flavones , such as chrysin , ( 5 , 7-dihydroxyflavone ) which have a partial agonistic effect on benzodiazepine receptors . central benzodiazepine receptor ( bdz - r ) agonists have been known to induce multiple effects including anxiolytic , myorelaxant , anticovulsant , hypnotic and amnestic effects ( 4446 ) . these two folk indications , anti - rheumatoid , anti - tetanus activities ( 40 ) , anxiolytic , myorelaxant effects acting on central benzodiazepine receptors of flavonoid found in e. angustifolia , may make this plant a suitable candidate for inducing muscle relaxant activity for sexual dysfunction . up to now however , most of these studies have focused on the prevalence and types of sexual disorders and have paid less attention to the treatment dimensions , including training , consultation and pharmacotherapy . hence , the present study aimed to compare the effects of e. angustifolia flower extract and sildenafil citrate on female orgasmic disorder in the women referring to selected gynecology clinics affiliated to shiraz university of medical sciences , shiraz , iran . based on the study objectives and the previous studies conducted on the issue , considering error rate of 5% , power of 80% , minimum mean difference of 0.6 , and variance of 0.92 . a sample size of 126-subjects overall , out of the 140 qualified women entered into the study , 125 ones ( 41 in the e. angustifolia group , 42 in the sildenafil citrate group , and 42 in the control group ) completed the study . the study population included 18 - 40 year old women suffering from sexual dysfunction . the health centers were selected through stratified sampling and convenience sampling was used to select the women at each center . then , the subjects were divided into e. angustifolia flower , sildenafil citrate , and control groups using stratified block randomization . the inclusion criteria of the study were obtaining scores more than 22 in female sexual function index ( fsfi ) , having normal menstruation , not being pregnant , not breastfeeding , without the history of heart attack , hypertension , and cardiovascular diseases , not consuming the medications which are effective in sexual function such as common antidepressants , not suffering from dyspareunia and vaginismus , not being menopausal , with the history of different types of headaches such as migraine , not using hormone drugs particularly oral contraceptive pills and lack of drug or alcohol abuse . on the other hand , the exclusion criteria of the study were showing allergic reactions to medicines and not being able to continue consuming the drugs for any reason . the data were collected through demographic information form , fsfi , and measurement of tsh and prolactin . fsfi which contains 19 items evaluates female sexual function in 6 domains of sexual desire , arousal , lubrication , orgasm , satisfaction and pain . the women were required to answer the questions according to their sexual desire and function during the past 4 weeks . in general , the scores 28 were considered as sexual dysfunction . nevertheless , since assessment of pain ( 6 points ) was omitted from the present study , score of 22 was considered instead of 28 . the reliability of the whole questionnaire and the subscales was confirmed by cronbach 's alpha 0.70 . moreover , investigation of the validity of the persian version of this questionnaire indicated a significant difference between the total mean score and the mean scores of the subscales in the two groups ( p0.001 ) ( 4748 ) . after obtaining written informed consents , tsh and prolactin were assessed in all the participants in order to reject thyroid and prolactin disorders which are the secondary causes of sexual dysfunction . in this study , the samples were selected through convenience sampling and were randomly allocated into two intervention groups and a control group . the first intervention group had to consume 4.5 gr of e. angustifolia extract in two divided doses ( 2 capsules every 12 hr ) for 35 days . the e. angustifolia capsule dosage was determined based on other studies conducted on the issue ( 4950 ) and a pharmacognosy advisor . in the second group , the dosage of sildenafil citrate was determined based on the previous studies most of which have used 50 and 100 g tablets ( 16 , 5153 ) . e. angustifolia capsules were made in the pharmacology department under the supervision of a professional counselor . moreover , the placebo was prepared from starch in similar packages to e. angustifolia capsules . the control group received the placebo which they were required to consume for 35 days ( 2 tablets every 12 hours ) . the participants were followed up twice a week through short message sending ( sms ) service and once a week through phone contact . after the intervention , the women completed fsfi . finally , the data were entered into the spss statistical software ( version 18 ) and analyzed using paired t - test , one - way anova , and bonferroni post - hoc test . besides , p<0.05 was considered statistically significant . this research project was approved by the local ethics committee of shiraz university of medical sciences and written informed consents were obtained from all the participants . in addition , the control group was provided with the results after completion of the study . this research project was approved by the local ethics committee of shiraz university of medical sciences and written informed consents were obtained from all the participants . in addition , the control group was provided with the results after completion of the study . most of the subjects ( 67.2% ) were between 30 and 40 years old . besides , the age of marriage in 44% and 4% of the subjects were below 20 and above 30 years old , respectively . in addition , the mean length of marriage was 21.59 4.61 years . moreover , most of the participants ( 43.2% ) had academic degrees and 33.6% had high school and diploma degrees . the results revealed no significant difference among the three groups regarding tsh ( p=0.448 ) and prolactin levels ( p=0.179 ) before the intervention . also , no significant difference was found among the three groups concerning orgasmic disorder before the intervention ( p=0.23 ) . however , a significant difference was observed among e. angustifolia , sildenafil citrate , and control groups in this regard after the intervention ( p=0.004 ) ( table 1 ) . comparison of frequency of sexual dysfunction ( orgasm disorders ) before and after intervention between the intervention and control groups according to the results of mcnemar test , no significant difference was found in the control group before and after the intervention ( 57.1% vs. 50% ; p>0.05 ) . also , no significant difference was observed in e. angustifolia group before and after the intervention ( 41.5% vs. 29.3% ; p=0.267 ) . on the other hand , a significant difference was found in the sildenafil citrate group before and after the intervention ( 40.5% vs. 29.3% ; p=0.006 ) ( table 1 ) . overall , e. angustifolia , sildenafil citrate , and control groups respectively showed 29.41% , 58.82% , and 12.5% reduction of sexual dysfunction score as compared to the condition before the intervention . the findings of the current study indicated a significant difference between the ea group and the control group ( p=0.001 ) and between the sildenafil citrate group and the control group ( p<0.001 ) regarding the mean score of orgasm ( table 2 ) . comparison of average score of sexual function ( in orgasm and sexual satisfaction area ) before and after intervention between the tests and control groups the study results revealed no significant difference among the three groups concerning sexual satisfaction before the intervention ( p=0.356 ) . however , a significant difference was observed among the three groups in this regard after the intervention ( p=0.03 ) ( table 3 ) . according to the results of mcnemar test , a significant difference was observed in e. angustifolia group before and after the intervention ( p<0.001 ) . on the other hand , no significant difference was found in the sildenafil citrate group before and after the intervention ( p=0.06 ) . comparison of frequency of sexual dysfunction ( sexual satisfaction area ) before and after intervention between the intervention and control groups overall , e. angustifolia and sildenafil citrate groups respectively showed 57.14% and 46.66% reduction sexual dysfunction score as compared to the condition before the intervention . also , a significant difference was observed between the ea group and the control group ( p<0.001 ) and between the sildenafil citrate and control groups ( p<0.001 ) regarding the mean score of sexual satisfaction ( table 2 ) . in this study , ea , sildenafil citrate , and control groups respectively showed 29.41% , 58.82% , and 12.5% reduction sexual dysfunction score as compared to the condition before the intervention . this difference was statistically significant in the sildenafil citrate group ( p=0.006 ) , but not in e. angustifolia group ( p=0.267 ) . nonetheless , a significant difference was observed between the two intervention groups and the control group regarding the mean score of orgasmic disorder ( p<0.001 ) . caroso ( 2002 ) performed a study on 68 women between 19 and 38 years old who had no symptoms of sexual dysfunction in order to assess the effectiveness of sildenafil citrate . the participants had to consume sildenafil citrate and placebo for 4 weeks and were entered into the study based on personal experiences questionnaire ( peq ) . the study results showed that sildenafil citrate improved arousal ( p<0.01 ) , orgasm ( p<0.001 ) and sexual satisfaction ( p<0.001 ) compared to the placebo group ( 22 ) . 2008 ) carried out a research on 22 menopausal women who suffered from orgasmic disorder . the participants received either 50 mg sildenafil citrate ( 11 subjects ) or placebo ( 11 subjects ) in a single dose for 15 days . the results revealed a considerable increase in the vaginal blood flow in the sildenafil citrate group as compared to the placebo group ( p<0.05 ) ( 18 ) . 2004 ) investigated the effectiveness of sildenafil citrate in treatment of the women suffering from sexual dysfunction due to multiple sclerosis ( ms ) . the study was conducted on 19 women with both sexual dysfunction and ms and the results showed that sildenafil citrate did not improve their sexual dysfunction ( p>0.441 ) ( 54 ) . similarly , ( 55 ) reported that sildenafil citrate had no positive effects on women 's sexual disability score , which is in contrast to the findings of the current study . sildenafil citrate is among the pde5 inhibitors which by preventing cyclic guanosin monophosphate ( cgmp ) catabolism , leads to an increase in nitric oxide ( no ) production , stimulation of no release and increase in vaginal blood flow . this , in turn , results in an increase in sexual desire , orgasm , tissue hyperpolarization and relaxation , increase in blood flow , hyperemia and swelling ( 12 ) , eventually leading to improvement of sexual dysfunction . up to now , no specific clinical scales have been used for diagnosis of female sexual dysfunction and no medications have been confirmed for its treatment . yet , two strategies , i.e. , androgens and pde5 inhibitors , are utilized for reduction of sexual disorders in women ( 56 ) . in the present study , e. angustifolia extract decreased the sexual dysfunction score as compared to the condition before the intervention and the control group ; however , the differences were not statistically significant . to date , no studies have been conducted on the effectiveness of e. angustifolia flower in sexual disorders . therefore , the study results were compared with those of the studies performed on other herbal medications used for sexual disorders . waynberg and brewer ( 2000 ) aimed to find an alternative for chemical medications used for treatment of female sexual dysfunction and consequently , assessed muira puama and ginkgo in 202 healthy women with low sexual desire . in that study , different aspects of the participants sexual life were investigated before and one month after the treatment . after consuming the supplements , the self - assessment questionnaire scores were higher than average in 65% of the participants . in addition , the results indicated a significant improvement in the frequency of sexual desires and fantasies , satisfaction with sexual life , intensity of sexual desires , ability to reach orgasm and intensity of orgasm ( p<0.001 ) ( 57 ) . similarly , ito ( 2006 ) reported that arginmax ( an herbal supplement ) improved sexual dysfunction particularly regarding sexual desire ( 51% vs. 8% in the placebo group ; p=0.008 ) ( 58 ) . e. angustifolia plant contains significant amount of flavonoids , terpenoids and sitosterol ( 5962 ) . research has shown that certain flavonoids and sitosterol have analgesic and anti - inflammatory effects ( 62 ) and lead to vascular smooth muscle relaxation ( 63 ) . evidence has shown that no , as an endogenous vascular dilator , plays a critical role in regulation of vascular tone ( 64 , 65 ) and is the main stimulant of cgmp production in smooth muscles ( 66 ) . increase in cgmp leads to an increase in the activity of kinase g protein which leads to vascular relaxation by phosphorylation of different molecules ( 67 ) . on the other hand , some experts believe that increase of cgmp results in smooth muscle vascular relaxation through other mechanisms ( 68 ) . for instance , cgmp may lead to relaxation of smooth muscles by decreasing the intracellular calcium concentration ( 69 ) . the positive effect of e. angustifolia on sexual dysfunction and sexual satisfaction in this study may be justified by the aforementioned mechanisms . the findings of the present study indicated a significant difference between the two intervention groups and the control group concerning the mean score of sexual satisfaction ( p<0.001 ) . other studies have also found a significant relationship between improvement of sexual performance and sexual satisfaction ( 15 , 57 ) . research has indicated that improvement of the quality of sexual relationships between husband and wife led to great satisfaction . it is , therefore , a decisive factor for marital and sexual satisfaction ( 2 , 7071 ) . overall , it seems that identification and treatment of sexual dysfunctions can increase satisfaction and thus can improve the families strength and survival and the community 's health . according to the previous studies , herbal medications , such as arginmax , ginseng , ginkgo biloba , and ethanol extract ( 7273 ) improved sexual dysfunction through an increase in no production . sexual dysfunction in women is a serious , complex , multi - factorial problem which might result from biological , psychological , cultural , social and hormonal factors . since the participants had normal menstrual cycles , hormones , and endocrine parameters , their sexual dysfunction might have occurred due to other factors , including insufficient stimulation and lack of preparation . phosphodiesterase - type 5 inhibitors ( sildenafil citrate ) and e. angustifolia flower also enhance vaginal smooth muscles relaxation , artery vasodilatation and swelling in the genital system , thereby improving sexual function . other factors associated with sexual dysfunction include relational problems , lifestyle , and type of food regimen that were not investigated in the current study . in this study , e. angustifolia , sildenafil citrate , and control groups respectively showed 29.41% , 58.82% and 12.5% reduction of changes as compared to the condition before the intervention . thus , it seems that sildenafil citrate has been more effective in improvement of sexual dysfunction as compared to e. angustifolia . the findings of the current study can be used for improvement of women 's and families sexual health . yet , further studies are recommended to be conducted using different doses of e.angustifolia at different times .

backgroundorgasmic disorder can create a feeling of deprivation and failure and provide mental problems , incompatibility and marital discord . this study aimed to compare the effects of elaeagnus angustifolia flower extract and sildenafil citrate on female orgasmic disorder in women in 2013.methodsin this randomized clinical trial , 125 women between 18 - 40 years old who suffered from orgasmic disorder were divided into three e. angustifolia , sildenafil citrate and control groups . the data were gathered using female sexual function index and through measurement of tsh and prolactin . the first intervention group had to consume 4.5 gr e. angustifolia extract in two divided doses for 35 days and the second one had to use 50 mg sildenafil citrate tablets for 4 weeks one hour before their sexual relationship . however , the control group had to consume the placebo . the data were analyzed using paired t - test , one - way anova , and bonferroni posthoc test and p<0.05 was considered significant.resultsthe frequency of orgasmic disorder before the intervention was 41.5% , 40.5% , and 57.1% in e. angustifolia , sildenafil citrate , and control groups , respectively ( p=0.23 ) . however , these measures were respectively 29.3% , 16.7% , and 50% after the intervention ( p=0.004 ) . a significant difference between the two groups regarding sexual satisfaction after the intervention ( p=0.003 ) compared to the beginning of the study ( p=0.356 ) . besides , the highest reduction of changes after the intervention ( 58.82% ) was observed in the sildenafil citrate group.conclusionboth e. angustifolia extract and sildenafil citrate were effective in reduction of the frequency of orgasmic disorder in women .

Introduction Methods Ethical considerations Results Discussion Conclusion Conflict of Interest

the cytosolic free ca concentration ( [ ca]i ) in eukaryotic cells is in the low nanomolar range ( ~100 nm ) , and transient modifications of this concentration serve as an intracellular mechanism for cell signaling because of the existence of a variety of effectors that are sensitive to such transient increases . for instance , in many cases the binding of extracellular ligands to their receptors , including g protein - coupled receptors ( gcprs ) located at the plasma membrane , triggers the phosphoinositide pathway by means of the activation of phospholipase c. this activation is responsible for the hydrolysis of phosphatidylinositol 4,5-bisphosphate and the subsequent generation of inositol 1,4,5-trisphosphate ( ins(1,4,5)p3 ) and 1,2-diacylglycerol ( dag ) . soluble ins(1,4,5)p3 binds to its receptor ( ins(1,4,5)p3r ) in the endoplasmic reticulum ( er ) , whereas the more lipophilic dag accumulates in the plasma membrane . ins(1,4,5)p3r is a receptor - operated ca channel that activates by binding to its endogenous ligand ins(1,4,5)p3 , leading to transient ca release from the er and thereby transiently increasing [ ca]i . many ca - sensitive effectors are found in the cytosol , which contribute to the diversity of the intracellular signaling initiated by the ca release from the er . alternatively , other ca channels located in the plasma membrane such as the ligand - gated plasma membrane p2x receptor and the voltage - operated ca channels families regulate the influx of extracellular ca to the cytosol upon biding to ligands or upon plasma membrane depolarization conditions , respectively . we highlight these examples to draw attention to the fact that there are two major sources of ca that may increase [ ca]i , - the extracellular milieu , and the intracellular compartments including the most important intracellular ca store , the er and the specialized sarcoplasmic reticulum ( sr ) in muscle cells . in all cases the increase of [ ca]i is transient , and the high cytosolic ca levels are rapidly cut down by the activation of ca pumps in the plasma membrane and the er . whereas the plasma membrane ca - atpase ( pmca ) extrudes ca into the extracellular milieu , the sarco(endo)plasmic ca - atpase ( serca ) pumps ca into the lumen of the er . other active ca transport systems are also involved in the restoration of cytosolic ca basal levels , such as the na / ca exchanger with a lower affinity for ca compared with ca pumps , and the mitochondrial ca uniporter ( mcu ) which transport ca into the mitochondria matrix . the combination of the activities of all ca transport systems defines specific ca transients in terms of amplitude , intensity , and duration which vary according to the initial stimulus and the cell type . thus , the number , type and molar ratios of the molecules involved in the origin and maintenance of the ca mobilization define the spatio - temporal pattern of ca signaling . for instance , ca signaling during fertilization of mammalian oocytes is initiated by the sperm - specific phospholipase c zeta ( plc ) that triggers the phosphoinositide pathway once plc has been released into the oocyte after the fusion with the sperm . this pathway induces an initial increase of [ ca]i in the oocyte which last for 12 min , which is followed by repetitive and transient increases of [ ca]i , required for the exit from cell cycle arrest of oocytes in the metaphase of the second meiotic division . another classical example of ca mobilization required to trigger downstream effects is found in skeletal muscle cells . depolarization of plasma membrane induces a conformational change in dihydropyridine receptors ( dhprs , voltage - dependent , l - type , plasma membrane ca channels ) which activate ryanodine receptors located in the sr , enabling the ca release from this intracellular store . these are just two classical examples of how ca fluxes regulate cell physiology , which also indicate the importance of intracellular ca stores for this signaling . to ensure the durability and maintenance of the signaling , a plasma membrane ca transport systems regulates intracellular ca stores refilling . this ca entry pathway is called store - operated ca entry ( soce ) , a ubiquitous mechanism and one of the most important pathways for ca entry in non - excitable cells . consistent with its descriptive name , soce is regulated by the ca concentration within the er . the mechanism that links luminal ca levels with plasma membrane ca entry is mediated by stim1 ( stromal interaction molecule 1 ) , a type i transmembrane protein that senses ca within the luminal space of the er , and activates plasma membrane ca channels ( socs ) upon ca depletion conditions in the intracellular stores . human stim1 protein consists of 685 aminoacids , with an sam domain ( sterile motif ) and an ef - hand domain that acts as a ca sensor within the luminal space of the er . upon depletion of ca within the er , the sam - ef domain mediates the oligomerization of stim1 , triggering this oligomerization in clusters of ~1 m diameter . the clustering favors the relocalization of stim1 in er - plasma membrane ( pm ) junctions , required for the activation of soce . stim1 directly binds to store - operated calcium channels ( socs ) , activating a highly ca - selective , non - voltage - gated , inwardly rectifying current known as the ca release activated ca current ( icrac ) . this current is reconstituted by both stim1 and orai1 ( also known as cracm1 ) , a plasma membrane protein that constitutes a major ca channel regulated by stim1 . in addition to orai1 , some of the transient receptor potential canonical ( trpc ) channels can function in a stim1-dependent mode . stim1 directly binds and activates trpc1 , trpc4 and trpc5 channels that can therefore act as socs . however , the stim1/trpc molar ratio determines the stim1-dependent or stim1-independent mode of action of the channels . in addition , trpc1 also associates with orai1 to produce trpc1-orai1-stim1 ternary complexes that act as socs . it was early found that the c - terminal portion of stim1 is sufficient for the activation of orai1 , and that both proteins physically interact through a coiled - coil domain in the c - terminus of orai1 . the minimal domain of stim1 that activates orai1 is a conserved crac activation domain ( cad ) of stim1 of ~100 amino acids that binds directly to orai1 . the cytosolic domain ( residues 234685 ) contains three coiled - coil domains , cc1 , cc2 , and cc3 . cc2 and cc3 overlap with the soar / cad region required to activate ca channels ( aminoacids 334442 ) . because cc1 interacts with cc2/cc3 at resting state , keeping stim1 in a closed conformation , the activation of stim1 by store depletion and the binding of stim1 with orai1 requires an intramolecular transition of stim1 into an open conformation , in order to expose cc2 and cc3 so they are able to activate orai1 by a physical interaction of coiled - coil domains . investigation of the mechanism of trpc1 gating led to the proposal that it involves an electrostatic mechanism through the interaction of two negative charged aspartates in trpc1 and the conserved polybasic domain , enriched in lysine residues , at the c - terminus of stim1 . an additional ca channel that is regulated by stim1 belongs to the voltage - operated ca channels family . stim1 binding to cav1.2 channels suppresses the activation of these channels , an action that is mediated also by the soar / cad domain of stim1 , causing long - term internalization of the channel from the membrane . thus , multiple actions are triggered by the cytosolic domain of stim1 , including the activation and inactivation of diverse channels in response to different stimuli . it is plausible that the cytosolic domain encompasses additional domains that modulate the ca channel - regulation activity of stim1 , by controlling stim1 subcellular localization and/or the selectivity of the ca channel to be activated or inhibited . in this regard , stim1 has a serine / proline - rich domain ( or s / p)-rich domain which could be acting as a modulator of the stim1 activity . stim1 is a phosphoprotein in which large - scale mass spectrometry studies have revealed potential phosphorylation sites . subsequent initial reports of detailed analyses of phosphoresidues from immunoprecipitated stim1 differed in their findings , mainly because the studies had focused on different cell cycle stages . during the m - phase , although yu et al . reported that stim1 is phosphorylated during meiosis of xenopus oocytes , substitution mutations of target residues to mimic constitutive phosphorylation or dephosphorylation do not modulate the clustering of stim1 in response to store depletion , an observation that supports the lack of any physiological function for stim1 phosphorylation during meiosis in xenopus oocytes . smyth et al . found that stim1 clustering is also inactivated during mitosis of mammalian cells , and they identified specific residues , such as ser602 and ser608 , that become dephosphorylated during that process . other sites were initially found to be constitutively phosphorylated ( ser575 , ser620 , and ser621 ) . ser668 belongs to a consensus sequence for cyclin - dependent kinase 1 ( cdk1 ) , and is phosphorylated by cdk1 in vitro . also , the expression of single alanine substitution mutations ( s668a or s486a ) does not rescue soce in mitotic cells . however , expression of a double mutant s486a / s668a does show soce responses in mitosis , confirming the role of stim1 phosphorylation at ser486 and ser668 in soce inactivation during mitosis . further evidence was that extracellular signal - regulated kinases 1/2 ( erk1/2 ) phosphorylate stim1 in vitro at ser575 , ser608 , and ser621 and that stim1 phosphorylation at erk1/2 target sites regulates soce in hek293 cells . the phosphorylation of stim1 at ser575 , ser608 , and ser621 was revealed by mass spectrometry using immunoprecipitated stim1 from asynchronous hek293 cells , and later with phospho - specific antibodies against phosphorylated residues . this latter strategy demonstrated that stim1 phosphorylation at erk1/2 target sites increases during soce activation , and consequently the alanine substitution mutation of these sites nullifies soce , whereas ser - to - glu mutation enhances ca entry . in contrast to the results reported in , phospho - specific antibodies against phosphoser575 , phosphoser608 , and phosphoser621 revealed a dynamic phosphorylation of stim1 that was strongly dependent on the ca store filling state . thus , ca store depletion is accompanied by an increase of stim1 phosphorylation at erk1/2 target sites , whereas ca store refilling triggers stim1 dephosphorylation at these sites . many aspects of the molecular mechanism by which the phosphorylation of stim1 regulates soce remain unclear , but the inhibition of stim1 phosphorylation decreases stim1 clustering in response to store depletion and impairs stim1-orai1 binding , as monitored by fluorescence resonance energy transfer ( fret ) and by co - immunoprecipitation . in an attempt to resolve the open question of the requirement of stim1 phosphorylation at ser575 , ser608 , and ser621 to activate soce in hek293 cells during interphase , we recently found that phosphorylation of stim1 at erk1/2 target sites regulates the association of stim1 with eb1 ( end - binding protein 1 ) , a regulator of growing microtubule ends . the role of the cytoskeleton in soce regulation has been studied in depth , and it was early established that stim1 colocalizes with tubulin and that the treatment of hek293 cells with nocodazole , which triggers microtubule depolymerization , severly reduces soce . later , sampieri et al . reported that stim1 travels through the er in association with eb1 under resting conditions , confirming the connection between stim1 and microtubules , and that the association ceases upon depletion of the er , facilitating the aggregation of stim1 into large clusters . those authors also observed that stim1 re - associates with eb1 when intracellular calcium stores are replenished , i.e. , stim1-eb1 dissociation is fully reversible . because stim1 binds to eb1 in the resting state , stim1 can be considered a microtubule plus - end - tracking protein ( + tip ) . there are two modes of direct interaction between eb1 and + tips - either through a cytoskeleton - associated protein glycine - rich ( cap - gly ) domain or by binding to a ser / thr - x - ile - pro ( s / txip ) consensus sequence . as a + tip , stim1 localization is dependent on microtubule formation , and a stim1 sequence encompassing residues 642645 ( thr - arg - ile - pro ) has been found to be essential for the binding to eb1 . for those + tips with an s / txip motif , examples of regulation by phosphorylation in the vicinity of this domain , but not within this sequence , have been reported . these examples include apc , mcak , and clasp2 , all of which are phosphorylated in the vicinity of the s / txip sequence , regulating their interaction with microtubules . in all cases , as with stim1 , sequences flanking the s / txip motif contain a high number of proline , serine , and basic residues , leading to a net positive charge in the surroundings of this eb1 binding domain . this observation might explain why phosphorylation in the vicinity of this sequence blocks the localization of + tips to microtubule ends . in this regard , the phosphorylation of stim1 at residues ser575 , ser608 , and ser621 triggers the dissociation of stim1 from eb1 . because refilling of ca stores is accompanied by dephosphorylation of these residues , this reversible phosphorylation constitutes a mechanism that fully explains the reversible interaction of stim1 and eb1 ( see figure 1 ) . indeed , ser - to - ala substitution mutants of stim1 do not dissociate from eb1 under store depletion conditons , and ser - to - glu mutants remain dissociated from eb1 even under resting conditions . in accordance with the proposed model described in figure 1 , those stimuli that induce store depletion and erk1/2 activation lead to phosphorylation of stim1 at ser575 , ser608 , and ser621 . this specific phosphorylation triggers the dissociation of stim1 from eb1 , which in turn facilitates stim1 clustering and the binding to socs to activate ca entry . replenisment of ca stores induces stim1 dephosphorylation by as yet to be described phosphatases , leading to the reassociation of stim1 with eb1 , and the microtubule localization pattern of stim1 ( fig . 1 ) . as has been reported for other + tips , the phosphor - regulation of stim1-eb1 interaction could be more complex , and other phosphoresidues might trigger a diversity of actions . for instance , the ser - to - ala substitution mutation of 10 residues in stim1 leads to er mislocalization by pulling er tubules into the mitotic spindle . because in wild - type stim1-expressing cells er tubules are excluded from this mitotic spindle , those results suggest that hyperphosphorylation of stim1 is required to maintain er normal structure in mitosis . however , it would be necessary to monitor the phosphorylation state of individual residues to discover which , if not all , of the residues are involved in this specific control of localization . finally , other phosphorylation sites at tyr residues could be important for the initial steps of soce activation , because an increase of total phosphorylated tyr has been detected in stim1 from platelets under store depletion conditions , and the inhibition of this phosphorylation decreases the binding of stim1 to orai1 and therefore soce . however , the identification of the specific modified residues and the molecular mechanism underlying this regulation remains to be clarified . with the recent studies of the role of stim1 phosphorylation on cell physiology , one has just started to understand the physiological relevance of this modulation . in addition to hek293 cells , stim1 phosphorylation , enhanced by thapsigargin , has been reported in neonatal rat ventricular myocytes . also , it is known that phosphorylation of stim1 at ser575 , one of the target sites of the erk1/2 activity , promotes the differentiation of cultured mouse c2c12 myoblasts to myocytes . in parallel , it has been shown that stim1 is necessary and sufficient for cardiomyocyte hypertrophy in vitro and in the adult heart in vivo . taken togeteher , these recent results indicate that the activity of stim1 as a ca channels regulator is important for skeletal and cardiac muscle cells development , and that stim1 phosphorylation constitutes a potential pharmacological target for the treatment of muscle hypertrophy . the mek - erk pathway was early suggested as being involved in the activation of soce in human platelets , probably as a downstream effector of ras proteins , and it has recently been demonstrated that constitutive dephosphorylation of stim1 at erk1/2 target sites impairs platelet adhesion to fibrinogen , supporting a physiological role for stim1 phosphorylation by this signaling pathway . in conclusion , phosphorylation regulates the activation of stim1 . however , the phosphorylation of different target residues has diverse effects . because a differential phosphorylation profile can trigger opposing actions , the phosphorylation state of target residues needs to be defined under different stimuli so that one can establish molecular models capable of predicting stim1 activities , particularly under pathophysiological conditions . in this scenario , phosphorylation target residues could be investigated as potential pharmacological targets for the treatment of diseases , since this would not alter upstream signaling cascades , and would therefore limit the intensity of side effects .

calcium ion ( ca2 + ) concentration plays a key role in cell signaling in eukaryotic cells . at the cellular level , ca2 + directly participates in such diverse cellular events as adhesion and migration , differentiation , contraction , secretion , synaptic transmission , fertilization , and cell death . as a consequence of these diverse actions , the cytosolic concentration of free ca2 + is tightly regulated by the coordinated activity of ca2 + channels , ca2 + pumps , and ca2 + -binding proteins . although many of these regulators have been studied in depth , other proteins have been described recently , and naturally far less is known about their contribution to cell physiology . within this last group of proteins , stim1 has emerged as a major contributor to ca2 + signaling by means of its activity as ca2 + channel regulator . stim1 is a protein resident mainly , but not exclusively , in the endoplasmic reticulum ( er ) , and activates a set of plasma membrane ca2 + channels termed store - operated calcium channels ( socs ) when the concentration of free ca2 + within the er drops transiently as a result of ca2 + release from this compartment . knowledge regarding the molecular architecture of stim1 has grown considerably during the last years , and several structural domains within stim1 have been reported to be required for the specific molecular interactions with other important players in ca2 + signaling , such as ca2 + channels and microtubules . within the modulators of stim1 , phosphorylation has been shown to both activate and inactivate stim1-dependent ca2 + entry depending on the cell type , cell cycle phase , and the specific residue that becomes modified . here we shall review current knowledge regarding the modulation of stim1 by phosphorylation .

Background STIM1 Regulates Ca Phosphorylation of STIM1 Regulates its Activity

assisting couples to achieve their desired family size ought to be the prime purpose of any family planning program . fertility control is facilitated when long - acting contraceptives are promoted and made available to all eligible women looking for long - term spacing . approximately 20% of currently married women between the ages of 15 and 49 in india have an unmet need for contraception , of whom 7.2% have an unmet need for spacing methods . intrauterine contraceptive device ( iucd ) is very effective ( 99% ) and an inexpensive family planning method which is reversible , requires little effort on the part of the user once inserted , and offers 510 years of protection against pregnancy . wider use of iucd would reduce the overall number of unintended pregnancies more than any other method . globally , about one of the five women in reproductive age group use iucd , while in india , the corresponding figure is about 3/100 women . the government of india launched postpartum iucd ( ppiucd ) services in the year 2000 in selected states , which were universalized in all the states by the year 2010 . under the program , postpartum period is generally considered to be an ideal time to introduce contraception since women are strongly motivated at this time . even though expulsion rate for ppiucd is higher , benefits of providing highly effective contraception immediately after delivery outweigh this disadvantage . the government of india provided iucd free of cost , nonetheless , it still was largely underutilized . hence , there is a need to identify factors that affect the acceptance of ppiucd provided through a public health approach . our aim was to measure the acceptance rate of ppiucd , when offered at the primary health center ( phc ) level , and factors associated with its acceptance . total population served by these two phcs was ~90,000 , spread over 28 villages ( in the year 201314 ) , and comprised the field practice area of the ballabgarh health and demographic surveillance system ( hdss ) . the village level multipurpose health workers ( mpws ) made fortnightly domiciliary visit to each household for service delivery and data collection . services that were provided included antenatal , postnatal checkups , 24 7 obstetric care , immunization , family planning , and referral services . in both we have discussed in detail , the profile of the pregnant women who deliver at these delivery huts where the ppiucd was offered . lady medical officers , who were also trained in ppiucd insertion , provided supportive supervision and training to the service providers . pregnant women were provided counseling regarding benefits and availability of ppiucd services during antenatal period by mpws . women coming for delivery at these two phcs were again motivated and counseled during the perinatal period . ppiucd was inserted either just after delivery of placenta or before discharge of mother or baby , usually within 48 h of delivery . women experiencing postpartum hemorrhage , premature rupture of membrane for more than 48 h before delivery , prolonged labor , and birth asphyxia in newborn were not offered ppiucd . recommended standard method was used for ppiucd insertion . after insertion , ppiucd acceptors were given a follow - up card , which had information about follow - up visits and warning symptoms . sociodemographic factors , such as caste , religion , family income , occupation and education of couple , obstetric history such as number of living children , gravida , number of antenatal visits , and sex of the newborn , were routinely captured at the time of delivery . variables such as type of follow - up ( clinic visit or telephonic ) , in situ status of iucd , side effects , and complications were captured during the follow - up visit at 6 weeks postpartum . results are expressed in proportions , and chi - square test was used wherever applicable . total population served by these two phcs was ~90,000 , spread over 28 villages ( in the year 201314 ) , and comprised the field practice area of the ballabgarh health and demographic surveillance system ( hdss ) . the village level multipurpose health workers ( mpws ) made fortnightly domiciliary visit to each household for service delivery and data collection . in the year 200506 , haryana government started establishing delivery huts by upgrading existing subcenters and phcs . services that were provided included antenatal , postnatal checkups , 24 7 obstetric care , immunization , family planning , and referral services . in both the study phcs we have discussed in detail , the profile of the pregnant women who deliver at these delivery huts where the ppiucd was offered . lady medical officers , who were also trained in ppiucd insertion , provided supportive supervision and training to the service providers . pregnant women were provided counseling regarding benefits and availability of ppiucd services during antenatal period by mpws . women coming for delivery at these two phcs were again motivated and counseled during the perinatal period . ppiucd was inserted either just after delivery of placenta or before discharge of mother or baby , usually within 48 h of delivery . women experiencing postpartum hemorrhage , premature rupture of membrane for more than 48 h before delivery , prolonged labor , and birth asphyxia in newborn were not offered ppiucd . recommended standard method was used for ppiucd insertion . after insertion , ppiucd acceptors were given a follow - up card , which had information about follow - up visits and warning symptoms . sociodemographic factors , such as caste , religion , family income , occupation and education of couple , obstetric history such as number of living children , gravida , number of antenatal visits , and sex of the newborn , were routinely captured at the time of delivery . variables such as type of follow - up ( clinic visit or telephonic ) , in situ status of iucd , side effects , and complications were captured during the follow - up visit at 6 weeks postpartum . data were entered in microsoft office excel worksheet . the analysis was done in stata version 12 ( statacorp . 2011 . results are expressed in proportions , and chi - square test was used wherever applicable . a total of 799 pregnant women reported at delivery hut of two phcs between may and december 2014 . of them , 151 were referred to a higher center per indications , and 648 delivered at the phc delivery hut . among them , 611 were eligible for ppiucd insertion . the acceptance rate of ppiucd was thus 38.9% ( 95% confidence interval [ ci ] : 35.142.9 ) . out of 238 , follow - up information could be gathered for 128 through either at clinic visits or through telephone . flowchart showing the postpartum intrauterine contraceptive device insertions and follow - up of pregnant women table 1 shows the acceptance rates by sociodemographic characteristics . ppiucd acceptance rate was lowest ( 33.2% ) when the women did not have any previous living child . it was 38.2% and 45.2% when number of the previous living children was 1 and 2 , respectively . profile of women who had accepted the postpartum intrauterine contraceptive device using multivariate binary logistic regression [ table 2 ] , the independent predictors of accepting ppiucd were , having a monthly family income of < usd75 ( odds ratio [ o.r . ] : 2.29 , 95% ci : 1.583.31 ) , and belonging to the muslim religion ( o.r . : 3.24 , 95% ci : 1.079.98 ) . probability of acceptance of postpartum intrauterine contraceptive device by eligible women out of 238 acceptors , follow - up information was available for 128 women ( 53.8% ) . out of the 128 , nearly two - thirds ( 63.3% ) had visited the phc for follow - up , whereas the rest ( 36.7% ) were followed - up over telephone [ figure 1 ] . on multivariate binary logistic regression [ table 3 ] , the independent predictors of being followed - up after the insertion of ppiucd ( either at health center or by telephone ) were , being literate ( o.r . : 2.34 , 95% ci : 1.194.60 ) , monthly family income of < usd75 $ ( o.r . : 2.34 , 95% ci : 1.314.16 ) , having at least one anc visit ( o.r . : 1.24 , 95% ci : 1.101.41 ) , and belonging to the general ( nonbackward ) caste ( o.r . : 0.56 , 95% ci : 0.320.98 ) . probability of receiving a follow - up after insertion of postpartum intrauterine contraceptive device ( either at health center or by telephone ) more than two - thirds of the women ( 68% ) had the device in situ with no complaints [ table 4 ] . the expulsion rate and removal rate by the time of follow - up visit were 18.0% and 13.0% , respectively . abdominal pain ( 16.4% ) , leukorrhea , and bleeding - per - vaginum ( 5.5% ) were the most common reported symptoms at the time of follow - up [ table 4 ] . complications among acceptors of postpartum intrauterine contraceptive device ( n=130 ) at the time of follow - up visit ( 6 weeks postpartum ) on univariate analysis , probability of expulsion ( spontaneous or removed ) by 6 weeks was associated with , age more than 25 years ( o.r . : 4.01 , 95% ci : 1.2812.56 ) , and a living previous child ( o.r . : 1.51 , 95% ci : 1.042.19 ) [ table 5 ] . probability of expulsion of postpartum intrauterine contraceptive device ( either spontaneous or removed ) by 6 weeks we measured the acceptance rate of ppiucd when offered at the phc level since no information is available from the primary care . we also described the probability of being followed - up , of having an expulsion of the device , and their independent predictors . these elements of analysis have so far been missing in existing literature , especially from a primary care setting from india . a study conducted in the district head quarters hospital of bolangir , odisha reported an overall acceptance rate of 17.5% . while the acceptance rate of ppiucd among women delivering in a tertiary care teaching hospital in western uttar pradesh , india , was reported as 14.4% . the acceptance rate of ppiucd insertions among eligible women , in our study , was 39% . we found a relatively higher rate of acceptance rate for ppiucd when compared with other published literature from india . lack of accurate and up to date information about ppiucd among health service providers and potential clients has been cited as one of the reasons for low acceptance rate of ppiucd . we had trained the health - care workers in counseling skills , which was utilized by them during anc provisions . this could be one of the reasons of higher acceptance rate of ppiucd in our study as compared to the previous published literature . we found that the acceptance rate of ppiucd was almost twice as high ( 48.6% ) among those belonging to poorer family as compared to those from richer families ( 26.1% ) . one of the reasons could be that women belonging to richer families had better knowledge and access to wider choice of alternative methods such as injectable hormonal contraceptives . hence , these women might have preferred contraceptive methods other than ppiucd . among the women practicing hindu religion , the ppiucd acceptance rate was 38% whereas it was almost twice as high ( 70.6% ) among women belonging to muslim religion . it is possible that due to small absolute numbers of muslim women ( n = 17 ) , the percentage figure was misleading . however , on the whole , the figures are in direct contrast to often held popular belief that due to religious injunction muslim women are often unwilling to accept contraceptive methods , especially iucd . the study findings suggest that given privacy , anonymity , and proper counseling , muslim women too are as likely to accept ppiucd as their hindu counterparts . often , women hear about ppiucd only when they are in labor , a time when it is difficult to make informed decision . in a study , we found that women who received anc visits were more likely to accept ppiucd after the delivery . it seems that such visits provided an opportunity for interaction with the health - care provider , repeated counseling , and clarification of doubts that women might have had . other indian studies have also reported that the acceptance rate of ppiucd was high when there was at least one living child . it is likely that couples already having living children may preferentially opt for one of the contraceptive methods . in a society that values highly a male child , it probably was reassuring to the woman that by having a male child a significant milestone has been achieved . contrary to popular belief , we found that sex of the newborn did not have any effect on decision to accept ppiucd . rather it was whether the woman already had one or more living children which was more important . it is possible that the desire to have more time to decide about third or higher order pregnancy inclined women to accept ppiucd irrespective of the sex of the newborn . the study by bhalerao and purandare reported that the acceptance of ppiucd was high among women who had at least one male child . hence , we were unable to assess the impact of sex of the previous children on decision to accept ppiucd . in our study , 128 women ( 53.8% ) could be followed - up . in their multicentric study in india of 2,733 women , reported a follow - up rate of 63.3% though they did not mentioned the type of health - care facilities included in their study . in the study done in district hospital of bolangir , odisha , similarly , a lost to follow - up of 21.4% was reported by shukla et al . in their study thus , our follow - up rate , although lower , reflected the possibility in a real - life situation at a primary care level . we found an expulsion rate of 18% which was higher than the expulsion rate of 10.7% at 6 months reported by shukla et al . in their study one recent study from turkey on ppiucd among women after c - section reported an expulsion rate of nearly 18% . another indian study conducted on 168 women reported 16.4% as iud expulsion rate in women undergoing postpuerperal iud insertion . pain abdomen and bleeding per vaginum were the most common side effects reported . the expulsion rate observed in our study was 17.7% . the expulsion rate of ppiucd is generally higher than the interval iucd , ( o.r . : early expulsion is more likely if the iucd is not placed high up in uterine fundus . our data pertained to the period when ppiucd insertion had been recently introduced at the phc level . it is likely that the inadequate skill of the health - care providers could have been a factor for relatively higher expulsion rate . upgrading skill by robust training and better supportive supervision can help in reducing the expulsion of ppiucd . since this was a secondary data analysis , we were not able to study other variables which might have affected ppiucd acceptance , for example , fear of side effects , quality of counseling , category of health - care worker who did the counseling , etc . , the reported complications rates of ppiucd need to be interpreted cautiously since the follow - up information were unavailable for two - fifths of the acceptors . it is possible that only those women who had complications of ppiucd reported to phc for remedial follow - up . in that case however , it is also likely that those women who had complications of ppiucd had lost faith in phc and had therefore availed services elsewhere . if this were true , then the observed rate of complications would be an underestimate . we can not speculate as to which of these two scenarios is likely to be true . hence , we recommend that a prospectively collected follow - up data would be able to find out the true picture . the acceptance rate of ppiucd was 39% and was higher than what has been reported in the literature . women from low - income family , having at least one living child , and having attended anc clinic were more likely to accept ppiucd .

introduction : acceptance rate of postpartum intrauterine contraceptive device ( ppiucd ) offered through a public health approach is unknown . our aim was to describe the acceptance rate , expulsion , and follow - up and factors associated with it when ppiucd was offered to women delivering at two primary health centers ( phcs).methods : we analyzed routine health data of deliveries at two phcs in district faridabad , india between may and december 2014 , having sociodemographic variables , obstetric history , and during the follow - up check - up at 6-weeks postpartum for in situ status of intrauterine contraceptive device , side effects , and complications.results:the overall acceptance rate among those eligible for ppiucd was 39% ( 95% confidence interval [ ci ] : 35.142.9 ) . independent predictor of acceptance was a monthly family income of < usd75 $ ( odds ratio [ o.r . ] : 2.29 , 95% ci : 1.583.31 ) . the expulsion rate , and removal rate at 6 weeks postpartum was 18.0% and 13.0% , respectively . expulsion by 6 weeks was associated with , age > 25 years ( o.r . : 2.21 , 95% ci : 1.034.73 ) , gravida 4 ( o.r . : 4.01 , 95% ci : 1.2812.56 ) , and a living previous - child ( o.r . : 1.51 , 95% ci : 1.042.19).conclusion : acceptance rate of ppiucd was higher than that reported in literature . women from lower income family , having at least one living child , and having attended antenatal care clinic were more likely to accept ppiucd .

Introduction Methods Study setting Delivery hut scheme Provision of postpartum intrauterine contraceptive device services at the study site Study variables Data entry and statistical analysis Results Discussion Conclusion Financial support and sponsorship Conflicts of interest

neurofibromatosis-1 ( nf1 ) , also known as von recklinghausen disease , is an inherited , autosomal dominant disease phenotypically characterized by multiple caf au lait spots , lisch nodules , freckling , and development of neurofibromas , and in some cases , their malignant derivatives 1 . also , gastrointestinal stromal tumors ( gists ) arise in nf1 patients 150 times more frequently than in the general population 2 . with an estimated birth incidence of 1:3000 in western countries , it was postulated that nf1 is caused by a loss - of - function of tumor suppressor gene leading to the inactivation of neurofibromin that regulates cell proliferation and cell differentiation through inhibitory regulation of the ras oncogene , a gtpase 3 - 7 . gastrointestinal stromal tumor ( gist ) is the most common mesenchymal neoplasm of the gastrointestinal tract . gists are believed to originate from the interstitial cells of cajal , pacemaker cells that regulate peristalsis in the digestive tract 8 . an oncogenic ( mutational ) activation of kit or pdgfra tyrosine kinases has been identified in a great majority of gists 9 . however , no gist - specific kit or pdgfra mutations were identified in gist associated with nf1 , carney triad ( ct ) , carney - stratakis syndrome ( css ) , in pediatric gists , or in a small subset ( < 10% ) of sporadic adult gists 10 - 14 . molecular mechanisms underlying the development of kit / pdgfra - wild type ( wt ) gists are not well understood . recently , gists associated with ct ( triad of gastric gist , pulmonary chondroma , and extra - adrenal paraganglioma ) and css ( hereditary gist paraganglioma syndrome ) have been shown to have a loss of succinate dehydrogenase subunit b ( sdhb ) expression 15 , 16 . sdhb , a mitochondrial protein , is one of the ubiquitously expressed proteins functioning at the interface of tricarboxylic acid cycle and electron transport chain . it is believed that loss of sdhb expression leads to a tumorigennic mechanism involving the stabilization of hypoxia inducing factors ( hif ) , which in turn upregulates genes that promote adaptation of cells in low - oxygen environments by inducing angiogenesis and glycolysis 17 - 20 . data on sdhb expression in nf1-associated gists are limited and only three cases were tested and turned to be sdhb- positive 15 . the aim of this study was to evaluate sdhb expression in a significant number of nf1-associated gists and test a hypothesis that a lack of sdhb expression , unlike in ct- and css - associated tumors and pediatric gists , is not involved in pathogenesis of nf1-associated gists . a total of 23 nf1-associated gists , from years 1970 - 1999 , were obtained from the armed forces institute of pathology ( afip ) tissue repository . immunostaining was performed with leica bond - max automatic immunostainer ( leica , bannockburn , il ) using a commercially available sdhb mouse monoclonal antibody 21a11 ( abcam ab14714 , cambridge , ma ) . heat induced epitope retrieval ( high ph , edta - based buffer , ph 9.0 , leica bond - max ) was applied for 25 minutes . leica bond - max avidin - biotin free polymer system was used in the detection according to the recommended procedure . diaminobenzidine was used as the chromogen , following the blocking of endogenous peroxidase with 3% hydrogen peroxide diluted in phosphate buffer . only stains with positive internal controls ( granular cytoplasmic staining in vascular endothelia and pericytes ) were considered valid . one case with unsuccesful immunostaining ( internal control negative ) was removed from the study . 1 ) . in the analyzed cohort , there were 8 ( 36.4% ) male and 14 ( 63.6% ) female patients . tumor sizes varied from 0.9 to 10 cm ( median , 3.5 cm ) , and 15 patients presented with multiple tumors . most of these gists had mitotic counts < 5 per 50 hpfs , and only two tumors contained more than 5 mitoses per 50 hpfs . gist - specific kit exon 9 , 11 , 13 , and 17 , and pdgfra exon 12 , 14 , and 18 mutations were not found as previously reported 11 . in this study , we demonstrate consistent sdhb expression in neurofibromatosis 1 ( nf1)-associated gists . clinicopathologic features of these gists include patient female predominance , small intestinal location , common multiplicity , kit and dog 1 expression , and lack of gist - specific kit and pdgfra mutations , as previously reported 10 , 11 . lack of gist - specific kit and pdgfra mutations has also been reported in pediatric gists , carney triad ( ct ) and carney - stratakis ( cs ) syndrome - associated gists , and in a small subset of adult gists 10 - 14 . lack of sdhb expression has been reported in carney triad ( ct ) and carney - stratakis ( cs ) syndrome - associated gists and more recently in pediatric and adult sporadic gists showing morphologic and clinical features similar to ct- and cs - gists . it has been proposed that based on sdhb expression , gists could be divided into 2 distinctive subtypes : sdhb- positive ( type 1 ) and sdhb - negative ( type 2 ) tumors 15 . type 1 gists can occur in adults anywhere in the gastrointestinal tract , have equal sex distribution , generally have kit or pdgfra mutations , and , in majority , respond to imatinib treatment . type 2 gists occur in childhood or young adulthood nearly exclusively in the stomach , show a striking predilection for the female sex and epitheliod morphology . these tumors lack kit or pdgfra mutations and show a limited response to imatinb inhibition 14 . based on sdhb - expression status , nf1-associated gists belong to type 1 category . however , they differ from type 1 gist characteristics by restriction of occurrence to intestines , common tumor multiplicity , female predominance , and consistent lack of gist - specific kit and pdgra mutations . similar to sdhb type 2 tumors , they do not respond well to imatinib treatment 21 - 23 . therefore , clinicopathologically nf1-associated gists deserve a separation from other type 1 ( sdhb - positive ) gists . although imatinib mesylate , kit and pdgfra tyrosine kinase inhibitor was successfully used for treatment of advanced and disseminated gists 24 , such a treatment is ineffective or minimally effective in kit / pdgfra - wild type gists , as well as those with certain mutations , such as pdgfra asp842val substitution 25 . thus , genotyping of gists is considered a standard clinical test in all disseminated gists and tumors with a substantial risk of developing metastatic disease . in conclusion , nf1-associated gists express sdhb , similar to a majority of adult gists . in view of different kit / pdgfra mutation status and prospect to targeted treatment , simple categorization of gists into type 1 and type 2 based on sdhb expression is inadequate . a classification based on both sdhb expression status and other clinicopatholopgic factors : kit / pdgfra mutation and nf1 syndrome status characterize gists more accurately allowing subdivision of sdhb - positive tumors into different categories based on these clinicopathologic factors ( table 1 ) .

gastrointestinal stromal tumor ( gist ) is the most common mesenchymal tumor of the digestive tract . gists develop with relatively high incidence in patients with neurofibromatosis-1 syndrome ( nf1 ) . mutational activation of kit or pdgfra is believed to be a driving force in the pathogenesis of familial and sporadic gists . unlike those tumors , nf1-associated gists do not have kit or pgdfra mutations . similarly , no mutational activation of kit or pdgfra has been identified in pediatric gists and in gists associated with carney triad and carney - stratakis syndrome . kit and pdgfra - wild type tumors are expected to have lesser response to imatinib treatment . recently , carney triad and carney - stratakis syndrome -associated gists and pediatric gists have been shown to have a loss of expression of succinate dehydrogenase subunit b ( sdhb ) , a krebs cycle / electron transport chain interface protein . it was proposed that gists can be divided into sdhb- positive ( type 1 ) , and sdhb - negative ( type 2 ) tumors because of similarities in clinical features and response to imatinib treatment . in this study , sdhb expression was examined immunohistochemically in 22 well - characterized nf1-associated gists . all analyzed tumors expressed sdhb . based on sdhb - expression status , nf1-associated gists belong to type 1 category ; however , similarly to sdhb type 2 tumors , they do not respond well to imatinib treatment . therefore , a simple categorization of gists into sdhb - positive and - negative seems to be incomplete . a classification based on both sdhb expression status and kit and pdgfra mutation status characterize gists more accurately and allow subdivision of sdhb - positive tumors into different clinico - genetic categories .

Introduction Materials and Methods Results Discussion

the prevalence of distal symmetric polyneuropathy ( dsp ) is greater than 3% and is even higher in the elderly ( 1,2 ) . this common condition causes significant morbidity and is associated with pain , falls , and reduced quality of life ( 3,4 ) . while diabetes is by far the number one cause , enhanced glucose control is only marginally effective at reducing the incidence in patients with type 2 diabetes ( 5 ) . one possible explanation for this small therapeutic effect is that other factors , such as the other components of the metabolic syndrome ( mets ) , may be significant contributors to nerve injury ( 6 ) . the other mets components may also be the underlying cause for some of the 30% of dsp patients currently labeled as idiopathic ( 710 ) . past studies have revealed an association between the mets and polyneuropathy ( 1114 ) . however , previous investigations have reported conflicting associations between the individual mets components and neuropathy ( 1521 ) . for example , while diabetes is a well - established risk factor for neuropathy , population studies investigating the effect of prediabetes have reached different conclusions ( 16,22,23 ) . to date , most studies investigating the association between the mets and polyneuropathy have been performed only in patients with diabetes , used cross - sectional designs , and lacked rigorous definitions of dsp such as only evaluating one confirmatory test ( 6 ) . the goal of this study is to address these limitations . in a large cohort of older adults , we aimed to determine whether the prevalence of symptomatic dsp , utilizing a rigorous definition , increases with increasing numbers of mets components independent of glycemic status . we also investigated the associations between the individual mets components and symptomatic dsp in cross - sectional and longitudinal analyses . data for our analysis came from participants in the health , aging , and body composition ( health abc ) study , which is an ongoing , prospective cohort study of 7079 year olds ( n = 3,075 ; 48.4% male ; 41.6% black ) followed since the baseline evaluation in 1997 or 1998 . the cohort consists of a random sample of age - eligible white medicare beneficiaries , all black medicare beneficiaries , and all community - dwelling black persons in pittsburgh and memphis . participants had to self - report having no difficulty with walking 1/4 mile , climbing 10 steps , or any basic activity of daily living ; be free of any life - threatening cancers ; and plan to remain in the study area for at least 3 years . this cohort has been followed annually for the past 17 years with questionnaires , biospecimens , anthropomorphic measures , blood pressure , and neuropathy outcome assessments at differing intervals . participants were included in the cross - sectional analysis if they had complete neuropathy outcome data from year 4 in 20002001 . participants were included in the longitudinal analysis if they had complete neuropathy outcome data from years 4 ( 20002001 ) and 11 ( 20072008 ) . all study protocols were approved by institutional review boards at the university of pittsburgh and university of tennessee health science center . diabetes ( fasting glucose 126 mg / dl or 2-h glucose 200 mg / dl ) and prediabetes ( fasting glucose 100 mg / dl or 2-h glucose 140 mg / dl ) were defined according to the guidelines of the expert committee on the diagnosis and classification of diabetes mellitus ( 24 ) . participants on diabetes medications were also considered to have diabetes . lipid panels and blood pressure were measured at years 1 , 2 , 4 , 6 , 8 , 10 , and 11 , with blood pressure also measured at years 3 and 5 . height , weight , and bmi were measured at years 1 , 4 , 6 , 8 , 10 , and 11 , whereas waist circumference was only measured at years 1 and 6 . the updated national cholesterol education program / adult treatment panel iii criteria were used to define the mets and its individual components , which takes into consideration medications for the different components ( 25 ) . specifically , the mets criteria were a waist circumference 102 cm in men and 88 cm in women , systolic blood pressure 130 mmhg or diastolic blood pressure 85 mmhg , triglycerides 150 mg / dl , and hdl < 40 mg / dl in men and < 50 mg / dl in women . in the cross - sectional analysis , metabolic data from year 1 were used . in the longitudinal analysis , baseline metabolic data were obtained from year 1 with the exception of hemoglobin a1c , which was only available starting in year 4 . our primary dsp outcome measure was determined by the combination of neuropathy symptoms as identified by at least one of two questionnaire items endorsed and at least one of three confirmatory neuropathy tests abnormal ; therefore , our primary outcome construct was symptomatic dsp . the questionnaire items were as follows : in the past 12 months , have you ever had numbness , an asleep feeling, a prickly feeling or tingling in your legs or feet ? and in the past 12 months , have you ever had a sudden stabbing or burning pain , or a deep aching in your legs or feet ? the three confirmatory neuropathy tests included a peroneal motor nerve conduction velocity ( cv ) < 40 m / s , a vibration threshold > 131 m , and inability to feel a 10-g ( heavy ) monofilament at the dorsum of the great toe in three out of four trials . in addition , the peroneal compound motor action potential ( cmap ) at the ankle and a 1.4-g ( light ) monofilament at the dorsum of the great toe ( abnormal if unable to feel in three of four trials ) were secondary outcomes . five of the six secondary dsp outcome measures did not require symptoms ; therefore , these measures had the potential to capture asymptomatic as well as symptomatic dsp . the peroneal motor nerve conduction studies were performed on a neuromax 8 ( xltek ) . vibration detection was performed at the bottom of the great toe using the vsa-3000 vibratory sensory analyzer ( medoc ) . feet were warmed to 30c , and measures were performed on the right unless contraindicated owing to knee replacement , amputation , trauma , ulcer , or recent surgery , in which case the left side was tested unless also contraindicated . one - way anova ( continuous variables ) and tests ( categorical variables ) were used to test for differences in demographics between the three glycemic groups . we determined the prevalence of neuropathy stratified by glycemic status and the number of mets components . we then applied the cochran - mantel - haenszel test to investigate the effect of the number of mets components on neuropathy prevalence , controlling for glycemic status . multivariable logistic regression was used to model the year 4 primary neuropathy outcome as a function of the year 1 mets components ( waist circumference , prediabetes , diabetes , hdl level , triglyceride level , systolic blood pressure ) after adjustment for baseline demographic factors ( age , sex , race , height , smoking , and alcohol consumption ) . weight , hemoglobin a1c , log homa of insulin resistance , and diastolic blood pressure were also considered as mets component variables but were not used in the final model ( akaike information criterion was used in model selection ) . secondary neuropathy outcomes including neuropathic symptoms , heavy monofilament , and light monofilament outcomes were also analyzed using multivariable logistic regression models . multivariable linear regression was used to analyze the continuous neuropathy measurements ( peroneal cv , peroneal cmap , vibration threshold ) as a function of mets components , adjusting for the same covariates . finally , we fit a separate multivariable logistic regression model to study the association of the number of mets components , excluding glycemic status , with our primary neuropathy outcome , adjusting for demographic factors and glycemic status . we first estimated the longitudinal change in each mets component ( hemoglobin a1c , weight , systolic blood pressure , triglycerides , and hdl ) by fitting a simple linear regression model over time for each subject . then , the estimated subject - specific slopes , along with the baseline measurements for each mets component , were incorporated into a multivariable logistic regression model to investigate the association of longitudinal change in mets components and incident symptomatic neuropathy ( those with neuropathy in year 11 but not at year 4 ) after adjustment for baseline demographics as described above . data for our analysis came from participants in the health , aging , and body composition ( health abc ) study , which is an ongoing , prospective cohort study of 7079 year olds ( n = 3,075 ; 48.4% male ; 41.6% black ) followed since the baseline evaluation in 1997 or 1998 . the cohort consists of a random sample of age - eligible white medicare beneficiaries , all black medicare beneficiaries , and all community - dwelling black persons in pittsburgh and memphis . participants had to self - report having no difficulty with walking 1/4 mile , climbing 10 steps , or any basic activity of daily living ; be free of any life - threatening cancers ; and plan to remain in the study area for at least 3 years . this cohort has been followed annually for the past 17 years with questionnaires , biospecimens , anthropomorphic measures , blood pressure , and neuropathy outcome assessments at differing intervals . participants were included in the cross - sectional analysis if they had complete neuropathy outcome data from year 4 in 20002001 . participants were included in the longitudinal analysis if they had complete neuropathy outcome data from years 4 ( 20002001 ) and 11 ( 20072008 ) . all study protocols were approved by institutional review boards at the university of pittsburgh and university of tennessee health science center . diabetes ( fasting glucose 126 mg / dl or 2-h glucose 200 mg / dl ) and prediabetes ( fasting glucose 100 mg / dl or 2-h glucose 140 mg / dl ) were defined according to the guidelines of the expert committee on the diagnosis and classification of diabetes mellitus ( 24 ) . participants on diabetes medications were also considered to have diabetes . lipid panels and blood pressure were measured at years 1 , 2 , 4 , 6 , 8 , 10 , and 11 , with blood pressure also measured at years 3 and 5 . height , weight , and bmi were measured at years 1 , 4 , 6 , 8 , 10 , and 11 , whereas waist circumference was only measured at years 1 and 6 . the updated national cholesterol education program / adult treatment panel iii criteria were used to define the mets and its individual components , which takes into consideration medications for the different components ( 25 ) . specifically , the mets criteria were a waist circumference 102 cm in men and 88 cm in women , systolic blood pressure 130 mmhg or diastolic blood pressure 85 mmhg , triglycerides 150 mg / dl , and hdl < 40 mg / dl in men and < 50 mg / dl in women . in the cross - sectional analysis , metabolic data from year 1 were used . in the longitudinal analysis , baseline metabolic data were obtained from year 1 with the exception of hemoglobin a1c , which was only available starting in year 4 . our primary dsp outcome measure was determined by the combination of neuropathy symptoms as identified by at least one of two questionnaire items endorsed and at least one of three confirmatory neuropathy tests abnormal ; therefore , our primary outcome construct was symptomatic dsp . the questionnaire items were as follows : in the past 12 months , have you ever had numbness , an asleep feeling, a prickly feeling or tingling in your legs or feet ? and in the past 12 months , have you ever had a sudden stabbing or burning pain , or a deep aching in your legs or feet ? the three confirmatory neuropathy tests included a peroneal motor nerve conduction velocity ( cv ) < 40 m / s , a vibration threshold > 131 m , and inability to feel a 10-g ( heavy ) monofilament at the dorsum of the great toe in three out of four trials . in addition , the peroneal compound motor action potential ( cmap ) at the ankle and a 1.4-g ( light ) monofilament at the dorsum of the great toe ( abnormal if unable to feel in three of four trials ) were secondary outcomes . five of the six secondary dsp outcome measures did not require symptoms ; therefore , these measures had the potential to capture asymptomatic as well as symptomatic dsp . the peroneal motor nerve conduction studies were performed on a neuromax 8 ( xltek ) . vibration detection was performed at the bottom of the great toe using the vsa-3000 vibratory sensory analyzer ( medoc ) . feet were warmed to 30c , and measures were performed on the right unless contraindicated owing to knee replacement , amputation , trauma , ulcer , or recent surgery , in which case the left side was tested unless also contraindicated . one - way anova ( continuous variables ) and tests ( categorical variables ) were used to test for differences in demographics between the three glycemic groups . we determined the prevalence of neuropathy stratified by glycemic status and the number of mets components . we then applied the cochran - mantel - haenszel test to investigate the effect of the number of mets components on neuropathy prevalence , controlling for glycemic status . multivariable logistic regression was used to model the year 4 primary neuropathy outcome as a function of the year 1 mets components ( waist circumference , prediabetes , diabetes , hdl level , triglyceride level , systolic blood pressure ) after adjustment for baseline demographic factors ( age , sex , race , height , smoking , and alcohol consumption ) . weight , hemoglobin a1c , log homa of insulin resistance , and diastolic blood pressure were also considered as mets component variables but were not used in the final model ( akaike information criterion was used in model selection ) . secondary neuropathy outcomes including neuropathic symptoms , heavy monofilament , and light monofilament outcomes were also analyzed using multivariable logistic regression models . multivariable linear regression was used to analyze the continuous neuropathy measurements ( peroneal cv , peroneal cmap , vibration threshold ) as a function of mets components , adjusting for the same covariates . finally , we fit a separate multivariable logistic regression model to study the association of the number of mets components , excluding glycemic status , with our primary neuropathy outcome , adjusting for demographic factors and glycemic status . we first estimated the longitudinal change in each mets component ( hemoglobin a1c , weight , systolic blood pressure , triglycerides , and hdl ) by fitting a simple linear regression model over time for each subject . then , the estimated subject - specific slopes , along with the baseline measurements for each mets component , were incorporated into a multivariable logistic regression model to investigate the association of longitudinal change in mets components and incident symptomatic neuropathy ( those with neuropathy in year 11 but not at year 4 ) after adjustment for baseline demographics as described above . of participants in the health abc cohort , 2,382 had complete neuropathy outcome data and were included in our cross - sectional analysis . of these 2,382 , 1,263 participants also had complete neuropathy outcome data at year 11 and were included in our longitudinal analysis . in a comparison of year 4 with year 11 , 184 subjects developed symptomatic neuropathy ( incident neuropathy ) , 948 remained neuropathy free , 67 continued with neuropathy , and 64 became neuropathy free . the mean ( sd ) age was 73.5 ( 2.9 ) , and 51.7% were female . participants with normoglycemia accounted for 49.1% of the population , prediabetes 29.9% , and diabetes 21.0% . demographics of the health abc cohort at year 1 stratified by glycemic status dbp , diastolic blood pressure ; sbp , systolic blood pressure . the prevalence of symptomatic dsp was significantly higher in participants with higher number of mets components ( p = 0.03 ) , controlling for glycemic status ( fig . the dsp prevalence was 8.5% in those with no mets components ( excluding hyperglycemia ) compared with 12.9% in those with four mets components . in those with normoglycemia , the dsp prevalence was 8.6% in those with no mets components compared with 11.4% in those with four mets components . in those with prediabetes , the dsp prevalence was 5.6% in those with no additional mets components compared with 10.6% in those with four additional mets components . in contrast , the dsp prevalence in participants with diabetes was similar between those with no additional mets components ( 16.7% ) and those with four additional mets components ( 17.1% ) . dsp was defined as those with neuropathic symptoms ( questionnaire ) plus at least 1 of 3 confirmatory tests ( heavy monofilament , peroneal cv , or vibration threshold ) in year 4 of the study . glycemic status was determined by the glucose tolerance test according to the expert committee on the diagnosis and classification of diabetes mellitus in year 1 . mets components were defined using the updated national cholesterol education program / adult treatment panel iii definition . in a multivariable logistic regression model investigating the individual mets components , diabetes ( odds ratio [ or ] 1.65 [ 95% ci 1.182.31 ] ) , height ( or 1.39 [ 95% ci 1.241.54 ] , unit = 5 cm ) , and male sex ( or 0.59 [ 95% ci 0.390.92 ] ) were significantly associated with the dsp primary outcome ( table 2 ) . prediabetes , waist circumference , systolic blood pressure , triglycerides , and hdl were not significantly associated with dsp . in a separate multivariable logistic regression model investigating the association of the number of mets components in addition to glycemic status , the number of mets components ( or 1.20 [ 95% ci 1.061.37 ] ) was significantly associated with symptomatic dsp . multivariable logistic regression evaluating the association of mets components ( year 1 ) and neuropathy ( year 4 ) ( cross - sectional ) data are or ( 95% ci ) . sbp , systolic blood pressure . in investigation of the association of mets components and the 6 secondary neuropathy outcomes , increasing age and height were significantly associated with all neuropathy outcome measures with the exception of neuropathic symptoms . increasing while systolic blood pressure and triglycerides were significantly associated with one and two neuropathy outcomes , respectively , the association was in the opposite direction of what was expected ( lower blood pressure and lower triglycerides ) . multivariable linear regression evaluating the association of mets components ( year 1 ) and continuous outcomes of neuropathy ( year 4 ) ( cross - sectional ) sbp , systolic blood pressure . in a multivariable logistic regression model evaluating the longitudinal change of mets components , only the baseline hemoglobin a1c ( year 4 or 1.42 [ 95% ci 1.151.75 ] ) and height ( or 1.26 [ 95% ci 1.091.46 ] ) were significantly associated with incident symptomatic neuropathy . baseline weight ( or 0.99 [ 95% ci 0.921.07 ] ) , systolic blood pressure ( or 1.06 [ 95% ci 0.971.17 ] ) , triglycerides ( or 0.99 [ 95% ci 0.841.16 ] ) , and hdl ( or 0.94 [ 95% ci 0.811.09 ] ) were not significantly associated with incident neuropathy . similarly , the slopes of the mets components ( hemoglobin a1c or 1.91 [ 95% ci 0.2812.95 ] , weight or 0.71 [ 95% ci 0.202.55 ] , systolic blood pressure or 1.54 [ 95% ci 0.623.86 ] , triglyceride or 0.79 [ 95% ci 0.134.85 ] , and hdl or 0.87 [ 95% ci 0.135.88 ] ) were not significantly associated with incident neuropathy . in a multivariable logistic regression model investigating the individual mets components , diabetes ( odds ratio [ or ] 1.65 [ 95% ci 1.182.31 ] ) , height ( or 1.39 [ 95% ci 1.241.54 ] , unit = 5 cm ) , and male sex ( or 0.59 [ 95% ci 0.390.92 ] ) were significantly associated with the dsp primary outcome ( table 2 ) . prediabetes , waist circumference , systolic blood pressure , triglycerides , and hdl were not significantly associated with dsp . in a separate multivariable logistic regression model investigating the association of the number of mets components in addition to glycemic status , the number of mets components ( or 1.20 [ 95% ci 1.061.37 ] ) was significantly associated with symptomatic dsp . multivariable logistic regression evaluating the association of mets components ( year 1 ) and neuropathy ( year 4 ) ( cross - sectional ) data are or ( 95% ci ) . sbp , systolic blood pressure . in investigation of the association of mets components and the 6 secondary neuropathy outcomes , increasing age and height were significantly associated with all neuropathy outcome measures with the exception of neuropathic symptoms . increasing while systolic blood pressure and triglycerides were significantly associated with one and two neuropathy outcomes , respectively , the association was in the opposite direction of what was expected ( lower blood pressure and lower triglycerides ) . multivariable linear regression evaluating the association of mets components ( year 1 ) and continuous outcomes of neuropathy ( year 4 ) ( cross - sectional ) sbp , systolic blood pressure . in a multivariable logistic regression model evaluating the longitudinal change of mets components , only the baseline hemoglobin a1c ( year 4 or 1.42 [ 95% ci 1.151.75 ] ) and height ( or 1.26 [ 95% ci 1.091.46 ] ) were significantly associated with incident symptomatic neuropathy . baseline weight ( or 0.99 [ 95% ci 0.921.07 ] ) , systolic blood pressure ( or 1.06 [ 95% ci 0.971.17 ] ) , triglycerides ( or 0.99 [ 95% ci 0.841.16 ] ) , and hdl ( or 0.94 [ 95% ci 0.811.09 ] ) were not significantly associated with incident neuropathy . similarly , the slopes of the mets components ( hemoglobin a1c or 1.91 [ 95% ci 0.2812.95 ] , weight or 0.71 [ 95% ci 0.202.55 ] , systolic blood pressure or 1.54 [ 95% ci 0.623.86 ] , triglyceride or 0.79 [ 95% ci 0.134.85 ] , and hdl or 0.87 [ 95% ci 0.135.88 ] ) were not significantly associated with incident neuropathy . in our large cohort of older black and white men and women , 21.0% of the population had diabetes and 52.8% met criteria for mets at baseline . these numbers are comparable with those in previous studies using national health and nutrition examination survey ( nhanes ) data , which have estimated a diabetes prevalence of 20% in those over 65 years of age and a mets prevalence of 4758% in those over 70 years of age ( 26,27 ) . in our well - functioning older cohort , a previous nhanes study revealed similar results with a symptomatic dsp prevalence of 3.1% in those over 40 years of age , which was three times higher in those age 7079 years ( 2 ) . a study in italy in those over the age of 55 years revealed a symptomatic dsp prevalence of 78% with an even higher prevalence in those > 75 years ( 1 ) . similarly , others have estimated a clinical neuropathy prevalence of 69% in those > 7079 years old ( 28 ) . in concert with previous studies , we demonstrate that the prevalence of diabetes , mets , and dsp is incredibly high in even a well - functioning older population . as a result , any causal link between mets and neuropathy , regardless of diabetes status , would have potential therapeutic implications for a large proportion of older adults . furthermore , previous studies have demonstrated that the peripheral nerve measures used in this study are associated with important functional outcomes in older populations including mobility , quadriceps strength , and other physical performance measures ( 2931 ) . therefore , therapeutic interventions to prevent and/or ameliorate dsp in this population may have a clinically meaningful effect . we found a statistically significant association between the number of mets components and symptomatic dsp when adjusting for glycemic status in two separate analyses . in fact , the dsp prevalence increased from 8.5 to 12.9% when we compared subjects with no mets components , excluding hyperglycemia , with those with all four components . the implication is that mets components may be important risk factors for the development of dsp independent of glycemic status . the magnitude of the effect of additional mets components on dsp prevalence ( 1.1% difference per component ) is not as high as the effect of diabetes ( 8.6% ) . however , given the high prevalence of mets components individually and in combination , this effect may be clinically important . our results are comparable with those of the recent prospective metabolism and islet cell evaluation ( promise ) study in patients at high risk of diabetes that demonstrated that the vibration threshold increased as the number of mets components increased ( 32 ) . however , in contrast to our analysis , this association did not persist after adjustment for participant demographics . interestingly , we found that the rise in dsp prevalence with additional mets components was most evident in those with normoglycemia and prediabetes . the number of mets components did not affect the dsp prevalence in those with diabetes . of note future studies investigating the effect of treatment of mets components on the prevention or treatment of dsp may need to target the normoglycemic populations and populations with prediabetes . the prevalence of symptomatic dsp in those with prediabetes ( 9.3% ) was comparable with the prevalence in those with normoglycemia ( 9.7% ) . furthermore , prediabetes was not associated with any of our neuropathic outcome measures in multivariable regression models . these observations add to other conflicting studies on the importance of prediabetes as a cause of dsp . the monica / kora ( multinational monitoring of trends and determinants in cardiovascular disease / cooperative health research in the region of augsburg ) , san luis valley , and promise ( all with dsp defined by neurologic examination ) studies all revealed an increased prevalence of dsp in those with impaired glucose tolerance compared with normoglycemia ( 16,23,32 ) . however , investigators using nhanes data reported a dsp prevalence ( based on monofilament testing ) comparable with that in our population with no significant difference between those with normoglycemia ( 10.5% ) and those with impaired fasting glucose ( 11.9% ) ( 22 ) . all of these studies used different definitions of dsp , were conducted in different populations , and included subjects of different ages . one possible explanation for the discrepant results is that the effect of prediabetes in older adults is less than in younger adults . alternatively , the neuropathy definition used in this study was insufficient to capture small fiber predominant neuropathy , which is typically seen in those with either prediabetes and/or mets ( 33 ) . another possibility is that prediabetes is not as important as the other mets components that tend to cluster with this condition . future studies are needed to further understand the role of prediabetes in dsp . a clinical trial investigating the effects of the treatment of prediabetes on neuropathy outcomes may be the only way to definitely address this question . diabetes was the only mets component that was significantly associated with the primary dsp outcome in both cross - sectional and longitudinal analyses . however , waist circumference ( four of six ) and hdl ( two of six ) were associated with some of the secondary neuropathy outcome measures . one potential reason for the lack of associations between mets components and dsp is that we did not have enough power to detect the associations , which likely have smaller effect sizes . the power of our secondary outcomes may have been more robust because most had the potential to capture asymptomatic dsp as well as symptomatic dsp . furthermore , we did not have adequate power to investigate interactions between glycemic status and the other mets components . interactions are likely important , as the prevalence of dsp only increased as the number of mets components increased in those with normoglycemia and prediabetes . another possibility is that the main drivers of dsp are not the mets components themselves but , rather , other factors that are associated with them . for example , perhaps abdominal obesity is not one of the main drivers of dsp and , instead , adipose tissue derived inflammatory markers , such as interleukin-6 levels or adiponectin - to - leptin ratios , are the true mediators ( 34 ) . further studies with larger samples sizes and more precise mets and neuropathy measurements are needed to further delineate the role of mets components in the development of dsp . limitations include the lack of a neurologic examination to determine neuropathy status and neuropathic symptoms based on a questionnaire . however , we were able to combine questionnaire items pertaining to neuropathic symptoms in the feet with well - validated neuropathy confirmatory studies . our sample size was not adequate to address small effect sizes or interaction between mets components and glycemic status . similarly , the sample size in the longitudinal analysis was smaller than the cross - sectional analysis . nevertheless , our study is one of the largest to date to investigate the association between mets components and dsp . our analysis was confined to those well - functioning 7079 year olds at the start of the study . how our results apply to other populations mets components are significantly associated with dsp independent of glycemic status , although the effect is less than that of the presence of diabetes . the precise components , or combinations of components , that drive the development of dsp remain unclear . larger studies with more precise metabolic measures are needed to guide the development of future intervention studies . given the high prevalence of both the mets and dsp , particularly in older adults , even small causal effects have the potential for a large impact on population health .

objectiveprevious studies demonstrate that the metabolic syndrome is associated with distal symmetric polyneuropathy ( dsp ) . we aimed to determine the magnitude of this effect and the precise components involved.research design and methodswe determined the symptomatic dsp prevalence in the health , aging , and body composition ( health abc ) study ( prospective cohort study , with subjects aged 7079 years at baseline ) , stratified by glycemic status ( glucose tolerance test ) and the number of additional metabolic syndrome components ( updated national cholesterol education program / adult treatment panel iii definition ) . dsp was defined as neuropathic symptoms ( questionnaire ) plus at least one of three confirmatory tests ( heavy monofilament , peroneal conduction velocity , and vibration threshold ) . multivariable logistic and linear regression evaluated the association of metabolic syndrome components with dsp in cross - sectional and longitudinal analyses.resultsof 2,382 participants with neuropathy measures ( mean age 73.5 2.9 years , 38.2% black , 51.7% women ) , 21.0% had diabetes , 29.9% prediabetes , 52.8% metabolic syndrome , and 11.1% dsp . stratified by glycemic status , dsp prevalence increased as the number of metabolic syndrome components increased ( p = 0.03 ) . diabetes ( cross - sectional model , odds ratio [ or ] 1.65 [ 95% ci 1.182.31 ] ) and baseline hemoglobin a1c ( longitudinal model , or 1.42 [ 95% ci 1.151.75 ] ) were the only metabolic syndrome measures significantly associated with dsp . waist circumference and hdl were significantly associated with multiple secondary neuropathy outcomes.conclusionsindependent of glycemic status , symptomatic dsp is more common in those with additional metabolic syndrome components . however , the issue of which metabolic syndrome components drive this association , in addition to hyperglycemia , remains unclear . larger waist circumference and low hdl may be associated with dsp , but larger studies with more precise metabolic measures are needed .

Introduction Research Design and Methods Population MetS Components DSP Definition Statistical Analysis Cross-sectional Analyses Longitudinal Analysis Results Cross-sectional Analyses Longitudinal Analysis Conclusions

zingiberaceae , one of the largest families of the plant kingdom , is an important natural resource that provides many useful food products , spices , and traditional medicines to treat a variety of diseases [ 1 , 2 ] . the consumption of herbal products for therapeutic purposes and to promote wellness is widely popular since people are greatly concerned about side effects of synthetic drugs . herbs are being increasingly used in the pharmaceutical industry as raw materials for the preparation of herbal medicines . arsenic is widely distributed in the earth 's crust and present at an average concentration of 2 mg / kg . around one - third of atmospheric flux of arsenic arsenic is one of the first chemicals designated as a group 1 carcinogen and well known to be poisonous to organisms . the inorganic arsenic species ( as(iii ) and as(v ) ) are the most toxic forms of arsenic present in food . previous studies have indicated that ingested inorganic arsenic is strongly associated with a wide spectrum of adverse health outcomes , primary cancers , and other chronic diseases . the daily intake of total arsenic from food and beverages is generally in the range of 20300 g / day . furthermore , joint food and agriculture organization / world health organization expert committee on food additives note that benchmark dose lower confidence limit for a 0.5% ( bmdl 0.5 ) of inorganic arsenic in human was 3.0 g / kg body weight per day . root or rhizome is a part of the plant that has high opportunity to contaminate with arsenic . current research demonstrated that plants absorbed heavy metals from soil [ 12 , 13 ] . the contaminants and residues of toxic metal arsenic may cause harm to the consumers of herbal medicines . plants that grow in an arsenic - affected area may have a high level of arsenic . the transfer of arsenic from soils to plants might be a key step in the route of arsenic entry into human body [ 14 , 15 ] . experimental data had shown that a variety of vegetable crops accumulate arsenic by root uptake from soil deposited on the leaves , and gulz et al . reported several edible plants grown in contaminated soils that accumulated high levels of arsenic . the who determined that arsenic concentration in plants grown in soils without arsenic - containing pesticides varied from 0.02 to 5 mg / kg ( dry weight ) , and in the arsenic soil contamination indicated root can contain higher levels of arsenic than other parts of plant . a previous study about rhizome of zingiber officinale ( ginger ) shows that arsenic level varied from not detected to 0.13 g / l . other arsenic contamination reports of karadas and kara show that arsenic levels in cumin and turmeric were 174 14 and 39 5 ng / g , dry wt , respectively . vegetable crops in the contaminated region were found high in arsenic level ( dry wt ) in arum ranged from 74.3 to 89.2 mg / kg , in cabbage from 27.12 to 39.39 mg / kg and in pumpkin from 17.28 to 22.05 mg / kg . baroni et al . analyzed sixty - four plant species , and the highest arsenic contents investigated high arsenic levels in cumin and turmeric ( 47.86209.75 and 297.33280.9 ng / g , res . ) moreover , dry ginger exhibited mean arsenic content of 77.9 8.7 ng / g . in general , the highest concentration of arsenic was found in plant roots , the intermediate level in vegetative tissues ( leaves and stems ) , and the lowest level in reproductive tissue ( fruits and seeds ) [ 23 , 24 ] . plants absorb arsenic fairly easily , so that high - ranking concentration may be present . among many of public researches , most studies had focused on foods and not much on the information that was available on plants especially in the part of rhizomes although it was a high opportunity for arsenic accumulation . for this reason , well - known rhizomes of zingiberaceae family that are used as food , dietary supplements and alternative medicines in thailand , that is , a. galanga , b. rotunda , c. longa , c. zedoaria , z. cassumunar , and z. officinale , were interesting to investigate their total and inorganic arsenic concentrations . standard reference material ( srm ) 1568a ( rice flour ) was purchased from the national institute of standards and technology ( gaithersburg , md , usa ) . nitric acid ( hno3 ) and hydrochloric acid ( hcl ) were purchased from merck chemicals ( darmstadt , germany ) ; dimethylarsinic acid ( dma ) , hydrazine sulfate , hydrobromic acid , and other chemicals were obtained from sigma - aldrich ( st . all standard solutions , reagents , and samples were prepared using deionized water ( 18 m cm ) throughout the study . to remove possible arsenic residue contamination , all glasswares were washed thoroughly with tap water , air - dried , soaked in 10% ( v / v ) hno3 for 2024 h , and washed three times with deionized water . six kinds of plants in the family zingiberaceae of which rhizomes are widely used for consumption and medication purposes were selected . ( kra - chaai ) , curcuma longa ( l. ) ( khamin - chan ) , curcuma zedoaria ( berg . ) roscoe ( khamin - oi ) , zingiber cassumunar roxb . sixteen samples of each rhizome species were collected during december 2011 and january 2012 from eight provinces ( chiang rai , lampang , khon kaen , ubon ratchathani , ratchaburi , samut prakan , krabi , and songkhla ) in north , northeast , central and south thailand . totally , 96 samples were collected for investigation . w. gritsanapan , and the voucher specimens ( ag111201 - 16 , br120101 - 16 , cl111201 - 16 , cz111201 - 16 , zc120101 - 16 , and zo120101 - 16 ) were deposited at department of pharmacognosy , faculty of pharmacy , mahidol university , thailand . freeze drying is used to dry sliced rhizomes to keep them in a stable condition . the dried samples were grinded into powder with a porcelain mortar and pestle and passed through a fine mesh sieve . powder of lyophilized samples was kept in air tight containers at 4c and protected from light until analysis . the lyophilized sample preparation for determination of total arsenic was performed by acid digestion procedure described by muoz et al . . an accurate weight ( 0.5 0.01 g ) of each lyophilized sample was mixed with 1 ml of an ashing suspension ( 20% ( w / v ) mg(no3)26h2o and 2% ( w / v ) mgo in water ) and 5 ml of 50% ( v / v ) hno3 . the mixture was evaporated on a hot plate to dryness and mineralized at 450c in a furnace . the resulting white ash was dissolved in 5 ml of 6 n hcl and 5 ml of a freshly prepared reducing solution ( 5% ( w / v ) ki and 5% ( w / v ) ascorbic acid ) . the solution was left for 30 min , and then 10 ml of 50% ( v / v ) hcl was added to the solution . the solution was filtered through a whatman no . 1 filter paper into a 25 ml volumetric flask and adjusted to volume with 50% ( v / v ) hcl . an accurate weight ( 1.0 0.01 g ) of each lyophilized sample was placed in a 50 ml screw - capped centrifuge tube ; 4.1 ml of water was added to the sample and mixed until completely moistened . in order to hydrolyze as(iii ) from the thiol groups of proteins , 18.4 ml of concentrated hcl was added to the moistened sample , shaken for 1 h , and left overnight ( 12 to 15 h ) . a reducing agent ( 1 ml of 1.5% ( w / v ) freshly prepared hydrazine sulfate and 2 ml of hydrobromic acid ) was added to the sample tube and vortexed for 2 min . for extraction of inorganic arsenic , 10 ml of chloroform was added to the tube , vortexed for 3 min , and inverted for 1 min . to break the emulsion formed during the extraction , the tube was centrifuged at 1,000 g ( measured in gravity force or g - force ) for 10 min using an eppendorf bench top centrifuge 5810 ( hamburg , germany ) . , the combined chloroform phase was filtered through a syringe filter with a 25 mm ptef membrane , pore size 0.45 m ( chrom tech , apple valley , mn , usa ) , into another tube . inorganic arsenic in the chloroform phase was back - extracted into an aqueous phase with 10 ml of 1 n hcl and centrifuged at 1,000 g for 10 min . the amount of inorganic arsenic in the combined acidic aqueous phase was quantified as described in section 2.4 , with the addition of 2.5 ml of the ashing suspension and 10 ml of 50% ( v / v ) hno3 . a perkinelmer ( waltham , ma , usa ) aanalyst 300 atomic absorption spectrometer ( norwalk , ct , usa ) interfaced with an as-90 autosampler and a fias-400 flow injection system was used to determine total and inorganic arsenic concentrations in the final solutions . the atomic absorption spectrophotometric conditions were as follows : wavelength , 193.7 nm ; slit width , 0.70 nm ; edl current , 380 ma ; loop sample , 0.5 ml . the hydride generation conditions were as follows : quartz cell , 16 cm path length , 90.7 cm i.d . , electrothermal heating , cell temperature , 900c , carrier gas ( argon ) flow rate , 50100 ml / min , reducing agent ( 0.2% ( w / v ) nabh4 in 0.05% ( w / v ) sodium hydroxide solution ) flow rate , 57 ml / min , and hcl flow rate , 911 ml / min . the q2b analytical procedure described by the us fda was used for determination of the limit of quantification ( loq ) . mixtures of lyophilized samples of all six species with equal weights were used for the determination of the limit of quantification ( loq ) of the method . for the determination of the loq for total arsenic , samples were fortified with a standard arsenic mixture ( as(iii ) : dimethylarsinic acid ( dma ) , 1 : 1 ( w / w ) ) equivalent to total arsenic at concentrations of 250 , 500 , 1,000 , and 2,500 ng / g ; blank samples were not fortified with arsenic . a total of twelve regression lines ( six regression lines each for intraday and interday analyses ) were obtained by the least - square linear regression analyses of the residual peak heights of standard total arsenic versus fortified total arsenic concentrations . the residual peak heights were peak heights of total arsenic - fortified samples minus that of blank sample . the loq of the method was calculated using the equation loq = 10/s , where is the standard deviation of y - intercepts and s is the slope of linear regression analysis . for the determination of the loq for inorganic arsenic , samples were fortified with an inorganic arsenic mixture ( as(iii ) : as(v ) 1 : 1 ( w / w ) ) at concentrations of 50 , 100 , 500 , and 1,000 ng / g ; blank samples were not fortified with inorganic arsenic . a total of twelve regression lines ( six regression lines each for intraday and interday analyses ) were obtained by the least - square linear regression analyses of the residual peak heights of standard inorganic arsenic versus fortified inorganic arsenic concentrations . the residual peak heights were the peak heights of fortified samples of inorganic arsenic minus the average peak height of blank sample . the loq of the method was calculated using the equation loq = 10/s , where is the standard deviation of y - intercepts and s is the slope of linear regression analysis . the accuracy of determination of total arsenic was assessed by analyzing srm 2568a ( rice flour ) , because no commercial rice standard reference materials for inorganic arsenic are available . the amount of inorganic arsenic in srm 1568a ( rice flour ) was determined and compared with the values previously reported . one - way analysis of variance and tukey 's test using the spss statistics version 17.0 software were performed to determine differences in concentrations of total arsenic , inorganic arsenic ( both on wet weight and dry weight basis ) , and percentages of inorganic arsenic with respect to total arsenic of six rhizome types . a significance level of p < 0.05 was accepted for all comparisons . the calculation for loqs was based on the standard deviation of y - intercepts of the linear regression analysis ( ) and the slope ( s ) by using the equation loq = 10/s . the loqs for total and inorganic arsenic in rhizome samples were 19.7 and 15.7 ng / g , respectively . concentrations of total arsenic and inorganic arsenic found in srm 1568a ( rice flour ) were 283 34 ng / g ( n = 10 , reference value of 290 30 ng / g ) and 102 3.7 ng / g ( n = 10 ) , respectively . the concentration of inorganic arsenic was in agreement with previous reports of 111 6 ng / g and 111 3 ng / g , which was analyzed by the same method . the accuracy and precision for the determination of total and inorganic arsenic in rhizome samples fortified with arsenic mixture at four concentrations are shown in table 1 . the accuracy was assessed as percent recovery from the analysis of fortified arsenic mixture in the rhizome samples . the average recoveries across the four concentrations of fortified arsenic mixtures were 95.6% and 95.4% for total and inorganic arsenic , respectively . the precision of the method expressed as percentage of relative standard deviation ( % rsd ) was calculated with the equation % rsd = 100sd/ x- , where sd is the standard deviation and x- is the mean of arsenic concentrations recovered from the arsenic - fortified samples . the % rsd ranged from 1.7 to 5.0 for total arsenic and from 1.1 to 5.5 for inorganic arsenic . table 2 summarizes concentrations of total arsenic , inorganic arsenic , and percentages of inorganic arsenic with respect to total arsenic in rhizomes of six plants in zingiberaceae family collected from four regions of eight areas in thailand . the highest content of inorganic arsenic was found in plai ( 71.2 11.6 ng / g , dry wt ) . the inorganic arsenic content in six dried rhizomes was expressed within the range of 20.4 to 92.8 ng / g , total arsenic levels ranged from 42.5 to 145.1 ng / g , and the percentages of inorganic arsenic with respect to total arsenic ranged from 28.5 to 83.2 ng / g . determined concentrations of total arsenic in dried ginger ( n = 3 ) from china . average concentration was 77.9 ng / g , whereas the value in this study was 69.3 ng / g . the indicated concentration may imply that the growth environments and soil condition for growing ginger in thailand ( 69.3 11.8 ng / g ) were safer than those in china ( 77.9 8.7 ng / g ) . the study of the arsenic - affected area in india reported higher levels of arsenic in fresh cumin and turmeric powder than our study . they reported that arsenic level ranged from 47.86 to 209.75 ng / g and from 297.33 to 280.9 ng / g , respectively . our study found that the total arsenic levels in khamin - chan ( wet wt and dry wt ) ranged from 5.8 to 21.3 ng / g and from 42.5 to 87.2 ng / g , respectively . these results clearly showed that the high amount of arsenic deposited in rhizomes is according to soil location . in six plants of zingiberaceae family in this study , the average concentrations of inorganic arsenic in the roots of c. longa and c. zedoaria were 4.3 1.5 and 6.2 1.5 ng / g ( wet wt ) , respectively . the inorganic arsenic contents in two curcuma species were not statistically different ( p > 0.05 ) but they were significantly different ( p < 0.05 ) from zingiber genus . the inorganic arsenic contents in z. cassumunar and z. officinale were 11.7 4.5 and 4.4 1.0 ng / g ( wet wt ) , respectively . the well - known species of alpinia genus , a. galangal , showed the concentration ( wet wt ) of total arsenic ( 15.9 5.1 ng / g ) and inorganic arsenic ( 8.4 3.0 ng / g ) . finally , kra - chaai in boesenbergia genus contained the total arsenic level of 20.8 4.6 ng / g , the inorganic arsenic level of 13.4 3.1 ng / g , and the percentage of inorganic arsenic of 64.4% . in this study , the total arsenic concentrations in fresh rhizomes of kra - chaai , plai , khaa , khamin - oi , khamin - chan , and ginger ranged from 11.5 to 27.3 , 7.9 to 26.8 , 10.0 to 28.3 , 6.9 to 21.8 , 5.8 to 21.3 , and 4.8 to 13.7 ng / g , respectively . these values are in accordance with the fresh vegetable arsenic concentration reported that ranged from 0 to 195 ng / g . other edible roots , such as carrot and potato were reported high levels of arsenic at 195 and 103 ng / g , respectively . reported that the total arsenic level for vegetable group ranged from 8 to 604 ng / g , and the total and inorganic arsenic concentrations in edible roots or rhizomes were higher than other organs of herbs . of the six medicinal rhizomes of zingiberaceae family analyzed in this study , both of kra - chaai and plai exhibited high mean levels ( wet weight ) of total arsenic ( 20.8 4.6 and 17.5 6.6 ng / g ) , inorganic arsenic ( 13.4 3.1 and 11.7 4.5 ng / g ) , and percentage of inorganic arsenic ( 64.4 and 67.4% ) , respectively . a possible explanation is that these high levels were related to their arsenic accumulative capacity in the rhizosphere soils associated with each species . in contrast , the low levels of inorganic arsenic in khamin - chan , khamin - oi , and ginger ( wet weight ) ranged from 2.6 to 8.2 , 2.5 to 8.4 , and 2.6 to 6.0 ng / g , respectively , whereas the average percentages of inorganic arsenic were 41.8 , 44.5 , and 56.9 , respectively . form the statistical analysis , comparative contents of total arsenic ( ng / g , dry wt ) among six species , khaa , kra - chaai , khamin - oi , and plai were significantly different from khamin - chan and ginger . from our study , the total arsenic level in ninety - six samples complied with thai regulatory limit ( 2 g / g ) and national limit for arsenic in herbal medicines ( 4 g / g ) [ 3032 ] . table 3 shows a comparison of total arsenic level in some rhizomes or roots from various countries . further investigations should be performed on other botanical rhizomes with incidence of high arsenic accumulation to crucially guarantee that plants are safe for dietary supplements for health and well - being . all investigated amounts of total arsenic concentrations meet the requirements at a national level . among six species we found that dried root of z. cassumunar showed a higher level of inorganic arsenic than other species . arsenic accumulation in rhizomes may be an important risk factor that needed to be taken into account . the results will be valuable for preliminary risk assessment in raw materials of natural products . therefore , long - term consumption of herbal products that comprised some particular zingiberaceous rhizomes might cause adverse health effects .

the arsenic accumulation in rhizomes of zingiberaceous plants was determined by atomic absorption spectrometry interfaced with hydride generation system ( hg - aas ) . the raw herbal materials , rhizomes , were collected from different regions of thailand between december 2011 and january 2012 . six well - known zingiberaceous plants , 16 samples from each and a total of 96 samples , were analyzed alpinia galanga ( khaa ) , boesenbergia rotunda ( kra - chaai ) , curcuma longa ( khamin - chan ) , curcuma zedoaria ( khamin - oi ) , zingiber cassumunar ( plai ) and zingiber officinale ( ginger ) . concentrations of total arsenic based on dry weight were 92.4 9.2 , 103.5 20.8 , 61.7 12.5 , 89.8 17.5 , 106.7 19.5 and 69.3 11.8 ng / g , respectively and inorganic arsenic were 48.8 7.0 , 66.3 12.7 , 25.5 5.0 , 38.7 4.7 , 71.2 11.6 , and 38.5 5.5 ng / g , respectively . among these , plai and kra - chaai exhibited the highest levels of total arsenic and inorganic arsenic accumulation that remind consumers to be aware of excess consuming of these rhizomes . on the contrary , the lowest value found in khamin - chan indicating natural dietary supplements and herbal medicines comprising kamin - chan are safe from arsenic poison . all investigated amounts of total and inorganic arsenic were much lower than limits recommended by thai food and drug administration .

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

cell polarity is maintained in part by targeted vesicular trafficking to the apical and basolateral membrane domains as well as the primary cilium of polarized cells . the cilium is a rod - like organelle featured on the apical plasma membrane domain on most epithelial cells in vertebrates , increasing evidence indicates that polarized cell homeostasis is strongly affected by the health of primary cilia , perhaps because cilia are needed for numerous cell signaling pathways such as polycystin , wnt , and hedgehog signaling . acute deciliation can result in the loss of polarity in cells , implying an important role for the cilium in maintaining cell polarity . furthermore , the loss of ciliary function is associated with cystic livers and kidneys as observed in bardet - biedl syndrome ( bbs ) and autosomal dominant polycystic kidney disease ( adpkd ) . the exocyst is a highly conserved complex with important roles in mediating targeted vesicular trafficking in polarized cells . the exocyst is an eight - subunit complex ( sec3 , sec5 , sec6 , sec8 , sec10 , sec15 , exo70 , and exo84 ) that was first identified as a requirement for exocytosis of vesicles at the bud tips of yeast . in addition , the small gtpase sec4 is essential for post - golgi trafficking in yeast , and the exocyst complex functions as an effector for sec4 . a current model of the exocyst delineates its function as a tethering complex in the trafficking of vesicles from a post - golgi compartment , the recycling endosome , to the basolateral plasma membrane in columnar epithelial cells . to accomplish this task , the exocyst associates with vesicles that contain the epithelial cell - specific clathrin adaptor complex ap-1b . this process is regulated by the small gtpases rab8 , rab10 , cdc42 , and rala . biochemical data indicate that both sec5 and exo84 interact with rala in its gtp - bound form [ 13 - 16 ] , and structural and biochemical work further reveals that sec5 and exo84 competitively bind to active rala , suggesting that rala plays an important role in regulating exocyst assembly . these data established a role for the exocyst in basolateral sorting of cargo in polarized epithelial cells ( figure 1 , arrow 1 ) . the sorting of biosynthetic cargos to different plasma membrane domains takes place either at the trans - golgi network ( tgn ) or in recycling endosomes in polarized epithelial cells . vesicular trafficking to the cilium may originate in recycling endosomes where rab8 or rab11 or both may regulate the organization of distinct subdomains . the exocyst has been shown to play a role in vesicular trafficking from the recycling endosomes ( re ) to the basolateral membrane ( arrow 1 ) as well as in trafficking to the cilium in association with rab8 , rabin8 , and rab11 ( arrow 2 ) . vesicles may be directed to the base of the cilium by the interaction of rabin8 and bbs1 . rab11 also has a known role in sorting to the apical membrane ( arrow 3 ) . over the last couple of years , there has been mounting evidence that the exocyst also plays a role in vesicular trafficking to the cilium . were the first to show localization of the exocyst subunits sec6/8 at the base of the primary cilium in addition to its known localization just below the tight junctions . they also demonstrated that sec6/8 are overexpressed in adpkd primary cell cultures and immortalized cell lines , suggesting a link between the ciliopathy adpkd and the exocyst . a recent study then demonstrated a direct role for the exocyst in primary ciliogenesis and cystogenesis in madin - darby canine kidney ( mdck ) cells . knockdown of sec10 in mdck cells resulted in a decrease in cilium length which could be rescued with exogenous sec10 expression . in addition , knockdown of sec10 resulted in decreased expression levels of sec8 and exo70 , indicating that sec10 may play a central role in exocyst organization at the cilium . moreover , knockdown of sec10 decreased the levels of the intraflagellar transport protein 88 ( ift88 ) , suggesting that trafficking of ift88 to cilia may be exocyst - dependent . in the future , it will be interesting to learn whether similar findings will be observed with genetic mutations of sec10 or other exocyst components . notably , the exocyst is not the only component with a dual function in targeting to the basolateral membrane and cilia . rab8a , which was described as a gtpase involved mainly in regulating cargo sorting to the basolateral membrane , was recently shown to localize to the primary cilium membrane in human retinal pigmented epithelial cells and to function in primary cilium formation . similarly , in cilia - derived sensory organelles , the rod outer segments , rab8 plays a role in docking and fusion of rhodopsin transport carriers ( rtcs ) , and rab8 co - localizes with sec8 in the vicinity of rtc fusion sites in rana berlandieri frog cells . interestingly , the rab8 guanine nucleotide exchange factor rabin8 interacts directly with the bbs1 subunit of a multimeric complex , the bbsome , that localizes at the base of the cilium and regulates ciliary membrane biogenesis . biochemical data indicate that rabin8 may interact with sec15 and gtp - loaded rab11 ( wei guo , personal communication ) . furthermore , rab11 has been shown to bind to the c - terminus of sec15 [ 26 - 28 ] , indicating a cooperative action between the exocyst and rab8 and rab11 gtpases in regulating vesicular trafficking to the primary cilium ( figure 1 , arrow 2 ) . the role of the exocyst in sorting to cilia or cilia - derived structures seems to be conserved from invertebrates to vertebrates , although the majority of epithelial cells in invertebrates do not contain cilia . however , drosophila photoreceptor cells have a light - sensing microtubule structure on their apical stalk membrane . sorting of rhodopsin1 to rhabdomeres and rhabdomere formation are also dependent on sec6 , sec15 , and rab11 function , highlighting the importance of the exocyst in cilia formation . , this should soon change with the identification of players involved in this pathway . in the effort to establish a model , certain questions become apparent . for example , it is intriguing to note that the exocyst and rab8 are used for both cilium outgrowth and sorting to the basolateral membrane , whereas rab11 plays a role in cilium biogenesis in addition to its known function in regulating apical delivery of the polymeric iga receptor as it transcytoses from the basolateral to the apical membrane ( figure 1 , arrow 3 ) . how are these different pathways delineated ? given the known localization of rab8 , rab11 , and the exocyst in different areas of recycling endosomes , it seems conceivable to assume that the pathway to the cilium involving these components may be originating in recycling endosomes . perhaps the recycling endosomes are organized in three subdomains , each regulating a specific trafficking step and thereby preventing missorting of apical or basolateral cargos into the cilium . indeed , the organization of recycling endosomes into apical and basolateral domains is so dramatic that they are often referred to as apical recycling endosomes and central recycling endosomes ( for a detailed discussion of this topic , see ) . perhaps rab8 and rab11 another unknown is where vesicles that are targeted to the cilia fuse with the ciliary membrane . perhaps they are directed first to the base of the cilium via the interaction between rabin8 and bbs1 . from the base of the cilium , vesicles may be directed into the cilium on microtubule tracks because sec10 stains the entire length of the cilia membrane , implying that fusion of vesicles takes place along the ciliary membrane ; however , it is not clear whether such fusion might occur as there is currently no evidence for the presence of transport vesicles in cilia . alternatively , fusion of vesicles may occur at the base of the cilium , thereby leading to the outgrowth of ciliary membrane . as researchers continue to make strides toward understanding the role of the exocyst in polarized epithelial cells , mechanisms of how one

the role of the exocyst complex has been studied mainly in the context of basolateral sorting of cargos in polarized cells . recent developments indicate an extended yet specific function of the exocyst in the outgrowth of the primary cilium from the apical membrane , thereby highlighting a role for the exocyst in ensuring membrane trafficking to important signaling stations in the cell , the tight junctions , and the cilia .

Introduction and context Major recent advances Future directions Competing interests

alexandrite laser ( 755 nm ) is one of the most common methods of hair removal . however , the disuse of protective goggles during the application of this laser is a serious problem . alexandrite laser light is visible , and it is known that ocular injuries may occur during the use of alexandrite laser when protective goggles are not worn [ 1 , 2 ] . still , foveal injuries by alexandrite laser are rare . herein , we reported a case with accidental foveal injury and subsequent macular edema secondary to alexandrite laser contact , treated with an intravitreal ozurdex implant . a 35-year - old male patient presented to our clinic complaining of a scotoma in the right eye , which had begun 2 h after looking at a laser probe for a few seconds in an esthetics clinic . fundus examination showed a pale , white and sharply circumscribed laser spot lesion localized at the inferior nasal foveal region , involving the central foveola . a visual field test ( humphrey , zeiss , germany ) displayed a scotoma consistent with the affected region . a hypofluorescent lesion , which was in accordance with the round laser spot , was detected in the early and late phases of fundus fluorescein angiography ( canon inc . , an intense increase in reflectance in the region of the spot was also identified on optic coherence tomography ( oct ; slo , optos , dunfermline , uk ) . there was no change in the findings at the first control visit after 1 week . however , at the second week , the bcva of the patient had decreased to 3/10 , and edema and hemorrhage had occurred around the lesion . oct demonstrated intra- and subretinal edema as well as tissue disruption within the injured area ( fig . ozurdex was implanted via the pars plana region with its specific cartridge in the operating room . an intraocular pressure elevation ( 35 mm hg ) which was successfully controlled by a topical antiglaucomatous agent occurred . one month after the injection , bcva was 20/30 despite a hemorrhage around the injured area on fundus examination ( fig . oct showed decreased macular edema and a hyperreflectance scar in the affected area ( fig . 4 ) . a visual field test demonstrated no change in the scotoma of the patient . it is known that alexandrite laser causes a temperature increase and subsequent coagulation necrosis in melanin - containing tissues , which is termed selective photothermolysis . this temperature rise might cause burns , pigmentation changes , and erythema in surrounding tissues . the first type are anterior segment complications such as uveitis , pupillary distortion , iris atrophy , and cataract , which can occur due to laser applications to the face with closed eyes . the second type are posterior segment complications such as macular hole and macular scar caused by the direct effect of the laser beam on the retina while the eyes are open . the ozurdex intravitreal implant is composed of a biodegradable copolymer of lactic and glycolic acids containing micronized dexamethasone . the drug - copolymer complex gradually releases dexamethasone that transforms into glycolic and lactic acids after the injection and eventually into water and carbon dioxide . after intravitreal injection , the ozurdex implant reduces the vitreous levels of several mediators which cause disruption of the blood - retinal barrier ( i.e. , vascular endothelial growth factor ) and accordingly diminishes the vascular permeability . ozurdex is frequently used in the treatment of macular edema due to diabetes mellitus , retinal vein occlusion , and noninfectious uveitis . in our case , we first used topical steroid drops to prevent complications after alexandrite laser injury ; however , this medication was ineffective . therefore , the inflammatory process secondary to the foveal injury and the subsequent macular edema were treated with ozurdex because of its rapid and potent antiedematous effect . in a case report by anaya - alaminos bcva was reported as 20/200 initially and 20/100 after a 1-year follow - up period in a patient with accidental foveal injury by alexandrite laser . there was no information about the treatment or whether macular edema had occurred during the follow - up period . we believe that the better final visual acuity in our case was achieved by swift resolution of macular edema by ozurdex . retrobulbar and oral steroid treatments have been used in the treatment of foveal injuries caused by femtosecond laser and green laser pointer [ 9 , 10 ] . bcva improved from 20/30 to 20/20 after treatment in a case with femtosecond laser injury , while it increased from 20/100 to 20/60 in a case with green laser pointer injury . the difference between the initial visual acuities and their increases might have been caused by the different energy levels , the durations of laser contact , and the distances of the lesions from the central foveola . the response to treatment was often greater among eyes with a shorter duration of macular edema . we preferred an ozurdex implant to systemic steroids due to its rapid and target - oriented effect and the lower systemic side effects for reducing macular edema . in summary , alexandrite laser injuries might cause macular injury and edema , and the ozurdex implant can be an effective treatment in these cases . the patient was informed about the risks of the treatment , and informed consent was obtained .

alexandrite laser is one of the most common methods of hair removal . its utilization is gradually increasing due to easy accessibility and high effectiveness . however , the disuse of protective goggles during the application of this laser is a serious problem . in this case report , we presented a 35-year - old male patient who had foveal injury by alexandrite laser . the inflammatory process secondary to the foveal injury and subsequent macular edema were treated with ozurdex because of its potent antiedematous effect .

Introduction Case Presentation Discussion Statement of Ethics Disclosure Statement

in the present world , the prime requisite is to maintain global sulphur balance in the environment including wadden sea and other associated ocean belts . this therefore would make a better and safe environment to dwell in . in the foremost stage , formation of thiosulphate ( s2o3 ) occurs from sulphide ( s ) in aerobic situations . at this step , the end product is such that sulphur has + 2 oxidation state but it needs to be transformed to its elemental form ( s ) . in this phase , microorganisms play a crucial role to completely oxidize thiosulphate ( the most abundant sulphur anion ) to maintain the proper sulphur cycle throughout the environment . . moreover , deep ocean bed surviving microorganisms use sulphides and such varied sulphur compounds for their chemolithoautotrophy [ 2 , 3 ] . one such microorganism is an epsilon - proteobacterium sulfurimonas denitrificans [ 2 , 3 ] . sox multienzyme complex within their genes coding for the necessary proteins or few of them follow the pathway for reverse sulphate reduction . the sox multienzyme complex organization encodes a cluster of genes ( sox operon , sulphur oxidizing operon ) and is the most widely predominant one [ 47 ] . nevertheless , it is worth mentioning that the epsilon - proteobacteria sulfurimonas denitrificans ( s. denitrificans ) retain a sox multienzyme complex . intriguingly , the explicit participation of soxyz complex , soxxa complex , soxcd complex , and the soxb protein entirely accomplishes the thiosulphate oxidation mechanism [ 6 , 9 ] . amongst them , the molybdenum - cofactor preserving protein is soxc , while soxd protein is a cytochrome protein of type c. it comprises duo - heme clusters : one is well known as d1 and the other one is known as d2 . altogether , soxcd aids in the performance of sulfane dehydrogenase . first and foremost , it is essential to study the interactive complex of soxcd protein from s. denitrificans . further after that , the interactive nature amongst soxy and soxz proteins is necessary to be explored . after that , the soxyzcd complex formation and the residual participation for thiosulphate oxidation are highly paramount to be examined . this is , moreover , based upon the wet - laboratory experiments and their documentation , which states soxyz to be crucial and to serve as an important interacting partner for soxcd to complete the thiosulphate oxidation process [ 8 , 9 ] . in the presence of soxyzcd , sox system is proficient to oxidize elemental sulphur , sulphide , and explicitly sulfane sulphur ( -s- ) in thiosulphate [ 4 , 6 , 7 , 9 ] . within the complex , further soxcd aids in recycling the thiosulphate - bound protein soxy from soxyz complex via releasing of the sulphate group from soxy protein , hydrolytically [ 4 , 6 , 7 , 9 ] . formerly , the modeling of soxc and soxd protein from sulfurimonas denitrificans was performed via simulation . but hitherto , there was no such molecular and residual disclosure for the analysis of the entire pattern of interaction in soxyzcd with thiosulphate for the oxidation of thiosulphate in wadden sea and deep ocean belts . in addition to that , even previously the analysis of the conformational switches in soxyz that benefits the recycling process along with the thermodynamic stability in soxyz for the fulfilment of the entire phenomena in wadden sea and associated ocean belts was not dealt with . so , the 3d protein structures of soxc and soxd proteins from s. denitrificans were developed through comparative modeling technique . additionally , soxy and soxz proteins from s. denitrificans were modeled efficiently using fold recognition technique . the individual modeled proteins were docked amongst themselves to obtain soxyz and soxcd complex , first . the models were individually optimized and simulated for a stable interactive investigation . furthermore , the simulated soxcd protein of s. denitrificans was picked to dock with the simulated soxyz protein from the same microorganism . moreover , to the final soxyz complex , thiosulphate was docked via protein - ligand docking procedure . the detailed interactive residues indulged in the interaction of the quadracomplex ; soxyzcd was further studied . the stability and conformational alterations in soxyz protein upon soxcd interaction for its recycling were additionally examined . therefore , this novel present scenario provides a way out to examine the fields of binding , stability , conformational switches , and the arrangement of interactions in soxyzcd protein from s. denitrificans ( epsilon - proteobacteria ) . this might cover up a massive part for thiosulphate oxidation in dark ocean beds for the chemolithoautotrophy of microorganisms . in our previous investigation , we analyzed the residual interaction pattern between two essential proteins , soxc and soxd , from sulfurimonas denitrificans for global sulphur oxidation . for that purpose , we modeled the 3d structures of soxc and soxd protein and further disclosed the residue - level binding strength between two modeled proteins . the comparative study was examined for the stability of soxc and soxd protein after the duo - protein was simulated . in this present study , first we have opted for an improved process for the preparation of the homology models of the two paramount proteins . the individual amino acid sequences of soxc and soxd from s. denitrificans were obtained from uniprotkb ( accession numbers q30nu7 and q30nu8 for soxc and soxd , resp . ) . the sequences were substantiated from ncbi protein sequence database too . for template selection , templates were obtained by swiss - model template searching procedure . with the usage of varied force fields , the explorations of templates for the individual proteins ( soxc and soxd ) were performed . the same templates were also obtained by the utilization of psi - blast with reference to pdb . psi - blast helps to bring the distantly related species into consideration and is also acknowledged to be more sensitive when compared to pblast . the respective templates for building the respective comparative ( homology ) models were from the x - ray crystal structure of(i)paracoccus pantotrophus for soxc ( pdb code : 2xts_a with 87.00% query coverage sharing 43.78% sequence identity , e - value : 2e 114),(ii)paracoccus pantotrophus for soxd ( pdb code : 2xts_b with 51.00% query coverage and sharing 37.82% sequence identity , e - value : 8e 38 ) . paracoccus pantotrophus for soxc ( pdb code : 2xts_a with 87.00% query coverage sharing 43.78% sequence identity , e - value : 2e 114 ) , paracoccus pantotrophus for soxd ( pdb code : 2xts_b with 51.00% query coverage and sharing 37.82% sequence identity , e - value : 8e 38 ) . soxc and soxd protein models were constructed utilizing homology modeling with the aid of swiss model software . the modeled proteins were selected from the gmqe ( global model quality estimation ) score . furthermore , the next step for preferring the best modeled protein is to examine the sequence identity of the target and template sequence . the gmqe values are 0.70 and 0.38 for soxc and soxd , respectively . additionally , the qmean score was also supportive with 3.56 and 3.45 for soxc and soxd , respectively . the individual homology models were superimposed upon their relevant crystal templates to observe the root mean square deviations ( rmsd ) with the aid of pymol . dalilite server ( analysis tool web services from the embl - ebi , 2013 ) from embl packages also helped to validate the results . 0.068 and 0.079 were the rmsd values with respect to c atoms for soxc and soxd , respectively . this implies that the models were potentially akin to their crystal template structures . for the purpose of modeling the soxy and soxz proteins , fold recognition technique was utilized by operating phyre2 , rather than utilizing traditional homology modeling technique . to overcome the certain specific problems , fold recognition technique ( threading ) fold recognition is a technique for modeling a specific protein that demonstrates the comparable fold with respect to the proteins of well - known tertiary structures . so , phyre2 was operated here for the execution of fold recognition . in this method , first , the query sequence is used to perform psi - blast to classify remotely related amino acid sequences . the usage of hidden markov model ( hmm ) was to produce a familiar sequence profile . template i d from fold library , chain , pdb i d , confidence score , and query coverage were c4uwqk , chain k , 4uwq_k , 100% , and 77% , respectively , for soxy protein . template i d from fold library , chain , pdb i d , confidence score , and coverage were d1v8ha1 , chain a , 1v8h_a , 100% , and 99% , respectively , for soxz protein . during homology modeling , when the sequence identity between the protein to be modeled and its competent template remains within the range of 30% to 50% , then certain disparities in loop conformations and fallacies might occur in the side - chain packing [ 1517 ] . so , optimization and remodeling of the loop regions are essential to accomplish the proper orientation of - angles . modloop via modeller was operated for the establishment of the loop optimization . for the refinement and energy optimization of the single loop optimized protein structures ( soxy , soxz , soxc , and soxz ) , an efficient atomic - level algorithm , modrefiner , was followed . the algorithm executes via a combination of two different force fields : physics - based and knowledge - based . in terms of the proper h - bonding , topology of backbone , and positioning of side chains , the proteins were well optimized by drawing them closer to their native states , where the highest state of interaction exists . the stereochemical properties of the four homology modeled structures were substantiated by saves server . to analyze the residue - level profile of individual amino acid from the respective protein 3d models , examination of ramachandran plots from procheck web server revealed that most of the residues occupied the most preferred regions with zilch amino acids in the disallowed regions [ 21 , 22 ] . first , soxyz and soxcd complexes were formed which was followed by the docking of the duo - protein complexes amongst themselves to form the soxyzcd complex . for this purpose , at each case , the entirely automated program cluspro 2.0 was operated [ 23 , 24 ] . for the individual duo - complexes ( soxyz and soxcd ) , both soxy and soxc , being longer , served to be the receptors while the other two modeled proteins were uploaded as ligands . but for docking the duo - complexes , soxcd served as receptor and soxyz as ligand . the unstructured residues were confiscated by the execution of the advanced option of cluspro 2.0 . in each of the cases , amongst all the 10 docked structures , the finest cluster sized model was opted for for further investigations . docking outcomes from zdock and gramm - x were shown to be inclusive of the results [ 25 , 26 ] . the modeled and docked complexes were energy optimized using charmm force field to diminish the net overall energy . it was also performed to alter the respective conformations of the dihedral angles to allow them to vary concurrently . the steepest descent technique preceded by conjugate gradient mechanism was executed through operation of discovery studio software . the phenomena continued till the modeled structures individually attained a final rms gradient of 0.0001 . to attain a stable conformation for the individual protein complexes with an overall optimization of the net energy , two - step molecular dynamics ( md ) simulation initially , by template information of modeled structures and based on the principle of md simulation , fragment - guided molecular dynamics ( fg - md ) was operated . it first disassembles ( fragments ) the provided docked protein structure and further assembles each fragment to accomplish the energy optimized protein modeled structure . the physical based potentials , except for amber99 , explicitly carried the decoys away from the native protein state . fg - md was extensively tested with experimentally validated x - ray crystal structures and demonstrated noteworthy potential in the protein structure refinements at the atomic level . the complete simulation process comprises mainly three steps : ( i ) identification and analysis of the fragment structure , ( ii ) utilization of the simulated annealing technique for md simulation , and ( iii ) selection of the final model . thus , every time the structure is brought closer to its native state with a higher standard of accuracy . further , these reconstructed optimized complexes from its fragments were simulated via the operation of the chiron energy minimization tool . charmm force field is pursued for the optimization of the whole docked complex at a time . further , a high temperature exchange rate for the complexes is utilized with subjection to simulations by molecular dynamics ( md ) . thus , finally , not only were the steric clashes purged rapidly but also minimal disconcertion of the backbone of the protein was achieved . with the aid of protein interaction calculator ( pic ) , several interactions like disulphide bonds , main - chain side - chain interactions , and hydrophobic , ionic , aromatic - aromatic , aromatic - sulphur , and cation- interactions were examined . analysis of the interactive docked complexes was also carried out using the analyze binding site tool from discovery studio ( ds ) package . this is because the ionic interaction predominantly plays a pivotal role in strengthening the interaction . for the examination of the stability of soxyz complex after participation of soxcd complex ( for the recycling of soxyz ) , free energy of folding for soxyz complex was assessed and compared ( before and after the interaction of soxcd complex ) . a descent in the free energy of folding implies better folds and a strong interactive protein complex . increment in the percentage of residues adopting turn - like and helical conformations led to stronger interaction with a better conformational stability in the protein [ 33 , 34 ] . furthermore , ascent in the residues involved in turn - like regions leads to firmer backbone flexibility which additionally aids the protein to interact with its partner proteins . moreover , it has been previously investigated with evidence that proteins exhibiting few 310 helices accompanied by the increased percentage for pure -helices are conformationally steadier than the proteins adopting only pure -helices . so , the secondary structure distribution in the soxyz protein complex was evaluated and compared utilizing dssp method , before and after its interaction with soxcd protein complex . the stability and strength of interaction of the soxyz complex were thus again perceived through the conformational switches . calculation , analysis , and comparison of the solvent accessible surface area for the essential interacting amino acids residues from soxyz protein complex were performed . the specific amino acid residues that take part in the interaction pattern were taken into consideration from the preinteractive complex of soxyz with soxcd and after the tetracomplex formation . it was observed to rationalize the aforementioned energy estimation outcomes . as the interaction grows stronger , the area available for solvent will keep descending . the literature suggests that more negative electrostatic potential value apprehends a better interaction of the protein complex of interest and thereby leads to an overall complex stability . the pictorial representation of the electrostatic mapping upon the protein surface was illustrated by the assistance of pymol ( molecular viewer using python language ) . so , the optimized complex structure and the preoptimized tetracomplex structure were further utilized for the electrostatic potential mapping upon the surface of the protein . electrostatic surface potential was evaluated via vacuum electrostatics in kt / e units , with the support of pymol . to examine and rationalize all the estimated outcomes through statistical significance , p value was evaluated through the paired t - test in each of the cases . in this t - test , the only assumption usually accepted is that the standard deviations ( sds ) are not approximately akin to both measurements for the individual cases ( before and after soxcd interaction ) . analysis of the interaction amongst the essential proteins upon the involvement of the sulphur anion , thiosulphate , was also essential . thiosulphate was obtained from pubchem and was docked as a ligand with the receptor ( final stable soxyzcd protein complex ) . autodock vina was operated for the purpose . not only the condition and declaration of the search space but also the specification of the binding region was provided . further again , the molecular surface of the protein - ligand complex was computed with the utilization of segmentation algorithm using patchdock . further , the best suited and finest model was opted for , depending upon the relevant data for interaction energy , that is , gibbs free energy ( g ) . the soxyzcd - thiosulphate model having the maximum and best suited ( ) g value is chosen for having the highest strong binding affinity . the obtained final complex of soxyzcd - thiosulphate was analyzed with the aid of pymol plugins and the interacting residues and binding patterns were thereby examined . thus , the complete model having four interactive proteins and a ligand ( thiosulphate ) was investigated with the establishment of the interactive residues for the purpose of recycling the thiosulphate - bound soxy protein from the soxyz complex with the aid of soxcd . the tertiary structure for the homology modeled soxc protein from sulfurimonas denitrificans was analogous to its template from paracoccus pantotrophus ( pdb code : 2xts ; a chain ) . the 391-residue - long protein comprises mainly 18 sets of -sheets and 10 sets of -helices interspersed with coil regions . 14.3% of the amino acid residues contributed to the respective 10 -helices , whereas 29.2% of the residues were responsible for the adoption of -sheet conformation in the protein . the modeled soxc structure is well illustrated in figure 1 with red , cyan , and magenta shades representing -sheets , -helices , and interspersing coils , respectively . the tertiary structure for the homology modeled soxd protein from sulfurimonas denitrificans was also analogous to its template from paracoccus pantotrophus ( pdb code : 2xts ; b chain ) . only two sets of antiparallel -sheets were observed in the 3d structure with the involvement of 2.8% of the amino acid residues . soxd structure is illustrated in figure 1 with -helices and coils in yellow and pink shades , respectively . the functional tertiary structure for the homology modeled soxy protein from sulfurimonas denitrificans was 122 amino acids long . soxy protein comprises mainly 8 sets of -sheets and 2 sets of -helix conformations at the n - terminal end ( residues : 220 and 5961 ) . the model is well presented in the interactive picture of figure 1 with -helix , -sheets , and turn - like regions in marine - blue shade . the functional tertiary structure for the homology modeled soxz protein from sulfurimonas denitrificans was 97 amino acid residues long . the -sheets occupy 47.82% of the entire structure . the model is well presented in the interactive picture of figure 1 with -sheets and -turns in lime - green shade . the exploration of the interactive residues with the binding patterns of the soxyzcd tetracomplex using pic web server and discovery studio platform from accelrys software displays the four proteins to participate strongly via 9 sets of ionic interactions . several h - bonding interactions were perceived indulging the main chains and the side chains of the four individual proteins . table 1 tabulates the ionic interactions in the optimized and simulated tetracomplex ( soxyzcd ) with a pictorial representation through figure 2 , showing few paramount residues for the firmer interaction . the polar negatively charged asp2 and glu31 from soxc protein participated strongly in the interaction pattern . again the polar positively charged lys34 and arg93 were observed to actively participate in the formation of two ionic interactions with glu8 and asp57 from soxz . lys4 , a polar positively charged residue from soxz , further formed two ionic interactions with glu44 and asp35 from the soxd protein . this was additionally supported by the interaction of polar negatively charged asp5 ( adjacently positioned to lys4 that formed two ionic interactions ) from soxz with lys50 from soxd for a better stability to the tetracomplex . furthermore , lys61 , arg67 , and asp37 from soxd participated strongly in building up a strong electrostatic interaction in the soxyzcd tetracomplex . altogether , these interactions helped to accommodate the partner proteins to interact firmly . to analyze and compare the stability of soxyz complex after interaction with soxcd complex , free energy of folding vadar 2.0 aided to disclose a decline in the value from 144.71 kcal / mol ( in soxyz complex ) to 577.31 kcal / mol for the soxyzcd complex ( table 2 ) . therefore , the reduction in the free energy of folding indicates spontaneous interaction with better folds in the soxyz protein complex after interaction with soxcd complex . on comparison and analysis of the conformational switches in the soxyz complex before and after interaction with soxcd complex , it was disclosed that both soxy and soxz underwent abrupt conformational alterations upon the participation of soxcd in recycling soxy protein . for soxy captivatingly , the presence of few 310 helices in soxy protein ( after soxcd interaction ) accompanied by the increased percentage of residues adopting pure -helices additionally led to a firmer and steadier conformation in the protein ( figure 3 ) . the increase in percentage of pure -helices with the presence of 310 helices and -sheet conformations in the soxy protein ( after soxcd interaction ) makes soxy exhibit a steadier and more interactive conformation [ 34 , 35 ] . again , the increment in the -turn conformations in soxz from 21.73% to 24.00% after soxcd interaction ( figure 4 ) affirms even soxz to exhibit a stronger conformation and better stability . thereby , the entire conformational shift in soxyz protein complex benefits the recycling process of soxy protein by soxcd . the pictorial representation with tabulation of the conformational switches is illustrated in figures 4 and 5 , respectively , for soxy and soxz proteins . herein , from table 2 , the net area available for solvent accessibility from the interacting residues in soxyz duo - protein complex in soxyzcd complex was perceived to get decreased from 486.92 to 153.92 , after interaction . there was even a decline in the overall value for net area of solvent accessibility ( considering the summation of the accessible area values from all the residues from soxy and soxz ) from soxyz protein . in kt / e units , electrostatic surface potential displayed the postoptimized and simulated soxyzcd protein complex to exhibit better and firmer interaction ( figure 5 ) . the blue region symbolizes the electropositive regions , while the red shaded ones are for the electronegative areas . a vivid illustration from figure 5 provides the notion that , from the tetraprotein complex , soxyz complex ( after optimization and simulation ) exhibited an enhanced stability . the alteration in the electrostatic potential on the soxyz surface was from 48.710 to 56.295 . the respective outcomes for the alterations in soxyz complex upon soxcd interaction were observed to be statistically significant with an evaluated p value of less than 5% in each of the calculations . the p values for the alterations in the free energy of folding and net area for solvent accessibility values were 0.021342 and 0.01218 , respectively . furthermore , again the conformational switches in soxy were observed to be statistically significant with a p value of 0.011327 and 0.012138 for the ascent in the helical conformations and for the decrease in the -sheet conformations ( to allow few residues to adopt 310 helices ) . p value of 0.021231 for the presence in the residues undergoing 310 helices , which accompanied the pure -helical conformations , also is statistically significant . additionally , even the conformational shifts in soxz ( having no helices ) were also perceived to be statistically significant with a p value of 0.013219 and 0.005136 for decrement in -sheets ( to allow few residues to exhibit an increase in the turn - like regions ) and increment in turn - like regions , respectively . along with the strong and stable ionic interactions within the tetraprotein complex ( soxyzcd ) , thiosulphate binding interactions were therefore analyzed . for the interaction between soxy and thiosulphate ( to get recycled ) , mainly his65 , his80 , and asp78 from soxy formed h - bonds as depicted in figure 6 . strong hydrogen bonds were accomplished by these aforementioned residues from soxy protein with the thiosulphate molecule . formation of a cavity in soxy by the positively charged environment of soxz helps to accommodate the thiosulphate ligand within the protein strongly . previously , other molecular level studies involving manifold molecular and computational probes were examined with several species of proteobacteria like allochromatium vinosum , p. pantotrophus , pseudaminobacter salicylatoxidans ( kct001 ) , and many more [ 57 , 41 , 42 ] . in addition to that , molecular level studies were also explored for the eradication of environmental pollutants like phenol . literature study documents the species ( mainly proteobacteria ) to perform thiosulphate oxidation , but a computational investigation into the oxidation of sulphur after the formation of the tetracomplex protein ( soxyzcd ) from s. denitrificans ( which is responsible for performing thiosulphate oxidation on a major part on this earth including deep ocean belts ) was nowhere dealt with . so this novel molecular analysis with the epsilon - proteobacteria sulfurimonas denitrificans was essential to be explored . figure 1 in supplementary material available online at http://dx.doi.org/10.1155/2016/8683713 . to summarize , the four relevant energy optimized sox proteins , soxy , soxz , soxc , and soxd , were modeled discretely after the satisfaction of their necessary stereochemical properties . firstly , the necessary duo - complexes ( soxyz and soxcd ) were formed which was further followed by the generation of the tetracomplex soxyzcd . the finally docked best modeled tetracomplex of soxyzcd was energy optimized and underwent two - step molecular dynamics simulation further . to examine and explore the residual participation and the binding pattern for the steady simulated soxyzcd complex , the predominant strengthening ionic interactions were disclosed . nine sets of ionic interactions in the entire soxyzcd complex made the complex a firmly interactive one . the polar negatively charged asp2 and glu31 from soxc protein participated strongly in the interaction pattern . several other paramount residues helped in the proper accommodation of soxcd protein complex through pocket formation in soxyz complex . furthermore , the thiosulphate molecule was observed to interact with soxy protein efficiently with strong h - bonds by the participation of asp78 , his65 , and his80 amino acids . overall interactions between the four proteins and the thiosulphate molecule ( with soxy ) aided in dragging thiosulphate - bound soxy protein for its recycling from its respective duo - complex ( soxyz ) . thus , these residual contribution and binding patterns help in the performance of sulphur oxidation mechanism potentially . from the biological point of view , it was observed that soxz protein interacted strongly with more ionic interactions to participate with soxcd complex . this joint effort therefore aided in recycling the soxy protein , only to which the thiosulphate molecule binds , for the recycling of the soxy - thiosulphate complex . evaluations from g values in free energy of folding , net area available for solvent molecules , and electrostatic potential upon soxyz protein surface ( an abrupt increase in the negative value ) displayed that the sole soxyz protein turned out to be more stable and firmer after interaction with soxcd complex . the conformational transitions in soxyz complex also help to analyze the improved conformational stability and steady interaction of soxyz complex in the presence of soxcd . in soxy , the statistically significant increase in the pure -helices accompanied by 3-ten helices and -sheets presents the structure to become a more stable and interactive one . likewise , soxz ( having no helical regions ) also experienced conformational alterations with increased -turn conformations . this led to the better backbone flexibility in the proteins permitting them to easily interact with their partner proteins . this further affirms the plausible recycling of soxy protein from the soxyz complex , by soxcd complex . so , herein , the deep seas residing microorganism ( s. denitrificans ) was explored for the first time . the formation of soxyz and soxcd and further soxyzcd complex formation as well as their residual disclosure with importance to their respective positions were disclosed . additionally , the conformational shifts and thermodynamic stability in soxyz protein complex for recycling soxy - thiosulphate for the progression of sulphur oxidation mechanism served to analyze the core basis for rendering a toxic - free environment . finally , the outcomes of this present study focused mainly on investigating the conformational transitions and stability of soxyz complex upon the interaction of soxcd protein complex from the epsilon - proteobacteria s. denitrificans . in addition to that , it finally also dealt with the interaction and cooperative participation of thiosulphate for recycling soxy - thiosulphate complex from the soxyz complex , with the aid of soxcd protein complex . the 3d homology modeled functional protein structures of soxy , soxz , soxc , and soxd were analyzed and demonstrated , followed by optimization . the relevant necessary interactions in the optimized and simulated soxyzcd protein complex were explored from the respective positions of the pertinent proteins . predominantly , 9 sets of interactions for this recycling phenomenon for thiosulphate oxidation were served by the ionic interactions from mainly soxy , soxc , soxd , and soxz . the stability and stronger conformation for soxyz protein upon interaction with soxcd protein complex were explored further with corroboration of statistical significance . additionally , in the presence of soxcd , soxyz protein complex also interacts firmly and steadily . further , on the interaction with thiosulphate , four strong h - bonding interactions were observed to strengthen the protein - ligand interaction . from this stable simulated tetracomplex formation , soxy - thiosulphate gets susceptible to being dragged out for getting recycled by soxcd protein complex . though there exist former investigations on the cooperation of soxyz and soxcd proteins from other proteobacteria species for thiosulphate oxidation , a comparative in silico examination into the interactive pattern , binding residues , and conformational switches for the recycling of soxy - thiosulphate to oxidize thiosulphate ( from the wadden sea and associated ocean belts ) by soxcd complex of epsilon - proteobacteria ( s. denitrificans ) was not delved into hitherto . thus , this present novel research presents a rational outlook to explore and analyze the soxcd complex participation with soxyz protein complex from sulfurimonas denitrificans , an epsilon - proteobacterium . this aids in maintaining a systemic sulphur cycle in the biota , even giving importance to the ocean belts . the study further instigates the computational analysis of the participation of other proteins from the same microorganism for the help in thiosulphate oxidation . it is also exciting to investigate any mutational alterations that might affect the interactive pattern among the proteins and thereby might alter the sequential cellular mechanism . the computational investigation for the sustainability of the global sulphur balance through essential microorganisms can also be provoked in future .

thiosulphate oxidation ( an essential mechanism ) serves to maintain the global sulphur cycle . earlier experimental and computational studies dealt with environmental thiosulphate oxidation but none dealt with thiosulphate oxidation from deep ocean belts . wet - laboratory experimental research shows that epsilon - proteobacteria sulfurimonas denitrificans possess sox ( sulphur - oxidizing ) operon and perform thiosulphate oxidation efficiently underneath the oceans . from this specific sox operon , soxcd complex recycles the thiosulphate - bound soxy from soxyz complex to balance the environmental sulphur cycle . so , four chief proteins were variedly modeled and relevant simulated interactive structures were obtained . the final simulated tetraprotein complex ( soxyzcd ) from docked soxyz and soxcd complexes was disclosed to be a highly interactive one with predominant ionic residues . free energy of folding , solvent accessibility , and conformational shifts ( coil - like conformation to helices and sheets ) were observed in soxyz complex after interacting with soxcd . the stability of the complex ( soxyzcd ) after simulation was also observed through the electrostatic surface potential values . these evaluations were rationalized via biostatistics . this aids soxcd for recycling soxy along with thiosulphate , which remains interconnected by four h - bonds with soxy . therefore , this novel exploration is endowed with the detailed molecular viewpoint for maintaining the sulphur cycle ( globally ) including the ocean belts .

1. Introduction 2. Methodology 3. Results and Discussion 4. Conclusion and Future Scope

chikungunya is a mosquito - borne virus ( family togaviridae , genus alphavirus ) , first identified more than 60 years ago in modern - day tanzania , which is responsible for outbreaks of acute febrile polyarthralgia . epidemics have been described in africa , the middle east , europe , india , southeast asia , and most recently in the americas , where large numbers of cases of disease have been reported from more than 20 countries in the caribbean and central and south america , . three genotypes of chikungunya virus ( chikv ) , called west african , east / central / south african ( ecsa ) , and asian , have been defined . sequence analysis revealed that the virus circulating in the caribbean is phylogenetically related to asian genotype strains recently circulating in indonesia , china , and the philippines . although the disease is not generally considered life - threatening and prior to 2005 severe clinical forms of the infection were rarely reported , the runion island outbreak revealed new and serious forms never described before . little clinical information exists on the occurrence of severe and/or lethal infections during the current epidemic of chikungunya fever ( chikf ) in the americas . here , we describe a series of four severely ill adult venezuelan patients seen in a short period of time . our report is intended to elicit clinicians awareness of the atypical and severe forms of the disease in our region , as a way to potentially improve the outcomes of such life - threatening conditions . a 75 year old patient with no known past medical illnesses developed chills and fever accompanied by generalized arthralgia and bilateral swelling in the hands and feet interphalangeal joints , ankles and knees . after 3 days , he was seen in an emergency room with the diagnosis of febrile exanthem probably caused chikungunya virus , severe sepsis with multi - organ failure and severe metabolic acidosis . on admission positive physical findings included a dry oral mucosa and a generalized erythematous macular rash which was more intense and confluent on feet ( fig . bilateral inflammatory edema was evident in the knee , ankle and interphalangeal joints with marked functional limitation of the latter . laboratory results revealed marked leukocytosis with neutrophilia , elevation of c reactive protein , elevated procalcitonin , and abnormal renal function tests ( table 1 ) . his clinical status deteriorated over 48 h with the onset of generalized soft tissue edema , distinctive palpebral and facial swelling ( fig . 2 ) , multiple bullae , hemodynamic instability , hepatic cholestasis , atrial fibrillation with rapid ventricular response , metabolic acidosis , and oliguric acute kidney failure . he was transferred to the icu , where his condition continued to worsen , further manifesting a septic shock syndrome with progressive multiple organ dysfunction . he died 24 h later due to cardiorespiratory arrest , in association with unresponsive ventricular fibrillation . a postmortem liver biopsy was obtained which revealed moderate acute hepatitis , hepatocyte necrosis with apoptosis , macrovesicular fat metamorphosis and diffuse edema . no granulomas were observed . repeated blood , urine and respiratory cultures failed to identify bacterial pathogens . serum samples for chikungunya virus were positive by conventional and real time pcr and negative for dengue virus . leptospirosis , malaria , typhoid fever , meningococcemia , and other severe bacterial , viral and parasitic diseases prevalent locally were ruled out . this patient represents the first documented lethal case of chikf in venezuela . a 53 year old female with a recent diagnosis of iron deficiency anemia presented with a 3 day history of fever , chills and rigors . she also complained of swelling of her face and extremities as well as severe , disabling arthralgias affecting all of the interphalangeal joints of the hands as well as the wrists , elbows and knees . on admission , the patient had fever , appeared ill , and the physical examination was most remarkable for a maculopapular erythematous rash affecting the four extremities and an anasarca - like appearance . relevant laboratory results on admission ( table 1 ) included severe leukocytosis with mild thrombocytopenia , increased prothrombin time , hyperglycemia , hypoalbuminemia , elevated bun and creatinine , ldh , ast , alt , ck - mb , crp and procalcitonin levels . over 48 h the patient developed acute respiratory distress , requiring intubation and ventilatory support . a conspicuous , sharply delineated and necrotic skin lesion over the entire nasal region appeared ( fig . the rash also became petechial affecting on the trunk , and distal areas of upper and lower limbs . serum samples were reported positive for chikungunya virus by conventional and real time pcr and negative for dengue virus . a 65 year old male with known history of hypertension treated with amlodipine and valsartan experienced 4 days of fever , malaise , generalized arthralgias and arthritis of all the arm and leg joints . 24 h prior to admission he noticed dark urine and a violaceous skin tone to his feet . upon arrival to the er , the patient appeared severely ill , and was tachycardic , tachypneic and hypotensive . his hands were swollen , hot , and extremely painful , exhibiting an intense violaceous erythematous rash ( fig . 4 ) . distinct , rapidly confluent cyanotic areas covered the philtral area , nostrils and later the entire nasal region ( fig . laboratory results ( table 1 ) revealed respiratory alkalosis , impaired renal function , moderate leukocytosis with neutrophilia , thrombocytopenia , prolonged ptt , hypoalbuminemia as well as elevated ldh , alt and procalcitonin serum levels . the patient was placed on norepinephrine , given bicarbonate iv infusions and volume expanders , and transferred to the icu , where he was intubated and placed on mechanical ventilation . serum samples were positive for chikungunya virus by conventional and real time pcr and negative for dengue virus . a 32 year old female suddenly experienced malaise , myalgias , arthralgias , headache and fever that improved with the use of acetaminophen . she had underwent splenectomy 14 years before due to hodgkin 's lymphoma and had been immunized against streptococcus pneumonae and neisseria meningitidis . the following day , she was feeling worse and was evaluated at a medical center feeling worse and found to be hypotensive and tachycardic . laboratory results ( table 1 ) showed thrombocytopenia , leukopenia , and , she was conscious but appeared severely ill and was tachycardic , hypotensive and tachypneic . large generalized purpuric lesions were evident on her limbs , face and oropharynx , as well as acrocyanosis in both hands and feet with localized necrosis of finger tips ( fig . empiric antimicrobial therapy was started with meropenem and vancomycin and she was given a pulse dose of methylprednisolone . her condition continued to steadily deteriorate , requiring intubation and ventilator support . rapidly expanding bullae developed on both upper and lower limbs ( fig . bilateral basal pulmonary infiltrates were seen on the chest x - ray and a repeat echocardiogram revealed global hypokinesia with a low ventricular ejection fraction . nevertheless , after 72 h she began to improve gradually , and was discharged after 12 days . conventional and real time pcr from serum samples were positive for chikv and negative for dengue virus . a 75 year old patient with no known past medical illnesses developed chills and fever accompanied by generalized arthralgia and bilateral swelling in the hands and feet interphalangeal joints , ankles and knees . after 3 days , he was seen in an emergency room with the diagnosis of febrile exanthem probably caused chikungunya virus , severe sepsis with multi - organ failure and severe metabolic acidosis . on admission positive physical findings included a dry oral mucosa and a generalized erythematous macular rash which was more intense and confluent on feet ( fig . bilateral inflammatory edema was evident in the knee , ankle and interphalangeal joints with marked functional limitation of the latter . laboratory results revealed marked leukocytosis with neutrophilia , elevation of c reactive protein , elevated procalcitonin , and abnormal renal function tests ( table 1 ) . his clinical status deteriorated over 48 h with the onset of generalized soft tissue edema , distinctive palpebral and facial swelling ( fig . 2 ) , multiple bullae , hemodynamic instability , hepatic cholestasis , atrial fibrillation with rapid ventricular response , metabolic acidosis , and oliguric acute kidney failure . he was transferred to the icu , where his condition continued to worsen , further manifesting a septic shock syndrome with progressive multiple organ dysfunction . he died 24 h later due to cardiorespiratory arrest , in association with unresponsive ventricular fibrillation . a postmortem liver biopsy was obtained which revealed moderate acute hepatitis , hepatocyte necrosis with apoptosis , macrovesicular fat metamorphosis and diffuse edema . no granulomas were observed . repeated blood , urine and respiratory cultures failed to identify bacterial pathogens . serum samples for chikungunya virus were positive by conventional and real time pcr and negative for dengue virus . leptospirosis , malaria , typhoid fever , meningococcemia , and other severe bacterial , viral and parasitic diseases prevalent locally were ruled out . a 53 year old female with a recent diagnosis of iron deficiency anemia presented with a 3 day history of fever , chills and rigors . she also complained of swelling of her face and extremities as well as severe , disabling arthralgias affecting all of the interphalangeal joints of the hands as well as the wrists , elbows and knees . on admission , the patient had fever , appeared ill , and the physical examination was most remarkable for a maculopapular erythematous rash affecting the four extremities and an anasarca - like appearance . relevant laboratory results on admission ( table 1 ) included severe leukocytosis with mild thrombocytopenia , increased prothrombin time , hyperglycemia , hypoalbuminemia , elevated bun and creatinine , ldh , ast , alt , ck - mb , crp and procalcitonin levels . over 48 h the patient developed acute respiratory distress , requiring intubation and ventilatory support . a conspicuous , sharply delineated and necrotic skin lesion over the entire nasal region appeared ( fig . the rash also became petechial affecting on the trunk , and distal areas of upper and lower limbs . serum samples were reported positive for chikungunya virus by conventional and real time pcr and negative for dengue virus . a 65 year old male with known history of hypertension treated with amlodipine and valsartan experienced 4 days of fever , malaise , generalized arthralgias and arthritis of all the arm and leg joints . 24 h prior to admission he noticed dark urine and a violaceous skin tone to his feet . upon arrival to the er his hands were swollen , hot , and extremely painful , exhibiting an intense violaceous erythematous rash ( fig . distinct , rapidly confluent cyanotic areas covered the philtral area , nostrils and later the entire nasal region ( fig . 5 ) . laboratory results ( table 1 ) revealed respiratory alkalosis , impaired renal function , moderate leukocytosis with neutrophilia , thrombocytopenia , prolonged ptt , hypoalbuminemia as well as elevated ldh , alt and procalcitonin serum levels . the patient was placed on norepinephrine , given bicarbonate iv infusions and volume expanders , and transferred to the icu , where he was intubated and placed on mechanical ventilation . serum samples were positive for chikungunya virus by conventional and real time pcr and negative for dengue virus . a 32 year old female suddenly experienced malaise , myalgias , arthralgias , headache and fever that improved with the use of acetaminophen . she had underwent splenectomy 14 years before due to hodgkin 's lymphoma and had been immunized against streptococcus pneumonae and neisseria meningitidis . the following day , she was feeling worse and was evaluated at a medical center feeling worse and found to be hypotensive and tachycardic . laboratory results ( table 1 ) showed thrombocytopenia , leukopenia , and increased serum levels of alt , bun , creatinine and procalcitonin . she was admitted to the icu , remaining hemodynamically instable . , she was conscious but appeared severely ill and was tachycardic , hypotensive and tachypneic . large generalized purpuric lesions were evident on her limbs , face and oropharynx , as well as acrocyanosis in both hands and feet with localized necrosis of finger tips ( fig . empiric antimicrobial therapy was started with meropenem and vancomycin and she was given a pulse dose of methylprednisolone . her condition continued to steadily deteriorate , requiring intubation and ventilator support . rapidly expanding bullae developed on both upper and lower limbs ( fig . bilateral basal pulmonary infiltrates were seen on the chest x - ray and a repeat echocardiogram revealed global hypokinesia with a low ventricular ejection fraction . nevertheless , after 72 h she began to improve gradually , and was discharged after 12 days . conventional and real time pcr from serum samples were positive for chikv and negative for dengue virus . typical presentation of chikf includes abrupt onset of fever , arthralgias , and occasionally a maculopapular rash . polyarticular arthritis and tenosynovitis can occur and may result in excruciating joint pain which can last for months to years . indeed , the virus and illness name is derived from the swahili word that means to walk bent over . in the past , rare instances of hepatitis , myocarditis , hemorrhagic manifestations , and meningitis or encephalitis were described . recent outbreaks of chikf involving large number of patients , such as the 2005 epidemic of the french island territory of runion which affected 34% of all inhabitants , have resulted in more detailed descriptions of clinical manifestations including rare or previously unknown complications . these include neurologic syndromes ( meningitis , encephalitis , and guillain - barr syndrome ) ; neuro - ophthalmologic findings such as retrobulbar neuritis ; cardiac complications ( pericarditis and myocarditis ) ; and maternal fetal transmission resulting in abortion and congenital illness which all have been reported recently in association with chikv infection , suggesting that these severe presentations may be more common than previously considered , . the proportion of atypical cases in all chikf patients in the runion experience was 0.3% with an overall case - fatality rate of 10.7% . case fatality rate ( cfr ) reached 29% , however , when only severe cases were considered . indeed , the lethality rate was more than 5 times higher in patients aged 65 years as compared to those aged < 45 years . chikv infection seems to be responsible for severe clinical presentations , not only in elderly patients or patients at high risk , but also in younger patients with an unremarkable medical history . the mortality of severe cases may as high as 48% and chikv has been strongly suspected to have neurologic , hepatic , and myocardial tropism , with dramatic complications , . severe clinical forms are associated with the presence of several underlying medical conditions in about 90% of the cases . pre - existing respiratory or cardiovascular diseases and hypertension have been recognized as risk factors of developing severe illness . until now , lethal cases of chikf have been almost exclusively associated with infections by the ecsa genotype , a clade of which was responsible for the large epidemics on islands in the indian ocean and the indian subcontinent , from where most atypical and severe cases have been reported , , , . however , as of november 26 , 2014 , according to the pan american health organization , at least 149 deaths out of 747,317 have been attributed to chikf in the caribbean region during the current ongoing epidemic . case fatality rates from martinique and guadaloupe islands have been 0.97 and 0.73 per 1000 , respectively . although no epidemiological data on the morbidity and mortality of the venezuelan outbreak has yet been provided by the health authorities , the authors are aware of at least 20 additional , virologically documented , lethal cases in the country . moreover , estimates of the approximate total number of cases of chikf , based on the number of unexplained acute febrile illness episodes seen at the outpatient clinics of the ministry of health , since june 2014 , when the first authochonous patients were confirmed , are above 1,900,000 cases . therefore , it is plausible that the recognized deaths are only a small fraction of the real number . it may be tempting to attribute the occurrence of an unexpected number of severe and lethal cases to a change in the virulence of the virus , the findings in a recent report characterizing the whole genome of the 2013 chikv detected on st . martin island and the phylogenetic tree generated from sequencing data of chikv from british virgin islands in 2014 show no significant differences with the asian genotype being closely related to strains recently isolated in china and the philippines . the partial sequencing of the virus obtained from our four patients and several other local severe and/or lethal cases , have not shown any evidence of mutations that could associate with increased virulence ( zoila moros , personal communication ) . this finding supports that a single homogeneous chikv strain of the asian genotype was recently introduced into the caribbean and is currently moving throughout the region . the unique necrotic cutaneous nasal lesion observed in three of the cases , to our knowledge , has not been observed before in patients with chikf and will be discussed in detail elsewhere . in countries where both diseases are endemic a characteristic finding of the latter , not observed in any of the discussed patients , is the occurrence of massive capillary leakage and hemoconcentration . moreover , dengue virus infection was ruled out in all the cases by both specific serology and pcr . in countries experiencing epidemics of chikf clinicians must be familiar with the possibility of occurrence of atypical and severe cases of the disease , as such knowledge may help to improve its future management and strengthen the strategy of mitigation .

a large epidemic of chikungunya fever currently affects the caribbean , central and south america . despite a high number of reported cases , little is known on the occurrence of severe clinical complications . we describe four venezuelan patients with a severe and/or lethal course who exhibit unusual manifestations of the disease.case 1 describes a 75 year - old man with rapid onset of septic shock and multi - organ failure . cases 2 and 3 describe two patients with rapid aggressive clinical course who developed shock , severe purpuric lesions and a distinct area large of necrosis in the nasal region . case 4 depicts a splenectomized woman with shock , generalized purpuric lesions , bullous dermatosis and acronecrosis of an upper limb.chikungunya fever in the western hemisphere may also associate with atypical and severe manifestations . some patients experience a life - threatening , aggressive clinical course , with rapid deterioration and death due to multisystem failure .

Introduction Case reports Case 1 Case 2 Case 3 Case 4 Discussion

early - onset dementia ( at age < 65 years ) is an uncommon but important group of disorders . it has devastating impact on the lives of patients and families and deserves attention of the clinicians . dementia in younger people usually has some demonstrable secondary etiology ( psychiatric illness , alcohol or drugs or metabolic disturbance , familial alzheimer 's disease , wilson 's disease , etc ) . cognitive deficits secondary to brain tumors rarely occur , and there is little description in the available literature where it has been described in association with low grade gliomas , gliomatosis cerebri ( gc ) , and primary brain lymphoma . it is most commonly present as a diffusely infiltrating glial tumor of the cerebral cortex involving more than two lobes and occasionally infiltrating infratentorial structures and the spinal cord . we describe a young woman with gc confined to a single lobe ( right frontal ) and presented primarily as a dementia . before the advent of magnetic resonance imaging ( mri ) , diagnosis was generally not established until autopsy was done . even with mri , however , diagnosis is difficult . management of gc is also difficult by virtue of their diffuse nature , surgery is not suitable and large field radiotherapy carries the risk of severe toxicity . as per our knowledge , only few cases have been reported that had involvement of single hemisphere and presented only with memory loss . a 31-year - old woman was admitted to our hospital with a 3-year history of progressive memory loss and headache . the patient mainly complained of difficulties doing her usual daily routine activity job for about 1 year and misplacing personal objects , she was having difficulties doing simple calculations and drawing simple paper patterns , resulting in erroneous calculations and wrong measurements along with decreasing ability to perform complex tasks such as arranging a meeting with her old friend , pay bills , and marketing . childhood and prior developments were apparently normal . her past medical history was uneventful and was negative for stroke , transient ischemic attack , chronic infection , or other chronic illness . except for incontinence and a slight slurring of speech , the physical examination revealed no further neurological deficits , especially no speech impairment , seizures or myoclonia were observed . the mini mental state examination score was suggestive of dementia ( 21 of 30 ) and very low on the cognitive efficiency profile . nevertheless , she experienced no changes in personality , appetite , or sleeping pattern noted , nor was she observed to be a snorer or have spells of apnoea , and there was no history of auditory or visual hallucinations . blood count , biochemical profile , sedimentation rate , coagulation profile , and the results of radiography of the chest were normal . results of venereal disease research laboratories testing were negative , and no malignant cells were identified by cytological examination . serum vitamin b12 , folic acid , and thyroid - stimulating hormone levels were within normal limits . mr imaging and proton mr spectroscopy were performed on a clinical whole body 1.5-t imaging unit . t2-weighted and fluid attenuated inversion recovery flair images revealed an ill defined area of signal intensity alteration in the left frontal region with involvement of rostrum of corpus callosum with minimal postcontrast enhancement . the magnetic resonance spectroscopic imaging results showed an increased choline peak , a decreased n - acetyl aspartate ( naa ) peak with an increased ch / cr ratio at the site of lesion . the patient was given supportive care for a period of 15 days following that she improved a little bit and the patient was given supportive care for a period of 15days following that she improved a little bit and discharged , and at discharge , her mmse was 22/30 and she was also referred to higher centre . ( a ) axial view t2 flair image demonstrating tumor - related infi ltration involving left frontal lobe involvement of rostrum of corpus callosum . ( b ) axial view t2 flair image shows extensive hyper intensity involving the left frontal lobe and rostrum of corpus callosum , with a mild mass effect . ( c ) the mr spectroscopic imaging results showed an increased choline peak , a decreased naa peak with an increased ch / cr ratio at the site of lesion . ( d ) the mr spectroscopic imaging results showed an increased choline peak , a decreased naa peak with an increased ch / cr ratio at the site of lesion the differential diagnosis of young - onset dementia is extensive , including sporadic hereditary etiologies , neurodegenerative diseases , adult presentations of inborn errors of metabolism , other metabolic or storage diseases , and other syndromic diagnoses . although investigations of the etiologies and prevalence of early - onset dementia have been performed , in some individuals , the etiology remains indeterminate even after brain biopsy . in young adults ( up to 40 years of age ) , it is very rare to develop dementia without other features of neurological disease , or without features of disease elsewhere in the body . most cases of progressive cognitive disturbance in this age group are caused by psychiatric illness , alcohol , or other drugs or metabolic disturbance . these include familial alzheimer 's disease , spinocerebellar ataxia 17 ( dominant inheritance ) , adrenoleukodystrophy ( x - linked ) , gaucher 's disease type 3 , metachromatic leukodystrophy , niemann - pick disease type c , pantothenate kinase - associated neurodegeneration , tay - sachs disease , and wilson 's disease ( all recessive ) . dementia secondary to intracranial infections deserves a special attention especially in tropical and developing countries . syphilis , hiv infection , lyme neuroborreliosis , herpes virus , toxoplasmosis , cryptococcus , cytomegalovirus , or more rarely , whipple diseases are few of the infectious causes of behavioral changes including dementia . besides few isolated reports of neurocysticercosis presenting with dementia , a recent controlled study reported dementia in 12.5% of the patients with active neurocysticercosis . brain tumors can cause dementia by mass effect on structures such as the hypothalamus or pituitary gland , which control hormone secretion and can also press directly on brain cells , damaging them . treating the tumor , either medically or surgically , can reverse the symptoms in some cases . as in our case , the clinical course of gc is usually slow and long , especially when the neoplastic infiltrate is of low histological grade . the clinical course of gc is variable and includes pyramidal deficit , dementia , headache , cranial nerve changes , intracranial hypertension , seizures , and others . however , the present patient had a rare and atypical presentation in the form of dementia only . before the inception of mri , gc was diagnosed strictly in autopsy studies . however , mr imaging findings may be nonspecific , leading to a differential diagnosis that includes neoplastic , inflammatory , and vascular lesions . mr spectroscopy may further help in narrowing the differential diagnosis in favor of a neoplastic lesion by revealing increased cho / cr and cho / na and variably decreased na / cr.[9101214 ] some non - neoplastic lesions such as encephalitis , demyelinating disease , and organizing hemorrhage , may mimic these spectral changes in rare instances . if cho / cr or cho / na is not elevated , mr 5 spectroscopy may still help narrow the differential diagnosis in favor of gc . specifically , m - ins / cr and m - ins / na should be measured because they can be elevated even when cho / cr is normal . as a diffuse tumor , surgical treatment is usually not recommended , and radiology over extensive areas may cause serious toxicity . this case highlights two atypical findings : ( 1 ) dementia in the young patient caused due to gc and ( 2 ) involvement of single lobe of brain which may be initial stage of growing gc , later on bilateral lobes may be involved .

dementia with the onset before the age of 65 years is classified as early - onset dementia . although uncommon , it has considerable impact on the lives of patients and care givers , alike . a substantial subset of patients may have underlying reversible causes . yet , many , especially those of the very young may be initially misdiagnosed . a case of young woman with rapid mental decay is described here . she was finally diagnosed with gliomatosis cerebri ( gc ) involving only right frontal lobe . this atypical radiological feature of gc with primary presentation as memory loss needs special attention and clinicians should be aware of such conditions .

INTRODUCTION CASE REPORT DISCUSSION

thrombotic thrombocytopenic purpura ( ttp ) is a life - threatening disorder classically presenting with fever , thrombocytopenia , microangiopathic hemolytic anemia , renal dysfunction , and neurological impairment as well as various multiorgan complications and a mortality rate reaching 95% for untreated cases.1 the survival rate is 80%90% with early diagnosis and treatment with plasma infusion and plasma exchange . a 39-year - old sudanese male presented to the emergency room with fever , jaundice , and decreasing level of consciousness ( inability to recognize relatives ) for 7 days . the following laboratory values were obtained : white blood cell ( wbc ) count , 8.6 10/l ; hemoglobin ( hb ) , 67 g / l ; platelet ( plt ) count , 8 10/l ; creatinine , 121 mol / l ; prothrombin time and partial thromboplastin time , normal ; lactate dehydrogenase level ( ldh ) , 1947 u / l ; increased reticulocyte count ; and blood smear showing schistocytes . a urine analysis showed the following : blood , + 4 ; ph 5.5 ; protein , + 2 ; red blood cell count , 1520 ; wbc count , 1 ; and specific gravity , 1.0170 . the presence of microangiopathic hemolytic anemia , thrombocytopenic purpura , neurologic abnormalities , fever , and renal disease confirmed the patient s diagnosis . ten days later , his ldh rose to 1270 u / l , with hb of 105 g / l and plt count of 14 10/l . the patient was also started on cryoprecipitate supernatant because of the suboptimal response to plasmapheresis with fresh frozen plasma . this was thought to be a relapse of ttp ; the condition was considered and managed as resistant ttp . two days later , intravenous immunoglobulin ( full dose ) was started . despite the treatment for resistant ttp , the patient worsened , requiring icu ( intensive care unit ) admission because of the development of uncontrolled seizures , which required intubation and ventilation . the patient developed left - sided weakness and hypotension . treatment with antibiotics and then antifungal therapy commenced ; the case was labeled as refractory ttp . repeated brain computed tomography scans were normal . during this process , the creatinine kinase ( ck ) levels increased to > 50,000 u / l , with worsening kidney function and decreased urine output . acute renal failure in this patient was diagnosed according to the rifle ( risk , injury , failure , loss , end stage ) criteria : a rise in creatinine of more than three - fold , and urine output < 0.3 ml / kg / h over 24 hours . urine alkalanization was started when creatine phosphokinase ( cpk ) started rising to a target urine ph of 8 , with several fluid boluses targeting a urine output of > 200 ml / h . the serum calcium concentration was monitored on a daily basis throughout the patient s stay in the icu and after his discharge to a step - down unit . continuous venovenous hemodialysis started , although the cpk levels did not decrease for 5 days . after maintaining the patient on dialysis for 2 weeks , the patient started to improve clinically and was more alert , with a ck level of 5360 u / l and plt count of 95 109/l ( see figure 1 ) . all possible differential diagnoses for rhabdomyolysis were investigated , including metabolism , viral serology , bacterial levels , toxins , and drugs . the difficulty in weaning the patient from the ventilator stemmed from critical illness polyneuropathy and the consequences of the tracheostomy . he was conscious and alert , with residual weakness / paresis of the lower limbs , which was managed with physiotherapy . potential causes include malignant hyperthermia , neuroleptic malignant syndrome , near drowning / hypothermia , hiv ( human immunodeficiency virus ) infection , snake bites , heritable muscle enzyme deficiencies , metabolic and inflammatory myopathies , status epilepticus , status asthmaticus , eclampsia , prolonged labor , alcohol and cocaine abuse , copper sulfate , zinc phosphide , and other drugs . traumatic and nontraumatic causes of rhabdomyolysis have been described in other reviews.2,3 acute renal failure in rhabdomyolysis can be attributed to many mechanisms , such as precipitated myoglobin that blocks the renal tubules , oxygen free - radical injury , and renal arteriolar vasoconstriction induced by myoglobin . acidic urine precipitates myoglobin into casts that can occlude the renal tubules , which blocks urine flow or output . the other mechanism of renal cellular injury is the peroxidation of lipids in the cellular membrane by the oxygen free radicals produced by iron - containing heme . rhabdomyolysis releases platelet - activating factor , endothelin , and prostaglandins , which cause vasoconstriction and affect the glomerular filtration rate.4 in this particular case , the cause of resistant , sustained rhabdomyolysis could be multifactorial , related most directly to the muscle necrosis caused by a microthrombotic process secondary to ttp . rare reports have cited hematological factors in association with or as the cause of rhabdomyolysis.5 in this patient , ttp was diagnosed based on the presence of thrombocytopenia , microangiopathic hemolytic anemia , and central neurological dysfunction . notably , rhabdomyolysis is implicated as the cause of approximately 5%25% of cases of acute renal failure.6 there are two case reports in the literature worth mentioning . the first describes a patient with polymyositis who developed ttp , acute renal failure , and rhabdomyolysis after a flare - up of polymyositis following radiation therapy administered for uterine cervical cancer.7 the second case was a patient with thrombotic microangiopathy who presented with fulminant rhabdomyolysis and multiorgan failure . thrombotic microangiopathy typically presents as fulminating rhabdomyolysis with multiorgan dysfunction.5 although the patient developed multiorgan failure with ttp and severe sepsis , aggressive and continuous interventions including plasma exchange , chemotherapy , antibiotics , and continuous dialysis all contributed to the patient s recovery , despite the lengthy stay in the icu . conventional treatment for ttp , including plasma exchange , was not effective in isolation . when combined with chemotherapy and steroid therapy , this approach facilitated recovery.8 in conclusion , early , proper treatment of ttp can reduce morbidity and mortality in such patients when administered in combination with the early introduction of interventions such as continuous dialysis , even after the normalization of kidney function . this very rare or less recognized presentation of a rare disease could contribute to a high mortality rate if not recognized and treated expediently . finally , it is important to note that the acute renal failure was persistent , possibly because of rhabdomyolysis and not because of ttp alone .

thrombotic thrombocytopenic purpura ( ttp ) is a rare , life - threatening disorder . this paper describes the case of a 39-year - old sudanese male who presented to the emergency room with fever , jaundice , decreased level of consciousness , and worsening kidney function for 7 days , a high lactate dehydrogenase level ( 1947 ) , severe thrombocytopenia ( platelets 8) , and numerous schistocytes in the peripheral blood smear . the patient was admitted with a diagnosis of ttp for plasma exchange . fourteen days later , his creatinine kinase ( ck ) level rose to > 50,000 iu ; rhabdomyolysis was suggested . continuous venovenous hemodialysis ( cvvhd ) was started . the patient s ck level remained high , despite cvvhd , until the 6th day , after which this parameter gradually started to decrease . this report highlights a resistant case of ttp that presented with concomitant severe rhabdomyolysis , which demanded aggressive , continuous intervention .

Introduction Case report Discussion

leptospirosis is one of the most important zoonoses infecting both developing and developed countries in the world . , leptospirosis was also associated with recreational activities through exposure to the contaminated soil or water [ 35 ] . an increasing number of people involved in outdoor activities have increased the chances of infection . high density of rats in the markets poses potential threats to visitors and town service workers . the expansion of housing areas also increases the opportunistic contact between humans and the infected wildlife . environments are frequently associated with nonpathogenic leptospires . however , a novel pathogenic species , l. kmetyi , has been isolated from environmental samples in malaysia . malaysia is a tropical country with high seasonal rainfall , warm temperatures , and wet and humid climate . it is common that floods occur following heavy rainfall during monsoon season in the east coast . the presence of leptospires in the environment during flooding may potentially cause an outbreak of leptospirosis . kelantan which is located in the east coast of malaysia is among the affected states . the number of cases increased during flooding from on average 20 cases to 31 cases . many inhabitants in this state are also involved in agricultural activities which pose the highest risk as 50 cases were reported from the workers in this sector . it was also reported that , in 2011 , one death was recorded out of 276 cases . previously , one death was reported from a waterfall in kelantan which is jeram pasu . in order to identify the potential threats of leptospirosis in the environments , the present study aims to detect and characterize leptospira spp . samplings were conducted from december 2012 to november 2013 at the selected recreational areas ( waterfalls with spots of running and still water ) and markets in the north - eastern state of malaysia , kelantan ( figure 1 ) . those sampling sites , which cover urban and rural areas of three districts , were selected based on previous reports of leptospirosis cases , rat infestations , and improper waste management . water samples in recreational areas were collected from shaded , suspected areas for the presence of animals and in - between rock areas . water samples were filtered through 0.2 m nalgene filter unit and 40 ml of the samples were transferred into centrifuge tubes and then centrifuged at 4000 g , 27c for 20 mins . two ml of the samples were inoculated into 5 ml of liquid emjh media with the addition of 5-fluorouracil ( 100 g / ml ) . for soil samples , sampling locations were selected from wet and shaded areas , from garbage sites , and in the places where spoiled food was spotted . in sterile 50 ml falcon tubes , the soil samples were mixed with sterile water and shaken vigorously . the suspension was allowed to settle for 510 minutes before being filtered using 0.2 m nalgene filter unit . several drops of the filtrates were inoculated into emjh media supplemented with antimicrobial agents ( 5-fluorouracil , 100 g / ml ) and incubated at 30c in a shaker incubator at 25 rpm to accelerate the growth of leptospires . the presence of leptospires was examined under dark - field microscopy using 20x and 40x magnification daily for 28 days . leptospires can be distinguished by other spirochetes based on their characteristic thin helical structures with prominent hooked ends and motility . in an event of contamination , 1000 l of contaminated cultures were transferred into fresh liquid emjh media supplemented with sulfamethoxazole and trimethoprim ( 40/8 g / ml ) , amphotericin b ( 5 g / ml ) , and 5-fluorouracil ( 100 g / ml ) according to chakraborty et al . the concentration of trimethoprim was changed from 20 g / ml to 8 g / ml and fosfomycin was not added to the media . the cultures were examined under dark - field microscopy using 20x and 40x magnification daily for 28 days . if the contaminants were still present , the cultures were diluted in sterile distilled water using a serial dilution technique starting from dilutions 10 , 10 , 10 , and 10 and then transferred into solid emjh media . the diluted culture was incubated for 3 weeks or until the leptospires colonies were observed on plates . single , isolated colonies were transferred from the plates into liquid media using sterile pasteur pipettes . the microscopic agglutination test ( mat ) was performed using a set of hyperimmune rabbit antisera purchased from the queensland health clinical and statewide services , australia , including autumnalis , tarassovi , pyrogenes , javanica , grippotyphosa , copenhageni , canicola , ballum , hardjoparjitno , celledoni , patoc , djasiman , icterohaemorrhagiae , pomona , australis , and hebdomadis using the available method the plates were gently shaken to mix contents , covered to exclude debris and prevent evaporation , and incubated at 30c for 2 hours . all of the isolates were screened for agglutination at titre 1 : 100 under dark - field microscope . agglutination of at least 50% of the leptospires was considered as positive . dna extraction was performed on isolates by using qiagen dneasy blood & tissue kit ( qiagen , usa ) according to the manufacturer 's protocols for gram negative bacteria and stored at 20c until use . a 20 l - pcr reaction mixture containing 1x pcr buffer , 2.5 mm mgcl2 , 0.16 mm dntp 's premixed , 0.04 m of each primer , and 0.75 units of taq polymerase was used in all pcr amplifications . amplification was performed in mj research thermocycler with an initial denaturation at 94c for 5 minutes , followed by 30 cycles of 94c for 30 seconds , annealing for 30 seconds at ta of the primers ( table 1 ) , and extension at 72c for 30 seconds . the primer sets sapro 1 and sapro 2 were used to detect the nonpathogenic leptospires . amplified products were characterized by electrophoresis of 5 l of each reaction on 1.5% agarose gel for 50 min at 90 v. the pcr product amplified by bak2 primer pair was purified using qiaquick pcr purification kit ( qiagen , usa ) and subsequently sequenced using sanger sequencing kits and instruments supplied by applied biosystems , us , performed by first base laboratories ( selangor , malaysia ) . the dna sequences were edited in bioedit and compared against the genbank database using blast . the 16s rrna gene partial sequences of all isolates were aligned with the 16s rrna sequences of the type strains obtained from genbank by using multiple sequence comparison by log - expectation ( muscle ) in mega 6 software . a total of 144 samples comprised of 72 water ( markets , n = 36 ; recreational areas , n = 36 ) and 72 soil ( markets , n = 36 ; recreational areas , n = 36 ) samples were collected . out of those samples , 33 were positive for leptospires based on their characteristic morphology and motility . among positive samples , 7 ( 5% ) were water samples and 26 ( 18% ) were soil samples . a total of 29 positive samples ( water , n = 7 ; soil , n = 22 ) were collected from markets and only four positive samples ( water , n = 0 ; soil , n = 4 ) were collected from recreational areas . all of the leptospiral isolates in this study were not agglutinated with any of the reference hyperimmune sera used in mat ( table 2 ) . out of 33 isolates , 18 isolates were detected as leptospira spp . using g1/g2 and b64-i / b64-ii primers ( table 2 ) . none of the isolates were found to contain the virulence gene . molecular identification by 16s rrna verified that , from 33 positive samples in this study , 31 isolates were identified as leptospira spp . and two isolates were identified as leptospiral closest relatives , leptonema illini . a pathogenic leptospire , l. alstonii ( 3% ) , was isolated from a soil sample in a market . eight isolates ( 24.2% ) were identified as intermediate pathogenic species comprised of seven l. wolffii and one l. licerasiae . a total of 22 isolates ( 66.7% ) were identified as nonpathogenic leptospires , l. meyeri . the phylogenetic tree constructed using the neighbour - joining method was illustrated in figure 2 . ls12 , ls18 , ss5 , ss6 , ws5 , ws6 , ws9 , and ws15 were grouped into intermediate pathogenic clade . ls1 , ls7 , ss4 , ws1 , ws2 , ws3 , ws4 , ws10 , ws11 , ws12 , ws13 , ws14 , ws16 , ws17 , ws18 , ww2 , ww3 , ww4 , ww5 , ww6 , ww7 , and ww8 were grouped into nonpathogenic clade . even though this is not the first study reporting the detection and characterization of leptospiral isolates from environmental samples , its isolation from recreational and market areas would give an impact to the community . reported that the prevalence of leptospires in water samples from markets was 67.9% . in the present study , the water was found to contain detergent and appeared oily , most probably due to its close proximity to the nearby food stalls . it has been reported that 30 ppm of detergent ( ceepryn , fixanol , and sapamine ) were lethal to leptospires in water in 5 minutes . the prevalence of leptospires in water samples from recreational areas in malaysia was 11.67% . in the current study , a lower prevalence of 5.56% was noted , which could be due to the effect of inappropriate choice of sampling points . all isolates recovered in this study showed negative reactions to 16 reference hyperimmune sera tested in mat . similarly , negative results of mat for environmental isolates were also reported from another study in which eight isolates from the selected urban sites in malaysia were negative for a total of 25 hyperimmune sera tested . the availability of the reference hyperimmune sera was very limited because none of local reference laboratories supplies the hyperimmune sera . since the sera used in this study were procured overseas , the available panel of sera may not cover the circulating local serovar . a total of 37 serovars of leptospira from 13 serogroups have been identified in malaysia . this highlights the importance of having locally produced hyperimmune sera available for local use . in our study , g1/g2 and b64-i / b64-ii primers detected 18 out of 33 isolates as leptospira spp . however , 16s rrna gene sequencing identified 31 out of 33 isolates as leptospira spp . g1/g2 and b64-i / b64-ii primers were not able to detect all strains of leptospira spp . the occurrence of new strains of leptospires in humans , animals , and environment has increased the need for new primers that are more specific than g1/g2 and b64-i / b64-ii . leptospiral putative virulence gene , lipl32 , rises as a new target for the detection of pathogenic leptospires though its role in virulence mechanisms remains unknown . this is further complicated by the absence of this gene in pathogenic and intermediate leptospires isolated in this study . this gene was either absence or undetectable by pcr because in some cases the lipl32 gene was not detectable in l. licerasiae by pcr but its product was detectable by both southern blot hybridization and western immunoblot . in the previous study by murgia et al . , primer sets sapro 1 and sapro 2 were used to detect the saprophytic leptospires . however , these primers were found to be unspecific as pathogenic leptospire , l. alstonii , was amplified by this primer in this study . in this study , a pathogenic species , l. alstonii , was isolated from a soil sample in market area . our finding was in line with previous studies that also isolated l. alstonii from the environment . exposure to the soil in market areas which harboured pathogenic strains may explain the high seropositivity of leptospiral antibodies among garbage collectors and town cleaners in kelantan . the isolation of leptospires from frogs specifically from kidney was reported as early as 1964 . several pathogenic serovars or serogroups such as bim , australis , and ballum have been isolated from frogs [ 27 , 28 ] . however , inoculation of frogs with pathogenic serovars to establish experimental leptospirosis led to unsatisfactory results as leptospires were not recovered in their organs . the intermediate species , l. wolffii and l. licerasiae , were isolated from markets and recreational areas . they are able to cause diseases in humans although less frequent . the presence of pathogenic and intermediate species warrants adherence to precautions and preventive measures among the visitors to those areas . it was reported that malaysians who were involved in recreational activities especially water related are 2.4 times more likely to acquire leptospirosis compared to those who were not involved in similar activities . leptospirosis was detected in wildlife in malaysia including wild mammals - monkeys , bats , squirrels , and mongoose . those animals could be the reservoirs for leptospires in recreational areas located in remote areas . a previous study reported high numbers of l. meyeri ( 88.1% ) from environmental samples . l. meyeri serovar ranarum icf which was previously considered as nonpathogenic was shown to be related to pathogenic strain suggesting that this species consisted of both pathogenic and nonpathogenic strains . this strain was also amplified by sets of primers specific for pathogenic leptospira and not the sets of primers specific for saprophytic strain . this disagreement demands a novel typing method of leptospira spp . which covers not only pathogenic species but also intermediate and nonpathogenic strains to resolve the uncertainties in species ' designation in the genus of leptospira . the accurate species designation and classification are crucial because the presence of clinically imperative leptospires in the environment would not be overlooked . phylogenetic analysis of the studied isolates using 16s rrna gene sequences concurred with the findings of previous report by morey et al . in 2006 isolates in the nonpathogenic group were closely related to both l. meyeri and l. yanagawa . it was reported that species within the nonpathogenic groups were separated by no more than 10 bp . using the 16s rrna gene , l. meyeri recovered in this study were not separated . in order to demonstrate intraspecies genetic variations , it was also suggested that a cut - off point of 1,000 base pairs ( bp ) was applied for leptospiral species identification . the phylogenetic analysis of environmental isolates using 16s rrna was also found to be better than using pfge profiles . the data presented in this study demonstrates the presence of a clinically significant pathogenic l. alstonii and the predominance of l. meyeri in the environment . the pathogenic strain was found to not contain one of the highly conserve putative leptospiral virulence genes . hence , continuous control and surveillance are essential in lowering the burden of the disease .

the presence of pathogenic leptospira spp . in the environment poses threats to human health . the aim of this study was to detect and characterize leptospira spp . from environmental samples . a total of 144 samples comprised of 72 soil and 72 water samples were collected from markets and recreational areas in a north - eastern state in malaysia . samples were cultured on ellinghausen and mccullough modified by johnson and harris media . leptospires were positive in 22.9% ( n = 33 ) of the isolates . based on partial sequences of 16s rrna , a pathogenic leptospire , leptospira alstonii ( n = 1/33 ) , was identified in 3% of the isolates followed by intermediate leptospire ( l. wolffii , n = 1/33 , and l. licerasiae , n = 7/33 ) and nonpathogenic leptospire , l. meyeri ( n = 22/33 ) in 24.2% and 66.7% , respectively . this study demonstrates the presence of a clinically significant pathogenic l. alstonii in the environments which could pose health risks to the occupants and visitors .

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

bovine dermatophilosis is a severe skin infection of tropical ruminants inducing a severe loss in productivity and a 15% mortality rate . this disease is caused by the actinomycete bacterium dermatophilus congolensis associated with the tick amblyomma variegatum . currently there are no prospects for a vaccine , and acaricide or antibiotic control is hampered by the development of chemoresistance . animal breeders have observed that dermatophilosis susceptibility seems to be determined genetically , and we previously identified a bola - drb3-dqb class ii haplotype marker for high ( r2 = 0.96 ) susceptibility to the disease . with this marker , we developed a successful eugenic selection procedure for zebu brahman cattle in martinique ( fwi ) . over a period of five years , a marked reduction in disease prevalence , from 0.76 to 0.02 was achieved , and this low level has been maintained over the last two years . the selection procedure , based on a genetic marker system targeting the highly polymorphic bola locus , eliminates only those individuals which are at the highest risk of contracting the disease . in the present work , we discuss the properties of this system , including the " heterozygote advantage " and the " frequency dependence " theories , and examine their involvement in the biological mechanisms at the host / pathogen interface . we speculate on the exact role of the mhc molecules in the control of the disease , how the natural selection pressure imposed by the pathogens selectively maintains mhc diversity , and how our results can be practically applied for integrated control of dermatophilosis in developing countries .

feeding practices in the first few days of life play an important role in neonatal health : studies in nepal , india , and ghana have reported that newborns who were first breastfed within 24 hours of birth had a lower risk of neonatal mortality relative to newborns for whom breastfeeding was initiated after the first day . several studies in south asia have drawn attention to harmful practices such as delayed breastfeeding , giving prelacteals , and discarding colostrum . colostrum is an important source of antibodies and lymphocytes and provides nutrition appropriate to the immature digestive system of the newborn . however , with the exception of demographic and health surveys ( dhs ) , little is known about feeding practices for neonates in sub - saharan africa , and particularly in francophone africa . globally , there is also limited understanding of the demographic , economic , and cultural factors influencing newborn feeding practices . burkina faso is a predominantly rural landlocked country in west africa that has high levels of under - five and maternal mortality and has an estimated neonatal mortality rate of 29 per 1000 live births . feeding practices are generally poor : in the 2010 dhs , less than 25% of infants 05 months were exclusively breastfed and 36% received something other than breastmilk in the first three days of life . the prevalence of malnutrition is correspondingly high : 6% of children under 5 years and 11% of those less than 6 months were severely wasted in 2010 ( weight for height z - score < a prospective study of exclusive breastfeeding promotion and mortality is underway in southwest burkina faso , but little is known about feeding practices in newborns or factors influencing those practices . given the poor coverage of positive feeding practices as well as the high levels of malnutrition and stagnant neonatal mortality rate in burkina faso , there is an urgent need to better understand those practices as well as factors influencing them in order to design effective interventions . a cross - sectional household survey to measure coverage of maternal , neonatal , and child health ( mnch ) interventions was conducted in 20102011 in the context of a program evaluation and included a set of questions addressing newborn care practices . we report on the coverage of newborn feeding practices measured in this survey as well as an analysis of the determinants of those practices . the survey was conducted in 16 health districts across seven regions of burkina faso with a population of 4 424 354 in 2010 , representing 28% of the country s population . nine of the survey districts were implementing a mnch program and were purposively selected by the ministry of health because of their high under five mortality rates and weak presence of health partners , relative to other regions of the country . the remaining seven districts , which serve as a comparison arm in the evaluation , were selected using an adapted approach to restricted randomization , in which the comparison and intervention districts were balanced on education , health systems , and socio - economic variables . the survey sample size was 18 000 households . based on previous household surveys , 0.42 women with a live birth in the previous year per household were expected , for a sample size of 7560 live births in the two years preceding the survey . this sample size would allow measurement of newborn feeding indicators with an absolute precision of 3 percentage points or less , assuming a design effect of 2 and alpha of 0.05 . all households in the survey districts were eligible to participate , with the exception of those residing in urban census enumeration areas ( eas ) , which were excluded from the sampling frame . households were selected using two - stage cluster sampling stratified by district . in each district , 34 rural census eas ( 68 per district in each of two districts implementing community case management of pneumonia ) were selected with probability proportional to population size , and 30 households were selected in each ea using systematic sampling . a household questionnaire , including a household listing , was administered to the head of household or other adult household member and used to identify eligible women of reproductive age ( 1549 years ) residing in the household . no other inclusion or exclusion criteria were used beyond willingness to participate in the survey . after being read an oral consent script and providing consent to participate , all women were asked to provide summary birth histories and basic socio - demographic data . women reporting one or more live births in the 24 months preceding the interview date were asked a series of questions about the most recent birth , including antenatal and delivery care , newborn characteristics , and postnatal care . we used standard dhs questions regarding prelacteals and the time to initiation of breastfeeding . the questions concerning which prelacteals were given and whether colostrum was given were adapted from a previous study in bangladesh . a 14-day training workshop that included training in local languages , role plays , and a field exercise was conducted with 113 potential interviewers , of whom 72 were selected as interviewers and 18 as team leaders based on performance measures . language experts from the institut national dalphabetisation ( ina ) translated the questionnaires into the five principal local languages in the survey area ( moor , fulfuld , gourmatchma , kassna , and nuni ) and trained the interviewers to administer the questionnaires in these languages . although there was no formal written back - translation , the local language training was conducted as a discussion between study staff , language experts , and interviewers in order to determine the most appropriate terminology and formulation for each question . interviewers had been selected for the training based on their language capacity . because local languages are primarily oral and can not be read by most people in burkina , interviewers were given french questionnaires with key terms noted in parentheses in the local language . interviewers practiced administering the questionnaires in the local language during the training to ensure that translation was standard across interviewers . team leaders and supervisors regularly observed interviews in the field to ensure that questions were being translated correctly . data were collected between august 26 and october 31 , 2010 , corresponding to the end of the rainy season in burkina . supplementary data collection was conducted from december 3 , 2010 to january 18 , 2011 in 31 clusters that had been inaccessible during the rainy season , as well as 55 clusters where data collection was not completed during the study period . in addition , 24 clusters covered during the main data collection period were re - surveyed ( households initially surveyed were re - interviewed by different teams ) , primarily because of low woman / household ratios , which raised questions of omission of eligible women . supervision was provided by the team leaders , who had received additional training in supervision methods , and by the study investigators . questionnaires returned to the central office were re - checked for inconsistencies , and open answers were coded . four feeding practices were measured : breastfeeding initiation within 1 hour of birth , breastfeeding initiation within 24 hours of birth , giving the colostrum , and giving prelacteals . to estimate coverage levels , the proportion of newborns receiving each feeding practice was calculated . for this analysis , missing and do nt know responses were included and analyzed as separate categories , as they provided some indication of data quality . when calculating coverage rates , newborns who did not have an opportunity to receive the care practice were excluded . for example , babies who died within 72 hours of birth , or who were less than 72 hours old at the time of the interview , were excluded from the numerator and denominator when calculating the proportion of newborns who received a prelacteal . time to breastfeeding initiation was measured as a continuous variable and categorized for analysis as < 1 hour , 111 hours , 1223 hours , 2447 hours , and 48 hours or more . two coverage indicators were calculated from these categories : newborns breastfed < 1 hours of birth , and newborns breastfed < 24 hours of birth . a newborn was considered to have received a prelacteal if the mother reported giving anything other than breastmilk in the first 3 days of life ( definition is consistent with dhs ) . mothers were asked whether they had given the newborn any colostrum , and their response was coded as a binary ( yes / no ) variable . maternal , newborn , and household - level characteristics were included in the model as explanatory variables . for newborns , we included sex , perceived size at birth , place of delivery ( home or facility ) , delivery complications , and anc attendance ( number of visits ) during the pregnancy . maternal characteristics included age categorized in 5 year intervals , education ( none , primary , or secondary ) , parity , previous death of a child , religion , and ethnicity . we also included household wealth quintile , which was determined by applying principal components analysis to a set of household asset variables including household goods , land ownership , and livestock ownership . complicated delivery was defined as an unplanned facility delivery , a delivery by cesarean section , or a delivery where women reported staying at the facility for at least 3 days post - birth . with the exception of delivery by cesarean section , which in rural burkina faso would be done exclusively in cases of obstructed labor or other complications , these measures are proxies for complicated deliveries ; by definition they include only facility deliveries . data on birthweight were not collected in this study because in a previous survey , most babies born in rural areas were not weighed at birth . instead , mothers were asked whether the baby was very large , larger than average , average , smaller than average , or very small at birth . data on birthweight and perceived size at birth were both collected in the national 2010 burkina faso dhs , in which 66% of live births had a measured birthweight . in the 2010 dhs , perceived size at birth had good specificity ( 94% ) but poor sensitivity ( 45% ) in detecting low birthweight ( < 2500 g ) . therefore , perceived size at birth in this analysis was used not as a proxy for low birthweight but rather as an indicator of the mother s perception of the newborn s size . bivariate and multivariate poisson regression models were used to estimate relative risk ratios comparing the risk of feeding behaviors ( early initiation of breastfeeding , giving colostrum , giving prelacteals ) by maternal / newborn characteristic . all variables hypothesized to be determinants , confounders , or effect modifiers were retained in the final analysis , whether statistically significant or not , as causal determinants might not show statistically significant associations with these feeding practices for reasons related to sample size , measurement , or model . variance inflation factors ( vif ) were calculated for variables hypothesized to be collinear . with the exception of maternal age and parity , which had a vif of 1.74 , the other variables had vifs near 1.00 . standard errors and confidence intervals were adjusted using the taylor linearization method to account for the effect of stratification and clustering . survey weights were used to account for unequal probability of selection and for non - response . all analyses were weighted , and weights were standardized by dividing the weight for each live birth by the mean weight . the study was reviewed and approved by the burkina faso national ethics committee for health research ( n 2009 - 67 ) , by the burkina faso national statistics council , and by the johns hopkins school of public health institutional review board ( irb2590 ) . the survey was conducted in 16 health districts across seven regions of burkina faso with a population of 4 424 354 in 2010 , representing 28% of the country s population . nine of the survey districts were implementing a mnch program and were purposively selected by the ministry of health because of their high under five mortality rates and weak presence of health partners , relative to other regions of the country . the remaining seven districts , which serve as a comparison arm in the evaluation , were selected using an adapted approach to restricted randomization , in which the comparison and intervention districts were balanced on education , health systems , and socio - economic variables . the survey sample size was 18 000 households . based on previous household surveys , 0.42 women with a live birth in the previous year per household were expected , for a sample size of 7560 live births in the two years preceding the survey . this sample size would allow measurement of newborn feeding indicators with an absolute precision of 3 percentage points or less , assuming a design effect of 2 and alpha of 0.05 . all households in the survey districts were eligible to participate , with the exception of those residing in urban census enumeration areas ( eas ) , which were excluded from the sampling frame . households were selected using two - stage cluster sampling stratified by district . in each district , 34 rural census eas ( 68 per district in each of two districts implementing community case management of pneumonia ) were selected with probability proportional to population size , and 30 households were selected in each ea using systematic sampling . a household questionnaire , including a household listing , was administered to the head of household or other adult household member and used to identify eligible women of reproductive age ( 1549 years ) residing in the household . no other inclusion or exclusion criteria were used beyond willingness to participate in the survey . after being read an oral consent script and providing consent to participate , all women were asked to provide summary birth histories and basic socio - demographic data . women reporting one or more live births in the 24 months preceding the interview date were asked a series of questions about the most recent birth , including antenatal and delivery care , newborn characteristics , and postnatal care . we used standard dhs questions regarding prelacteals and the time to initiation of breastfeeding . the questions concerning which prelacteals were given and whether colostrum was given were adapted from a previous study in bangladesh . a 14-day training workshop that included training in local languages , role plays , and a field exercise was conducted with 113 potential interviewers , of whom 72 were selected as interviewers and 18 as team leaders based on performance measures . language experts from the institut national dalphabetisation ( ina ) translated the questionnaires into the five principal local languages in the survey area ( moor , fulfuld , gourmatchma , kassna , and nuni ) and trained the interviewers to administer the questionnaires in these languages . although there was no formal written back - translation , the local language training was conducted as a discussion between study staff , language experts , and interviewers in order to determine the most appropriate terminology and formulation for each question . interviewers had been selected for the training based on their language capacity . because local languages are primarily oral and can not be read by most people in burkina , interviewers were given french questionnaires with key terms noted in parentheses in the local language . interviewers practiced administering the questionnaires in the local language during the training to ensure that translation was standard across interviewers . team leaders and supervisors regularly observed interviews in the field to ensure that questions were being translated correctly . data were collected between august 26 and october 31 , 2010 , corresponding to the end of the rainy season in burkina . supplementary data collection was conducted from december 3 , 2010 to january 18 , 2011 in 31 clusters that had been inaccessible during the rainy season , as well as 55 clusters where data collection was not completed during the study period . in addition , 24 clusters covered during the main data collection period were re - surveyed ( households initially surveyed were re - interviewed by different teams ) , primarily because of low woman / household ratios , which raised questions of omission of eligible women . supervision was provided by the team leaders , who had received additional training in supervision methods , and by the study investigators . questionnaires returned to the central office were re - checked for inconsistencies , and open answers were coded . four feeding practices were measured : breastfeeding initiation within 1 hour of birth , breastfeeding initiation within 24 hours of birth , giving the colostrum , and giving prelacteals . to estimate coverage levels , the proportion of newborns receiving each feeding practice was calculated . for this analysis , missing and do nt know responses were included and analyzed as separate categories , as they provided some indication of data quality . when calculating coverage rates , newborns who did not have an opportunity to receive the care practice were excluded . for example , babies who died within 72 hours of birth , or who were less than 72 hours old at the time of the interview , were excluded from the numerator and denominator when calculating the proportion of newborns who received a prelacteal . time to breastfeeding initiation was measured as a continuous variable and categorized for analysis as < 1 hour , 111 hours , 1223 hours , 2447 hours , and 48 hours or more . two coverage indicators were calculated from these categories : newborns breastfed < 1 hours of birth , and newborns breastfed < 24 hours of birth . a newborn was considered to have received a prelacteal if the mother reported giving anything other than breastmilk in the first 3 days of life ( definition is consistent with dhs ) . mothers were asked whether they had given the newborn any colostrum , and their response was coded as a binary ( yes / no ) variable . maternal , newborn , and household - level characteristics were included in the model as explanatory variables . for newborns , we included sex , perceived size at birth , place of delivery ( home or facility ) , delivery complications , and anc attendance ( number of visits ) during the pregnancy . maternal characteristics included age categorized in 5 year intervals , education ( none , primary , or secondary ) , parity , previous death of a child , religion , and ethnicity . we also included household wealth quintile , which was determined by applying principal components analysis to a set of household asset variables including household goods , land ownership , and livestock ownership . complicated delivery was defined as an unplanned facility delivery , a delivery by cesarean section , or a delivery where women reported staying at the facility for at least 3 days post - birth . with the exception of delivery by cesarean section , which in rural burkina faso would be done exclusively in cases of obstructed labor or other complications , these measures are proxies for complicated deliveries ; by definition they include only facility deliveries . data on birthweight were not collected in this study because in a previous survey , most babies born in rural areas were not weighed at birth . instead , mothers were asked whether the baby was very large , larger than average , average , smaller than average , or very small at birth . data on birthweight and perceived size at birth were both collected in the national 2010 burkina faso dhs , in which 66% of live births had a measured birthweight . in the 2010 dhs , perceived size at birth had good specificity ( 94% ) but poor sensitivity ( 45% ) in detecting low birthweight ( < 2500 g ) . therefore , perceived size at birth in this analysis was used not as a proxy for low birthweight but rather as an indicator of the mother s perception of the newborn s size . bivariate and multivariate poisson regression models were used to estimate relative risk ratios comparing the risk of feeding behaviors ( early initiation of breastfeeding , giving colostrum , giving prelacteals ) by maternal / newborn characteristic . all variables hypothesized to be determinants , confounders , or effect modifiers were retained in the final analysis , whether statistically significant or not , as causal determinants might not show statistically significant associations with these feeding practices for reasons related to sample size , measurement , or model . variance inflation factors ( vif ) were calculated for variables hypothesized to be collinear . with the exception of maternal age and parity , which had a vif of 1.74 , the other variables had vifs near 1.00 . standard errors and confidence intervals were adjusted using the taylor linearization method to account for the effect of stratification and clustering . survey weights were used to account for unequal probability of selection and for non - response . all analyses were weighted , and weights were standardized by dividing the weight for each live birth by the mean weight . the study was reviewed and approved by the burkina faso national ethics committee for health research ( n 2009 - 67 ) , by the burkina faso national statistics council , and by the johns hopkins school of public health institutional review board ( irb2590 ) . from august 26 , 2010 to january 18 , 2011 , 18 360 households across 16 health districts in burkina faso were visited by study interviewers . interviewers completed 17 139 ( 93% ) household interviews identifying 23 134 women of reproductive age , of whom 21 953 ( 95% ) completed the interview . the main reason for household and woman non - response was the absence of the household or of the woman . data collection was largely completed during the rainy season and harvest period , when individuals and households frequently move away from their compounds to be closer to their fields . among the women interviewed , 7 878 reported a live birth in the preceding two years . these women were young ( median age 27 years , range 1549 years ) , but of high parity ( median 4 live births , range 115 ) , indicative of the high fertility in this population . the vast majority of the women ( 94% ) had no formal education . despite declining under - five mortality rates in burkina faso , mortality was high : after excluding primiparas , approximately 45% of all women with a live birth in the two years before the survey reported a death of a prior live born child . respondents reported frequent health service contacts , with 45% reporting 4 or more antenatal care ( anc ) visits during the pregnancy and over 70% delivering in a health facility . breastfeeding was very common in this population : 0.5% ( n=36 ) of live births were reported as never breastfed , and 64% ( 23 of 36 ) of these non - breastfed babies died in the first seven days after birth . one - quarter of newborns were put to the breast within one hour of birth , and three - quarters were breastfed in the first 24 hours . most mothers reported initiating breastfeeding either in the first few hours of life , or after 24 hours ; only 2% reported initiating breastfeeding 1223 hours after birth . colostrum was given to 84% of newborns , and 21% of those surviving to 3 days were given something other than breastmilk in the first 3 days of life ( table 1 ) . water , sugar - water , and teas were the most common prelacteals ( table 2 ) . receipt of colostrum was more frequent among those breastfed in the first 24 hours : 88% of those breastfed within 24 hours received the colostrum , as compared to 75% among those who began breastfeeding at 1 day or later ( arr=1.16 ) . receipt of prelacteals was more frequent among babies first breastfed after 24 hours relative to those breastfed in the first day ( 42% vs. 15% , arr=2.67 ) . birth in a health facility was associated with receipt of positive feeding practices : facility - born babies were slightly more likely to be breastfed within 24 hours ( arr=1.06 95% ci : 1.021.10 ) and to receive colostrum ( arr=1.07 , 95% ci : 1.041.11 ) , relative to those born at home . facility - born babies were also 34% less likely to be given prelacteals ( arr=0.66 , 95% ci : 0.570.75 ) . however , there was a slight negative association between facility birth and breastfeeding within one hour of birth ( arr=0.81 , 95% ci : 0.710.94 ) . more frequent anc attendance was associated with an increased probability of giving colostrum ( arr=1.12 , 95% ci : 1.011.24 for 4 or more anc visits as compared to no anc ) , and decreased risk of giving prelacteals ( arr=0.57 , 95% ci : 0.450.73 for 4 or more anc visits ) . newborn sex and maternal education , age , parity , and death of a child were not consistently associated with breastfeeding practices , except that women with a secondary education , relative to no education , were more likely to give colostrum to the newborn ( arr=1.13 , 95% ci : 1.081.19 ) . maternal ethnicity and religion , however , were strongly associated with all feeding practices , although the direction of these associations varied . bissa ethnicity was associated with protective breastfeeding practices , relative to the majority mossi ethnic group ( arr=2.47 95% ci : 2.042.98 for breastfeeding within one hour , arr=1.12 , 95% ci : 1.061.17 for colostrum , arr=0.32 , 95% ci : 0.150.66for prelacteals ) . women of the gourmatch ethnic group were slightly more likely than the mossi to initiate breastfeeding within 24 hours ( arr=1.16 , 95% ci : 1.111.21 ) and to give colostrum to the newborn ( arr=1.11 , 95% ci : 1.051.16 ) , but less likely to give prelacteals ( arr=0.67 , 95% ci : 0.490.91 ) . traditional or animist religion was negatively associated with initiation of breastfeeding within one hour of birth ( arr=0.74 , 95% ci : 0.590.94 ) , and with giving the colostrum ( arr=0.94 , 95% ci : 0.881.00 ) , and positively associated with giving prelacteals ( arr=1.57 , 95% ci : 1.301.90 ) relative to muslim religion . from august 26 , 2010 to january 18 , 2011 , 18 360 households across 16 health districts in burkina faso were visited by study interviewers . interviewers completed 17 139 ( 93% ) household interviews identifying 23 134 women of reproductive age , of whom 21 953 ( 95% ) completed the interview . the main reason for household and woman non - response was the absence of the household or of the woman . data collection was largely completed during the rainy season and harvest period , when individuals and households frequently move away from their compounds to be closer to their fields . among the women interviewed , 7 878 reported a live birth in the preceding two years . these women were young ( median age 27 years , range 1549 years ) , but of high parity ( median 4 live births , range 115 ) , indicative of the high fertility in this population . the vast majority of the women ( 94% ) had no formal education . despite declining under - five mortality rates in burkina faso , mortality was high : after excluding primiparas , approximately 45% of all women with a live birth in the two years before the survey reported a death of a prior live born child . respondents reported frequent health service contacts , with 45% reporting 4 or more antenatal care ( anc ) visits during the pregnancy and over 70% delivering in a health facility . breastfeeding was very common in this population : 0.5% ( n=36 ) of live births were reported as never breastfed , and 64% ( 23 of 36 ) of these non - breastfed babies died in the first seven days after birth . one - quarter of newborns were put to the breast within one hour of birth , and three - quarters were breastfed in the first 24 hours . most mothers reported initiating breastfeeding either in the first few hours of life , or after 24 hours ; only 2% reported initiating breastfeeding 1223 hours after birth . colostrum was given to 84% of newborns , and 21% of those surviving to 3 days were given something other than breastmilk in the first 3 days of life ( table 1 ) . water , sugar - water , and teas were the most common prelacteals ( table 2 ) . receipt of colostrum was more frequent among those breastfed in the first 24 hours : 88% of those breastfed within 24 hours received the colostrum , as compared to 75% among those who began breastfeeding at 1 day or later ( arr=1.16 ) . receipt of prelacteals was more frequent among babies first breastfed after 24 hours relative to those breastfed in the first day ( 42% vs. 15% , arr=2.67 ) . birth in a health facility was associated with receipt of positive feeding practices : facility - born babies were slightly more likely to be breastfed within 24 hours ( arr=1.06 95% ci : 1.021.10 ) and to receive colostrum ( arr=1.07 , 95% ci : 1.041.11 ) , relative to those born at home . facility - born babies were also 34% less likely to be given prelacteals ( arr=0.66 , 95% ci : 0.570.75 ) . however , there was a slight negative association between facility birth and breastfeeding within one hour of birth ( arr=0.81 , 95% ci : 0.710.94 ) . more frequent anc attendance was associated with an increased probability of giving colostrum ( arr=1.12 , 95% ci : 1.011.24 for 4 or more anc visits as compared to no anc ) , and decreased risk of giving prelacteals ( arr=0.57 , 95% ci : 0.450.73 for 4 or more anc visits ) . newborn sex and maternal education , age , parity , and death of a child were not consistently associated with breastfeeding practices , except that women with a secondary education , relative to no education , were more likely to give colostrum to the newborn ( arr=1.13 , 95% ci : 1.081.19 ) . maternal ethnicity and religion , however , were strongly associated with all feeding practices , although the direction of these associations varied . bissa ethnicity was associated with protective breastfeeding practices , relative to the majority mossi ethnic group ( arr=2.47 95% ci : 2.042.98 for breastfeeding within one hour , arr=1.12 , 95% ci : 1.061.17 for colostrum , arr=0.32 , 95% ci : 0.150.66for prelacteals ) . women of the gourmatch ethnic group were slightly more likely than the mossi to initiate breastfeeding within 24 hours ( arr=1.16 , 95% ci : 1.111.21 ) and to give colostrum to the newborn ( arr=1.11 , 95% ci : 1.051.16 ) , but less likely to give prelacteals ( arr=0.67 , 95% ci : 0.490.91 ) . traditional or animist religion was negatively associated with initiation of breastfeeding within one hour of birth ( arr=0.74 , 95% ci : 0.590.94 ) , and with giving the colostrum ( arr=0.94 , 95% ci : 0.881.00 ) , and positively associated with giving prelacteals ( arr=1.57 , 95% ci : 1.301.90 ) relative to muslim religion . this paper describes current feeding practices in the early neonatal period in rural burkina faso , and identifies factors associated with positive feeding practices . breastfeeding was almost universal , and initiation of breastfeeding occurred in the first 24 hours for 76% of newborns , which is similar to breastfeeding initiation patterns reported in northern ghana ( 71% within 24 hours ) . studies of breastfeeding initiation in south asia have reported a wide range of coverage rates for breastfeeding initiation within 24 hours ( 15% 90% ) . consistent with the high coverage for breastfeeding initiation in the first day , most women in our survey also reported giving colostrum to their newborns . although colostrum has traditionally been perceived as dirty or old milk and was often discarded in many settings 4 , 5 , 19 , recent studies in india and the gambia have also documented increased acceptance of the practice of giving colostrum . harmful practices such as giving prelacteals were reported for only 21% of newborns . in this study , after accounting for confounders , early initiation of breastfeeding was associated with a reduced risk of receiving prelacteals , perhaps because early initiation of breastfeeding reduced the perceived need for prelacteals , or because mothers had received counseling on both practices . this finding contradicts previous qualitative studies in india and the gambia that have reported that prelacteals are given to newborns even when breastfeeding is initiated soon after birth , but is consistent with a study in rural nepal that found an eight - fold increase in the odds of being exclusively breastfed among newborns breastfed within 24 hours of birth relative to those for whom breastfeeding was initiated after 24 hours . the practice of giving water as a prelacteal echoes the findings from studies in other african settings , where water was perceived to be necessary for breastfed infants in order to prevent dehydration . in mozambique and the gambia , for example , caregivers interviewed were resistant to exclusive breastfeeding , believing that children needed to be given water even in the newborn period . addressing this belief through mass media campaigns and/or counseling during antenatal and postnatal visits could reduce the use of prelacteals and increase the prevalence of exclusive breastfeeding . in an analysis of dhs data in bangladesh , secondary education was positively associated with early initiation of breastfeeding . in this study , however , secondary education was positively associated with giving colostrum but not with any other practice , possibly because the proportion of women with any secondary education was very small ( 1.5% in burkina faso vs. 34% in bangladesh ) . surprisingly , maternal characteristics other than ethnicity , religion , and education showed no association with feeding practices after adjusting for confounders . the association with ethnicity suggests that feeding behavior may be largely determined by established practices within the mother s ethnic group . to date , educational and behavior change campaigns related to newborn feeding practices in this setting have been limited and not explicitly designed to meet the varying needs of specific ethnic groups , which may also explain our failure to find associations between feeding practices and maternal age or education . newborns perceived to have been smaller than average or very small at birth were less likely to have been breastfed within 24 hours of birth , relative to newborns perceived to have been of average or larger than average size . studies in brazil and sri lanka have also reported that breastfeeding initiation was delayed among low birthweight newborns relative to normal birthweight newborns . it should be noted that our analysis used maternal perception of size at birth , which may not be a good proxy for birthweight . of concern , however , is that even when mothers perceived a newborn to be smaller than average , he or she was no more likely to receive positive feeding practices than larger newborns . further exploring breastfeeding practices among vulnerable babies ( low birth weight , preterm ) is critically important given the suspected high prevalence of low birthweight in this population ( 19% , based on adjusted dhs data ) and the vulnerability of these babies . an important limitation to this study is that the indicators are based on maternal recall of events soon after birth , with the recall period extending up to 2 years after the birth . standard survey questions were used where possible , both to increase comparability with other surveys and because the questions had already been tested in various settings . as is true of most mnch survey questions , in addition , there were limitations to some of our explanatory variables , such as complicated delivery , which was based on imperfect proxies . some misclassification is therefore likely to have occurred for both feeding practices and explanatory variables . to the extent that the misclassification was non - differential , . however , differential misclassification by explanatory variables ( such as place of delivery ) is possible and would have resulted in biased relative risk estimates , though the direction of the bias is difficult to predict . the effectiveness of early initiation of breastfeeding may be dependent on its timing in relation to birth 1 , 2 , 3 . recall of the interval between birth and initiation of breastfeeding is likely to be difficult for women in many settings ; in this study population an additional complication was that most of the women in the sample had no formal education and had difficulty providing a numeric response when asked to recall time intervals . if women were not accurately able to gauge or recall these time intervals , misclassification is likely to have occurred . if the ability to recall or gauge time was differential ( e.g. , women with some education were able to recall or estimate time more accurately ) , it may have biased observed associations between maternal characteristics and feeding practices . we considered asking about the timing of breastfeeding initiation in relation to an event such as the delivery of the placenta ; however , the use of this as an anchoring time point is not recommended , given the substantial natural variability in time to placental delivery . in addition to misclassification , social desirability bias may have led women to over - report practices like early initiation of breastfeeding , if they perceived those practices to be positive . however , this risk may be minimal in this study given the lack of educational activities or campaigns about newborn feeding practices . positive early feeding practices may positively influence breastfeeding duration and exclusivity later in infancy : delayed breastfeeding initiation and use of prelacteals have been associated with shorter duration of breastfeeding , relative to newborns breastfed within 12 hours of birth and who received breastmilk as their first feed . the prevalence of positive feeding practices in the newborn period is therefore of critical importance , particularly in settings with a high prevalence of malnutrition , such as burkina faso . the results of this study indicate that positive feeding practices , including breastfeeding initiation within 24 hours , giving colostrum , and not giving prelacteals , are relatively common in the study population , although more progress is needed , particularly with respect to breastfeeding initiation . the data also suggest that early initiation of breastfeeding led to reduced use of prelacteals and a higher likelihood of receiving the colostrum . the finding that facility - born neonates are more likely to receive positive feeding practices is encouraging , especially given the increasing coverage of facility birth in rural burkina . however , initiation of breastfeeding appeared to be somewhat delayed in facility births . this delay may have been due to other activities surrounding the birth ( weighing of the newborn , monitoring of the third stage of labor ) and suggests the need for additional promotion of immediate breastfeeding among auxiliary midwives and nurses who are in the delivery room . these results suggest several avenues for improving feeding practices for newborns in burkina faso , such as through counselling during postnatal health checks after home births , and increased efforts to improve facility practices around breastfeeding initiation . the findings also highlight the need for a qualitative assessment of factors influencing feeding practices in the neonatal period . if water is given after birth in an effort to prevent dehydration , for example , then addressing this understanding will be essential to reducing the use of prelacteals . it could also increase early initiation of breastfeeding , if breastfeeding was promoted as a means of preventing dehydration . research is also needed to understand the role ethnicity plays in determining feeding practices in burkina in the neonatal period and beyond , and to assess what interventions might be effective in changing these practices . an important limitation to this study is that the indicators are based on maternal recall of events soon after birth , with the recall period extending up to 2 years after the birth . standard survey questions were used where possible , both to increase comparability with other surveys and because the questions had already been tested in various settings . as is true of most mnch survey questions , in addition , there were limitations to some of our explanatory variables , such as complicated delivery , which was based on imperfect proxies . some misclassification is therefore likely to have occurred for both feeding practices and explanatory variables . to the extent that the misclassification was non - differential , it would have biased relative risk estimates towards the null . however , differential misclassification by explanatory variables ( such as place of delivery ) is possible and would have resulted in biased relative risk estimates , though the direction of the bias is difficult to predict . the effectiveness of early initiation of breastfeeding may be dependent on its timing in relation to birth 1 , 2 , 3 . recall of the interval between birth and initiation of breastfeeding is likely to be difficult for women in many settings ; in this study population an additional complication was that most of the women in the sample had no formal education and had difficulty providing a numeric response when asked to recall time intervals . if women were not accurately able to gauge or recall these time intervals , misclassification is likely to have occurred . if the ability to recall or gauge time was differential ( e.g. , women with some education were able to recall or estimate time more accurately ) , it may have biased observed associations between maternal characteristics and feeding practices . we considered asking about the timing of breastfeeding initiation in relation to an event such as the delivery of the placenta ; however , the use of this as an anchoring time point is not recommended , given the substantial natural variability in time to placental delivery . in addition to misclassification , social desirability bias may have led women to over - report practices like early initiation of breastfeeding , if they perceived those practices to be positive . however , this risk may be minimal in this study given the lack of educational activities or campaigns about newborn feeding practices . positive early feeding practices may positively influence breastfeeding duration and exclusivity later in infancy : delayed breastfeeding initiation and use of prelacteals have been associated with shorter duration of breastfeeding , relative to newborns breastfed within 12 hours of birth and who received breastmilk as their first feed . the prevalence of positive feeding practices in the newborn period is therefore of critical importance , particularly in settings with a high prevalence of malnutrition , such as burkina faso . the results of this study indicate that positive feeding practices , including breastfeeding initiation within 24 hours , giving colostrum , and not giving prelacteals , are relatively common in the study population , although more progress is needed , particularly with respect to breastfeeding initiation . the data also suggest that early initiation of breastfeeding led to reduced use of prelacteals and a higher likelihood of receiving the colostrum . the finding that facility - born neonates are more likely to receive positive feeding practices is encouraging , especially given the increasing coverage of facility birth in rural burkina . this delay may have been due to other activities surrounding the birth ( weighing of the newborn , monitoring of the third stage of labor ) and suggests the need for additional promotion of immediate breastfeeding among auxiliary midwives and nurses who are in the delivery room . these results suggest several avenues for improving feeding practices for newborns in burkina faso , such as through counselling during postnatal health checks after home births , and increased efforts to improve facility practices around breastfeeding initiation . the findings also highlight the need for a qualitative assessment of factors influencing feeding practices in the neonatal period . if water is given after birth in an effort to prevent dehydration , for example , then addressing this understanding will be essential to reducing the use of prelacteals . it could also increase early initiation of breastfeeding , if breastfeeding was promoted as a means of preventing dehydration . research is also needed to understand the role ethnicity plays in determining feeding practices in burkina in the neonatal period and beyond , and to assess what interventions might be effective in changing these practices .

objectivesnewborn feeding practices are important to neonatal health and survival , but understudied in sub - saharan africa . we assessed the prevalence and determinants of newborn feeding practices in burkina faso.study designan 18 000 household survey was conducted in rural burkina faso in 20102011 . women of reproductive age were asked about antenatal , delivery , and newborn care practices for their most recent live birth . coverage of newborn feeding practices was estimated and multivariate regression was used to assess determinants of these practices.resultsseventy-six percent of live births were breastfed within 24 hours of birth , 84% were given colostrum , and 21% received prelacteals . facility delivery and antenatal care attendance were associated with positive feeding practices.conclusionspositive newborn feeding practices were common in rural burkina faso , relative to other low - income settings . interventions are needed to improve feeding practices among home - born babies , and to encourage earlier initiation of breastfeeding among facility - born newborns .

Background Methods Setting Sample size Selection of households, women, and births Data collection Data management and analysis Ethical and administrative clearance Results Study population Coverage of newborn care practices Determinants of breastfeeding practices Discussion Limitations Conclusions Supplementary Material

diabetic neuropathy is a common complication of diabetes which affects around 50 percent of patients . there is much debate about the pathogenesis of this condition but a lot of studies consider hyperglycemia as the main initiating factor ( 2 - 5 ) . hyperglycemia may lead to neuropathy as a consequence of increasing oxidative stress ( 6 - 8 ) , formation of glycation end products ( 9 , 10 ) , accumulation of polyols ( 11 , 12 ) , nerve ischemia due to vasculopathies ( 13 - 15 ) , etc . peripheral diabetic neuropathy ( pdn ) may alter pain sensation ( hyper- , hypoalgesia , allodynia ) or leave it unchanged ( normalgesia)(16 ) . it is shown that intraneural ca concentration is an important underlying cause that may participate in pnd by changing intracellular calcium signaling ( 17 - 20 ) . intracellular calcium concentration is a variable which is , depending on the specific cell type , affected by the integration of many signaling pathways . elevated ca may lead to neuronal excitability ( sensitization ) ( 21 ) or death ( 22 ) , both responsible for diabetic neuropathies ( 16 ) . trpv1 is a nonselective cation channel that belongs to the transient receptor potential ( trp ) family of ion channels . it is activated by capsaicin and is responsible for the painful sensation of red peppers . it senses a vast range of stimuli ( temperature , low extracellular ph and some lipid derivatives ) and plays role in many physiologic and pathophysiologic conditions . many studies show that it has a crucial role in regulating calcium signaling in many cultured cell lines ( 23 - 27 ) . cb1 is a g protein - coupled receptor ( gpcr ) that is activated by the natural compound ( -)--tetrahydrocannabinol ( -thc ) and the endogenous endocannabinoids . the role of cb1 receptors in a variety of conditions such as anxiety , memory , immune function , depression , schizophrenia , parkinson 's disease , etc . is widely investigated . many studies show that trpv1 and cb1 receptors , expressed in a single cell , have opposing effects ( 29 - 33 ) . there is some evidence to support the idea that changes in cb1/trpv1 receptor signaling alters the cell response to anandamide or other exogenous more specific ligands ( 34 , 35 ) . in our study , we have hypothesized that hyperglycemic medium for pc12 cells would cause an imbalance in cb1/trpv1 signaling in these cells . we test this hypothesis by determining trpv1 and cb1 mrna expression in cells grown in normal and hyperglycemic conditions . we also measured toxic effects of capsaicin and protective effects of win55 212 - 2 in different glycemic conditions . the usefulness of pc12 cell line for studying functions and/or expression of trpv1 and cb1 is discussed elsewhere ( 34 , 36 ) . rat adrenal pheochromocytoma pc12 cells were grown in rpmi 1640 medium with sodium bicarbonate and l - glutamine ( sigma - aldrich ) with 10% ( v / v ) fetal calf serum , penicillin / streptomycin ( 100 u / ml and 100 g / ml , biochrom ag ) . d - glucose ( sigma - aldrich ) solutions were added to the medium to achieve final glucose concentrations of 5.5 ( low glucose ) and 50 mm ( high glucose ) , simulating low - glycemic and hyper - glycemic conditions , respectively . d - mannitol ( 50 mm ) was added to some 5.5 mm glucose medium samples to compensate for the higher osmolality . cells were cultured for 5 days in 6 well dishes and medium was replaced every 2 days . cells were then transferred to 96 well plates ( 5000 per well ) and incubated for 24 hrs . after settlement of the cells the medium was aspirated and new low or high glucose media enriched with the proper concentration of the drugs were added to the cells . capsaicin , capsazepine , win55 212 - 2 and am251 were obtained from alexis biochemicals . all drugs were dissolved in dimethyl sulfoxide ( dmso ) and added to the media with dmso concentration not more than 1% . drug enriched media were added to the cells 24 hr before the mtt assay . to find the minimum toxic concentration of capsaicin the drug was used at 10 , 100 , and 200 m in low and high glucose medium . cytoprotective effects of win 55,212 ( at 1 , 5 , 20 m ) were determined on the minimum toxic concentration of capsaicin ( 10 m ) in low and high glucose conditions . am251 ( 10 m ) , capsazepine ( 10 m ) , capsaicin ( 10 m ) and win55 212 - 2 ( 10 m ) were coadministered to see the possible interaction of drugs with receptors . cell viability was measured using the colorimetric assay in which live cells can reduce 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ( mtt ) to a blue formazan crystal . 24 hr after the treatments cells were incubated with mtt ( 0.5 mg / ml , sigma - aldrich ) for 4.5 hr at 37c and then the wells were aspirated and the blue formazan crystals were dissolved in dmso for 5 min . the plate was read in a elx808 microplate reader ( biotek ) at 550 nm . total rna was extracted ( n=6 ) using a high pure rna isolation kit ( roche applied science , indianapolis , in , usa ) according to kit instructions . a total volume of 20 l containing : total rna 10 l ( 0.1 g/l ) , dntps ( 10 mm ) 2 l , expand reverse transcriptase ( roche ) 1 l , oligo ( dt ) 15 ( 20 pmol ) 1 l , dithiothreitol ( dtt ) 2 l and buffer 4 l was used for the reverse transcription procedure . cdna amplification was done in steponeplus real time pcr system ( applied biosystems , ca , usa ) . the internal standard was beta - actin and the specific primers for cb1 and trpv1 were as follows : cb1 forward primer 5'-cgt - cgt - tca- agg - aga - atg - agg-3 ' , cb1 reverse primer 5'-tgc - cga - tga - agt - ggt - agg - aag-3 ' , trpv1 forward primer 5'-gcg - agt - tca - aag - acc - cag - a-3 ' and trpv1 reverse primer 5'- att - ctc - cac - caa - gag - ggt - ca-3'(37 ) . the reaction contained the following compounds : 6 l of power sybr green pcr master mix 2x ( applied biosystems , ca , usa ) , 20 pmol of the primers ( 0.24 l ) , 1.2 l of template , 4.56 l rnase free water . the amplification profile for the genes was as follows : the holding stage ( 95c for 15 min ) , the cycling stage ( denaturation 15 sec in 95c , combined annealing / extension 60 sec in 60c ) . the influence of the medium glucose concentration on capsaicin toxicity was analyzed using two - way analysis of variance ( anova ) . student t - test was used to compare the effect of individual drugs with each other where appropriate . the results of real time pcr were analyzed by the software rest 2008 ( corbett research pty , v 2.0.7 ) . this software uses the equation 2 ( relative quantification method ) to calculate the fold change expression ratio of genes . rat adrenal pheochromocytoma pc12 cells were grown in rpmi 1640 medium with sodium bicarbonate and l - glutamine ( sigma - aldrich ) with 10% ( v / v ) fetal calf serum , penicillin / streptomycin ( 100 u / ml and 100 g / ml , biochrom ag ) . d - glucose ( sigma - aldrich ) solutions were added to the medium to achieve final glucose concentrations of 5.5 ( low glucose ) and 50 mm ( high glucose ) , simulating low - glycemic and hyper - glycemic conditions , respectively . d - mannitol ( 50 mm ) was added to some 5.5 mm glucose medium samples to compensate for the higher osmolality . cells were cultured for 5 days in 6 well dishes and medium was replaced every 2 days . cells were then transferred to 96 well plates ( 5000 per well ) and incubated for 24 hrs . after settlement of the cells the medium was aspirated and new low or high glucose media enriched with the proper concentration of the drugs were added to the cells . capsaicin , capsazepine , win55 212 - 2 and am251 were obtained from alexis biochemicals . all drugs were dissolved in dimethyl sulfoxide ( dmso ) and added to the media with dmso concentration not more than 1% . drug enriched media were added to the cells 24 hr before the mtt assay . to find the minimum toxic concentration of capsaicin the drug was used at 10 , 100 , and 200 m in low and high glucose medium . cytoprotective effects of win 55,212 ( at 1 , 5 , 20 m ) were determined on the minimum toxic concentration of capsaicin ( 10 m ) in low and high glucose conditions . am251 ( 10 m ) , capsazepine ( 10 m ) , capsaicin ( 10 m ) and win55 212 - 2 ( 10 m ) were coadministered to see the possible interaction of drugs with receptors . cell viability was measured using the colorimetric assay in which live cells can reduce 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ( mtt ) to a blue formazan crystal . 24 hr after the treatments cells were incubated with mtt ( 0.5 mg / ml , sigma - aldrich ) for 4.5 hr at 37c and then the wells were aspirated and the blue formazan crystals were dissolved in dmso for 5 min . the plate was read in a elx808 microplate reader ( biotek ) at 550 nm . total rna was extracted ( n=6 ) using a high pure rna isolation kit ( roche applied science , indianapolis , in , usa ) according to kit instructions . a total volume of 20 l containing : total rna 10 l ( 0.1 g/l ) , dntps ( 10 mm ) 2 l , expand reverse transcriptase ( roche ) 1 l , oligo ( dt ) 15 ( 20 pmol ) 1 l , dithiothreitol ( dtt ) 2 l and buffer 4 l was used for the reverse transcription procedure . cdna amplification was done in steponeplus real time pcr system ( applied biosystems , ca , usa ) . the internal standard was beta - actin and the specific primers for cb1 and trpv1 were as follows : cb1 forward primer 5'-cgt - cgt - tca- agg - aga - atg - agg-3 ' , cb1 reverse primer 5'-tgc - cga - tga - agt - ggt - agg - aag-3 ' , trpv1 forward primer 5'-gcg - agt - tca - aag - acc - cag - a-3 ' and trpv1 reverse primer 5'- att - ctc - cac - caa - gag - ggt - ca-3'(37 ) . the reaction contained the following compounds : 6 l of power sybr green pcr master mix 2x ( applied biosystems , ca , usa ) , 20 pmol of the primers ( 0.24 l ) , 1.2 l of template , 4.56 l rnase free water . the amplification profile for the genes was as follows : the holding stage ( 95c for 15 min ) , the cycling stage ( denaturation 15 sec in 95c , combined annealing / extension 60 sec in 60c ) . the number of cycles was 40 . the influence of the medium glucose concentration on capsaicin toxicity was analyzed using two - way analysis of variance ( anova ) . student t - test was used to compare the effect of individual drugs with each other where appropriate . the results of real time pcr were analyzed by the software rest 2008 ( corbett research pty , v 2.0.7 ) . this software uses the equation 2 ( relative quantification method ) to calculate the fold change expression ratio of genes . pc12 cells were incubated for 24 hr with doses of 0 , 10 , 100 , 200 m of capsaicin which were grown in a low ( 5.5 mm ) or high ( 50 mm ) glucose medium for 5 days . two way anova results showed that viability was decreased in high glucose compared to low glucose medium ( glucose , f ( 1,63)=136.4 , p<0.001 ) . viability was decreased at higher doses of the drug both in low and high glucose medium ( dose , f ( 3,63)=134.3 , p<0.001 ) and decrement of viability was greater in the medium with higher glucose concentration ( dose medium , f ( 3,63)=41.5 , p<0.001 ) . to find out if a cb1 agonist has protective effects on toxicity induced by capsaicin , win55 212 - 2 ( 0 , 1 , 5 , 20 m ) and capsaicin ( 10 m ) were added to low and high glucose media 24 hr before mtt assay ( figure 2 ) . the results show that in the low glucose medium the viability of cells is statistically different ( p < 0.05 ) when win55 212 - 2 concentrations are 1 , 5 m compared to when they are grown in win55 212 - 2-free medium . in high glucose conditions viability is only improved ( p<0.05 ) when the concentration of win55 212 - 2 is 20 m . to see the effects of drug antagonism capsazepine ( 10 m ) , as a trpv1 antagonist was co - administered with capsaicin ( 10 m ) 24 hr before mtt assay . in another experiment , am251 ( 10 m ) , as a cb1 antagonist , was co - administered with capsaicin ( 10 m ) and win55 212 - 2 ( 10 m ) . tests were done in cells which were grown in low ( 5.5 mm ) and high ( 50 mm ) glucose media for 5 days ( figure 3 ) . t - test results show that in both media co - administration of capsazepine can improve ( p<0.05 ) viability of cells compared to capsaicin alone . the results also show that when a cb1 antagonist , am251 ( 5 m ) , is added to capsaicin and win55 212 - 2 the improving effects of win55 212 - 2 is ablated and the viability of cells is not statistically significant ( p>0.05 ) with capsaicin . data analysis indicates that mrna for cb1 is down - regulated in high glucose compared to low glucose medium . trpv1 mrna is up - regulated and the fold change in expression ratio is 8.7 ( p<0.01 ) ( table 1 ) . pc12 cells were incubated for 24 hr with doses of 0 , 10 , 100 , 200 m of capsaicin which were grown in a low ( 5.5 mm ) or high ( 50 mm ) glucose medium for 5 days . two way anova results showed that viability was decreased in high glucose compared to low glucose medium ( glucose , f ( 1,63)=136.4 , p<0.001 ) . viability was decreased at higher doses of the drug both in low and high glucose medium ( dose , f ( 3,63)=134.3 , p<0.001 ) and decrement of viability was greater in the medium with higher glucose concentration ( dose medium , f ( 3,63)=41.5 , p<0.001 ) . to find out if a cb1 agonist has protective effects on toxicity induced by capsaicin , win55 212 - 2 ( 0 , 1 , 5 , 20 m ) and capsaicin ( 10 m ) were added to low and high glucose media 24 hr before mtt assay ( figure 2 ) . the results show that in the low glucose medium the viability of cells is statistically different ( p < 0.05 ) when win55 212 - 2 concentrations are 1 , 5 m compared to when they are grown in win55 212 - 2-free medium . in high glucose conditions viability is only improved ( p<0.05 ) when the concentration of win55 212 - 2 is 20 m . to see the effects of drug antagonism capsazepine ( 10 m ) , as a trpv1 antagonist was co - administered with capsaicin ( 10 m ) 24 hr before mtt assay . in another experiment , am251 ( 10 m ) , as a cb1 antagonist , was co - administered with capsaicin ( 10 m ) and win55 212 - 2 ( 10 m ) . tests were done in cells which were grown in low ( 5.5 mm ) and high ( 50 mm ) glucose media for 5 days ( figure 3 ) . t - test results show that in both media co - administration of capsazepine can improve ( p<0.05 ) viability of cells compared to capsaicin alone . the results also show that when a cb1 antagonist , am251 ( 5 m ) , is added to capsaicin and win55 212 - 2 the improving effects of win55 212 - 2 is ablated and the viability of cells is not statistically significant ( p>0.05 ) with capsaicin . data analysis indicates that mrna for cb1 is down - regulated in high glucose compared to low glucose medium . trpv1 mrna is up - regulated and the fold change in expression ratio is 8.7 ( p<0.01 ) ( table 1 ) . this study provides evidence that hyperglycemia diminishes expression of cb1 and enhances that of trpv1 receptors in pc12 cell line and shows that this change has negative effects on cell viability in the face of a trpv1 agonist . the increasing number of diabetic patients in the world and the related economic and human losses has made diabetes as one of the most important research fields in medicine . cb1 and trpv1 are two important endovanilliod receptors that recently are the subject of many research articles . many ongoing studies are conducted to understand the changing pattern of the endovanilloid system in the course of diabetes and there are hopes that modification of its components would open new ways to encounter long term complications of the disease . neuropathies are some late complications of diabetes that are caused by the increased excitability or death of peripheral or central neurons ( 16 ) . our results show that the high glucose medium has decreased mrna expression of cb1 receptors ( table 1 ) . effects of glucose in down - regulation of cb1 receptor in pc12 cells and drg neurons ( isolated from streptozotocin - induced diabetic rats ) is reported in previous studies ( 34 ) . a number of in vivo or cell culture based studies show that cb1 is a cytoprotective receptor but the mechanism of this protection is less known ( 39 - 42 ) . in spite of cb1 receptors , hyperglycemia has increased rna transcripts for trpv1 gene ( table 1 ) . trpv1 is a nonspecific cation channel with high permeability for calcium ions ( 27 ) . impairment of calcium homeostasis in sensory neurons is an important factor in the development of diabetic neuropathies ( 19 , 20 , 43 , 44 ) . trpv1 channels play a major role in neuronal sensitization and chronic pain sensation ( 45 ) . prolong activation of trpv1 channels may even lead to neuronal cell apoptosis ( 23 , 46 ) . in our experiments trpv1 receptor is up regulated ( by the fold change expression ratio of 8.7 ) in cells grown in hyperglycemic media which is supportive for the higher toxicity of capsaicin in this condition ( figure 1 ) . in low glucose medium win55 212 - 2 has reverted capsaicin ( 10 m ) toxicity in the minimum concentration of 5 m while in high glucose medium a minimum concentration of 20 m is needed ( figure 2 ) . reduced protective effects of cb1 agonist may be explained by reduced expression of cb1 ( by the fold change expression ratio of 0.04 ) and enhanced expression of trpv1 genes . these results suggest that one mechanism for cytoprotection of the cb1 receptors is their counteraction with trpv1 receptors . osmolality plays no role because there were no significant differences between glucose and manitol osmotic controls ( data not shown ) . in both low and high glucose media the effects of capsaicin ( 10 m ) is reverted by using a trpv1 antagonist , capsazepine ( 10 m ) , showing that toxicity is receptor mediated . co - administration of a cb1 antagonist , am251 ( 5 m ) , has also prevented the protective effects of win55 212 - 2 ( 5 m ) indicative of the role of cb1 receptors in the process ( figure 3 ) . previous studies show that inflammation is an important underlying cause for enhanced signaling of trpv1 ( 20 , 44 , 45 , 47 ) . enhanced signaling is reported to be related to increased oligomerization , phosphorylation , and/or targeted expression of trpv1 proteins on the cell surface membrane ( 47 ) , enhanced cooperative functional expression of cav3.2 t - type calcium and trpv1 channels ( 20 ) , increased translation and transport of trpv1 ion channels without changing its transcription ( 44 ) and enhanced trpv1 mrna expression ( 45 ) . the study by evans et al ( 2007 ) has shown that there is a crosstalk between these two receptors and cb1 differently affects trpv1 signaling at low and high concentrations of nerve growth factor ( ngf)(48 ) so modification of the interaction between receptors may be another reason for increased signaling of trpv1 channels . in our study we have demonstrated that higher glucose concentrations are responsible for more toxic effects of trpv1 activation and less protective effects of a cb1 agonist . in support of these results real time pcr assay shows an increase for trpv1 and a decrease for cb1 receptors . it is also probable that hyperglycemia would pave the way for more toxic effects of trpv1 agonist by changing the crosstalk between trpv1 and cb1 receptors . since anandamide and some other lipid derivatives in the body are agonists for both trpv1 and cb1 receptors detailed understanding of the changes of these receptors and neural cell response to their endogenous ligands in diabetic patients may help to better understand the pathophysiologic basis of diabetic neuropathy and open ways to introduce new drugs for the prevention and/or treatment of this condition .

objective ( s ) : hyperglycemia is widely recognized as the underlying cause for some debilitating conditions in diabetic patients . the role of cannabinoid cb1 and vanilloid trpv1 receptors and their endogenous agonists , endovanilloids , in diabetic neuropathy is shown in many studies . here we have used pc12 cell line to investigate the possible influence of glucose concentration in culture medium on cytoprotective or toxic effects of a cb1 [ win55 212 - 2 ( win ) ] , or trpv1 [ capsaicin ( cas ) ] agonist . materials and methods : cell viability was tested using the mtt assay . we have also measured trpv1 and cb1 transcripts by real time reverse transcription - polymerase chain reaction while cells were grown in low ( 5.5 mm ) and high ( 50 mm ) glucose concentrations.results : real time pcr results indicated that high glucose medium increased ( p<0.01 ) trpv1 mrna and decreased ( p < 0.001 ) that of cb1 . cell culture tests show that hyperglycemic cells are more vulnerable ( dose medium , f ( 3,63)=41.5 , p<0.001 ) to the toxic effects of capsaicin compared to those grown in low glucose medium.conclusion : these findings propose that hyperglycemic conditions may result in neuronal cell death because of inducing a counterbalance between cytotoxic trpv1 and cytoprotective cb1 receptors .

Introduction Materials and Methods Cell culture Drugs Cell viability (MTT) assay RNA extraction Reverse transcription Real time polymerase chain reaction Data analysis Results Toxic effects of capsaicin on PC12 cells in low and high glucose medium Cytoprotective effects of WIN55 212-2 on toxic effects of capsaicin Effect of TRPV1 and CB1 antagonist on toxic and protective effects of TRPV1 and CB1 agonists Effects of low and high glucose concentration on mRNA expression of CB1 and TRPV1 Discussion Conclusion

the rapidly destructive coxarthrosis was first reported by postel and kerboull in 1970 and has been a rare disease ever since . until the present day , the exact etiology has not been established123 ) . initially , pain is the only symptom while the plain radiograph shows no abnormal findings . narrowing of involved joint space proceeds more than 2 mm or more than 50% of the entire joint space within 6 to 12 months45 ) . eventually , total obliteration of joint space accompanied by necrosis and segmentation of femoral head and subchondral bone of acetabulum , and joint subluxation leads to joint destruction ( fig . 1 , 2)3678910 ) . such radiologic findings are accompanied by severe pain in the hip joint and claudication28 ) . similar to the osteonecrosis of the femoral head , the total hip arthroplasty is often the treatment of choice for rapidly destructive coxarthrosis . charrois et al.11 ) have mentioned that more amount of transfusion is expected in rapidly destructive coxarthrosis than that in the osteonecrosis of the femoral head . the purpose of this study is to compare the perioperative blood loss in primary non - cemented total hip arthroplasties for rapidly destructive coxarthrosis and typical osteonecrosis of the femoral head . the participants were selected among the patients who had received the primary non - cemented total hip arthroplasty from january of 2000 to december of 2013 . the first group consisted of 24 patients who were diagnosed with the rapidly destructive coxarthrosis . the first group consisted of patients whose joint space got narrowed more than 2 mm or 50% within 6 to 12 months45 ) . all of them were surgically and pathologically confirmed with femoral head necrosis and joint destruction . one hundred seventy - one patients , who had suffered the ficat stage iv osteonecrosis of the femoral head , were included in the second group . the patients with preoperative usage of steroid or anticoagulant therapy and hemodynamic abnormality on the blood tests were excluded from the study ( table 1 ) . exclusion was based on the fact that those patients had tendency to bleed more , and might require transfusion due to difficulty of coagulation . after exclusion , total number of 19 patients was enrolled in the rapidly destructive coxarthrosis group ( group 1 ) and 40 patients were enrolled in the osteonecrosis of the femoral head group ( group 2 ) . there was no patient with loss to follow - up in our study . for comorbidities , group 1 had 1 patient diagnosed with diabetes mellitus , 6 with hypertension , 1 with asthma , 1 with dyslipidemia and 2 patients had history of cerebrovascular accident . group 2 had 5 patients diagnosed with diabetes mellitus and 8 with hypertension ( table 2 ) . all operations were conducted under general endotracheal anesthesia and performed by a single surgeon with posterolateral approach . physical exercises including tilting table , continuous passive motion , and parallel bar gaiting were prescribed three days after operation date . the patients of group 1 ( male : female , 13:6 ) had a mean age of 63.3 years ( standard deviation [ sd ] , 13.4 ) , a mean weight of 59.3 kg ( sd , 6.0 ) , mean body mass index ( bmi ) of 21.8 kg / m ( sd , 1.6 ) , and mean surgery time of 188 minutes ( sd , 30.0 ) , total blood volume of 4,028 ml ( sd , 493 ) . the patients of group 2 ( male : female , 27:13 ) had a mean age of 59.3 years ( sd , 12.9 ) , a mean weight of 62.5 kg ( sd , 9.2 ) , mean bmi of 23.1 kg / m ( sd , 2.6 ) , and mean surgery time of 162 minutes ( sd , 31 ) , total blood volume of 4,114 ml ( sd , 618 ) . measurement of the blood loss was done by the method proposed by mercuriali and inghilleri12 ) , and nadler et al13 ) . it was calculated by comparing the hematocrits of 1 day before the operation and that of 5 days after the operation and adding the compensated blood loss and non - compensated blood loss . for the statistical analysis of the variables , chi - square test , unpaired t - test , and analysis of covariance ( ancova ) test all the statistical processing of data was done with the help of the department of statistics in college of medicine , the catholic university of korea , and sas version 9.1 statistical software ( sas institute , cary , nc , usa ) was used . the non - compensated blood loss and compensated blood loss were 362 ml ( sd , 187 ; range , 77 - 675 ) and 630 ml ( sd , 180 ; range , 380 - 760 ) in group 1 , respectively . in group 2 , the non - compensated blood loss was 180 ml ( sd , 145 ; range , 53 - 519 ) and the compensated blood loss was 503 ml ( sd , 260 ; range , 190 - 1,505 ) . the total blood loss was 992 ml ( sd , 300 ; range , 457 - 1,434 ) in group 1 and 683 ml ( sd , 360 ; range , 226 - 1,975 ) in group 2 showing a significant difference ( p=0.002 , table 4 ) . parameters other than bmi and operation time did not show significant difference with chi - square test and unpaired t - test ( different implant used ratio , p=0.924 ; sex difference ratio , p=0.056 ; age , p=0.262 ; body weight , p=0.179 ; bmi , p=0.053 ; surgery time , p=0.003 ; estimated total blood volume , p=0.594 ) . thus , post - hoc analysis with ancova test was conducted to evaluate whether bmi and operation time have any influence on different blood loss between two groups . after adjustment with ancova test , influence of bmi and operation time on the fact that group 1 had larger amount of blood loss than group 2 did not have statistical significance ( bmi , p=0.9466 ; operation time , p=0.721 ) . we had tried to calculate relatively accurate blood loss in this study . compared to group 2 , the blood loss occurring from the primary non - cemented total hip arthroplasty first , the magnetic resonance imaging and other radiologic studies have revealed more extensive marrow edema in group 1 , some extending to the peritrochanteric area ( fig . 2 ) . boutry et al.14 ) have mentioned that rapidly destructive coxarthrosis had more extensive bone marrow edema at peritrochanteric area , when compared with osteonecrosis of femoral head . these findings indicate the increased marrow pressure , and the blood loss may be a result of such fact15 ) . this may have been caused by the superolateral displacement of femur head and the inferior displacement of hyaline body within the joint , creating stimulation to the synovium , and it can be speculated that neo - angiogenesis and activation of inflammatory cytokines had increased the hemorrhage . komiya et al.6 ) had revealed that the activation of interleukin-1 and metalloproteinase-2 and 3 were significantly higher in the synovial fluid of the rapidly destructive coxarthrosis . also , matsumoto et al.16 ) have pointed out that decreased level of tissue inhibitor of matrix metalloproteinases in serum and joint fluid may indirectly proliferate pathogenesis of rapidly destructive coxarthrosis . lastly , the preoperative and postoperative transfusion may have contributed to the increased hemorrhage . in this study , the average packed red blood cell units received were 3.4 units in group 1 , and 2.7 units in group 2 . a previous study had reported that increased transfusion to patients with the primary total hip arthroplasty was related with more blood loss through the drain tube , and that finding corresponds with the relationship between the amount of transfusion and the blood loss in this study17 ) . this means that while the transfusion keeps the homeostasis of the blood , it is related with increased blood loss after the surgery . aforementioned three hypotheses may have been the triggering factors for the increased blood loss in group 1 than in group 2 , and it is our opinion that this study can contribute to estimating the blood loss of the two groups during and after the surgery and in developing the transfusion plan to the patients . further studies should be conducted to evaluate relationship between these hypotheses and blood loss with statistical analysis . limitation of this study is that the number of enrolled patients with rapidly destructive coxarthrosis is quite few , due to its rarity . moreover , comorbidities like diabetes and hypertension are not taken into consideration which might have influence on results . compared to the osteonecrosis of the femoral head group , the rapidly destructive coxarthrosis group had more blood loss when the primary non - cement total hip arthroplasty was performed . this may have been caused by marrow edema extended up to peritrochanteric area , severe synovitis , and increased blood transfusion during and after the surgery . our results will make a great contribution to the estimation of blood loss during and after the surgery and the establishment of transfusion plan .

purposethe purpose of this study is to compare the perioperative blood loss in primary non - cemented total hip arthroplasty ( tha ) performed for rapidly destructive coxarthrosis ( rdc ) with the perioperative blood loss in primary non - cemented tha for typical osteonecrosis of the femoral head ( onfh).materials and methodsfrom january 2000 to december 2013 , 19 patients were diagnosed with rdc ( group 1 ) and 40 patients were diagnosed typical ficat stage iv onfh ( group 2 ) , comparison of perioperative blood loss between group 1 and group 2 in primary noncemented tha was done . patients with preoperative usage of steroid or anticoagulants medication and with hemodynamic abnormal blood test results were excluded . the blood loss was measured up to the fifth post operation day and calculated with formula proposed by mercuriali , inghilleri and nadler.resultsnon-compensated blood loss calculated in milliliters of red blood cells was 362 ml ( standard deviation [ sd ] , 187 ; range , 77 - 675 ) in group 1 and 180 ml ( sd , 145 ; range , 53 - 519 ) in group 2 . compensated blood loss was 630 ml ( sd , 180 ; range , 380 - 760 ) in group 1 and 503 ml ( sd , 260 ; range , 190 - 1 , 505 ) in group 2 . the total blood loss after primary non - cemented tha is greater when surgery is performed for rdc than for onfh , with the volume of 992 ml ( sd , 300 ; range , 457 - 1 , 434 ) in group 1 and 683 ml ( sd , 360 ; range , 226 - 1 , 975 ) in group 2 respectively.conclusiontotal perioperative blood loss was significantly greater in rdc than in onfh in primary non - cemented tha .

INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION

the major adverse consequences of hearing impairment for an individual 's social , emotional , cognitive , behavioral , and economic well - being and overall quality of life are well documented in the literature17 . however , these devices may not fully compensate for severe and/or profound hearing loss in which the damage to the auditory system is extensive3 . the use of cochlear implants ( cis ) is indicated for both children and adults in such cases . in recent years , the benefits conferred by the use of cis , especially with regard to speech perception , have proven to surpass those produced by the use of hearing aids , making cis a highly efficient resource for patients with severe / profound hearing loss . however , few studies so far have assessed the satisfaction of adult users of cis . research on patients ' perceptions of the health services that they receive has been a constant concern of researchers and professionals responsible for health care . the definition of satisfaction may vary in order to address different research objectives ( e.g. , to assess quality or to understand user perception ) and different aspects of the service , from the institution 's amenities of care , such as the food , cleanliness , and comfort , to the technical quality of the services6 . satisfaction reflects the perspective of the patient and thus depends not only on the performance of the ci but also on the patient 's perceptions and attitudes . therefore , it is very important to know what factors are related to patients ' satisfaction and how these factors influence patient perception in order to refine the objectives of rehabilitation in response to the patient 's needs . however , there is currently no questionnaire or inventory that was developed specifically to evaluate the satisfaction of ci users . in a study conducted by ou , et al.12 ( 2008 ) , the satisfaction with amplification in daily life ( sadl ) questionnaire developed by cox and alexander4 ( 1999 ) , which assesses the satisfaction of users of hearing aids , was adapted for use in ci recipients and reportedly proved appropriate for this population . no study using this tool to evaluate the satisfaction of users of cis has been conducted in brazil . therefore , this study aimed to evaluate the degree of satisfaction of adult users of cis and the relationship thereof with their levels of speech perception . this was a prospective cross - sectional study conducted in the audiological research center ( cpa ) of the hospital of craniofacial anomalies , university of so paulo ( hrac / usp ) , in bauru , so paulo , brazil . the project was approved by the ethics committee of hrac / usp bauru under case no . this study comprised a sample of 12 adult users of cis who were patients at the audiological research center of the hospital for rehabilitation of craniofacial anomalies / university of so paulo ( cpa , hrac / usp ) , all of whom used ci models and technologies available in brazil . the age of these patients ranged from 19 to 59 years and their durations of ci use from 1 to 15 years . all patients who visited for routine monitoring from april through july 2012 were invited to participate in the study , and the sample consisted of those who spontaneously agreed to enroll . the participants were invited from among patients who visited for routine monitoring from april through july 2012 . the inclusion criteria for this study were age e18 years , use of unilateral cis with or without the use of contralateral hearing aids , and systematic use of cis . individuals who had severe limitations in understanding and expression that interfered with their responding to the questionnaire and individuals who did not agree to participate were excluded from the study . to evaluate satisfaction , we culturally adapted the satisfaction with amplification in daily life ( sadl ) questionnaire to brazilian portuguese11 . for use in this study , the questionnaire was modified to exclude questions 7 and 14 , which relate to the cost of cis and therefore do not apply in brazil12 . a previously trained examiner administered the questionnaire and addressed any doubts expressed by the participants without interfering in any way with their answers . this questionnaire has 4 subscales : positive effects ( questions 1 , 3 , 5 , 6 , 9 , and 10 ) : 6 items related to acoustic and psychological benefits service and cost ( questions 12 , 14 , and 15 ) : 3 items related to professional capacity , product price , and number of repairs negative factors ( questions 2 , 7 , and 11 ) : 3 items related to environmental noise amplification , acoustic feedback , and telephone use personal image ( questions 4 , 8 , and 13 ) : 3 items related to aesthetics and the stigma of using hearing aids . the 14 questions about satisfaction were answered on a 7-point scale of equal intervals corresponding to a categorical scale ranging from not a bit to very much satisfied . the score was calculated for each subscale , and higher scores represented better user perception of satisfaction . to facilitate the subjects ' understanding of the range of responses , this scale displayed representative figures for the 7 levels of responses ( figures 1 and 2 ) . scale of responses to questions 1 , 3 , 5 , 6 , 8 , 9 , 10 , 11 , 12 , 14 , and 15 . visual support scale adapted to the scale of responses to the satisfaction with amplification in daily life questionnaire ( sadl ) for questions with non - inverted scores . visual support scale adapted to the scale of responses to the sadl for questions with inverted scores . the answers to the questions related to each subscale were averaged to generate a score for each of the 4 subscales . all subscales must have been scored for the score to be considered valid and used in the analysis . the 4 subscales of the sadl represent 4 different areas that contribute to the overall satisfaction . speech perception under quiet conditions was assessed by testing the recognition of separate lists of monosyllabic words , nonsense syllables , and sentences . perception of speech in the presence of background noise was evaluated using a version of the hearing in noise test ( hint ) adapted to brazilian portuguese2 under free field conditions , both in quiet and with background noise , using 2 lists ( list 5 in the quiet and list 6 in the noisy environment ) of 20 sentences each . we opted to use hint under free field conditions due to the participants ' use of cis , which precluded the use of headphones . the hint results were expressed by sentence recognition threshold ( srt / hint ) values . under quiet conditions , this threshold corresponded to the intensity in decibels at which the individual exhibited 50% sentence recognition ; in the presence of background noise , this threshold corresponded to the s / r ratio in decibels at which the individual exhibited 50% sentence recognition . we performed a descriptive analysis of the results in order to characterize the subjects ' responses . the spearman correlation test was also used to verify the correlations between the overall satisfaction level and each sadl subscale with the characteristics of the participants ( e.g. , duration of ci use ) and the speech perception results under quiet and noisy conditions . a significance level of 5% this study comprised a sample of 12 adult users of cis who were patients at the audiological research center of the hospital for rehabilitation of craniofacial anomalies / university of so paulo ( cpa , hrac / usp ) , all of whom used ci models and technologies available in brazil . the age of these patients ranged from 19 to 59 years and their durations of ci use from 1 to 15 years . all patients who visited for routine monitoring from april through july 2012 were invited to participate in the study , and the sample consisted of those who spontaneously agreed to enroll . the participants were invited from among patients who visited for routine monitoring from april through july 2012 . the inclusion criteria for this study were age e18 years , use of unilateral cis with or without the use of contralateral hearing aids , and systematic use of cis . individuals who had severe limitations in understanding and expression that interfered with their responding to the questionnaire and individuals who did not agree to participate were excluded from the study . to evaluate satisfaction , we culturally adapted the satisfaction with amplification in daily life ( sadl ) questionnaire to brazilian portuguese11 . for use in this study , the questionnaire was modified to exclude questions 7 and 14 , which relate to the cost of cis and therefore do not apply in brazil12 . a previously trained examiner administered the questionnaire and addressed any doubts expressed by the participants without interfering in any way with their answers . this questionnaire has 4 subscales : positive effects ( questions 1 , 3 , 5 , 6 , 9 , and 10 ) : 6 items related to acoustic and psychological benefits service and cost ( questions 12 , 14 , and 15 ) : 3 items related to professional capacity , product price , and number of repairs negative factors ( questions 2 , 7 , and 11 ) : 3 items related to environmental noise amplification , acoustic feedback , and telephone use personal image ( questions 4 , 8 , and 13 ) : 3 items related to aesthetics and the stigma of using hearing aids . the 14 questions about satisfaction were answered on a 7-point scale of equal intervals corresponding to a categorical scale ranging from not a bit to very much satisfied . the score was calculated for each subscale , and higher scores represented better user perception of satisfaction . to facilitate the subjects ' understanding of the range of responses , a visual support type of scale this scale displayed representative figures for the 7 levels of responses ( figures 1 and 2 ) . scale of responses to questions 1 , 3 , 5 , 6 , 8 , 9 , 10 , 11 , 12 , 14 , and 15 . visual support scale adapted to the scale of responses to the satisfaction with amplification in daily life questionnaire ( sadl ) for questions with non - inverted scores . scale of responses to questions 2 , 4 , 7 , and 13 . visual support scale adapted to the scale of responses to the sadl for questions with inverted scores . the answers to the questions related to each subscale were averaged to generate a score for each of the 4 subscales . all subscales must have been scored for the score to be considered valid and used in the analysis . the 4 subscales of the sadl represent 4 different areas that contribute to the overall satisfaction . speech perception under quiet conditions was assessed by testing the recognition of separate lists of monosyllabic words , nonsense syllables , and sentences . perception of speech in the presence of background noise was evaluated using a version of the hearing in noise test ( hint ) adapted to brazilian portuguese2 under free field conditions , both in quiet and with background noise , using 2 lists ( list 5 in the quiet and list 6 in the noisy environment ) of 20 sentences each . we opted to use hint under free field conditions due to the participants ' use of cis , which precluded the use of headphones . the hint results were expressed by sentence recognition threshold ( srt / hint ) values . under quiet conditions , this threshold corresponded to the intensity in decibels at which the individual exhibited 50% sentence recognition ; in the presence of background noise , this threshold corresponded to the s / r ratio in decibels at which the individual exhibited 50% sentence recognition . we performed a descriptive analysis of the results in order to characterize the subjects ' responses . the spearman correlation test was also used to verify the correlations between the overall satisfaction level and each sadl subscale with the characteristics of the participants ( e.g. , duration of ci use ) and the speech perception results under quiet and noisy conditions . a significance level of 5% the study comprised 12 adult users of cis from the audiological research center of the hospital for rehabilitation of craniofacial anomalies ( hrac / usp ) . no potential subject was excluded during the selection stage within the period determined for the study . on average , the participants had undergone ci surgery at 36 years of age and had used their devices for 3 years ( table 1 ) . the participants in the study were on the whole satisfied with their devices and recorded high scores both overall and on the subscales of the sadl questionnaire ( table 2 ) . examination of the results for speech perception under quiet and noisy conditions for correlations with the satisfaction scores revealed a correlation between the degree of satisfaction and the level of speech perception under quiet conditions ( table 3 ) . * p < 0.05 = statistically significant only 6 ( 50% ) participants performed the hint speech perception test under quiet conditions , and only 5 ( 41% ) performed it under noisy conditions ( hint ) ( table 4 ) . no correlation was found between the satisfaction scores and the hint results ( table 5 ) . this study aimed to evaluate the degree of satisfaction of adult users of cis and examine its relationship with their levels of speech perception . the participants in the study were satisfied with their devices , and the degree of satisfaction correlated positively with the ability to recognize monosyllabic words under quiet conditions , that is , the better a person 's performance on this test , the more satisfied the participant tended to be with his or her device . the duration of use of the device did not influence the satisfaction scores ; this contrasts with the results of tyler and summerfield16 ( 1996 ) , who reported a correlation between the duration of use and satisfaction . it is noteworthy that those authors used a different assessment of satisfaction than employed in the current study . the mean scores on the sadl questionnaire were lower for this population than in the study conducted by ou , et al.12 ( 2008 ) , who found an overall score of 5.6 . those authors used the sadl questionnaire to assess satisfaction among post - lingual users of ci , i.e. , those who had established oral language before they developed hearing loss . only 1 participant in our study the highest score was recorded for the service and cost subscale , showing that ci users were satisfied with the services received and ci maintenance . the cost of the device is known to be able to impact user satisfaction , and in the case of ci , which are high - tech and costly devices , the need for a great deal of maintenance could also impact these results , as there is currently no federal guideline ensuring the repair and maintenance of such devices . the lowest score was recorded for the negative factors subscale , which was related to the amplification of background noise and telephone use . despite the benefits that the ci confers on its user with respect to the perception of sound , recognition of speech in noisy environments remains a very challenging situation even for adults with implants14 . studies have shown that the use of advanced technological systems associated with cis can reduce the adverse effects of noisy environments5 . the systems currently available to minimize the effects of noise on the speech perception of ci users include new strategies for pre - processing , frequency modulation ( fm ) systems , directional microphones , and a larger input dynamic range ( idr)14 . therefore , it can be anticipated that ci users will soon attain understanding of speech even in unfavorable communication situations , satisfactory musical perception , and effective communication on the telephone8 . there are few studies in the literature concerning the perception of satisfaction among adult users of ci users , hampering the comparison of our findings with those of others . zwolan , kileny , and telian18 ( 1996 ) evaluated the satisfaction of 12 pre - lingual adult users of ci using a questionnaire that they had developed and found that satisfaction correlated more strongly with the perception of environmental sounds than with speech perception . tateya , et al.15 ( 2000 ) conducted a study of 37 ci users and found that speech understanding was one of the most important factors related to satisfaction . on the other hand , the level of satisfaction of ci users does not always correspond with the degree of improvement in their speech perception scores9 - 15 . humes , halling , and coughlin7 ( 1996 ) noted that speech perception scores correlate weakly overall with the benefit , satisfaction , or use of electronic devices . therefore , although better speech perception may be associated with greater user satisfaction , other factors also contribute to the perception of satisfaction10 , preventing us from predicting the degree of satisfaction based solely on the level of speech perception . this can be explained by the fact that different people use very different criteria to judge whether they are satisfied with their ci . at one extreme , some patients have unrealistic expectations of what the ci can do and hope that it will essentially eliminate the psychosocial , vocational , and communicative consequences of their hearing loss . their satisfaction may depend on our success at reducing their expectations to a realistic level . in contrast , there are also patients who judge the success of the intervention process by comparing their comprehension skills with and without the ci . these are the individuals who can be satisfied with their devices even if the instrument they use provides less objective benefit than could possibly have been achieved . furthermore , satisfaction is a dynamic rather than a static measure of intervention and may fluctuate over time13 . therefore , the assessment of satisfaction may help professionals to predict which other aspects in addition to speech perception may affect the satisfaction of ci users and reorganize the intervention process accordingly in order to improve patients ' quality of life . overall , the participants were satisfied with their ci and the service received . the user 's degree of satisfaction with the device correlated with the level of speech perception under quiet conditions .

summary introduction : in recent years , the benefits associated with the use of cochlear implants ( cis ) , especially with regard to speech perception , have proven to surpass those produced by the use of hearing aids , making cis a highly efficient resource for patients with severe / profound hearing loss . however , few studies so far have assessed the satisfaction of adult users of cis . objective : to analyze the relationship between the level of speech perception and degree of satisfaction of adult users of ci . method : this was a prospective cross - sectional study conducted in the audiological research center ( cpa ) of the hospital of craniofacial anomalies , university of so paulo ( hrac / usp ) , in bauru , so paulo , brazil . a total of 12 users of cis with pre - lingual or post - lingual hearing loss participated in this study . the following tools were used in the assessment : a questionnaire , satisfaction with amplification in daily life ( sadl ) , culturally adapted to brazilian portuguese , as well as its relationship with the speech perception results ; a speech perception test under quiet conditions ; and the hearing in noise test ( hint)brazil under free field conditions . results : the participants in the study were on the whole satisfied with their devices , and the degree of satisfaction correlated positively with the ability to perceive monosyllabic words under quiet conditions . the satisfaction did not correlate with the level of speech perception in noisy environments . conclusion : assessments of satisfaction may help professionals to predict what other factors , in addition to speech perception , may contribute to the satisfaction of ci users in order to reorganize the intervention process to improve the users ' quality of life .

Introduction Methods Selection of participants Procedures Statistical Analysis: Results Discussion Conclusion

hypertensive crisis is defined as a sudden and abrupt elevation in blood pressure ( bp ) . it is subclassified into hypertensive emergency and hypertensive urgency based on the presence or absence of end - organ damage . renal parenchymal disease , renal artery stenosis , coarctation of the aorta , and neuro - endocrine tumors need to be considered in its etiology . adult patients with hypertensive histories commonly develop severe hypertension while under general anesthesia , which can be successfully treated by anesthesiologists . however , in children with severe anesthesia - associated hypertension , management may be delayed and variable . the anesthetic management of a child with hypertensive crisis during general anesthesia has not been reported . we therefore present a rare case of hypertensive crisis that developed in a three - year - old girl with undiagnosed renovascular hypertension due to left renal artery stenosis , who underwent general anesthesia for preauricular fistulectomy . a three - year - old girl was admitted to the hospital for right preauricular fistulectomy under general anesthesia . she was well - grown with a weight of 13 kg ( 25th percentile ) and a height of 95.7 cm ( 50th percentile ) and she had no medical history . preoperative laboratory data including complete blood cell , coagulation test , and renal and liver function test were all within normal limits . vital signs also were all unremarkable ; heart rate ( hr ) was 114 /min and regular , bp was 90/60 mmhg , and temperature was normal . on arrival in the operating room , electrocardiogram , pulse oxymetry , and non - invasive bp were established . anesthesia was induced with intravenous thiopental 5 mg / kg and rocuronium 0.6 mg / kg . while she was manually ventilated with 2% sevoflurane and 100% oxygen via face mask , initial bp was measured 143/90 mmhg on the right arm . we administered fentanyl 10 g intravenously and intubated with a 4.5 mm i d cuffed endotracheal tube without difficulty . after intubation , bp increased as high as 180/100 mmhg and hr was 160/min . because we suggested the cause of high bp was due to inadequate anesthetic depth , sevoflurane concentration was increased to 3% and intravenous fentanyl 10 g was injected two more times . hr was stabilized to 105 /min , but bp showed only slight decrease to averaging 140 mmhg of systolic blood pressure and was still high throughout the surgery . immediately before extubation , bp increased up to 190/105 mmhg and hr was 98 /min . intravenous nicardipine 0.1 mg was given and she was sent to the recovery room . in the recovery room , intravenous fentanyl 10 g was given one more time , but bp did not decrease and was sometimes unmeasurable . she had no hypertension - related symptoms like headache or dizziness , but showed irritability and agitation that were n't effectively controlled by opioids . we promptly consulted with the pediatrician and decided to transfer her to the pediatric department . immediate antihypertensive treatment was started with nicardipine 0.5 g / kg / min in the recovery room . in the pediatric ward , an additional labetalol 0.4 - 3 mg / kg / h iv infusion was started , and diltiazem 3.5 mg / kg / day , atenolol 1 mg / kg / day , and hydralazine 7.2 mg / kg / day were given orally . administration of these drugs was effective , but bp still showed fluctuation from 95/45 to 145/75 mmhg . she had no past medical history and physical examinations were unremarkable . in laboratory findings , plasma renin activity was elevated up to 24.2 ng / ml / h ( normal range : 0.29 - 3.7 ng / ml / h ) . plasma epinephrine and norepinephrine soared to 411.66 pg / ml ( normal range : 0 - 120 pg / ml ) and 1,063.31 pg / ml ( normal range : 0 - 410 pg / ml ) respectively . in imaging analyses , renal ultrasound showed a decrease in the size of the left kidney ( 5.7 cm in length ) and no abnormalities were detected in the right kidney . abdominal ct - angiography scan showed severe stenosis of inner diameter of less than 1 mm at proximal portion of left renal artery , hypoplasia , and decreased perfusion in left kidney ( fig . the options for treatment were balloon angioplasty or renal revascularization or nephrectomy . in this case , left nephrectomy was recommended due to the patient 's age and the size of involved renal artery . therefore , left laparoscopic nephrectomy was planned and she was transferred to the genitourinary department for surgery . intravenous infusion of nicardipine 0.5 g / kg / min and labetalol 1 mg / kg / h were continued until the day of surgery , bp measured in ward was stable at 100/41 mmhg , and hr was 92 /min . bp showed a mild elevation up to 130/85 mmhg and intravenous infusion of nicardipine 0.5 g / kg / min and labetalol 1 mg / kg / h were continued during induction and the early period of surgery . since bp was stabilized during the mid - period of surgery and hr fell to 68 /min , intravenous infusion was stopped . bp was stable in the recovery room without medication and the child was sent to the ward with bp of 108/45 mmhg . although the drugs which were given preoperatively were continued , bp showed a certain fluctuation from 100/40 to 150/70 mmhg postoperatively . enalapril 0.4 mg / kg / day was added orally with careful monitoring of renal function , and then bp was stabilized . after changing all intravenous drugs to oral medications ( amlodipine , carvedilol and enalapril ) , bp remained stable . after 6 months of follow - up , the patient 's bp was normalized with the aid of oral antihypertensive medications . we describe a three - year - old girl with hypertensive crisis that developed during induction of anesthesia and was exacerbated during emergence . hypertensive crisis in early and mid - childhood is rare and is associated with significant morbidity and mortality . hypertensive crisis in adults is defined as acute , severe elevation of blood pressure above 180/120 mmhg ( about 20 mmhg above stage 2 hypertension ) ; however , hypertensive crisis in children has not been clearly defined . a child is diagnosed with hypertension if the average systolic and diastolic blood pressures are greater than the 95th percentile for age , gender , and height . stage 1 hypertension is the designation for bp levels that range from 95th percentile to the 99th percentile plus 5 mmhg . stage 2 hypertension is the designation for bp levels that are above the 99th percentile plus 5 mmhg . our patient was three - year - old girl with a height of 95.7 cm ( 50th percentile ) , and stage 2 hypertension ( average bp > 119/79 mmhg ) . yang et al . reported stage 2 hypertension may serve as a critical threshold for a high risk of hypertensive crisis in children . renovascular hypertension is caused by an arterial lesion that compromises blood flow in one or both kidneys , and represents 10% of all cases of secondary hypertension in children . renal artery stenosis is one of the most common abnormalities observed in children with secondary hypertension . the initial treatment of renovascular hypertension is medical therapy with antihypertensive drugs , the side effects of which can be exacerbated by rapid normalization of bp or drugs acting on the renin - angiotensin axis that reduce renal blood flow . medications commonly used to treat hypertensive crisis in children are nicardipine , labetalol , and sodium nitroprusside . our patient was given intravenous infusions of nicardipine and labetalol , as well as oral diltiazem , atenolol , and hydralazine . as medical treatment alone is often insufficient to control hypertension , leaving patients at risk for life - threatening events such as cerebrovascular accidents and cardiac failure , more aggressive treatments are required . management of hypertension can be quite challenging , especially in very young children and children with intra - renal artery involvement . depending on the size of the vessels involved , endovascular or surgical intervention may not be possible . nephrectomy may be indicated when the source of hypertension is a small , poorly functioning kidney . the precipitating factors of hypertension during general anesthesia are most commonly related to drugs , excessive surgical stimulation , light anesthesia , or equipment . if no obvious cause could be identified , it tends to be due to a combination of light anesthesia and/or excessive surgical stimulation and anesthesiologists often increase the depth of anesthesia . once hypertension is identified and confirmed , its rapid control by the careful use of a volatile anesthetic agent , intravenous opioids , or rapid - acting antihypertensives will usually avoid serious morbidity . if hypertension is unresponsive to the treatment indicated , an unusual etiology should be considered . our patient presented with normal blood pressure and no signs and symptoms suggestive of hypertension preoperatively . she appeared anxious and irritable during the induction , despite having been premedicated with midazolam . in such cases of anxiety in young children we had difficulty to recognize high bp after endotracheal intubation as hypertensive crisis because maintenance of high concentration of sevoflurane and administration of opioids decreased bp in some extent . however , bp soared to 220/115 mmhg in the recovery room and she was postoperatively diagnosed as renovascular hypertension . our patient showed emergence agitation , unmeasurable blood pressure , and a hypertensive crisis with systolic blood pressure of more than 200 mmhg in the postoperative period . hypertensive crisis not only represents severely elevated blood pressure with end - organ dysfunction , also results from a complex cascade linking neuro - hormonal and hemodynamic pathways , causing blood pressure to spiral out of control . renin - angiotensin - aldosterone activation , oxidative and mechanical damage to the microvasculature , fluid overload , severe renal dysfunction , sympathetic overstimulation , and endothelial dysfunction may all contribute to the pathology of hypertensive crisis . the trigger factors in our patient are assumed to be sympathetic overstimulation due to endotracheal intubation , surgical stimuli , and postoperative pain , with her underlying compromised renal blood flow acting in synergy . in conclusion , hypertensive crisis during the induction of anesthesia and postoperative period is rare in children , and anesthetic management can be difficult when there is no evident cause . blood pressure measurement in perioperative period may be difficult due to uncooperative and restless infants and young children , but it is important nonetheless in diagnosing unexpected severe hypertension . we suggest that blood pressure should be monitored in infants and young children perioperatively , and that anesthesiologists should be aware of the management of the unexpected hypertensive crisis and the possibility of secondary hypertension in children .

pediatric hypertensive crisis is a potentially life threatening medical emergency , usually secondary to an underlying disease . hypertension commonly occurs during general anesthesia , and is usually promptly and appropriately treated by anesthesiologists . however in children with severe , unexplained , or refractory hypertension , it has the potential to cause morbidity and even mortality in susceptible patients . we report an anesthetic management of an unexpected hypertensive crisis that developed during general anesthesia in a three - year - old girl with undiagnosed severe left renal artery stenosis .

Introduction Case Report Discussion

unanticipated difficult laryngoscopic intubation has been a major concern for anesthesiologists , and the incidence ranges from 0.05% to 18% . failure to maintain the patency of the airway after induction of anesthesia can lead to catastrophic sequelae , such as irreversible brain damage and death . suggested a simple screening test for difficultly in airway assessment that is widely used today in its modified form introduced by samsoon and young . several authors have found considerable interobserver variation when using the mallampati test ( m.t . ) , some of which could be attributed to the execution of the test . because mallampati et al . in their original paper did not specify whether the patient should phonate or not , subsequently , phonation has been shown to affect classification . owing to the fact that no clear - cut direction exists regarding the utilization or non - utilization of phonation during the test performance , we designed this study to test our hypothesis that the mallampati test could well be performed in the supine position without phonation with equally good results . in this regard , we compared the effect of phonation and no phonation on the mallampati test in supine and upright positions in an attempt to arrive at the best possible situation for the assessment of the mallampati test in predicting difficultly in laryngoscopy and intubation . in this cross - sectional study , 661 patients aged 16 - 60 years were recruited during the years 2011 to 2012 . an ethical approval was waivered by the ethics committee of our institute as no intervention infringing patient 's safety was involved ; however , all patients who gave an informed consent entered the study . patients entered the study without any randomization of positions because each patient had to be subjected to all the four situations . exclusion criteria included asa class higher than ii , urgency of the situation , facial , mouth , throat and airway anomalies , pregnancy and awake intubation . mallampati class was assessed with the head in full extension , the mouth wide open and the tongue extruded in the upright position with and without phonation , and again in the supine position with phonation and in the absence of phonation . while conducting the m.t.in the supine position , the head was placed on a pillow that could elevate the head approximately 10 cm above the horizontal level . the oropharyngeal structure during each of the four categories was classified as below : class 0 : the ability to visualize any part of the epiglottis on mouth opening.class i : soft palate , fauces , uvula and pillars seen.class ii : soft palate , fauces and uvula seen.class iii : soft palate and base of the uvula seen.class iv : soft palate not visible at all . class 0 : the ability to visualize any part of the epiglottis on mouth opening . mallampati classes 0 , i and ii were declared to be easy and classes iii and iv were considered to be difficult . during intubation , a blinded observer with at least 5 years experience in clinical anesthesia graded the laryngoscopy grade according to the cormack lehane grading ( clg ) score as below : grade i : full view of the glottisgrade ii : partial view of the glottis is arytenoidsgrade iii : only epiglottis visiblegrade iv : neither glottis nor epiglottis is visible grade i : full view of the glottis grade ii : partial view of the glottis is arytenoids grade iii : only epiglottis visible grade iv : neither glottis nor epiglottis is visible clg scores i and ii were classified as easy intubations whereas grades iii and iv were classified as difficult intubations . if three attempts failed to provide a good glottis exposure , alternative measures for intubation were initiated , such as the use of a bougie or employing a fiberscope . a patient was dubbed as a case of difficult intubation if the insertion of the tube took more than 10 min and/or required more than three attempts by an experienced anesthesiologist . the anesthesia protocol was the same for all patients and comprised of midazolam 0.03 mg / kg and fentanyl 2 /kg as premedication drugs , an induction dose of thiopental sodium in the range of 3 - 5 mg / kg and 0.5 mg / kg of atracurium for skeletal muscle relaxation . laryngoscopy with subsequent intubation was attempted when a train of four stimulations showed disappearance of the second twitch . maximum , minimum and mean sd and for qualitative data , the number ( percentage ) were reported . kappa agreement , sensitivity , specificity , positive and negative predictive values and accuracy were calculated for each of the situations . a p - value < 0.05 was considered to be significant . difficultly in laryngoscopy was found in 28 ( 4.2% ) of the patients , and nine ( 1.4% ) patients were found to have difficult intubation . demographic data of the patients se , sp , ppv and npv of the mallampati test in different positions and phonation status regarding laryngoscopy sesitivity(se ) , specificity(sp ) , positive predictive value ( ppv ) and negative predictive value ( npv ) of the mallampati test during different positions as assessed during laryngoscopy revealed the best kappa in both the upright and the supine positions with accompanying phonations . as depicted in table 3 , the se , sp and npv were the highest in the upright position without phonation compared with the other positions . se , sp , ppv and npv of the mallampati test in different positions and phonation status as regards endotracheal intubation we found that the mallampati test both during laryngoscopy and during intubation fared well in both the supine and upright positions without phonation and fetched the highest se , whereas this se was lower when phonation was attempted by the patient in the said positions [ tables 2 and 3 ] . the test had the highest sp both during laryngoscopy and during intubation in both the upright and the supine positions with phonation , but it was found to be the lowest in both supine and upright positions not accompanied with phonation [ tables 2 and 3 ] . the sp was found to be high and acceptable in all the different positions , but the highest figures were seen in the presence of phonation . on the other hand , the npv tested in all the four situations was above 95% , and it had a good correlation with the test of agreement ( kappa ) . however , the kappa agreement test revealed that the best correlation was in the supine plus phonation and upright plus phonation positions . the kappa agreement for the mallampati test in different positions and statuses could reveal that the best correlation was in the upright position with phonation and supine position with phonation . the highest correlation was with the supine plus phonation position in the prediction of difficult laryngoscopy and intubation , but , compared with the other situations , the correlation was not significant . the kappa agreement regarding se , sp , ppv and npv in all the different positions for laryngoscopy and intubation regarding other variables such as age gender , asa class , body mass index ( bmi ) and diseases such as diabetes and rheumatoid arthritis failed to show a significant difference ( p > 0.05 ) . this study shows that the mallampati class that went high in the supine phonation caused an improvement in the mallampati class , i.e. changing a higher class to a lower class . anatomical factors and characteristics are of considerable importance in predicting difficultly in intubation but have limitations because of their variability from one person to another and in the same person at different periods of life . this interobserver variability has been considered to be a major limitation of the mallampati classification . the original paper of mallampati et al . did not explicitly mention whether the patient should phonate while the test is being performed or not ; subsequently , it has been observed that phonation affects the classification . lewis et al . found that the results of the mallampati classification are more reproducible when the tongue is protruded during phonation . on the contrary , many others studies have reported that gagging or phonation results in unpredictable motion of the pharynx and should therefore be avoided as it may obstruct the view . in the present study , the mallampati test as compared with the laryngoscopic and intubation exposures had the highest se in the supine plus no phonation and upright plus no phonation situations . the highest sp on the other hand was seen in the upright plus phonation and supine plus phonation positions . the lowest se was found in the upright plus phonation situation and the lowest sp was seen in the upright plus no phonation and supine plus no phonation situations . our findings show that the mallampati test correctly depicts difficultly in intubation when the test is performed without phonation . during phonation , the number of false - negatives increases or , in other words , the test erroneously depicts an increase in the number of easy intubations that are in fact difficult . this would lead to an increase of apprehension among the anesthesiologists as difficult intubations would be predicted as easy intubations . our findings corroborate with that of other studies that state that gagging or phonation affects the mallampati class and should best be avoided . these findings are in sharp contrast to those of lewis et al . , who found that phonation did not influence the overall accuracy of the mallampati test . tram et al . failed to find a significant difference in the mallampati classification in the supine and upright positions . such variations in the conduct of mallampati tests may contribute to some of the heterogeneity of the results seen in systematic reviews . showed that variations in patient 's position can affect the mallampati class , and found that the mallampati score was improved by one or two classes in the supine position rather than in the upright position . bindra et al . in their study showed that phonation significantly improved the mallampati class and that change in position from upright to supine could only bring a very small change in the oropharyngeal view . our findings also revealed that there was an improvement in the mallampati class in the supine position with phonation and a higher class was changed into a lower class . the ppv was found to be low in all the positions ; however , compared with other positions , it was negligibly higher in the supine plus phonation position . there is exceedingly low probability that the test would prove to be reliable in predicting difficultly in intubation , and this conforms with several studies conducted so far . this could confirm that the test would prove to be of value in predicting easy intubations and , regarding this variable , one could rely on its validity . in this study , the se , sp , ppv and npv in all the mallampati test positions and situations showed a suitable kappa coefficient in evaluation with laryngoscopy and intubation . the best kappa , however , was seen in the upright plus phonation and supine plus phonation situations . also , the highest level of kappa was seen in the supine position along with phonation . but , the overall difference between the four mallampati test situations for kappa was not significant . our study demonstrated that se , sp , ppv and npv in all the mallampati test positions had suitable kappa coefficient in evaluation with laryngoscopy and intubation as regards the other variables such as gender , age , asa class , bmi and coexisting diseases such as diabetes mellitus and rheumatoid arthritis failed to show any significant difference . our study supports our hypothesis that phonation affects the mallampati class and , to prevent false mallampati negatives , it seems that mallampati class could fetch the best results if performed without phonation . in other words , patients who did not phonate had a higher se . compared with the upright position without phonation , the se of the test was equally high in the supine position , giving credence to our claim that the test would prove to be of value in patients who can not adopt the upright posture . apart from the limitations inherent in conducting the mallampati test , there were no other limitations . however , we presume that there might be some among the elderly who would perhaps fail to follow the directions , leading to false outcomes . our findings reveal that the mallampati class was high in the supine position and that the mallampati class had improved in the supine position coupled with phonation . the test had poor results in predicting difficultly in laryngoscopy and intubation and had high false - positive and false - negative outcome . moreover , the test conducted in the supine position with phonation had a better kappa and agreement in determining difficultly in laryngoscopy and intubation .

background and aims : difficult ventilation and intubation have been recognized as the forerunners of hypoxic brain damage during anesthesia . to overcome catastrophic events during anesthesia , an assessment of the airway before induction is of paramount importance . we designed this study to compare the effect of phonation on the mallampati test in supine and upright positions as against the traditionally employed test without phonation in serving to predict difficult laryngoscopy and intubation.material and methods : in this cross - sectional study , 661 patients aged 16 - 60 years were recruited during the years 2011 to 2012 . the mallampati test was conducted on patients with and without phonation in both the sitting and supine positions . a blinded observer then performed laryngoscopy and intubation . difficult intubation was assessed according to the cormack - lehane grading scale . statistical analysis used : diagnostic statistical measures for each of the four situations sensitivity , specificity , positive and negative predictive values and accuracy were calculated.results:in this study , 28 patients ( 4.2% ) had difficult laryngoscopy and nine patients ( 1.4% ) had difficult intubation . there was no difference in the sensitivity of the mallampati test as regards prediction of laryngoscopy and intubation in the four different positions , but the upright position along with phonation had the highest specificity . the negative predictive value was above 95% in all the positions ; however , the positive predictive value was the highest in the supine position along with phonation.conclusion:based on our results , the supine position along with phonation had the best correlation in the prediction of difficult laryngoscopy and intubation . we further conclude that phonation significantly improved the mallampati class in the supine position compared with the upright position .

Introduction Material and Methods Results Discussion Conclusion

obesity is an epidemic and a significant health threat both in the united states and all around the world . poor dietary habits and lack of physical activity are the two main contributors to this growing health crisis . new smartphone applications and research projects aim at helping people track their daily food intake and activities . a growing number of smartphone apps moderate and vigorous physical activity and lifestyle changes can lead to health promotion and disease prevention , while aerobic exercise alone has not shown to be effective . additionally , increased portion sizes and high caloric intake are important contributors for developing obesity . provision of tools to accurately measure ee would allow people to actively track expenditure of calories relative to the amount of calories ingested , creating awareness of personal habits that can be modified to promote personal health . however , it is generally very difficult to know exactly how many calories people expend during daily physical activity as it depends on the age , gender , weight , height , type and intensity of activity . indirect calorimetry is a commonly used reference method to estimate energy expenditure . a cosmed k4b2 calorimeter uses pulmonary gas exchange to measure caloric expenditure with a very high correlation of 98.2% . such techniques are not exact and measure pulmonary gas exchange ( they measure oxygen exchange vo2 , not actual energy expenditure ) . the factors such as ratio of macro - nutrients burnt and any lactate accumulation during more intense exercises affect ee values but are not accounted in ee estimation using pulmonary gas exchange . accounting for this deficiency , indirect calorimetry is the closest we can get to actual ee values in an ambulatory setting . however , such a calorimeter is still impractical for use in daily life settings because of the high cost , complexity and difficulty of use . many wearable devices , such as fitbit , jawbone up and nike + fuelband provide a practical solution to monitor the dynamic energy expenditure by unobtrusively collecting data from wearable sensors to estimate ee . additionally , individuals will need to purchase and carry these devices with them all the time to get a comprehensive assessment of energy expenditure value . however , the main shortcoming of pedometers or any step - counting algorithms is their poor accuracy in detecting steps at slow speed and insensitivity to gait differences such as the length of the stride . another approach uses the accelerometer values directly , and attempt to find an empirical relation between accelerometer data and energy expenditure data measured by a calorimeter , e.g. cosmed k4b2 , . with smartphones becoming ubiquitous devices , they are conveniently suited personal devices to measure ee , rather than using dedicated wristbands , heart rate monitors or other tracking devices . users have to remember to carry them , not lose them and they are prone to damage . although the cost of commercially - available sensor products are coming down , many of these sensors cost $ 100 or more at this time and can be prohibitive for some of the population . on the other hand , people already have smartphones and the habit of carrying them along . however , further work needs to be done to improve ee accuracy using smartphone sensors . in this study , our focus is more on ambulatory activities a person engages in during the course of an individual s daily life such as walking , climbing upstairs or downstairs etc . currently existing smartphone apps utilize only the accelerometer data to estimate steps and converts them into mets ( metabolic equivalent values ) and calories estimates . however , this approach is quite inaccurate . new smartphones such as galaxy s3 , galaxy nexus , iphone 5 and later models have an integrated barometer sensor in the phone which passively measures atmospheric pressure . slight variations in atmospheric pressures can be used by these apps / algorithms to detect the work done against gravity , hence improving the results . however , caution should be exercised to not rely on absolute barometer values since these can vary depending on environmental factors as well as differences among devices . despite these limitations , variations of sensor readings within a time period and extracted features can be useful . barometer has been used for aiding gps , the main reason behind its introduction into smartphones . due to its excellent relative accuracy , differential in barometer has been used for floor - change detection , and activity classification . existing accelerometry equations require heavy computations or require high sampling frequency , both of which will drain the battery of smartphones quickly . the main contributions of this paper are as follows : 1)we propose and advance the use of machine learning techniques for ee . we have developed an initial linear regression , ann - based and bagged - regression tree based regression model to obtain a 96% correlation ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \rho $ \end{document } ) accuracy . we demonstrate high accuracy and low error ( root mean square error , rmse = 0.73).2)trials were conducted on 12 individuals and validated against reference ee data ( provided by cosmed k4b2 calorimeter ) . we can demonstrate high correlation using basic features and low sampling frequency , which will lead to battery efficiency.3)we demonstrate the benefit of incorporating barometer smartphone sensor in addition to accelerometer to improve ee estimation accuracy . unlike many of the currently available calorimetry equations , or usage of complex feature sets ( computationally unfeasible on smartphones ) , our approach uses simple features extracted from barometer and accelerometer sensors , fed to machine learning algorithms to obtain high accuracy and low rmse values . we demonstrate that using barometer sensor , in addition to accelerometer , improves correlation without computational overhead . we propose and advance the use of machine learning techniques for ee . we have developed an initial linear regression , ann - based and bagged - regression tree based regression model to obtain a 96% correlation ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \rho $ \end{document } ) accuracy . we demonstrate high accuracy and low error ( root mean square error , rmse = 0.73 ) . trials were conducted on 12 individuals and validated against reference ee data ( provided by cosmed k4b2 calorimeter ) . we can demonstrate high correlation using basic features and low sampling frequency , which will lead to battery efficiency . we demonstrate the benefit of incorporating barometer smartphone sensor in addition to accelerometer to improve ee estimation accuracy . unlike many of the currently available calorimetry equations , or usage of complex feature sets ( computationally unfeasible on smartphones ) , our approach uses simple features extracted from barometer and accelerometer sensors , fed to machine learning algorithms to obtain high accuracy and low rmse values . we demonstrate that using barometer sensor , in addition to accelerometer , improves correlation without computational overhead . we would also like to point out the scopes and limitations of our described model . first and foremost , our analysis have been built and analyzed on the basis of the most basic activities of otherwise normal and healthy human beings . the results can be extended to other physical activities like running , biking , etc , however , these will need further validation testing . secondly , our proposed model requires an individual to carry a smartphone at all times . this can be problematic as a smartphone may not always be carried by individuals and the sensor location will not always be known . recognizing the activity type with a non - fixed location of sensor on the body is complex task that will require further work . the rest of the paper is organized as follows : section 2 provide an overview of related works in this area . section 4 gives a brief summary of the prediction models used in the paper followed by experimental results in section 5 . numerous methods have been proposed to measure short- and long - term exercise energy expenditure . most are compared to either doubly - labeled water ( dlw ) or indirect calorimetry ( using a device such as cosmed k4b2 calorimeter ) as reference to actual ee values . pedometer estimates of ee are weak and have a weak correlation to actual values ( 0.530.61% , ) . similarly , the activity monitor estimates of ee are also low ( correlation 0.480.60 ) . it is possible to improve ee accuracy using multiple body sensors ( correlation of 0.90.95 ) , but it becomes inconvenient to use multiple body sensors in daily living conditions . multiple calorimetry equations from accelerometry data have been developed in research literature , but all tend to over- or under - estimate ee depending on type and intensity of the activity . a group of investigators evaluated machine - learning based approaches to estimate ee but considered only treadmill walking and leaves out activities of daily living . however , in prior work , the authors determine that height , weight and bmi ( body mass index , defined later ) are the best indicators for personalized ee for each individuals . fitbit is a highly popular commercial device which uses accelerometer and altimeter sensors to capture personal activity , a significant improvement over traditional pedometers . however , some experiments have demonstrated that fitbit is not very accurate as it lacks activity - classification algorithms . existing body - sensor based ee estimation employs a body - worn accelerometer and performs signal analysis to estimate calories expended in real - time using regression formulas . however , using a single sensor on the body is not enough to provide accurate measurement for body movement . instead , we found many of these devices were accurate in step counts but inaccurate in ee estimation . step - count based algorithms show high degrees of correlation with ee in scenarios such as walking , running and standing . however , active lifestyle often involves climbing up or down stairs . in these scenarios , for example - in a sample trial we asked some study subjects to climb up 4 flight of stairs and then to climb down the same number of stairs . the ee estimate obtained using commercial products such as fitbit and nike + fuelband ( which use pedometer based approach ) are shown in table 1 . it is counter - intuitive that one will spend more calories climbing down than upwards . existing algorithms used in these devices appear to count steps and speed of the movement and attribute higher expenditure based on these variables . given that our volunteers moved faster when climbing down stairs versus up stairs these devices measured higher caloric expenditure for the less vigorous activity of climbing down versus up . moreover , the two devices have huge variations in their measurements , leaving user to wonder about their accuracy . table 1.average measurement of ee ( cal ) and step counts using commercial devices ( nike + fuelband and fitbit one ) over volunteers performing climbing task . the devices report more work done in climbing down than up , and also have huge disparity in measurements.activity in stairs \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \rightarrow $ \end{document}updownsteps ( nike+)54.46 22.5859.83 11.45ee ( nike+)3.32 2.154.38 0.67steps ( fitbit)61.96 20.4471.38 13.18ee ( fitbit)7.82 2.859.14 2.92 heart rate monitors have also been used as stand - alone devices or along with accelerometer sensors to collect data and predict energy expenditure . some devices such as wahoo heart rate monitor , acquire heart rate data by measuring pulse rate and use a linear relation between heart rate and oxygen uptake to predict energy expenditure . however , heart rate monitors have low accuracy during sedentary behavior and require individual calibration , . calfit is a widely used android application that tracks time , location and physical activity patterns of users for health and wellness studies . it uses smartphones gps receiver to get the location information and the accelerometer for obtaining motion data . it uses a prior algorithm to estimate energy expenditure strictly based on accelerometer data . another previous work shows how smartphones , along with gps data , can be used to effectively estimate ee of individuals during biking . it is also used as pressure sensor which measures relative and absolute altitude through the analysis of changing atmospheric pressure . the barometer sensor can be used for motion detection , but it is mostly used by location - based applications to evaluate elevation . have first introduced the concept of combining barometer with accelerometer for detecting ambulatory movements , where authors embed a barometer sensor into a portable device to evaluate daily physical activity and classify the activity type . uses barometer in addition to accelerometer , but consider only linear regression models , thus limiting its accuracy . our primary aim was to build an application capable of accurately estimating ee without leveraging significant computational resources on the smartphones . low computational and power requirements will make such an algorithm more usable and attractive to consumers . there are three components of a system developed to estimte ee : ( a ) sampling and sensing of signal ( b ) feature calculation and ( c ) the machine learning algorithm itself . although the machine learning algorithm is computationally expensive in training , its complexity is very low for testing and practical use . therefore , we have focused on the other two aspects in this paper : ( a ) we have used a very low sampling rate of 2hz and ( b ) we choose a subset of features which give high correlation to ee but have minimal computational requirements . however , studies have shown that 0.120 hz is appropriate for most human activities . in this study , however , we restrict our measurements to the default smartphone sampling rate of 2hz which corresponds to low battery consumption and processing overhead . both accelerometer and barometer sensors are sampled at 2hz ( corresponding to 2 samples per second ) . we use a window of time equivalent to 4 seconds ( 8 samples ) to obtain different feature vectors required for our analysis . the basic features involve direct calculations of mean values from the tri - axial accelerometer and barometer sensors and these computations are simple . the derived features are obtained from basic accelerometer data and selected from existing studies in this domain , which we believe will improve the accuracy of our algorithm . we also collect anthropomorphic inputs about the users and use them as feature vector in our machine learning algorithm . the study involved 12 otherwise healthy volunteer subjects each performing a specified protocol of human activities . the participants are students in computer science department and were recruited based on open announcement in research group meetings . participants were made fully aware of the nature of the study , the purpose and any risks / concerns involved . the range ( minimum and maximum ) are mentioned in parenthesis.attribute\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mu ~\pm ~\sigma $ \end{document } ( min , max)age25.98 3.3 ( 22,34)height ( m)1.7621 0.06 ( 1.60,1.84)weight ( kg)73.01 17.58 ( 52,109)bmi ( kg/\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ m^{2}$ \end{document}23.44 5.32 ( 18.08,35.59 ) one critical task is to measure reference ee values which are used for training and testing the machine learning algorithm . direct calorimeter requires observations in a confined metabolic chamber and is therefore impractical in our scenario . doubly labeled water techniques are also inappropriate in our seting because they calculate ee over a long duration instead of for a single activity . to calibrate energy expenditure values over small time intervals , we used cosmed k4b2 indirect calorimeter , which is portable and can be used with our setup . we used samsung galaxy nexus smartphones to record observations of barometer and smartphone sensors . during each person s exercise protocol in the laboratory , smartphone - based tri - axial accelerometer and barometer were measured and recorded simultaneously along with the cosmed k4b2 system . data were downloaded from the devices after the prescribed physical activity protocol and merged based on time stamps for comparison . before each test , the oxygen and carbon dioxide analyzers and the flow turbine in the cosmed k4b2 were calibrated according to the manufacturer s instructions . subjects were asked to perform a series of activities in our exercise laboratory while being simultaneously monitored by the cosmed k4b2 portable metabolic system and a smartphone with built - in accelerometer and barometer sensor ( a galaxy nexus android - based phone running a customized software to record and extract accelerometer readings for ee estimation ) . , subjects were asked to be well hydrated and to rest or perform only light exercise the day before the test . subjects were advised to not perform any vigorous exercise and take at a normal meal 23 hours prior to testing . for each subject , we recorded age , gender , body height and body mass . body mass index ( bmi ) was calculated according to the formula : body mass ( kg ) divided by square of height ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ m^{2}$ \end{document } ) . during each person s exercise protocol in the laboratory , smartphone - based tri - axial accelerometry and barometer data were downloaded from the devices after the prescribed physical activity protocol and merged based on time stamps for comparison . heart rate , oxygen consumption , carbon dioxide production , respiratory exchange ratio ( rer ) and ventilation rate were continuously monitored . , the oxygen and carbon dioxide analyzers and the flow turbine in the cosmed k4b2 were calibrated according to the manufacturers instructions . energy expenditure was calculated using the following equation : cosmed k4b2 ee ( kcalmin1 ) = ( 3.815 kcallo\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ 2- 1+$ \end{document } ( 1.232 kcallo\documentclass[12pt]{minimal } 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\usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ bmi$ \end{document } : body to mass index of the person , calculated by dividing the weight with the square of height and measured in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ kg / m^{2}$ \end{document}. we use the following feature vectors ( fv 69 ) obtained from the accelerometer sensor and barometer values over a window . \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mu a_{x}$ \end{document } : mean of x component of accelerometer signal.\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mu a_{y}$ \end{document } : mean of y component of accelerometer signal . x and y refer to horizontal components of accelerometer signal , which are fixed to the local coordinates of smartphone.\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mu a_{z}$ \end{document } : mean of z component of accelerometer signal . this refers to the up - down movement of the human body.\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mu _ { p}$ \end{document } mean of barometer signal . \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mu a_{x}$ \end{document } : mean of x component of accelerometer signal . \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mu a_{y}$ \end{document } : mean of y component of accelerometer signal . x and y refer to horizontal components of accelerometer signal , which are fixed to the local coordinates of smartphone . \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mu a_{z}$ \end{document } : mean of z component of accelerometer signal . \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mu _ { p}$ \end{document } mean of barometer signal . the fv above are calculated easily from sensor data and are referred to as basic fvs . next , we define the additional fvs we derived from tri - axial accelerometer data . these features have been useful in human activity recognition and possibly also improve accuracy in our scenario . these features are calculated from the data collected from accelerometer and barometer , which are treated as time series signal . here , the word energy refers to energy of time - series signal and not the physical ee values . \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mu ac a_{x}$ \end{document } , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mu ac a_{y}$ \end{document } , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mu ac a_{z}$ \end{document } : absolute mean of energy deviation from average of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ a_{x}$ \end{document } , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ a_{y}$ \end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ a_{z}$ \end{document } signals.(for example , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mu ac a_{x}$ \end{document } = mean of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ |a_{x}-\mu a_{x}|$ \end{document})\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ svm$ \end{document } : signal vector magnitude is the root mean square value of ac component along all three axis.\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } 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variance of energy deviation from average energy of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ a_{x}$ \end{document } , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ a_{y}$ \end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ a_{z}$ \end{document } signals.(for example , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } 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( for example , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mu ac a_{x}$ \end{document } = mean of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ |a_{x}-\mu a_{x}|$ \end{document } ) \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ svm$ \end{document } : signal vector magnitude is the root mean square value of ac component along all three axis . \documentclass[12pt]{minimal } \usepackage{amsmath 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\usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ h_{x}$ \end{document } , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ h_{y}$ \end{document } , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ h_{z}$ \end{document } : entropy of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ a_{x}$ \end{document } , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ a_{y}$ \end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ a_{z}$ \end{document } signals in given window . \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \sigma _ { p}^{2}$ \end{document } : variance of barometer signal . \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ ran_{p}$ \end{document } : range of barometer signal ( in given window ) . \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ mgh$ \end{document } : work done against gravity . \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ mgh = ran_{p}\times wei$ \end{document}. the activity counts or acceleration values collected using accelerometer can be combined with demographic information and regression techniques , or physical models of the human body to produce energy expenditure estimates . this model is also deployed in calfit used by researchers in california to assess associations between the built environment and physical activity in many case studies . ee estimates given by this method uses the following heuristic relation:\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \begin{equation * } \overbrace { ee}= a a_{h}^{k } + b a_{z}^{m } , \end{equation*}\end{document } where , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ a_{h}= ( a_{x}^{2 } + a_{\vphantom { r_{j_{j}}}y}^{2})^{0.5}$ \end{document}\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \textrm { a}=.01281^{*}\textit { wei } + 0.84322 $ \end{document}\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \textrm { b}=.0389^{*}\textit { wei } - 0.68244^{*}\textit { gen } + 0.69250 $ \end{document}\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \textrm { k}=.0266^{*}\textit { wei } + 0.14672 $ \end{document}\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \textrm { m}=-0.00285^{*}\textit { wei } + 0.96828 $ \end{document } \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ a_{h}= ( a_{x}^{2 } + a_{\vphantom { r_{j_{j}}}y}^{2})^{0.5}$ \end{document } \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \textrm { a}=.01281^{*}\textit { wei } + 0.84322 $ \end{document } \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \textrm { b}=.0389^{*}\textit { wei } - 0.68244^{*}\textit { gen } + 0.69250 $ \end{document } \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \textrm { k}=.0266^{*}\textit { wei } + 0.14672 $ \end{document } \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \textrm { m}=-0.00285^{*}\textit { wei } + 0.96828 $ \end{document } researchers have reported 6095% correlation using equation 1 for ambulatory activities such as walking or running . artificial neural networks are a family of statistical learning models , which have excellent prediction ability . we also use an ensemble technique bagging in conjunction with regression trees with reduced - error - pruning ( rep ) . pruning is used to avoid over - fitting . bagging technique ensembles or merges the output of multiple such models to obtain the final prediction . the relative advantage of bagged rep regression trees is in low computational complexity and ability to identify high information attributes . the smartphone sensors logged their data using a native android app into a csv file while cosmed k4b2 calorimeter was used to validate the readings and measure actual ee . the smartphone was kept in waist pouch by the participants , as shown in figure 1 . unlike , activity specific classification and ee estimation algorithms , our focus here is on designing a single robust ee algorithm , that can be applied to a combination of all regular ambulatory activities in a combined manner . table 3 gives the performance results using artificial neural networks and simple linear regression models . \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \rho $ \end{document } indicates correlation between predicted output and actual ee values . raw accelerometer means that only the mean accelerometer values are provided as inputs to machine learning algorithm . all fv refer to the case when all 35 fvs mentioned earlier are included in ann.table 3.regression results indicate superior performance of machine learning algorithms over linear regression in both cases ( a ) using only accelerometer mean values and ( b ) all features.model used\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \rho $ \end{document}rmsemaebagged regression treeraw accelerometer ( only)0.90041.03020.5201all feature vectors ( fv)0.94680.79100.4012artificial neural networkraw accelerometer ( only)0.71891.62351.2244all feature vectors ( fv)0.87941.12660.7347linear regressionraw accelerometer ( only)0.60281.82511.4611all fvs0.58071.86431.4797demographic features ( only)0.60401.82311.4425 it can be clearly seen that linear regression gives very poor performance with only accelerometer as well as all fvs . in both cases there is no improvement in linear regression performance with increase in feature vectors . on the other hand , brt ( bagged rep - regression trees ) and ann ( artificial neural networks ) thus , the utility of using non - linear models for regression is clear . using ann model , we are able to achieve 72% correlation with actual ee values with a rmse of 1.62 using only accelerometer equations . when all fvs are used , correlation increases to 88% and rmse reduces to 1.13 . the rmse reduces to 0.79 when building brt with all features , while it is 1.03 when using only raw accelerometer values . regression trees are built using information - theoretic criterion and pick high entropy features on the top . reduced - error - pruning algorithm prunes the redundant leaves of the tree to guard against over - fitting . bagging is a meta - ensemble algorithm and the results of multiple rep - regression trees are pooled to obtain a higher accuracy than a single rep - regression tree . thus , we observe that ensemble technique gives higher correlation than anns . here , we observe that using all fvs does not improve the accuracy of linear regression model for ee over raw accelerometry values , because their impact on ee is non - linear . machine learning algorithms are able to capture the non - linearity and thus the correlation is higher ( and consequently rmse is low ) when we see the case of neural networks and bagged regression trees . figure 2 shows the results when we compare the rmse for leave one out sampling . in leave - one - out sampling , the model is trained with \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ n-1 $ \end{document } users and tested on 1 user , and this procedure is repeated for each user . leave - one - out technique is therefore good to find out the generality of our technique on new users , for whom the model has not been trained . the figure shows that bagging ( brt ) gives lesser rmse than ann and linear regression techniques . the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ r^{2}$ \end{document } statistics gives the coefficient of determination ( range usually 0 to 1 ) . the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ r^{2}$ \end{document } coefficient for ann was 0.87 , but was 0.96 ( close to 1 ) for brt . figure 2.plot of rmse ( root mean square error ) comparison of three techniques using leave - one - out sampling . the bars show the mean rmse value while error bars show the variance in values . plot of rmse ( root mean square error ) comparison of three techniques using leave - one - out sampling . the bars show the mean rmse value while error bars show the variance in values . the models generated using linear regression technique and single accelerometer can be used as a baseline ee algorithm . it obtains 60.28% correlation with output ee values and the root mean square error ( rmse ) is around 1.8251 . to serve as another useful baseline , we also tried to study the results when using only a model with demographic information of the participants . it evaluates the worth of a fv by measuring the gain ratio with respect to the class . the information gain is equal to the total entropy for an attribute if for each of the attribute values a unique classification can be made for the result attribute . in this case the relative entropy subtracted from the total entropy are 0.\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \begin{equation * } \textrm { gainratio}(c , fv ) = ( h(c ) - h(c|fv ) ) / h(fv ) . \end{equation * } \end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ c$ \end{document } is set of all training examples , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ h()$ \end{document } denotes entropy . figure 3.relative information gain ratio of input feature vectors ( fvs ) obtained using gainratio criterion . relative information gain ratio of input feature vectors ( fvs ) obtained using gainratio criterion . we can observe that many input features have high information gain ratio in the range of 0.700.80 . while collecting data , we had annotated different set of activities using manual markers . here we find that the gainratio for annotations is pretty low ( 0.08 ) indicating that knowledge of activity - type has little correlation to ee values . however , since most of the features such as mean , variance , range , entropy and correlation have high gainratio , we argue that many of them are correlated and hence redundant . next , we use a twofold approach to reduce the attributes to be used in the model . first , we used feature selection algorithms to find the independent features which can give high predictive power . feature selection aims at reducing the number of attributes to be used in the model , while trying to retain the predictive power of the original set of attributes in the pre - processed data . we use the correlation feature selection ( cfs ) strategy to identify a subset of attributes which were highly correlated with the outcome variable while having low inter - correlation amongst themselves . the cfs technique was used in conjunction with a greedy stepwise search to find the subset \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ s$ \end{document } with the best average merit , which is given by:\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \begin{equation * } merit_{s}=\frac { n\overline { r_{fo}}}{\sqrt { n+n(n-1)\overline { r_{ff } } } } \end{equation * } \end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ n$ \end{document } is the number of features in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ s$ \end{document } , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \overline { r_{fo}}$ \end{document } is the average value of feature - outcome correlations , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \overline { r_{ff}}$ \end{document } is the average value of all feature - feature correlations . we used the entire dataset for feature selection , which can potentially bias the results and should be avoided in general ( cross - validation should be used for feature selection as well ) . the reason to use the entire dataset for feature selection process is our multi - step strategy , which included a manual screening to eliminate computationally - inefficient coefficients . using cross - validation for cfs would give slightly different subsets for each fold , which would complicate the manual screening step , and each resulting subset would again give different subsets after the second round of cfs . to simplify the process , we used the entire data at each step and got a single subset of features for the final model . extracting each feature vector from raw sensor inputs particularly , on an embedded device like a smartphone , such operations may drain the battery . since the exact speed of computation is device dependent , we report relative speed ( time of execution relative to time of execution of raw acc . values ( to compute the mean ) . let \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ t_{acc}$ \end{document } denote the time required to process accelerometer values along one dimension . for a feature \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ f_{i}$ \end{document } the computation ratio is calculated as follows:\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \begin{equation * } r_{f_{i}}=\log _ { 10}\left({\frac { t_{f_{i}}}{t_{acc}}}\right ) \end{equation * } \end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ t_{f_{i}}$ \end{document } refers to time taken by computation of feature \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ f_{i}$ \end{document } on same device . these computations are performed with a desktop processor running at 2.6 ghz and averaged over 200k computations . the relative computation time is same for mobile processors , but desktop values are reported because we could average them over large sample size . next , our goal is to prune the fvs with higher computational cost without sacrificing the accuracy of ee estimation . as shown in figure 4 , correlation , pitch and range have an - order of magnitude higher computational cost than mean , energy and entropy . different features have different cost of computation , making low - cost features such as energy , svm , entropy and mean preferred to other features . the y - axis shows \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ r_{f_{i}}$ \end{document } metric which is zero for feature \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ f_{i}$ \end{document } if the computational cost is same as that of mean of accelerometer values ( simplest operation ) . \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ r_{f_{i}}$ \end{document } is in logarithmic scale , indicating that increase by 1 indicates \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ 10\times $ \end{document } increase in computational requirement on mobile devices . different features have different cost of computation , making low - cost features such as energy , svm , entropy and mean preferred to other features . the y - axis shows \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ r_{f_{i}}$ \end{document } metric which is zero for feature \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ f_{i}$ \end{document } if the computational cost is same as that of mean of accelerometer values ( simplest operation ) . \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ r_{f_{i}}$ \end{document } is in logarithmic scale , indicating that increase by 1 indicates \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ 10\times $ \end{document } increase in computational requirement on mobile devices . we would desire to use the features with least computation cost and high predictive power to form the ee estimation model . using cfs strategy in conjunction with our knowledge of computational cost , we obtain the following reduced 6 fvs in our final model : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mu a_{z}$ \end{document } , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ age$ \end{document } , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ wt$ \end{document } , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ bmi$ \end{document } , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mu p$ \end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \sigma p$ \end{document}. we note that all derived fvs for acceleromter data are pruned by the cfs algorithm . both mean and variance of barometer reading is used for ee estimation . generating a bagged rep - regression tree ( brt ) from these fvs the tree is pruned and only the top attributes , used in tree - branching are shown . it can be seen that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mu a_{z}$ \end{document } is most significant predictor followed by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ age$ \end{document } , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ wt$ \end{document } and barometer readings ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mu p$ \end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \sigma p$ \end{document } ) . we can clearly see mean barometric values ( meanp ) in the third level of hierarchy , implying clear contribution of barometer sensor in precision of regression tree ( brt ) . we can clearly see mean barometric values ( meanp ) in the third level of hierarchy , implying clear contribution of barometer sensor in precision of regression tree ( brt ) . figure 6 shows the actual ee values and the values estimated using brt ( final model ) . there is a close agreement between the values . figure 6.sample plot of ee estimates ( using bagged regression tree ( brt ) , a machine learning scheme ( final model ) . ) with actual ee values ( cosmed ) . sample plot of ee estimates ( using bagged regression tree ( brt ) , a machine learning scheme ( final model ) . ) with actual ee values ( cosmed ) . calorimetry equations ( ce ) proposed in literature , such as the one used in and have very high computational complexity as they involve fractional arithmetic and are not efficient on smartphone processors . we want to quantify the impact of these calculations on the accuracy of ann and brt models . although ce estimates roughly followed the trend for most activities , the average correlation was low . estimation error ( along the range of activities ) was 89.9788% and correlation was found to be 0.5027 . the correlation was particularly low for climbing activities while it was close to 0.65 for other activities , . next , we ran the models with and without this equation along with accelerometer ( basic ) features . it can be seen that including ce has no ( or negative ) impact on the accuracy of ann/ brt model with raw acc . table 4.impact of barometer values on ee prediction using machine learning approaches.model used\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \rho $ \end{document}rmsemaeartificial neural networks ( ann)raw accelerometer ( only)0.77791.65281.2321raw acc . + ce0.91010.97010.4920final model ( brt reduced)0.95600.73000.4015 the experimental results validated our assertion that barometric sensor ( bar . ) has high correlation with ee accuracy . appending the mean of barometer values ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mu _ { p}$ \end{document } ) improve the correlation of ee to actual energy expenditure for both ann and brt as shown in table 4 . it is not possible to obtain second by second ee from commercial devices such as fitbit or nike + fuel band . however , we did calibrate these values before and after each trial and normalized for each subject s weight . the errors in individual measurements sum up and ce algorithm ( calorimetry equation used in calfit ) presents an estimate which is within 25% of the cosmed values . ann and brt values have same mean as the cosmed values . however , variance of error is smaller in brt . we can see that nike + fuelband tends to underestimate the ee for each individual while fitbit tends to overestimate the value . the error bars in the figure show the standard deviation for each device / algorithm . our algorithm has a smaller deviation over the population considered , which is comparable to actual cosmed values . figure 7.overall absolute ee comparison of cosmed , brt and ann with nike + , fitbit and calorimetry equation for two tasks across subjects . all three have same mean estimates but the variances are larger with ann as against brt . the variance in cosmed readings reflect range across subjects . the calorimetry equations and nike + product underestimates while fitbit overestimates the results . overall absolute ee comparison of cosmed , brt and ann with nike + , fitbit and calorimetry equation for two tasks across subjects . all three have same mean estimates but the variances are larger with ann as against brt . the variance in cosmed readings reflect range across subjects . the calorimetry equations and nike + product underestimates while fitbit overestimates the results . one of the most power consuming operation in ee estimation is sampling of smartphone sensors . unlike recent works that use gyroscope sensor in addition to accelerometer , we directly used the accelerometer and barometer sensor , and use machine learning algorithms to estimate ee . in next set of experiments , we tried to quantify the power savings in making this choice . we used monsoon power monitor to measure the energy consumption of sensors on the smartphone , turned off network interfaces and display and keep all system settings same during the experiments . the sampling rate was kept to be low ( at 2 hz ) and the results are averaged over few minutes of observations . figure 8 shows the relative power expenditure of these sensors ( when the smartphone os is on ) . these readings clearly demonstrate how accelerometer and barometer sensor has very low power consumption than gyroscope . figure 8.power consumption of smartphone ( with display , network and other apps off ) reading individual sensor signal at 2 hz . power consumption of smartphone ( with display , network and other apps off ) reading individual sensor signal at 2 hz . next , we justify our use of low sampling rates to obtain power - efficiency . the impact of different sampling rates on power consumption can be done using the emulation method described in and . figure 9.power consumption of smartphone ( with display , network and other apps off ) reading individual accelerometer sensor signal at different sampling rates . power consumption of smartphone ( with display , network and other apps off ) reading individual accelerometer sensor signal at different sampling rates . the training of machine learning algorithm is a power consuming task , but it can be done offline in the cloud . overall , our framework can be easily implemented in smartphones and has a power consumption less than 30 mw ( which will vary a bit with smartphone model and operating system ) . in this work , we proposed a technique for accurate ee estimation in ambulatory settings utilizing machine learning techniques and combining the sensor readings from both the accelerometer and barometer sensors of smart phones . to emulate a practical low - power setting , we used a smartphone and sampled the accelerometer and barometer sensorss at 2hz only . we then used these values to extract feature vectors ( fvs ) and fit a bagged regression tree which can yield up to 96% correlation and rmse of 0.70 with very small computational overhead . we observed significant benefits in fusing the input of barometer sensor to an accelerometer sensor as it allows , with use of simpler fvs , achievement of higher correlation and accuracy to reference ee values . improved ee estimation will provide important information regarding energy balance for individuals during various physical activities in both clinical and community living settings . in general , they rely on step counts and are expensive . building accurate ee models is helpful for the masses to measure their caloric expenditure using their already purchased smartphones and make healthy decisions about lifestyles . it is also of great interest to researchers , who can use smartphone - sensed data of individuals to gain insight into ee and gait features as well as trends . commercial devices such as fitbit have shared apis but do not allow access to sensor data , limiting clinical research explorations . motivated by the present study s encouraging results , we plan to collect a more extensive dataset , using a larger sample size of study subjects , along with other physical activities like biking , running , walking / running on inclines , and everyday chores such as gardening / cleaning . in the future , using this larger data set with wider range of activities , we wish to build a more representative model for ee ( using bagged regression trees ) . we then envision building a smartphone application which can be used to obtain accurate ee without significantly impacting the battery life of smartphones .

energy expenditure ( ee ) estimation is an important factor in tracking personal activity and preventing chronic diseases , such as obesity and diabetes . accurate and real - time ee estimation utilizing small wearable sensors is a difficult task , primarily because the most existing schemes work offline or use heuristics . in this paper , we focus on accurate ee estimation for tracking ambulatory activities ( walking , standing , climbing upstairs , or downstairs ) of a typical smartphone user . we used built - in smartphone sensors ( accelerometer and barometer sensor ) , sampled at low frequency , to accurately estimate ee . using a barometer sensor , in addition to an accelerometer sensor , greatly increases the accuracy of ee estimation . using bagged regression trees , a machine learning technique , we developed a generic regression model for ee estimation that yields upto 96% correlation with actual ee . we compare our results against the state - of - the - art calorimetry equations and consumer electronics devices ( fitbit and nike+ fuelband ) . the newly developed ee estimation algorithm demonstrated superior accuracy compared with currently available methods . the results were calibrated against cosmed k4b2 calorimeter readings .

Introduction Related Work Methodology Prediction Models Experiments and Results Discussions and Future Work

cognitive treatments using computers , which began with memory training , are being widely used these days1 . computer - assisted cognitive rehabilitation has advantages , in that it provides personalized treatment based on a subject s neuropsychological pattern to stimulate impaired areas2 . a computer - assisted cognitive rehabilitation training program consists of exercises focused on visual reaction , visual scanning , attention , information processing speed , memory , and problem solving . these exercises can not only provide flexibility and adjustment within a treatment regimen , but may also shorten treatment time . they also provide a means for objectively measuring subject s performance as well as providing instant feedback3 . the causes of decreasing cognitive information processing speeds among the elderly include the decrease in the number of brain cells , the weakening of motor nerve cells , and a decrease in general activity4 . cognitive dysfunction begins with memory decline and is accompanied by miscalculation , disorientation , misjudgment , and comprehension disability , all of which greatly affect daily life5 . balance is the ability to maintain the body s center of gravity within the support base with minimal sway6 . the sensory process undertaken during balancing refers to the interaction among the somatic senses , including proprioception , visual sense , and stereotactic input from the vestibular system7 . one study of cognition and balance reported that the reduction of balance ability due to aging is associated with cognitive function8 . furthermore , in a study of senses and balance , with patients divided into different age groups , colledge et al.9 reported that the reduction of balance ability with aging was associated with the slowing of central information processing speed . with the above in mind , this study was conducted to investigate the effects of computer - assisted cognitive rehabilitation training on cognition and balance ability , an area closely related to daily living activities of the elderly , and to present a therapeutic program for reduced cognitive function and a safe therapeutic approach for elderly people who have difficulty engaging in physical exercise . thirty typical elderly people between the ages of 65 and 80 were randomly assigned to a computer - assisted cognitive rehabilitation training group of 15 subjects or a control group of 15 subjects . a description of the purpose and methods of the study was provided to all the participants , and the experiments were conducted after the participants had read and signed an informed consent form . general characteristics of the subjectssex ( n)age ( yr)height ( cm)weight ( kg)training group ( n=15)male : 6 female : 972.8 3.8160.3 7.359.6 8.9control group ( n=15)male : 7 female : 871.7 5.6160.3 8.161.8 9.3mean sd . the intervention used by the computer - assisted cognitive rehabilitation group was the visual interruption training found in the rehacom program and the visual construction ability program of the attention training program . the subjects performed these exercises for 30 minutes per session , three sessions per week , for six weeks . for measurement purposes , their cognitive function was measured using the korean version of the mini mental state examination ( mmse - k ) , and their static balance was measured with the balance measurement system , biorescue ap 153 ( rmingenierie , france ) . data analyses were undertaken using the independent t - test to analyze the characteristics of the subjects and the differences between the two groups . this was done using spss 12.0 for windows . in order to compare the foot pressure training before and after the program , the mmse - k scores and balance ability at the start and end of the intervention are shown in table 2table 2 . comparison of variables between pre- and post - intervention in each groupvariabletraining groupcontrol groupmmse - k ( score)*pre26.0 2.226.1 1.6post29.3 0.7 * 26.6 1.8sway area ( mm)*pre65.6 7.066.5 35.0post43.8 31.3 * 62.9 27.2sway path length ( mm)*pre28.3 5.929.2 5.9post23.8 2.0 * 27.5 5.5mean sd . the mmse - k , sway area and sway path length of the training group had significantly improved after the intervention , compared with their respective values before the intervention ( p<0.05 ) . however , the values of the control group were not significantly different ( p>0.05 ) . a comparison of the variable between the training group and the control group after the training showed a significant difference ( p<0.05 ) ( table 2 ) . the cognitive function of the elderly plays a key role in the independent performance of functional activities including daily living abilities10 . the evaluation and treatment of the reduced cognitive function of typical elderly people due to normal aging , and the cognitive damage due to neurological diseases such as dementia , is critical for the maintenance of independent living and the quality of life of elderly people . damage to a person s attention , concentration , and memory affect his or her problem - solving and inference abilities11 . in general , clinical therapies for the cognitive functions of the elderly include psychological approaches such as music therapy and reminiscence therapy , cognitive aids to compensate for cognitive impairment and computer - assisted cognitive rehabilitation training12 . the effects of computer - assisted cognitive rehabilitation training programs used in clinical settings for cognitive rehabilitation of brain - injury patients , the elderly , and dementia patients have been confirmed in many studies12 , 13 . according to gunther et al.14 , some of the effects that appear immediately after computer - assisted cognitive rehabilitation training continue even after five months , and they proposed the use of computer - assisted cognitive rehabilitation programs to treat and prevent cognitive defects among the elderly . on the other hand , chen et al.15 reported that the application of a computer - assisted cognitive rehabilitation program achieved significant improvement in various cognitive areas of traumatic brain - injury patients , but no difference was found in a control group which received a traditional therapy . in this study , a six - week - long computer - assisted cognitive rehabilitation training program was carried out using elderly subjects , and its effects on the subjects cognition and balance were investigated . the mmse - k score for the period of treatment showed significant differences in the training group , but not in the control group . to sustain the ability to balance , an appropriate response to environmental changes needs be followed instantaneously , and a proper response can be made possible by quick information processing abilities9 , which heavily rely on selective attention . for this reason , the sway area and distance also decreased significantly in the training group , but not in the control group . based on these results , we conclude that a computer - assisted cognitive rehabilitation program can be used as a therapeutic approach restoring the cognitive functions and balance abilities of elderly people who have limitations on their physical activities due to aging . furthermore , this approach can be used as an alternative clinical program for preventing the decline of cognitive function of , and falls by , the elderly .

[ purpose ] the purpose of this study was to investigate the effects of a six - week - long computer - assisted cognitive rehabilitation training program on the improvement of cognition and balance abilities of the elderly . [ subjects ] thirty healthy elderly people , aged 65 to 80 , were randomly assigned either to the training group ( n=15 ) or the control group ( n=15 ) . [ methods ] cognitive functions were evaluated using mmse - k , and the biorescue ap 153 ( rmingenierie , france ) was used to examine subjects changes in static balance . [ results ] the mmse - k score showed a significant change over the course of the treatment period in the training group , but not in the control group . the sway area and sway path length decreased significantly in the training group , but it did not show any changes in the control group . [ conclusion ] computer - assisted cognitive rehabilitation training is an effective intervention method for the improvement of the cognition and balance abilities of the elderly .

INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION

insomnia , agitation , and anxiety are common presentations to primary care clinicians , with these frequently being treated with anxiolytic and hypnotic medications ( defined by the british national formulary [ bnf ] chapters 4.1.1 and 4.1.2 ) . these medications may also be used as part of a program of alcohol withdrawal , treatment for epilepsy or muscle spasms although this represents a minority of prescriptions in clinical practice . it is recognized that the long - term use of these medications in general practice is generally not appropriate and should be limited to short - term prescriptions and kept off repeat prescription . the combination of perceived effectiveness by patients and risks associated with long - term use such as dependence and tolerance make this medication group challenging to manage in primary care . indeed , there are ongoing concerns about the rates of prescribing of this medication group in clinical practice , with particular concerns voiced for patients over 65 including falls and cognitive impairment . over the last few years , these medications have had their clinical efficacy questioned in the context of increasing concerns regarding dependence , tolerance , and alongside other adverse effects . a 2012 meta - analysis and systematic review of the us food and drug administration data reported that after reviewing 13 studies containing 65 z - drug - placebo comparisons , z - drugs produce only slight improvements in subjective polysomnographic sleep latency , regardless of the type of drug used . the authors noted that although the drug effect and placebo response were small and of uncertain clinical significance , the two together produced reasonable clinical response . furthermore , a retrospective cohort study published in 2014 of 34,727 aged 16 years old and older attending the uk primary care reported that anxiolytic and hypnotic drugs were associated with significantly increased risk of mortality over a 7-year period , after adjusting for a range of potential confounders . there has been recent work noting that there is a clear evidence that the use of hypnotic and anxiolytic medications are associated with an increased risk falls and hip fractures in older people . furthermore , these medications may lead to cognitive problems in older patients , with a meta - analysis reporting increased rates of memory problems , confusion , and disorientation more common in patients receiving benzodiazepines and z - drugs . there have even been reports that benzodiazepine use increases the risk of developing alzheimer 's disease , with this association increasing with prolonged exposure . it remains unclear how these concerns have impacted clinical prescribing practice on the coalface of primary care clinical practice . this is particularly relevant as they have been changes in guidelines in 2014 , which have promoted an active approach to reducing the prescription of these medications . this was adopted locally by a clinic b monitoring program where patients on repeat doses of these medications were actively reviewed and either had doses reduced or switched to less harmful agents . this study aimed to assess changes in prescribing practice of hypnotic and anxiolytic medications between 2007 and 2015 which would encompass local and national changes in clinical practice using routinely collected prescribing data in a single primary care practice in arbroath ( scotland ) . the analysis used community - dispensed prescribing data for patients from the east practice , springfield medical centre in arbroath in scotland held by nhs tayside and community prescribing bodies in 2007 , 2011 , and 2015 . the east practice springfield medical centre is one of four practices providing primary care services to the population of arbroath and is staffed with three - partner general practitioners ( gps ) : one nurse practitioner , one practice nurse , and one health - care assistant alongside support staff . the practice serves approximately 4000 patients in a densely populated town with marked deprivation , based on the scottish index of multiple deprivation quintile ( simd ) . the simd is based on information from major population surveys in scotland and allows comparison between the most deprived and the rest of the population in scotland in numerous domains . in 2007 , there was a local process of coding patients being prescribed benzodiazepines ( particularly those on repeat ) to aid gp review of their prescriptions and facilitate reduction in benzodiazepine prescriptions . for each individual , all community - dispensed prescriptions for anxiolytic and hypnotic medications were extracted between january 01 and december 31 for the study years of 2007 , 2011 , and 2015 . the prescriptions were reviewed on a four yearly basis to assess for changes following the introduction of clinic b monitoring in 2007 with subsequent update and review in 2011 and adjusted national guideline recommendations in late 2014/early 2015 . there were also difficulties in obtaining other years as a result of local information technology challenges , so these year groups represented a pragmatic approach . anxiolytic and hypnotic medications were defined in accordance to bnf drug groupings , hypnotics ( drugs defined in bnf chapter 4.1.1 ) , and anxiolytics ( bnf chapter 4.1.2 ) . information regarding the patients age , gender , medication name , and number of prescriptions in this drug class over the course of the year were extracted . over the course of the year , all prescriptions were reviewed with the number of different drug classes documented with the maximal number being 5 ( benzodiazepines , nonbenzodiazepine hypnotics , sedative antihistamines , azapirones , and melatonin ) . these data were fully anonymized , and any patient identifiable information was removed before analysis . baseline data were compared using one - way anova for normally distributed continuous variables , kruskal wallis test for nonnormally distributed variables , and chi - squared test for categorical variables . p values reported note the significance of differences between the 3 years groups in each of the data comparisons . as a result of the small sample size , medications were compared between years as an overall drug class ( e.g. , benzodiazepines ) rather than individual agents . this study was deemed not to require ethical approval as it entailed analysis of routinely collected clinical data . the east practice springfield medical centre is one of four practices providing primary care services to the population of arbroath and is staffed with three - partner general practitioners ( gps ) : one nurse practitioner , one practice nurse , and one health - care assistant alongside support staff . the practice serves approximately 4000 patients in a densely populated town with marked deprivation , based on the scottish index of multiple deprivation quintile ( simd ) . the simd is based on information from major population surveys in scotland and allows comparison between the most deprived and the rest of the population in scotland in numerous domains . in 2007 , there was a local process of coding patients being prescribed benzodiazepines ( particularly those on repeat ) to aid gp review of their prescriptions and facilitate reduction in benzodiazepine prescriptions . for each individual , all community - dispensed prescriptions for anxiolytic and hypnotic medications were extracted between january 01 and december 31 for the study years of 2007 , 2011 , and 2015 . the prescriptions were reviewed on a four yearly basis to assess for changes following the introduction of clinic b monitoring in 2007 with subsequent update and review in 2011 and adjusted national guideline recommendations in late 2014/early 2015 . there were also difficulties in obtaining other years as a result of local information technology challenges , so these year groups represented a pragmatic approach . anxiolytic and hypnotic medications were defined in accordance to bnf drug groupings , hypnotics ( drugs defined in bnf chapter 4.1.1 ) , and anxiolytics ( bnf chapter 4.1.2 ) . information regarding the patients age , gender , medication name , and number of prescriptions in this drug class over the course of the year were extracted . over the course of the year , all prescriptions were reviewed with the number of different drug classes documented with the maximal number being 5 ( benzodiazepines , nonbenzodiazepine hypnotics , sedative antihistamines , azapirones , and melatonin ) . these data were fully anonymized , and any patient identifiable information was removed before analysis . baseline data were compared using one - way anova for normally distributed continuous variables , kruskal wallis test for nonnormally distributed variables , and chi - squared test for categorical variables . p values reported note the significance of differences between the 3 years groups in each of the data comparisons . as a result of the small sample size , medications were compared between years as an overall drug class ( e.g. , benzodiazepines ) rather than individual agents . this study was deemed not to require ethical approval as it entailed analysis of routinely collected clinical data . there were 4155 patients , 4239 patients , and 4255 patients registered at east practice in 2007 , 2011 , and 2015 , respectively . of this group , 3.1% ( n = 130 ) , 4.1% ( n = 173 ) , and 6.3% ( n = 267 ) were prescribed a hypnotic or anxiolytic medication at least once over the course of 2007 , 2011 , and 2015 , respectively ( p = 0.375 ) . baseline characteristics and prevalence of hypnotic & anxiolytic prescribing the mean number of prescriptions of anxiolytic and hypnotic drugs over the course of the year in 2007 was 5.7 , in 2011 was 4.83 , and in 2015 was 5.33 ( p = 0.526 ) . the mean number of different anxiolytic and hypnotic drugs were 1.14 , 1.2 , and 1.2 in 2007 , 2011 , and 2015 , respectively ( p = 0.319 ) . the proportion of patients prescribed a benzodiazepine medication decreased between 2007 and 2015 : 83.8% ( n = 109 ) in 2007 , 70.5% ( n = 122 ) in 2011 , and 51.7% ( n = 138 ) in 2015 ( p = 0.006 ) . the percentage of patients prescribed diazepam reduced from 55.4% ( n = 72 ) of all prescribed hypnotic and anxiolytic medications in 2007 to 43.1% ( n = 115 ) in 2015 . the proportion of these patients prescribed a nonbenzodiazepine drug increased between 2007 and 2015 : 30% ( n = 39 ) in 2007 , 46.2% ( n = 80 ) in 2011 , and 52.4% ( n = 140 ) in 2015 ( p = 0.001 ) . the vast majority of these prescriptions in this drug class were for zopiclone , 97% in 2007 , 93.7% in 2011 , and 90% in 2015 . the proportion of patients prescribed melatonin also increased : 3.1% ( n = 4 ) in 2007 , 3.5% ( n = 6 ) in 2011 , and 5.6% ( n = 15 ) in 2015 ( p = 0.020 ) . sedative antihistamines and azapirones were prescribed in very small numbers , with between zero and two patients receiving the medication in each of the study years . between 2007 and 2015 , the percentage of patients prescribed anxiolytic and/or hypnotic medications increased from 3.1% of the practice population to 6.3% of the practice population although this did not reach statistical significance . when looking at individual drug classes , there was a statistically significant reduction in benzodiazepine prescriptions in primary care alongside increases in nonbenzodiazepine and melatonin prescribing . during each of the study time points , males made up approximately a third of the group prescribed hypnotic and/or anxiolytic medications . patients received between a mean of 5.7 and 4.83 prescriptions of anxiolytic and/or hypnotic medications per year , with a mean of between 1.14 and 1.2 different hypnotic and anxiolytic medications over the course of the year . the mean number of prescriptions of hypnotic or anxiolytic medications has remained stable between 5.7 and 4.83 over the study period , and for most of these medications , more than three prescriptions would suggest prescriptions more frequent than recommended guidelines . this study reports that males have been consistently been prescribed less hypnotic and anxiolytic medications . this is in line with the current evidence , with a norwegian study of approximately 15,000 middle - aged adults with a mean 18-year follow - up reported that the proportion of anxiolytic or hypnotic drug users was 6.6% among men and 16.2% among women . furthermore , women are more likely to be treated for a mental health problem that men ( 29% vs. 17% ) and are more likely attend the primary care physician for management of their mental health diagnoses . it is pertinent to note that there is a national strategy for women 's mental health but no equivalent for men although there is a new drive to manage suicide risk in young men . finally , men are more likely to have mental health disorders such as alcohol and substance misuse where prescription of these medications is not as commonly utilized . over the course of the review , there was an increase in the percentage of patients prescribed hypnotic and/or anxiolytic medications to 6.3% , which is similar albeit lower when compared to a larger population - based studies in scotland that have placed hypnotic / anxiolytic prescriptions at between 7.5% and 8.1% . rates of hypnotic and anxiolytic prescriptions are lower when compared to other nations including norway , australia , and france . east practice appears to have lower rates of hypnotic and anxiolytic prescribing although the rates are increasing closer to the published scottish prevalence for these agents . the reduction in benzodiazepine prescribing is likely to have been combination of nationally driven targets for reducing benzodiazepine prescribing , locally driven targeted intervention for patients on benzodiazepine through clinic b monitoring and increasing recent research linking benzodiazepines to the development of alzheimer 's disease , falls and fractures in older patients and overall all - cause mortality changing prescribing practice . there is a good body of evidence that some of the newer antidepressants can manage symptoms with anxiety , with nondrug options for managing anxiety and insomnia having a strong evidence basis . however , it should be noted that a meta - analysis reviewing the use of antidepressant medications ( selective serotonin reuptake inhibitors [ ssris ] ) and benzodiazepines for anxiety disorder reported that the change in the prescribing pattern favoring newer ssris over benzodiazepines in the treatment of anxiety disorders has occurred without supporting evidence and direct comparison is recommended . the reported increase in nonbenzodiazepine may reflect patients being prescribed short - courses of these agents rather than short - acting benzodiazepines . there has been recent concern about the possible medicalization of sleep disorders which may explain increases in these prescriptions . the expectation of uninterrupted sleep by patients with the availability of new medications has certainly impacted clinical practice . indeed , an american study reported a large increase in patient complaints of sleeplessness with associated increases in the use of benzodiazepine and nonbenzodiazepine hypnotic medications between 1993 and 2007 . indeed this us study suggested that life problems may be being treated with medical solutions , after reporting that there was a 21 fold increase in non - benzodiazepine medications between 1993 and 2007 in the context of only a 5 times increase in imsomnia diagnoses made by clinicians . the current national institute for clinical excellence guidelines only recommended hypnotic drug therapy is used for the management of severe insomnia interfering with normal daily life only after due consideration of the use of nonpharmacological measures for short periods of time only . as there is no evidence suggesting superiority of one hypnotic drug to another , patients should be prescribed the medication with lowest purchase cost and patients experiencing side effects from one agent or experiencing lack of benefit from one agent should not be trailed on other hypnotic agents ( excluding melatonin ) . crucially , hypnotics are not particularly effective with high number needed to treat to obtain benefit in the context of high rates of adverse effects . reported that for 13 people taking a hypnotic for 1 week , twelve people 's sleep would either improve or not irrespective of whether they had taken a hypnotic or a placebo and one person would experience sleep improvement ; two patients would experience an adverse event . the increase in this drug group should be seen in context with a reduction in benzodiazepines , but it is a concern that this group of medication is consistently increasing . it is postulated that the medicalization of insomnia and patient expectations are leading to clinician pressure to prescribe hypnotic agents for patients . efforts are going to be made locally to provide advice and information about nondrug approaches to managing sleep problems , which aims to reduce the use of these medications in the coming years . there was a statistically significant increase in the number of patients being prescribed melatonin over the course of the 8 years . melatonin is an endogenous hormone produced in the body in response to darkness that is important in regulating circadian rhythms . levels are known to be reduced in some middle - aged and elderly patients with insomnia , and studies to date have reported a benign side effect profile compared to other agents . a recent meta - analysis reported that melatonin decreases sleep onset latency , increases total sleep time , and improves overall sleep quality without any major side effect reported . the benefits noted did not decrease with prolonged use unlike other hypnotic agents , and although the absolute benefits were small given the favorable side effect profile , this agent may have a role for middle - aged and elderly patients . it appears that the increase in data regarding the safety and role of melatonin in primary sleep disorders , alongside the possible role in adolescent , has led to a small increase in prescribing which is likely to increase in time as the cost of the medication decreases and further studies are published . first , the paper reports patients prescribed an anxiolytic and/or hypnotic medications at three time points ( 2007 , 2011 , and 2015 ) . therefore , the data obtained does not allow us to fully ascertain the changes in prescribing practice over this time . however , such work does provide a useful platform for discussing general changes in prescribing and considering for these changes . second , this was a single center study of a general practice with a small patient list in an urban - deprived area . these results are not necessarily generalizable to other areas of the united kingdom or further afield . however , the proportion of patients prescribed anxiolytic and hypnotic medications were similar to published scottish data . third , due to the small sample size , we were unable to break down each of the cohorts into high - risk patient groups such as patients with multi - morbidity or elderly patients . finally , the study did not include larger number of different drug groups in the analyses due to the nature of the data set . it is possible that by focusing on only two classes of medications , we may have missed broader trends in psychoactive prescribing . despite the limitations of this research paper , this study has provided an opportunity to review and assess a single gp practices prescriptions of hypnotic and anxiolytic medications . following work on the data used for analyses , it is planned to perform further work looking at prescribing rates of psychoactive medications more broadly involving more local gp practices to assess different patient subgroups . there has been published work noting large - scale variation in anxiolytic and hypnotic prescribing by gps , with demographic factors more powerful determinants of this . however , high prescribing practices were less well developed , in that their quality and outcomes framework scores were lower and they were less likely to be training practices . it is hoped that further work will allow further data be obtained with regard to gp prescribing variation . between 2007 and 2015 , the percentage of patients prescribed anxiolytic and/or hypnotic medications increased from 3.1% of the practice population to 6.3% of the practice population although this did not reach statistical significance . when looking at individual drug classes , there was a statistically significant reduction in benzodiazepine prescriptions in primary care alongside increases in nonbenzodiazepine and melatonin prescribing . during each of the study time points , males made up approximately a third of the group prescribed hypnotic and/or anxiolytic medications . patients received between a mean of 5.7 and 4.83 prescriptions of anxiolytic and/or hypnotic medications per year , with a mean of between 1.14 and 1.2 different hypnotic and anxiolytic medications over the course of the year . the mean number of prescriptions of hypnotic or anxiolytic medications has remained stable between 5.7 and 4.83 over the study period , and for most of these medications , more than three prescriptions would suggest prescriptions more frequent than recommended guidelines . this study reports that males have been consistently been prescribed less hypnotic and anxiolytic medications . this is in line with the current evidence , with a norwegian study of approximately 15,000 middle - aged adults with a mean 18-year follow - up reported that the proportion of anxiolytic or hypnotic drug users was 6.6% among men and 16.2% among women . furthermore , women are more likely to be treated for a mental health problem that men ( 29% vs. 17% ) and are more likely attend the primary care physician for management of their mental health diagnoses . it is pertinent to note that there is a national strategy for women 's mental health but no equivalent for men although there is a new drive to manage suicide risk in young men . finally , men are more likely to have mental health disorders such as alcohol and substance misuse where prescription of these medications is not as commonly utilized . over the course of the review , there was an increase in the percentage of patients prescribed hypnotic and/or anxiolytic medications to 6.3% , which is similar albeit lower when compared to a larger population - based studies in scotland that have placed hypnotic / anxiolytic prescriptions at between 7.5% and 8.1% . rates of hypnotic and anxiolytic prescriptions are lower when compared to other nations including norway , australia , and france . east practice appears to have lower rates of hypnotic and anxiolytic prescribing although the rates are increasing closer to the published scottish prevalence for these agents . the reduction in benzodiazepine prescribing is likely to have been combination of nationally driven targets for reducing benzodiazepine prescribing , locally driven targeted intervention for patients on benzodiazepine through clinic b monitoring and increasing recent research linking benzodiazepines to the development of alzheimer 's disease , falls and fractures in older patients and overall all - cause mortality changing prescribing practice . there is a good body of evidence that some of the newer antidepressants can manage symptoms with anxiety , with nondrug options for managing anxiety and insomnia having a strong evidence basis . however , it should be noted that a meta - analysis reviewing the use of antidepressant medications ( selective serotonin reuptake inhibitors [ ssris ] ) and benzodiazepines for anxiety disorder reported that the change in the prescribing pattern favoring newer ssris over benzodiazepines in the treatment of anxiety disorders has occurred without supporting evidence and direct comparison is recommended . the reported increase in nonbenzodiazepine may reflect patients being prescribed short - courses of these agents rather than short - acting benzodiazepines . there has been recent concern about the possible medicalization of sleep disorders which may explain increases in these prescriptions . the expectation of uninterrupted sleep by patients with the availability of new medications has certainly impacted clinical practice . indeed , an american study reported a large increase in patient complaints of sleeplessness with associated increases in the use of benzodiazepine and nonbenzodiazepine hypnotic medications between 1993 and 2007 . indeed this us study suggested that life problems may be being treated with medical solutions , after reporting that there was a 21 fold increase in non - benzodiazepine medications between 1993 and 2007 in the context of only a 5 times increase in imsomnia diagnoses made by clinicians . the current national institute for clinical excellence guidelines only recommended hypnotic drug therapy is used for the management of severe insomnia interfering with normal daily life only after due consideration of the use of nonpharmacological measures for short periods of time only . as there is no evidence suggesting superiority of one hypnotic drug to another , patients should be prescribed the medication with lowest purchase cost and patients experiencing side effects from one agent or experiencing lack of benefit from one agent should not be trailed on other hypnotic agents ( excluding melatonin ) . crucially , hypnotics are not particularly effective with high number needed to treat to obtain benefit in the context of high rates of adverse effects . reported that for 13 people taking a hypnotic for 1 week , twelve people 's sleep would either improve or not irrespective of whether they had taken a hypnotic or a placebo and one person would experience sleep improvement ; two patients would experience an adverse event . the increase in this drug group should be seen in context with a reduction in benzodiazepines , but it is a concern that this group of medication is consistently increasing . it is postulated that the medicalization of insomnia and patient expectations are leading to clinician pressure to prescribe hypnotic agents for patients . efforts are going to be made locally to provide advice and information about nondrug approaches to managing sleep problems , which aims to reduce the use of these medications in the coming years . there was a statistically significant increase in the number of patients being prescribed melatonin over the course of the 8 years . melatonin is an endogenous hormone produced in the body in response to darkness that is important in regulating circadian rhythms . levels are known to be reduced in some middle - aged and elderly patients with insomnia , and studies to date have reported a benign side effect profile compared to other agents . a recent meta - analysis reported that melatonin decreases sleep onset latency , increases total sleep time , and improves overall sleep quality without any major side effect reported . the benefits noted did not decrease with prolonged use unlike other hypnotic agents , and although the absolute benefits were small given the favorable side effect profile , this agent may have a role for middle - aged and elderly patients . it appears that the increase in data regarding the safety and role of melatonin in primary sleep disorders , alongside the possible role in adolescent , has led to a small increase in prescribing which is likely to increase in time as the cost of the medication decreases and further studies are published . first , the paper reports patients prescribed an anxiolytic and/or hypnotic medications at three time points ( 2007 , 2011 , and 2015 ) . therefore , the data obtained does not allow us to fully ascertain the changes in prescribing practice over this time . however , such work does provide a useful platform for discussing general changes in prescribing and considering for these changes . second , this was a single center study of a general practice with a small patient list in an urban - deprived area . these results are not necessarily generalizable to other areas of the united kingdom or further afield . however , the proportion of patients prescribed anxiolytic and hypnotic medications were similar to published scottish data . third , due to the small sample size , we were unable to break down each of the cohorts into high - risk patient groups such as patients with multi - morbidity or elderly patients . finally , the study did not include larger number of different drug groups in the analyses due to the nature of the data set . it is possible that by focusing on only two classes of medications , we may have missed broader trends in psychoactive prescribing . despite the limitations of this research paper , this study has provided an opportunity to review and assess a single gp practices prescriptions of hypnotic and anxiolytic medications . following work on the data used for analyses , it is planned to perform further work looking at prescribing rates of psychoactive medications more broadly involving more local gp practices to assess different patient subgroups . there has been published work noting large - scale variation in anxiolytic and hypnotic prescribing by gps , with demographic factors more powerful determinants of this . however , high prescribing practices were less well developed , in that their quality and outcomes framework scores were lower and they were less likely to be training practices . it is hoped that further work will allow further data be obtained with regard to gp prescribing variation . this study reports a reduction in benzodiazepine prescriptions in primary care alongside increases in nonbenzodiazepine and melatonin prescribing , with an increase in prescribing rates of this drug class overall . changes in this prescribing practice may reflect the medicalization of insomnia , local changes in prescribing practice , and alongside national recommendations . this clinical paper provides a useful platform for discussing community - based prescribing for this challenging group of medications and reports that locally available scottish prescribing data can be utilized to look in more detail in primary care prescribing practice at a single practice level . this study will be the basis for future work in this area with an increase in the number of practices involved to allow targeted analysis at high - risk patients for the adverse side effects of psychoactive medications alongside other high - risk medications .

introduction : over the last few years , hypnotic and anxiolytic medications have had their clinical efficacy questioned in the context of concerns regarding dependence , tolerance alongside other adverse effects . it remains unclear how these concerns have impacted clinical prescribing practice.materials and methods : this is a study reviewing community - dispensed prescribing data for patients on the east practice medical center list in arbroath , scotland , in 2007 , 2011 and 2015 . anxiolytic and hypnotic medications were defined in accordance with the british national formulary chapter 4.1.1 and chapter 4.1.2 . all patients receiving a drug within this class in any of the study years were collated and anonymized using primary care prescribing data . the patients age , gender , name of the prescribed drug(s ) , and total number of prescriptions in this class over the year were extracted.results:the proportion of patients prescribed a benzodiazepine medication decreased between 2007 and 2015 : 83.8% ( n = 109 ) in 2007 , 70.5% ( n = 122 ) in 2011 , and 51.7% ( n = 138 ) in 2015 ( p = 0.006 ) . the proportion of these patients prescribed a nonbenzodiazepine drug increased between 2007 and 2015 : 30% ( n = 39 ) in 2007 , 46.2% ( n = 80 ) in 2011 , and 52.4% ( n = 140 ) in 2015 ( p = 0.001 ) . there was a significant increase in the number of patients prescribed melatonin ( p = 0.020).discussion : this study reports a reduction in benzodiazepine prescriptions in primary care alongside increases in nonbenzodiazepine and melatonin prescribing , with an increase in prescribing rates of this drug class overall.conclusion:changes in this prescribing practice may reflect the medicalization of insomnia , local changes in prescribing practice and alongside national recommendations .

Introduction Materials and Methods Service characteristics Data collected and analysis Results Discussion Key findings Prescribing by gender Changes anxiolytic and hypnotic drug class as a whole Changes in specific drug classes Limitations and further work Conclusions Financial support and sponsorship Conflicts of interest

during the islamic scientific golden age that started in the 9th century , islamic medicine greatly influenced european science , an effect that continued until the renaissance . islamic scientists not only accumulated the existing information , but added to this knowledge through their own observations , trials and skills ( 13 ) . persian physicians had a great role in this period of innovation , commentary and systematization , and the dominant medical figures of that period were of persian origin : ali ibn sahl rabban al - tabari ( 807 870 ad , 194 257 hijra ) , muhammad ibn zakariya al - razi ( 865 925 ad , 252 312 hijra ) , ali ibn al - abas al - majusi ( 930 994 ad , 317 381 hijra ) and abu - ali al - husayn ibn abdallah ibn sina ( 981 1037 ad , 368 424 hijra ) . all their great works , however , have been written in arabic , as it was the lingua franca of the period ( 1 , 2 ) . the samanid dynasty ( 875 999 ad , 262 386 hijra ) established an autonomic state and nurtured the revival of persian literature and traditions . persian language that had lost its official status after the arab conquest once again started to be used in the arts and sciences with increasing nationalistic vigor ( 1 , 2 ) . undoubtedly the most substantial medical book of this period belongs to abubakr rabi ibn ahmad al - akhawayni al - bokhari ( died 983 ad , circa 370 hijra ) , and his book , hidayat al - mutallimin fi - al - tibb ( learner s guide to medicine ) . the hidayat consists of 185 chapters ( bab ) , the first six chapters are on the humors ( akhlat ) and faculties ( quwa ) , followed by 28 chapters on anatomy , 21 on physiology , symptoms and comments on diet and hygiene , and 130 chapters on the diseases of various organs . hidayat al - mutaallemin fi - al - tibb ( learner s guide to medicine ) is the oldest treatise on medicine written in persian . some years after the death of akhawayni , three known manuscripts of the hidayat were copied . the oldest one , copied in 1058 , is the manuscript in the bodleian library , university of oxford . jalal matini , a contemporary persian writer , compared the three existing manuscripts and published a literary edition of the hidayat in 1965 that has been published by mashhad university press ( 4 ) . it is an attempt to increase our understanding of the dental problems and their management in medieval persia . we intend to introduce specific chapters of this book to dentists and other specialists who are interested in the subject . this would increase our historical understanding of the dental problems that existed more than one thousand years ago , and some of those old remedies and herbal products may have certain clinical benefits and could reenter modern medicine . what we did in this research at first was to find chapters that are related to dental and oral medicine . our criteria were to select those chapters that contained more clear descriptions of diseases and related therapies . we tried to understand the basic concepts and meanings of those chapters , and in order to preserve the genuine form of those passages , we tried to translate some segments word for word . in the discussion part we have tried to clarify the concepts and to compare them with those of akhawayni s predecessors and successors , and modern medicine ideas . there are two chapters related to oral and dental diseases in akhawayni s book : a chapter on dental pain and one on buccal pain . what follows is a translation of some parts of these two chapters ( 4 ) . at first we will present the original text and then try to explain them with a modern outlook . we have tried to translate the original passages word for word , and in those instances where it was difficult to convey the concept through existing words , we added some words or sentences in brackets . in the following passage there is an interesting elaboration of the role of the dental nerve in generation of pain , and also considerations on the periodontal soft tissue inflammation , and infections that could mimic dental pain . the implementation of natural antiseptics such as vinegar and some other probable antiseptic and anti - inflammatory herbal and nonorganic remedies are also interesting . nerve cautery has also been discussed and dental extraction has been mentioned as the last therapeutic option . there are also interesting prescriptions of some natural breath fresheners and the need for differentiation between different causes of this symptom including dental or gastrointestinal origins . if the tooth is painful , it means that the nerve that is under the tooth and around it [ is painful ] ; if the pain is accompanied by inflamed gum that is red and burning , it could be alleviated with cold objects and gurgling with oxime , and chewing plantago psyllium soaked in vinegar and the tooth is not painful unless [ because of ] a faulty meal and if those incompatible matters remain there , they create inflammation and if they remain within the dental body , they decay the tooth and blacken the tooth and if the pain does not improve with oil of flower ( salix babilonia ) and pistacia lentiscus , [ in the second stage ] you should use the vinegar and salt frequently until those faulty materials dry out . and if the pain is without inflammation , it is phlegmatic and the patient should gargle [ a mixture of ] muscari comosum mill and tar , and if the pain does not improve [ then ] boil the citrullus colocynthis schrad with vinegar and gargle until the phlegm is removed or boil dried leaves of ruscus aculeatus and dried pine tree wood and two cloves of garlic with vinegar and gargle this mixture when it is still warm or grind and mix the anthemis pyrethrum with the skin of the root of capparis spinosa and then boil with vinegar and gargle the mixture . if the pain does not subside then incise the periphery of the tooth with a lancet to separate the nerve from the tooth , and if [ this method ] is ineffective then one deramsang ( 4.6 grams ) of the seed of black hyoscyamus niger and two deramsangs of wet styrax officinale should be ground , mixed , burned and the fume [ of the burned mixture ] should be conducted by a tube to the painful tooth to anesthetize it . if you did all and none was effective and the pain does not improve , then the tooth should be extracted or cauterized , and if it is hollow , extraction is inevitable .... under each tooth there is a nerve a tooth that does not have the nerve ( sense ) does not feel the pain the nerve that is under the tooth feels the pain ( is painful ) .... when the tooth is painful , the nerve that is under the tooth or within the tooth is painful [ page 298 ] ... and if the teeth are yellow , they should be whitened with sea - salt and sea - foam ( meerschaum ) and ghozareh chini ( we were unable to find the exact meaning of this substance ) and khashar ( cuminum cyminum ? ) all ground together , and you should brush the teeth with this mixture and you should be careful not to damage the gum ... it is obvious in the above passages that akhawayni had an anatomical insight into the diseases of the teeth and treated them based on a somehow logical framework . his methods of treatment included herbal gargles , pastilles , fumigations and purges as well as cautery and surgery . akhawayni has mentioned that if application of herbal remedies with different methods fails then the physician should consider the cauterization of the area with hot iron , and if this step also fails , tooth extraction should be considered . akhawayni and medieval persian physicians were also aware that dental pain is directly related to the nerve and if we separate the nerve from the painful tooth it would nt be painful anymore . anesthetizing the tooth by conducting a fume directly to the painful point is a very interesting method that has been discussed by akhawayni . he prescribed sea - foam ( magnesium silicate , mg4si6o15 ( oh)26h2o ) for discolored teeth ( 5 , 6 ) . and the cause that is named malus spiritus ( bad breath ) could be from three sources . : from stomach and [ it is ] accompanied by a high fever from the teeth that should be extracted , and it is possible that it originates from the lung ( page 300 ) and its treatment is the tablet of mask , [ that includes ] skin of dried bergamot , caryophyllus aromaticus , valeriana celtic , vinegar and aquilaria malaccensis lamk , one deramsang ( 4.6 grams ) from each should be mixed and ground with a half dangsang ( 2.3 grams ) of moschus moschiferus . the tablet should be taken in mouth every morning and painful oral cavity or cavitatis oris has three causes ; one is because of bilious blood and [ the oral cavity ] is erythematous and burning ; and it is possible that painful oral cavity is accompanied by [ mucosal ] whiteness that is worse than the previous case ; and it is also possible that painful oral cavity is because of corrosive ulcer , and the mouth and teeth are blackened and putrefied and the tooth may be destroyed and lost . [ page 302 ] and this [ situation ] needs foldfiun tablet , [ which is made by ] grinding and mixing yellow auripigmentum lime , oak apple and aluminum sulphate together and then drenching [ the mixture ] in old vinegar for one week and then [ foldfiun ] tablets are made [ from this mixture] . if it is necessary , brush the mouth [ gum ] with a rough cloth ( sackcloth ) and vinegar until it bleeds , and [ as a second stage ] wash the mouth with vinegar and apply this medicine [ powdered foldfiun tablet ] morning and evening until the condition improves . i have treated many of these patients and what i have mentioned here are methods that i have experienced myself and i did not mention those that i have not examined in the above passages akhawayni discusses the diseases of the mouth . he maintains that bad breath could be due to an infected tooth that should be extracted . he categorizes them into conditions that are accompanied with erythema , conditions that are accompanied with malakoplakia , and a corrosive and gangrenous ulcer of buccal mucosa . for treatment of gingivitis gums should be rubbed with a strong linen cloth until they bleed in order to remove the rotten flesh , and then the mouth should be rinsed with vinegar and specific remedies may be applied ( 5 , 6 ) . in the following passage there is an interesting elaboration of the role of the dental nerve in generation of pain , and also considerations on the periodontal soft tissue inflammation , and infections that could mimic dental pain . the implementation of natural antiseptics such as vinegar and some other probable antiseptic and anti - inflammatory herbal and nonorganic remedies are also interesting . nerve cautery has also been discussed and dental extraction has been mentioned as the last therapeutic option . there are also interesting prescriptions of some natural breath fresheners and the need for differentiation between different causes of this symptom including dental or gastrointestinal origins . if the tooth is painful , it means that the nerve that is under the tooth and around it [ is painful ] ; if the pain is accompanied by inflamed gum that is red and burning , it could be alleviated with cold objects and gurgling with oxime , and chewing plantago psyllium soaked in vinegar and the tooth is not painful unless [ because of ] a faulty meal and if those incompatible matters remain there , they create inflammation and if they remain within the dental body , they decay the tooth and blacken the tooth and if the pain does not improve with oil of flower ( salix babilonia ) and pistacia lentiscus , [ in the second stage ] you should use the vinegar and salt frequently until those faulty materials dry out . and if the pain is without inflammation , it is phlegmatic and the patient should gargle [ a mixture of ] muscari comosum mill and tar , and if the pain does not improve [ then ] boil the citrullus colocynthis schrad with vinegar and gargle until the phlegm is removed or boil dried leaves of ruscus aculeatus and dried pine tree wood and two cloves of garlic with vinegar and gargle this mixture when it is still warm or grind and mix the anthemis pyrethrum with the skin of the root of capparis spinosa and then boil with vinegar and gargle the mixture . if the pain does not subside then incise the periphery of the tooth with a lancet to separate the nerve from the tooth , and if [ this method ] is ineffective then one deramsang ( 4.6 grams ) of the seed of black hyoscyamus niger and two deramsangs of wet styrax officinale should be ground , mixed , burned and the fume [ of the burned mixture ] should be conducted by a tube to the painful tooth to anesthetize it . if you did all and none was effective and the pain does not improve , then the tooth should be extracted or cauterized , and if it is hollow , extraction is inevitable .... under each tooth there is a nerve a tooth that does not have the nerve ( sense ) does not feel the pain the nerve that is under the tooth feels the pain ( is painful ) .... when the tooth is painful , the nerve that is under the tooth or within the tooth is painful [ page 298 ] ... and if the teeth are yellow , they should be whitened with sea - salt and sea - foam ( meerschaum ) and ghozareh chini ( we were unable to find the exact meaning of this substance ) and khashar ( cuminum cyminum ? ) all ground together , and you should brush the teeth with this mixture and you should be careful not to damage the gum ... it is obvious in the above passages that akhawayni had an anatomical insight into the diseases of the teeth and treated them based on a somehow logical framework . his methods of treatment included herbal gargles , pastilles , fumigations and purges as well as cautery and surgery . akhawayni has mentioned that if application of herbal remedies with different methods fails then the physician should consider the cauterization of the area with hot iron , and if this step also fails , tooth extraction should be considered . akhawayni and medieval persian physicians were also aware that dental pain is directly related to the nerve and if we separate the nerve from the painful tooth it would nt be painful anymore . anesthetizing the tooth by conducting a fume directly to the painful point is a very interesting method that has been discussed by akhawayni . he prescribed sea - foam ( magnesium silicate , mg4si6o15 ( oh)26h2o ) for discolored teeth ( 5 , 6 ) . and the cause that is named malus spiritus ( bad breath ) could be from three sources . : from stomach and [ it is ] accompanied by a high fever from the teeth that should be extracted , and it is possible that it originates from the lung ( page 300 ) and its treatment is the tablet of mask , [ that includes ] skin of dried bergamot , caryophyllus aromaticus , valeriana celtic , vinegar and aquilaria malaccensis lamk , one deramsang ( 4.6 grams ) from each should be mixed and ground with a half dangsang ( 2.3 grams ) of moschus moschiferus . the tablet should be taken in mouth every morning and painful oral cavity or cavitatis oris has three causes ; one is because of bilious blood and [ the oral cavity ] is erythematous and burning ; and it is possible that painful oral cavity is accompanied by [ mucosal ] whiteness that is worse than the previous case ; and it is also possible that painful oral cavity is because of corrosive ulcer , and the mouth and teeth are blackened and putrefied and the tooth may be destroyed and lost . [ page 302 ] and this [ situation ] needs foldfiun tablet , [ which is made by ] grinding and mixing yellow auripigmentum lime , oak apple and aluminum sulphate together and then drenching [ the mixture ] in old vinegar for one week and then [ foldfiun ] tablets are made [ from this mixture] . if it is necessary , brush the mouth [ gum ] with a rough cloth ( sackcloth ) and vinegar until it bleeds , and [ as a second stage ] wash the mouth with vinegar and apply this medicine [ powdered foldfiun tablet ] morning and evening until the condition improves . i have treated many of these patients and what i have mentioned here are methods that i have experienced myself and i did not mention those that i have not examined in the above passages akhawayni discusses the diseases of the mouth . he maintains that bad breath could be due to an infected tooth that should be extracted . he categorizes them into conditions that are accompanied with erythema , conditions that are accompanied with malakoplakia , and a corrosive and gangrenous ulcer of buccal mucosa . for treatment of gingivitis gums should be rubbed with a strong linen cloth until they bleed in order to remove the rotten flesh , and then the mouth should be rinsed with vinegar and specific remedies may be applied ( 5 , 6 ) . it is obvious in the above passages that akhawayni had an anatomical insight into the diseases of the teeth and treated them based on a somehow logical framework . his methods of treatment included herbal gargles , pastilles , fumigations and purges as well as cautery and surgery . akhawayni has mentioned that if application of herbal remedies with different methods fails then the physician should consider the cauterization of the area with hot iron , and if this step also fails , tooth extraction should be considered . akhawayni and medieval persian physicians were also aware that dental pain is directly related to the nerve and if we separate the nerve from the painful tooth it would nt be painful anymore . anesthetizing the tooth by conducting a fume directly to the painful point is a very interesting method that has been discussed by akhawayni . he prescribed sea - foam ( magnesium silicate , mg4si6o15 ( oh)26h2o ) for discolored teeth ( 5 , 6 ) . and the cause that is named malus spiritus ( bad breath ) could be from three sources . : from stomach and [ it is ] accompanied by a high fever from the teeth that should be extracted , and it is possible that it originates from the lung ( page 300 ) and its treatment is the tablet of mask , [ that includes ] skin of dried bergamot , caryophyllus aromaticus , valeriana celtic , vinegar and aquilaria malaccensis lamk , one deramsang ( 4.6 grams ) from each should be mixed and ground with a half dangsang ( 2.3 grams ) of moschus moschiferus . the tablet should be taken in mouth every morning and painful oral cavity or cavitatis oris has three causes ; one is because of bilious blood and [ the oral cavity ] is erythematous and burning ; and it is possible that painful oral cavity is accompanied by [ mucosal ] whiteness that is worse than the previous case ; and it is also possible that painful oral cavity is because of corrosive ulcer , and the mouth and teeth are blackened and putrefied and the tooth may be destroyed and lost . [ page 302 ] and this [ situation ] needs foldfiun tablet , [ which is made by ] grinding and mixing yellow auripigmentum lime , oak apple and aluminum sulphate together and then drenching [ the mixture ] in old vinegar for one week and then [ foldfiun ] tablets are made [ from this mixture] . if it is necessary , brush the mouth [ gum ] with a rough cloth ( sackcloth ) and vinegar until it bleeds , and [ as a second stage ] wash the mouth with vinegar and apply this medicine [ powdered foldfiun tablet ] morning and evening until the condition improves . i have treated many of these patients and what i have mentioned here are methods that i have experienced myself and i did not mention those that i have not examined he maintains that bad breath could be due to an infected tooth that should be extracted . he categorizes them into conditions that are accompanied with erythema , conditions that are accompanied with malakoplakia , and a corrosive and gangrenous ulcer of buccal mucosa . for treatment of gingivitis gums should be rubbed with a strong linen cloth until they bleed in order to remove the rotten flesh , and then the mouth should be rinsed with vinegar and specific remedies may be applied ( 5 , 6 ) . dental diseases have been mentioned in sumerian records quite vividly . during late antiquity , dental care and even prosthetic dentistry reached a high degree of development in the roman period , but fell into decadence when western roman empire began to decline and christianity rose ( 7 ) . it appears that the major dental problems mentioned by akhawayni are toothaches related to dental caries , oral ulcer and gingival disease . he starts with noninvasive methods mostly using herbal remedies , and if those are ineffective , then surgical interventions are applied . he reiterates that he has examined his methods of treatments many times and believes in their effectiveness . akhawayni s treatments are mainly based on anatomical principles and less influenced by humoral theory , and there is no mention of charms , magic and amulets . false ideas of dental worms can not be seen in his chapter on dental disease ( 4 ) . the idea of tooth worm that was first mentioned in sumerian scripts continued to be discussed by romans and arabs , as well as in medieval europe and even late anglo - saxon documents ( 7 , 8) . akhawayni describes the dental anatomy very minutely , and interestingly there is no mention of tooth worm gnawing away the dental substance . he believes in an incompatible material that remains within the dental body and decays the tooth ( 4 ) . the medieval cure for tooth worm was , inhaling the smoke of burned hyoscyamus niger as mentioned by roman doctors ( 7 , 8) . akhawayni prescribes the smoke of hyoscyamus niger and styrax officinale for toothaches but there is no mention of tooth worm . interestingly we can see this false idea entering persian medical texts showing the weakness of scientific thinking and lack of innovation until the world stepped toward the modern period . akhawayni subdivides toothaches into those that are associated with inflammation , redness and burning , and those that are not associated with severe inflammation . he prescribes gurgling with oxime and chewing plantago psyllium soaked in vinegar for the first group . those without severe inflammation are due to a retained phlegm that should be removed through gargling muscari comosum mill and a mixture of citrullus colocynthis schrad and vinegar and gargling until the phlegm is removed ( 4 ) . medieval european manuscripts of gilbert anglicus and guy de chauliac ( 13 century ad ) have mentioned effective pain relief using papaver somniferum and oil of caryophyllus aromaticus . alum and punica granata have been mentioned by roger of frugard as ingredients in a lotion to overcome suppuration ( 8) . akhawayni s recommendations for periodontal disease and gingivitis include scarification of the gum to bleed and then application of vinegar and other solutions with probable antiseptic characteristics . he believes that tooth extraction is the last recourse when every other attempt has proven useless , a method that no doubt deserves high praise . in akhawayni s writings we can not find any direct description of drilling and emptying the painful teeth as described by avicenna and late byzantine - roman physician archigenes ( 7 ) . akhawayni is one of the few authors who have mentioned the cutting of the dental nerve for relieving the pain . he describes a method of gently pricking a lancet around the tooth and cutting the nerve ( 4 ) . however , because akhawayni wrote his book in persian , which was not the lingua franca of the islamic world in that period , it did not receive the credit that it deserved . we should consider that akhawayni was indirectly a student of razes who created the great medical encyclopedia of alhavi , which covers most subjects and principles of medical science . one of those subjects is dentistry , which contains definitions , diagnosis , treatment and prognosis of dental and oral cavity diseases and associated medical care leading to oral health and management ( 911 ) . alli ibn al - abbas al - ahvazi , the great pillar of the third century , also was a great pillar of medicine with very interesting ideas on dental and oral medicine , as he believed bad breath could originate from the stomach . differentiation between gingival- and dental - originated pain is paramount because in the first case there is no need for the tooth to be extracted . avicenna ( 370 428 ) also gathered the ideas of his great predecessors such as razes al ahvazi and akhawayni and with his novel outlooks became one of the remaining figures of science worldwide ( 911 ) . it is interesting that in the more recent medical books we can see the entrance of false ideas such as the tooth worm , as well as some novel ideas such as using anthemis pyrethrum ( anacyclus prethrun ) in vinegar for loosening the painful tooth for its easier extraction ( 12 ) . our article was an attempt to introduce hidayat al - mutallimin fi - al - tibb ( learner s guide to medicine ) as an important and forgotten 10 century persian medical treasure . the passage on dental and oral disease shows the importance of oral and dental care in medieval persia . it was very interesting that we found great considerations of anatomical based diagnosis and therapeutic considerations . it shows that medieval islamic - iranian physicians were aware of the importance of the dental nerve as a root of sensation , and the oral soft tissue and periodontal inflammation that can very easily be confused with dental originated pain .

persian physicians had a great role in assimilation and expansion of medical sciences during the medieval period and islamic golden age . in fact the dominant medical figures of that period were of persian origin such as avicenna and razes , but their works have been written in arabic that was the lingua franca of the period . undoubtedly the most substantial medical book of that period that has been written in persian belongs to abubakr rabi ibn ahmad al - akhawayni al - bokhari and his book , hidayat al - mutallimin fi - al - tibb ( learner s guide to medicine).there are two chapters related to oral and dental diseases in the hidayat , a chapter on dental pain and a chapter on bouccal pain . akhawayni s views on dental diseases and treatments are mainly based on anatomical principles and less influenced by humeral theory and no mention about the charms , magic and amulets . false idea of dental worm can not be seen among his writings . cutting of the dental nerve for relieving the pain , using the anesthetizing fume , using the natural antiseptic and keeping the tooth extraction as the last recourse deserves high praise .

Introduction Method Passages and Descriptions Description 1 Description 2 Discussion Conclusion

understanding of the daily rhythmicity in nociception is important for the standardization of studies of analgesic drugs . yet , few studies have investigated the daily rhythmicity in nociception . although studies on rats and golden hamsters have indicated the occurrence of greater pain sensitivity during the dark phase of the light - dark cycle , another study on rats indicated the occurrence of greater sensitivity during the light phase , and a study on mice indicated the occurrence of two daily peaks in sensitivity , one during the light phase and one during the dark phase . they were housed in microlon cages maintained at 25 1c under an l12:d12 light - dark cycle . nociception was evaluated by eddy 's hot plate method , tail immersion method , and tail clip method . the latency between the noxious stimulus and the animal 's response was recorded as reaction time . rats previously adapted to an l12:d12 light - dark cycle were divided into 7 groups of 6 animals and tested at one of 7 times of day 4 hours apart . the same groups of animals were retested a week later with the same protocol , except that the animals initially tested first during the light phase of the light - dark cycle were tested first during the dark phase , and vice versa . reaction time ( average of the three methods ) was longest at the transition from light to darkness and shortest a few hours before the transition from darkness to light . this suggests greater pain sensitivity late in the dark phase , which is in agreement with previous studies in rats and golden hamsters . another study in rats suggested the occurrence of greater sensitivity during the light phase , but this was probably an artifact of the experimental procedure , as only two time points during the day were reported . a study on mice suggested the occurrence of two daily peaks in sensitivity , one during the light phase and one during the dark phase . the amplitude of the daily variation in latencies was much smaller in that study than in ours , and it is possible that random oscillations were interpreted as a daily rhythm . daily rhythmicity of nociception in rats the figure shows the daily variation in reaction time to nociceptive stimulation . the horizontal bar at the top indicates the timing of the light - dark cycle . sensitivity to pain is highest late in the dark phase of the light - dark cycle and lowest at the light - dark transition . we , the authors declare that we have not received funds from any agency or organization for carrying out this work . carried out the study cv carried out the replicate study nm and sjp evaluated the data statistically

backgroundmany behavioral and physiological variables exhibit daily rhythmicity . few investigations of the daily rhythmicity in nociception have been conducted , and conflicting results have been obtained . the present study evaluated the daily rhythmicity in nociception in wistar rats.methodsnociception was investigated by eddy 's hot plate method , tail immersion method , and tail clip method . the latency between the noxious stimulus and the animal 's response was recorded as reaction time . separate groups of rats were tested in 4-hour intervals for 24 hours.resultsthere was clear daily variation in response latency . reaction time was shortest a few hours before lights - on and longest at the light - dark transition.conclusionnociception exhibits robust daily rhythmicity in rats . sensitivity to pain is highest late in the dark phase of the light - dark cycle and lowest at the light - dark transition .

Background Methods Results and discussion Conclusion Competing interests Authors' contributions Acknowledgement

a 30-year - old male presented in the emergency department with a history of bilateral blurring of vision of one - day duration . since his systemic hypertension was not well controlled with this drug his physician had switched over to a combination of atenelol 50 mg with chlorthalidone 12.5 mg ( tenoric 50 , ipca pharmaceuticals ) . refraction was done and his visual acuity was improving to 20/20 in both eyes with a spherical correction of -5 diopter sphere ( ds ) . a cycloplegic refraction revealed a value of -3 ds/-0.5 diopter cylinder ( dc ) x 80 in the right eye and -3.75 ds/-1.0 dc x 130 in the left eye . b - scan ultrasonography showed shallow peripheral serous choroidal detachment [ fig . 2 ] . an optical coherence tomogram ( oct - stratus version 4.0 , zeiss ophthalmics ) was also done since retinal striae were present , which showed a mild thickening of the macula . hematological evaluation including renal parameters and computerized tomogram of the head were within normal limits . he was reviewed after five days and his visual acuity had improved to 20/20 without any refractive error . fundus examination revealed disappearance of retinal striae at the macula and peripheral choroidal effusion had also resolved [ figs . a change analysis was done using the software in stratus oct version 4.0 which revealed definite reduction in the thickness of the macula . we report a case of acute myopia induced by oral consumption of chlorthalidone for systemic hypertension . acute transient myopia may be induced by several drugs and different mechanisms have been described . the exact mechanism producing acute myopia is not very clear even though it has been postulated to be due to ciliary body effusion,1 peripheral uveal effusion2 and ciliary spasm3 and lens swelling . oral sulphonamides cause transient myopia as a result of forward displacement of the lens due to allergic ciliary body edema and rotation.3,4 intravenous administration of equine anti lymphocytic globulins causes transient myopia due to ciliary spasm.7 some of the other drugs that have been described to cause transient myopia include indapamide,5 hydrochlorothiazide,2 triamterene2 and topiramate.8,9 in this case a combination of ciliary spasm and peripheral choroidal effusion explain the induced myopia . since the cycloplegic refraction showed a decrease in myopic shift by about 2 diopter it is evident that there was an element of ciliary spasm . retinal striae have been reported to occur with acute myopia after drug intake.10 ciliary body rotation and edema resulting in forward movement of iris lens diaphragm has been reported as a possible cause for induced myopia by some authors.1,2,3,11 in this case there was no definite shallowing of the anterior chamber . we feel that this mechanism may not explain the pathogenesis of myopia in this case . to our knowledge since this is a common diuretic used in the treatment of systemic hypertension , we feel that it is important to be aware of this possible idiosyncratic adverse effect .

we report a case of sudden loss of vision due to the development of acute myopia after the intake of chlorthalidone used for treating systemic hypertension . clinically this was associated with ciliary spasm , shallow peripheral choroidal effusion and retinal striae at the macula with increase in macular thickness seen on optical coherence tomography . all these findings were reversed completely once the drug was discontinued . development of acute myopia should be kept in mind as an adverse effect of a commonly used antihypertensive drug , namely chlorthalidone .

Case Report Discussion

the obstructive sleep apnea syndrome ( osas ) is a highly prevalent sleep disorder characterized by recurrent episodes of upper airway obstruction and subsequent recurrent arousal during sleep . it is estimated that up to 5% of adults in western countries have osas . according to published data emerging studies suggest that the repetitive episodes of hypoxia and reoxygenation , in a manner similar to that of the ischemia / reperfusion injury model , promote the activation of proinflammatory pathways and disrupt normal endothelial function . brachial artery flow - mediated dilation ( fmd ) is a validated and widely used research tool for the quantification of endothelial function . it is a sensitive marker of inflammation and an important marker of future cardiovascular risk . elevated crp levels have been reported by several researchers in osas patients [ 1416 ] . we hypothesized that the proper use of cpap should also help improve the levels of fmd and crp and thus reduce the risk for adverse cardiovascular events . the aim of this study was to assess the effect of 3-month cpap therapy on fmd and plasma crp levels in patients with osas . this study was conducted in the sleep laboratory of the department of clinical therapeutics at alexandra hospital ; athens medical school . informed consent was obtained from each patient and approval was also obtained for the conduct of this study from the hospital ethics committee . male patients with suspected osas , receiving no medication , and known not to suffer from diabetes mellitus , arterial hypertension , dyslipidemia , or inflammatory , cardiovascular , neuromuscular or pulmonary diseases were recruited into the study . all patients underwent full overnight polysomnography . according to the standard criteria of the american academy of sleep medicine : 1 ) apnea was defined as a complete cessation of airflow for at least 10 seconds ; 2 ) hypopnea was defined as a reduction in airflow of at least 50% , a < 50% reduction associated with electroencephalographic arousals , or a > 3% decrease in oxygen saturation ; and 3 ) the apnea - hypopnea index ( ahi ) referred to the average number of apneas and hypopneas per hour of sleep . subjects with an ahi 15 were assigned to receive cpap treatment and formed our study group . subjects with an ahi<5 were defined as not having osas and were included in the control group . patients with an ahi 515 were not eligible for cpap treatment and were excluded from the study analysis . a high - resolution 12.0-mhz transducer ultrasound was used to measure brachial artery diameter at rest and during reactive hyperemia . the assessment was carried out in a quiet room , with a stable temperature at 22 to 24c , by an experienced physician who was blinded to the patient s sleep recordings and blood analysis . reactive hyperemia was induced by inflation of a blood pressure cuff placed on the lower part of the arm and inflated to 250 mm hg , followed by release after 5 minutes . flow - mediated dilation ( fmd ) was calculated as the ratio of change in diameter ( maximum of 2 measurements baseline ) over baseline value . patients smoking status , body mass index ( bmi ) ( calculated as kilograms per meter squared ) , arterial blood pressure , and heart rate were recorded at baseline and 3 months after cpap treatment . non - smokers were defined as those patients who had never smoked . furthermore blood analysis including : glucose , total cholesterol , low - density lipoprotein cholesterol ( ldl ) , high - density lipoprotein cholesterol ( hdl ) , triglycerides and crp , a standard statistical software package spss ( spss inc , chicago , il ) was used in the analysis . difference regarding smoking status between osa and control subjects was assessed with the chi - square test . the t test was used to detect differences in age , bmi , blood pressure , heart rate , lipidemic profile , sleep parameters , fmd and crp values between osa patients and control subjects . the association between baseline crp and fmd values with age , bmi , and sleep parameters was assessed with the pearson correlation test . paired - samples t test was used for comparison of the patients fmd and crp values at baseline and 3 months after cpap therapy . ten patients had an ahi between 5 and 15 and were excluded from the study , 18 subjects were included in the control group ( ahi<5 ) , and 20 patients with ahi 15 formed the osas group in need of cpap treatment . there were no significant differences between control subjects and osas patients with regard to the mean age , bmi , blood pressure , heart rate , lipid markers , glucose and smoking status . compared to the controls , the osas group had significantly lower fmd values ( 6.720.86 vs. 9.591.15 ) and higher plasma crp levels ( 0.820.16 vs. 0.290.14 ) ( p=0.00 ) . baseline fmd values were negatively correlated with age , bmi , ahi , dsi,% of time < 90% sa02 , and crp ( p<0.05 ) . plasma crp values were positively correlated with bmi , ahi , dsi , and% of time < 90% sa02 ( p<0.05 ) ( table 2 ) . after adjustment for age and bmi , fmd values retained their correlation with ahi ( b=0.08 , p=0.00 ) , dsi ( b=0.10 , p=0.00 ) and% of time < 90% sa02 ( b=0.28 , p=0.00 ) . after adjustment for bmi , crp values also retained their correlation with% of time < 90% sao2 ( b=0.04 , p=0.00 ) , dsi ( b=0.01 , p=0.00 ) and ahi ( b=0.01 , p=0.00 ) . out of 20 patients in the osas group , 6 failed to comply with the cpap treatment and returned the devise within 2 weeks . during the study period , patients bmi and smoking status did not change . in the group of patients who complied with the cpap treatment , there was a significant increase in the fmd values ( 9.180.55 vs. 6.270.50 ) and a decrease in the levels of crp ( 0.670.15 vs. 0.840.18 ) ( p=0.00 ) . no changes were detected in the group of patients who failed to comply with cpap ( figures 1 , 2 ) . the measurement of endothelium - dependent dilation in response to reactive hyperemia is a non - invasive and validated method for the assessment of the endothelial function . alterations in endothelium dependent dilatation have been documented in patients with coronary artery disease and diabetes and , recently , osas [ 1922 ] . the regulation of vasomotor tone , as measured by the change in the forearm blood flow after transient ischemia , appears to be regulated by the availability of nitric oxide . researchers have shown that in the osas the pathophysiologic stressors resulting from repetitive episodes of hypoxemia / reoxygenation downregulate the activity of the endothelial nitric oxide synthase and upregulate the expression of various vasoactive substances such as endothelin-1 and angiotensin ii [ 2325 ] . in this study , fmd values appeared to be significantly higher in the control group compared to the osas patients , and were negatively correlated with the ahi , dsi and% of time < 90% sa02 . in accordance with our results , previous studies have also shown that endothelial dysfunction , as measured by fmd , was correlated with the severity of osas [ 911 ] . furthermore , fmd values were negatively correlated with age . in a recent study by yim - yeh et al . recently , data have emerged regarding the potential beneficial effects of cpap on endothelial function . a study by ip et al . demonstrated that fmd was significantly improved after 4 weeks of cpap treatment , while bayram et al . found that the improvement in the endothelial function was sustained after 6 months of treatment in complaint patients . our results also confirm the beneficial role of cpap on fmd values after 3 months of treatment . recently , a large amount of evidence has demonstrated the pivotal role of inflammation in cardiovascular disease . in view of this , c - reactive protein has gained increasing attention as an independent risk factor for coronary disease . in patients with osas , circulating levels of inflammatory markers appeared to be elevated , suggesting the presence of systemic inflammation . in agreement with previous reports , we showed that crp levels were positively correlated with the osas severity [ 1416 ] . furthermore , crp was correlated with bmi , an observation consistent with the ability of adipose tissue to release certain cytokines , including interleukin-6 , and to stimulate crp synthesis . although both fmd and crp are involved in the pathophysiology of cardiovascular complications , the association between these 2 parameters in osas remains elusive . nevertheless , a study by nystrom et al in patients with coronary artery disease revealed a correlation between endothelial dysfunction ( measured with fmd ) and the levels of crp . these discrepancies may be attributed to differences in methodology and inclusion criteria among published studies . this study also demonstrated that appropriate use of cpap therapy can significantly decrease the levels of crp . previous studies have also presented similar results , thus denoting the possible beneficial role of cpap in reducing systemic inflammation and cardiovascular risk in osas patients [ 3539 ] . the study population consisted of patients with no prior history of cardiovascular or pulmonary disease , thus the study results should be interpreted with caution in patients beyond this particular group . furthermore , female patients were excluded from the study analysis to avoid potential sex - based differences in hormone secretion that could affect both crp and endothelial function . this single - center study demonstrated that male osas patients with no prior history of cardiovascular disease had significantly higher crp levels and lower fmd values compared to control subjects a finding indicative of the possible presence of subclinical atherosclerosis and subsequent increased risk for developing cardiovascular disease . appropriate cpap therapy improved both crp and fmd values , suggesting its potentially beneficial role in reducing cardiovascular morbidity and mortality in osas patients . further well - designed prospective studies are needed to elucidate the effect of cpap on the reduction of cardiovascular risk .

summarybackgroundcontinuous positive airway pressure ( cpap ) is the most effective method for treating obstructive sleep apnea syndrome ( osas ) and alleviating symptoms . improved sleep quality with effective cpap therapy might also contribute to attenuated systemic inflammation and improved endothelial function , with subsequent reduction of cardiovascular risk.the aim of this study was to assess the effect of 3-month cpap therapy on brachial artery flow - mediated dilation ( fmd ) and plasma c - reactive protein ( crp ) levels in patients with osas.material/methodsour study group consisted of 38 male patients with no prior history of cardiovascular disease . twenty patients with an apnea - hypopnea index ( ahi ) 15 were assigned to receive cpap treatment and 18 subjects with an ahi<5 were included in the control group . six patients failed to comply with the cpap treatment . measurement of fmd and blood analysis was performed at baseline and 3 months after cpap therapy.resultsbaseline fmd values were negatively correlated with age , bmi , ahi , dsi,% of time < 90% sa02 , and crp ( p<0.05 ) . plasma crp values were positively correlated with bmi , ahi , dsi and% of time < 90% sa02 ( p<0.05 ) . in the group of patients who complied with the cpap treatment , there was a significant increase in the fmd values ( 9.180.55 vs. 6.270.50 ) and a decrease in the levels of crp ( 0.670.15 vs. 0.840.18 ) ( p<0.05).conclusionsappropriate cpap therapy improved both crp and fmd values , suggesting its potentially beneficial role in reducing cardiovascular risk in osas patients .

Background Material and Methods Results Discussion Conclusions

hemoglobin oxygen desaturation in the absence of acute illness is common in children with sickle cell anemia ( sca ) , and is associated with higher cerebral blood flow velocities [ 1 , 2 ] , and with risk of complications including stroke . the underlying mechanisms of hemoglobin oxygen desaturation in sca are poorly understood but may involve the severity of anemia as well as differences in hemoglobin oxygen affinity compared to hemoglobin a ( hba ) , with increased expression of 2.3 dpg in hemoglobin s ( hbs ) resulting in a right - shifted hemoglobin oxygen affinity curve and other differences in red cell physiology . other potential causes include a history of acute chest syndrome and reduced pulmonary and cardiac function . coinheritance of alpha - thalassemia deletions and glucose-6-phosphate deficiency ( g6pd ) may affect the degree of anemia [ 8 , 9 ] whilst alpha - thalassemia status modifies red cell indices [ 1012 ] and rheology , as can iron status . we therefore investigated potential hematological , genetic , and nutritional predictors of daytime hemoglobin oxygen saturation in tanzanian pediatric patients homozygous for hbs ( sca ) and in non - sca local controls . ethical permission was granted by the muhimbili university of health and allied sciences ethics committee ( mu / rp / aecnoi.xii/77 ) . written informed consent was obtained from parents or guardians in their own language . children ( less than 17 years ) with sca ( hbss genotype ) were enrolled in the scd cohort study at muhimbili national hospital , dar - es - salaam . resting pulse oximetry data ( masimo radical , masimo corporation , usa ) and blood samples were collected at routine outpatient clinic visits between november 2007 and december 2008 . analysis was limited to data collected at a single steady - state time point . a strict definition of steady state was employed ( temperature < 37.5c , no malaria parasitaemia , no reported pain , no blood transfusion within 90 days or hospital admission within 30 days on either side of the selected time point ) and determined to be clinically well by the attending doctor . all cohort children are routinely prescribed folate supplementation ( 5 mg / day ) . non - sca children were those who presented for sickle testing between october 2004 and december 2008 but who had hbaa or hbas by hemoglobin electrophoresis . none of the children had malaria parasitaemia or fever ( temperature > 37.4c ) and all were clinically well . full blood counts were performed using an automated cell counter ( pentra 60 , horiba abx , kyoto , japan ) . serum iron and total iron binding capacity were measured in serum samples stored at 80c ( architect c8000 , abbott , new york , usa ) . lactate dehydrogenase ( ldh ) and bilirubin ( total and conjugated ) were measured in fresh serum samples ( architect c8000 , abbott , new york ) by muhimbili central pathology laboratory . children attending screening for sickle status were typed for hbs by alkaline hb electrophoresis ( helena , sunderland , tyne and wear , uk ) . children enrolled in the muhimbili sickle cohort also had hemoglobin fractions , including hbf , quantified by hplc using the -thalassemia short programme on the variant analyzer ( biorad , hercules , ca , usa ) . in addition , children enrolled in the muhimbili sickle cohort had hbss status confirmed by genotype and were genotyped for the 3.7 alpha - thalassemia deletion using a pcr - based method and agarose gel visualization as per published methods and the 202- and 376-single nucleotide polymorphisms ( snps ) for glucose 6-phosphate dehydrogenase deficiency ( g6pd ) and hbs using multiplex sequenom . data were analyzed using stata 11-ic ( statacorp , college station , tx , usa ) . daytime spo2 is not normally distributed due to an excess of observations having the maximum possible value of 100% and a skewed distribution towards the lower values . thus we investigated the use of negative binomial regression of count data using a new variable of spo2 - 100 compared to zero - inflated negative binomial regression . vuong tests indicated no strong consistent preference for either models across the explanatory variables tested . as we had no prior hypotheses that mechanisms to predict 100% versus < 100% spo2 may differ from mechanisms underlying the degree of desaturation , we selected the negative binomial regression model . we next compared the results of these models to those from simple linear regression of the nontransformed data and observed no major differences in the results . whilst analysis of the residuals from the linear regression models indicated some skew , this was not judged to be sufficient to render the results invalid , which were in agreement with those from the better fitting negative binomial regression models . thus for ease of presentation and interpretation the results of the linear regression models are presented . complete hematological , genetic , and iron status ( transferrin saturation ) data were available for 458 sca children . in addition , complete hematological and pulse oximetry data were available for 394 non - sca children , although transferrin saturation data were only available in a small subset of 62 children and genotyping was not conducted in these controls . pulse oximetry , hematological and iron status data for the two groups of children are summarized in table 1 . of the 458 sca children , 239 were boys ( 52% ) with a mean age of 9.7 y ( sd 4.3 y ) compared to 212 boys ( 54% ) in the 394 non - sca children with a mean age 7.0 y ( sd 4.7 y ) . in the sca children median daytime spo2 was 97% ( iq range 9499% ) ( table 1 ) , significantly lower ( wilcoxon rank sum test ; p < 0.0001 ) than in the non - sca children ( median 99% ; iq range 98100% ) ( table 1 and figure 1 ) . hemoglobin concentration , red cell indices ( red blood cell count ( rbc ) , mean cell hemoglobin ( mch ) , and mean cell hemoglobin concentration ( mchc ) , mean cell volume ( mcv ) ) , and markers of hemolysis ( ldh and unconjugated bilirubin ) were significantly different between the sca and non - sca groups ( table 1 ) . the proportion of sca children with low transferrin saturation ( < 16% ) indicating probable iron deficiency , was 28% ( 126/458 ) but was lower than that in non - sca tanzanian control children ( 32/62 , 52% ) ( table 1 ) . only 9 ( 2% ) of the sca and none of the non - sca patients had high transferrin saturation ( > 55% ) which might indicate iron over - load . forty - one percent of the sca children ( 192/458 ) were heterozygous for the 3.7 alpha - thalassemia deletion and 17% were homozygous ( 78/458 ) . eleven percent of sca children were heterozygous females for both the 202 and 376 g6pd single nucleotide polymorphisms ( snps ) which results in a moderate phenotype , whilst a further 12% ( 54/458 ) of sca children were affected homozygous females or affected males . predictors of spo2 in sca are shown in table 2 . within the sca population all of the hematological variables higher hemoglobin , rbc , and hbf% were associated with greater spo2 , whilst higher mch , mchc , and mcv were associated with lower spo2 . mch was the single biggest contributor to the variation in spo2 as indicated by the still modest adjusted r value of 0.09 . there was weak evidence of a negative association between transferrin saturation and spo2 . in the non - sca population the same hematological variables were tested for associations with spo2 , as well as hb phenotype , as versus aa . however , the only predictor of spo2 in this group was a positive association for age ( beta coefficient 0.05 , p = 0.024 ) with no evidence to suggest even weak effects of any of the hematological indices . in the sca population for whom genotypes were available , coinheritance of the alpha - thalassemia 3.7 genotype was associated with increased spo2 , whilst co - inheritance of g6pd deficiency was associated with decreased spo2 . pair - wise associations between the different explanatory variables for daytime spo2 within the sca group were investigated and are presented in full in supplementary table 1 available online at http://dx.doi.org/10.1155/2013/472909 . alpha - thalassemia genotype was highly associated with all of the hematological variables , with the number of 3.7 deletions being associated with higher hemoglobin ( pearson correlation coefficient 0.23 ) ; rbc count ( 0.55 ) ; lower mcv ( 0.24 ) , mchc ( 0.32 ) , and mch ( 0.60 ) ; and also lower hemolytic markers , particularly unconjugated bilirubin ( 0.26 ) , all with p values < 0.0001 . interestingly , presence of the 3.7 alpha - thalassemia deletion was also associated with improved nutritional status as determined by body - mass - index z - score . higher mch and mcv were associated with lower hemoglobin in the sca population whilst hbf% was associated with higher hemoglobin . transferrin saturation was positively associated with hemoglobin ( 0.17 ) and also higher mch ( 0.33 ) , both p values < 0.0005 , but was not associated with mcv . correlations between the hematological variables were similar in the non - sca population compared to sca ( data not shown ) except that higher mcv was correlated with higher hemoglobin ( 0.64 , p < 0.001 ) but was not associated with rbc count and no association was apparent between hemoglobin and mchc . in multivariable models predicting spo2 , the inclusion of mch with alpha - thalassemia genotype resulted in a diminished and nonsignificant association for alpha - thalassemia . however , in models including either mch or alpha - thalassemia there were an independent effect of hbf% and weak evidence of an independent effect of g6pd limited to affected males and homozygote females ( supplementary table 2 ) , but with the greatest variation explained by a model including mch and hbf% ( adjusted r = 0.15 , p < 0.001 , n = 315 ) . few studies have attempted to determine the hematological or genetic correlates of daytime spo2 . in this study we report that higher daytime spo2 in children with sca is associated with higher red cell count and hemoglobin , but with lower mch or mchc . coinheritance of the alpha - thalassemia 3.7 deletion in our sca population is strongly associated with increased hemoglobin , red cell count and decreased mch and mchc and also with increased spo2 , but not independently of these factors . thus the basis of the association between alpha - thalassemia genotype and spo2 would appear to be via its effects on red cell indices . in kenyan nonsickle populations , alpha - thalassemia 3.7 deletion copy number is also associated with slightly increased hemoglobin levels and increased red cell counts , but effects on anaemia appear to be modulated by sickle trait status . this suggests that the decreased anemia and increased red cells observed in sca plus alpha - thalassemia results from decreased rate of red cell destruction , proposed to be due to lower hbs concentration in red cells , and thus reduced hbs polymerization which is concentration dependent as well as being increased by desaturation . this is supported by the observation that alpha thalassemia was associated with lower levels of ldh and unconjugated bilirubin as markers of hemolysis . red cell count and hemoglobin were not associated with spo2 when adjusting for mch and thus anemia does not appear to be a direct cause of low spo2 in our population . a beneficial effect of alpha thalassemia on daytime spo2 has also been observed in jamaican children with sca , but in contrast to our study , there were also independent effects of hemoglobin , whilst neither mch , or mchc was associated with spo2 . it is interesting to note that within the non - sca population none of the tested variables were associated with spo2 . this may have been the result of the reduced variability in spo2 in the non - sca population , or reflect sickle - specific effects of red cell indices on spo2 . again , this contrasts with observations in older jamaican children who were hbaa ( 1518 years ) in whom hemoglobin and mcv ( but not mch or mchc ) were associated with spo2 . our observation of a beneficial effect of co - inheritance of alpha - thalassemia on daytime spo2 in children with sca appears to be in direct contrast to our previous observation of a negative effect on mean overnight spo2 , measured in a small group ( n = 30 ) of similar aged tanzanian children with sca . however , this effect of alpha - thalassemia appeared to be confounded by the strong negative association between transferrin saturation and mean overnight spo2 . we concluded that this association was most likely the result of reverse causality from nocturnal chronic and intermittent hemoglobin desaturation causing increased transferrin saturation due to upregulation of hypoxia - inducible factor , which has downstream effects of increasing iron absorption , which may also be increased by alpha - thalassemia . thus we suggest that in a larger dataset , alpha - thalassemia may also have a beneficial effect on mean overnight spo2 . similar to jamaican and american sca children , hbf% was positively and independently associated with spo2 . it is not possible in the current study to determine if an effect of hbf% is via direct effects on increased hemoglobin oxygen affinity or through indirect effects on rates of sickling or red cell rheology . interestingly alpha - thalassemia was also associated with increased hbf% , perhaps through increased rate of release of immature red cells from ineffective erythropoiesis . the co - inheritance of g6pd deficiency was negatively associated with spo2 . as there was no evidence of an association between g6pd and levels of hemolytic markers , as has been observed previously in sca , the effect on spo2 is likely through another mechanism . this includes elevated cerebral blood flow velocities [ 1 , 2 ] and an increased risk of stroke [ 3 , 23 ] . co - inheritance of alpha - thalassemia is protective against elevated cerebral blood flow velocities in children with sca [ 24 , 25 ] and is associated with reduced stroke risk , whilst g6pd increases the risk . thus the mechanisms behind these associations may , at least in part , be through effects on hemoglobin oxygen saturation and subsequent endothelial dysfunction [ 27 , 28 ] as well as other potential effects of red cell deformability and endothelial adhesion . in conclusion , in our population of tanzanian children with sca , the strongest predictor of lower spo2 is increased mean cell hemoglobin , which is strongly correlated with a decreased copy number of the 3.7 alpha - thalassemia deletion . the degree of anemia , red cell count , and hemolytic markers do not independently correlate with spo2 , thus suggesting that effects of mean cell hemoglobin and the alpha - thalassemia genotype on spo2 are mediated through effects on red cell rheology .

low hemoglobin oxygen saturation ( spo2 ) is common in sickle cell anemia ( sca ) and associated with complications including stroke , although determinants remain unknown . we investigated potential hematological , genetic , and nutritional predictors of daytime spo2 in tanzanian children with sca and compared them with non - sca controls . steady - state resting pulse oximetry , full blood count , transferrin saturation , and clinical chemistry were measured . median daytime spo2 was 97% ( iq range 9499% ) in sca ( n = 458 ) , lower ( p < 0.0001 ) than non - sca ( median 99% , iq range 98100% ; n = 394 ) . within sca , associations with spo2 were observed for hematological variables , transferrin saturation , body - mass - index z - score , hemoglobin f ( hbf% ) , genotypes , and hemolytic markers ; mean cell hemoglobin ( mch ) explained most variability ( p < 0.001 , adj r 2 = 0.09 ) . in non - sca only age correlated with spo2 . -thalassemia 3.7 deletion highly correlated with decreased mch ( pearson correlation coefficient 0.60 , p < 0.0001 ) . in multivariable models , lower spo2 correlated with higher mch ( -coefficient 0.32 , p < 0.001 ) or with decreased copies of -thalassemia 3.7 deletion ( -coefficient 1.1 , p < 0.001 ) , and independently in both models with lower hbf% ( -coefficient 0.15 , p < 0.001 ) and glucose-6-phosphate dehydrogenase genotype ( -coefficient 1.12 , p = 0.012 ) . this study provides evidence to support the hypothesis that effects on red cell rheology are important in determining spo2 in children with sca . potential mechanisms and implications are discussed .

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

single - port laparoscopic surgery ( spls ) is a development in the field of minimally invasive surgery that aims to minimize the surgical access trauma by reducing the number of abdominal incisions to a single incision . advocates of spls claimed potential advantages for this approach when compared to standard multitrocar laparoscopic surgery , such as better cosmetic results , decreased postoperative pain , or faster recovery , but proof for this is lacking . spls has been shown to be feasible in colorectal surgery in a rapidly increasing number of publications [ 14 ] . various procedures in colonic surgery have been performed in spls technique : both right and left hemicolectomies , sigmoid resections , and proctocolectomies with formation of an ileum - j - pouch have been reported ( review in [ 57 ] ) . in these studies , indications for spls colonic operations included chronic diverticulitis , crohn 's disease , ulcerative colitis , familial adenomatous polyposis , large adenoma , and carcinoma of the colon . most of these reports were limited to small patient series , demonstrating the technical feasibility of the spls procedure . in contrast , comparative studies of the spls technique with traditional laparoscopic or open surgery in larger series of patients are rare . therefore , the true value of the spls technique in colonic surgery remains unclear at present . nevertheless , the spls - technique might be interesting , especially in patients with benign disorders such as inflammatory bowel disease ( ibd ) . however , the surgical treatment of patients with ibd remains challenging , since many patients present with fistulizing disease , abscesses , cachexia , recurrent disease , and compromised healing capacity following the application of immunosuppressive drugs . the aim of this systematic review was to analyze the currently available literature on single - port laparoscopic surgery in patients with ibd with respect to feasibility , reported techniques , and safety and to identify potential benefits of this new technique in this particular group of patients . a systematic query was performed using the data bases pubmed , medline , and web of science . search terms included single - port laparoscopic surgery , colorectal surgery , single access , single incision , spls ; sas , spa ; sils , less , miss , silc , opus , simple , colon , bowel , small bowel , crohn 's disease , ulcerative colitis , and ibd . original articles , case reports , and technical notes were considered , whereas experimental studies in animal models , review articles , editorials , abstracts , and congress reports were excluded . studies combining spls with other access routes or using a robotic approach were also excluded . . publications describing spls in a mixed cohort undergoing small or large bowel surgery were considered only for the reported ibd patients , whereas those patients with appendicitis , benign large , or small bowel conditions other than ibd , or with malignant colorectal disease were excluded from analysis . data were extracted by one surgeon , experienced in both single - port and standard laparoscopic colorectal surgery . suitable articles were divided into different study types such as case reports , case series , or case - controlled studies . the studies were assessed for the following criteria : indication , spls - procedure , spls - port used , spls - port position , incision length , specimen extraction site , technical equipment , previous abdominal surgery , operation time , conversions , complications , wound infections , length of hospital stay , reoperations , and readmissions . the primary search found 155 potentially relevant studies . after eliminating studies in which the access route to the abdomen was not per spls or the organ studied was not small or large bowel , 108 studies remained . of these , 34 studies reported on spls in patients with ibd ( figure 1 ) . the selected studies were comprised of 5 case reports , 19 case series , and 10 case - controlled studies . the 34 selected studies reported on 1023 spls patients in total , including 301 patients with ibd . among these , there were 150 patients with crohn 's disease and 151 patients with ulcerative colitis . 8 studies described data of 10 or more ibd patients . however , since 5 groups of surgeons contributed more than one ( 24 ) publication to the final selection , quite a number of individuals might have been repeatedly reported , substantially reducing the actual number of reported ibd patients treated by spls technique . in contrast , 19 studies originated from researchers with only one publication on spls including ibd patients . 14 studies were restricted to spls in ibd patients only , whereas the other 20 studies included ibd patients in a mixed cohort of spls colorectal surgery . among the 14 ibd - only studies , there were 5 case reports , 6 case series including more than one ibd patient , and 3 case - controlled studies . the selected studies were published in the years 2010 ( n = 8) and 2011 ( n = 21 ) , and 2012 ( n = 5 ) , including those studies that were published online ahead of print . the reported spls procedures in ibd patients included 117 ileocolic resections ( ileocecal resection , right hemicolectomy , and ileocolic resection for recurrent crohn 's disease ) , 13 sigmoid resections , 3 left hemicolectomies , 77 subtotal colectomies with end ileostomy , 3 colectomies with ileorectal anastomosis , and 52 restorative proctocolectomies with ileum - pouch reconstruction ( tables 13 ) . furthermore , spls small bowel resections and stricturoplasties for crohn 's disease were reported . several studies that report on spls colorectal surgery in larger mixed cohorts did not specify whether the single procedures were performed in patients with ibd or in patients with other specific diagnoses [ 813 ] . 20 studies were restricted to a single type of resection , whereas 14 studies reported more than one kind of resection . 31 studies specified the type of port applied , of which 7 studies reported 24 different types of ports applied in their particular series . applied spls - ports were sils ( covidien , norwalk , ct ) in 20 studies , triport ( olympus , southend , uk and advanced surgical concepts , wicklow , ireland ) in 7 studies , quadport ( olympus america , center valley , pa and advanced surgical concepts , wicklow , ireland ) in 3 studies , gelport respectively gelpoint ( applied medical , rancho santa margarita , ca ) in 11 studies , ssl ( ethicon endosurgery , cincinnati , oh ) in 4 studies , and spider surgical system ( transenterix , durham , nc ) in 1 study . 1 study inserted 3 trocars trough a single incision tightened by a purse string , whereas other authors placed multiple trocars through the fascia separately trough a single skin incision secured by soft tissue flaps [ 4 , 10 ] . 14 studies reported the use of one or more additional trocars apart from the single port in some cases when difficulties occurred intraoperatively . the umbilicus was the most frequent site of abdominal access in spls procedures ( 20/34 ) . three authors used a paraumbilical access in patients with crohn 's disease [ 12 , 15 , 16 ] . in ibd patients undergoing a procedure with the need for an ileostomy , such as colectomy , the ileostomy site was used for insertion of the spls - port in 15 studies . other authors reported the use of the left iliac fossa as access site , whereas four authors also reported a suprapubic insertion site for the spls port [ 8 , 9 , 12 , 14 ] . 31/34 studies reported extraction of the specimen using the spls - port site , which had to be enlarged in several cases . three authors also reported transanal specimen delivery in some cases [ 1820 ] and one study reported transvaginal extraction of the excised colon . another study reported specimen delivery in a scar located at mcburney 's site in a case of enterocutaneous fistula . in studies reporting right - sided resections , ileocolic anastomoses were performed extracorporeally in most cases ( 19/22 ) and intracorporeally in one , while the method was not specified in two studies . reconstruction after left - sided colonic resection was performed transanally ( 17/20 ) using double stapling in the vast majority of studies and was only in rare cases handsewn . 24 of 34 studies reported the use of standard laparoscopy instruments for spls - procedures , whereas only three authors stated the use of specially adjusted curved spls instruments [ 9 , 21 , 23 ] . the optical systems used were flexible tip cameras in 7 studies , straight 5 mm 30 optics in 15 studies , straight 10 mm 30 optics in 9 studies , straight 5 mm 0 optic in two studies , and a straight 10 mm 0 optic in 1 study . the vast majority of the spls procedures in ibd were selected cases in a nonemergency setting . 13 studies reported exclusion criteria for spls procedures in patients with ibd : these were in particular : body habitus , respectively , bmi > 36 kg / m [ 1113 , 2327 ] , asa - classification > 3 , respectively , significant associated comorbidities [ 24 , 25 , 28 ] , hemodynamic instability , extensive previous abdominal surgery [ 2330 ] , previous history of peritonitis [ 12 , 13 ] , emergency surgery such as colonic perforation and toxic megacolon [ 8 , 12 , 13 , 23 , 26 , 28 , 30 ] , colonic dysplasia or malignancy [ 11 , 26 ] , respectively , low rectal malignancy , and pregnancy . 22 studies described spls right hemicolectomies or ileocecal resections in patients with crohn 's disease ( table 1 ) , including 4 case reports [ 817 , 2023 , 27 , 29 , 3136 ] . the predominant technique was a medial - to - lateral approach with cephaled dissection of the mesentery to the duodenum with a thermal sealing device and/or an endoscopic stapler [ 9 , 12 , 23 , 29 , 30 , 33 , 36 ] . other authors applied a posterior approach to mobilize the colon prior to mesenteric dissection [ 16 , 35 ] . the ileum and the colon were transected either intra- or extraperitoneally [ 9 , 12 , 16 ] . after extraction of the specimen at the spls port site , a side - to - side ileocolic anastomosis was performed using a stapling technique in an open extracorporeal fashion in the vast majority of the studies . some authors created a loop ileostomy in cases of complicated crohn 's disease [ 34 , 35 ] . spls subtotal colectomies with terminal ileostomy in patients with ibd were reported in 14 studies ( table 2 ) [ 8 , 11 , 13 , 17 , 19 , 20 , 2428 , 30 , 32 , 37 ] . . spls port insertion was usually accomplished at the previously marked ileostomy site [ 24 , 25 , 28 , 37 ] . for spls colectomy , most authors commenced dissection at the right hemicolon , arguing this part to be the most difficult and associated with the highest risk for conversion , followed by further clockwise dissection [ 20 , 2426 , 37 ] . other authors , however , reported an early transsection of the distal sigmoid at the level of the promontory , followed by a distal to proximal dissection of the colon close to the bowel wall . dissection of the mesocolon was performed using sealing devices and endo - staplers were applied for transsection of the rectum in all selected studies . extraction of the colon occurred at the ileostomy site followed by extracorporeal transsection of the terminal ileum , which was then turned into a terminal stoma after correct orientation of the small bowel . spls restorative proctocolectomies in patients with ulcerative colitis were reported in 12 studies [ 4 , 8 , 13 , 1720 , 26 , 27 , 3840 ] . in most of these , the spls port was inserted at the site chosen for the loop ileostomy in the right iliac fossa , while other studies reported insertion of the spls port at the umbilicus , using the ileostomy site or drain site for additional 512 mm ports in some cases [ 20 , 38 ] . in patients with previous subtotal colectomy , spls was successfully performed using the stoma site after prior mobilization of the terminal stoma . a medial to lateral approach was performed in most studies , and most authors began dissecting at the right hemicolon [ 18 , 20 , 38 ] . the entire colon was divided using sealing devices and divided at the level of the pelvic floor with an endo stapler in an anterior - posterior direction , introduced via the spls port . extraction of the colon was carried out via the port site or transanally [ 18 , 20 ] . the ileal j - pouch was constructed extracorporeally by linear staplers with a limb length of 1520 cm and reinserted into the abdomen via the port site . pouch - anal anastomosis was performed intracorporeally by double stapling [ 18 , 38 ] or , in cases of proctomucosectomy , handsewn transanally [ 18 , 20 ] . results from the literature for spls ileocecal resections and spls right hemicolectomies in crohn 's disease are depicted in table 1 . results for spls subtotal colectomies for ulcerative colitis and crohn 's disease are shown in table 2 , and results for spls restorative proctocolectomies in ulcerative colitis are demonstrated in table 3 . it is noteworthy that authors reporting on mixed cohorts of different procedures in large series of patients often do not give data for specific procedures . specific data were presented wherever possible and mixed data are indicated . reported mean or median operation times for ileocolic resections varied from 77 to 155 min , for subtotal colectomy with end ileostomy from 112 to 206 min , and for reconstructive proctocolectomy with ileal pouch from 153 to 300 min . reported median incision length was 35 ( 2055 ) mm . several authors reported widening the initial incision for extraction of the specimen in crohn 's disease patients with enlarged mesentery . for all spls procedures in ibd , cases of conversions to multiport surgery were reported in 14 studies and cases of conversion to open surgery were reported in 10 studies . reasons for conversions were medically related issues such as intraoperative bleeding , firm adhesions and previous surgery [ 12 , 20 , 27 , 29 ] , fistulizing disease ( interenteric fistula , conglomerate tumors , or masses [ 8 , 16 , 20 ] , friability of the inflamed mesentery , obesity [ 8 , 30 ] , or technically related aspects such as gas leak , instable port placement , inappropriate traction [ 8 , 12 , 29 ] , difficulties in flexure mobilization , and time constraints . these complications included anastomotic leakage , bleeding , ileus , bowel obstruction , intraabdominal abscesses , wound infections , delayed thermal injury to bowel , peristomal emphysema , ejaculation dysfunction , acute urine retention , incisional hernia , stenoses , and cardiovascular , pulmonary , and thromboembolic events ( tables 13 ) . mortality was reported in 4 studies [ 8 , 12 , 29 , 36 ] and specified in 3 of them . one case of mortality was reported after substantial intraoperative bleeding during externalization of the colon for an extracorporeal anastomosis after right hemicolectomy . another case of mortality due to pulmonary embolism was found in one study , although it remains unclear whether this was a patient with ibd . a third case of mortality due to cardiopulmonary failure was reported in a patient undergoing spls sigmoidectomy for complicated diverticulitis . the current review of the literature shows that single - port laparoscopic surgery has gained entrance into the surgical treatment of patients with inflammatory bowel disease . the number of publications on the subject is growing at a fast pace : whereas first case reports arose in 2010 , larger case series from specialized centers are now available that demonstrate the feasibility of spls in ibd . additionally , some comparative studies have been published lately , mostly comparing spls to historical cohorts of patients with traditional multiport laparoscopic surgery . evidence from prospectively designed , randomized studies concerning spls in ibd is not presently available . most of the currently available studies on the application of spls in colorectal surgery which include ibd patients are not restricted to single procedures in single pathological conditions , but rather describe mixed cohorts . as a consequence , it is not yet possible to perform a proper meta - analysis in order to evaluate the techniques in detail . however , it appears that nearly all ibd - related procedures that can be performed by standard multiport laparoscopy have now been performed in single - port technique as well . although this has mostly been done by specialized surgeons , it demonstrates the general feasibility of spls in ibd . the spls procedures include stricturoplasties , small bowel resections , ileocolic resections , sigmoid resections , subtotal colectomies with terminal ileostomies , and reconstructive proctocolectomies with ileal pouches . spls proctocolectomy for ulcerative colitis has been reported in minors , too . however , from the available literature , it becomes apparent that most authors applied spls predominantly in selected patients , and therefore spls is currently still far from becoming a routine procedure in ibd patients . emergency cases were excluded from spls in the vast majority of publications [ 16 , 2426 , 30 ] . from a technical point of view , most authors favor regular laparoscopic instruments , although a special 5 mm optic with a flexible tip seems to be rewarding in spls colorectal procedures . most authors applied commercially available spls ports , which were inserted through the umbilicus , paraumbilically , at the ileostomy site , or suprapubically depending on the specific procedure and the surgeon 's preference . spls was performed for ibd in patients with prior ( limited ) abdominal surgery , but also in patients with recurrent crohn 's disease [ 14 , 34 , 35 ] or enterocutaneous fistula and abscesses [ 22 , 35 ] . spls in experienced hands may therefore be a feasible approach even in complex patients . limitations of spls in ibd patients appear to be similar to those encountered in standard multitrocar laparoscopy . reasons for conversions were stated as occurrence of intraoperative bleeding , bowel injury , firm adhesions , intraenteral fistula , and masses . these reasons were also stated in the literature for ibd patients undergoing conversion during standard laparoscopic resections [ 4145 ] . in terms of patient safety , postoperative complications reported include anastomotic leakage , bleeding , bowel obstruction , and intraabdominal abscesses . these are typical complications of colorectal surgery in ibd as seen in both standard multitrocar laparoscopic and open surgery [ 46 , 47 ] . in contrast , delayed thermal injury as reported in two studies indicates inappropriate instrument handling in spls . a reduction of the frequency of wound infections by reducing the number of incisions using spls is not likely to occur . the incidence of late complications such as incisional hernia should be objectified in future studies on the long - term outcome of spls patients . furthermore , ibd - specific long - term complications such as recurrence of stenoses in crohn 's disease or pouchitis in ulcerative colitis are not likely to be influenced by the technique used for access to the abdomen in the primary operation . a reduction of peritoneal adhesions and consecutive bowel obstruction was postulated to be achieved by spls , but there are no long - term studies available so far which confirm this hypothesis . surgery in patients with ibd does not differ substantially from surgery for other conditions , but the patients undergoing these procedures are often complex and challenging due to a previous history of the disease , nutritional status , septic manifestations such as fistulas and abscesses , and/or immunosuppresive drugs . in the present review of the literature , no specific data on the patient 's exposure to immunosuppressive drugs could be retrieved . some of the selected studies , however , reported preoperative administration of azathioprine , steroids , or biologicals [ 8 , 16 , 24 , 25 , 28 , 35 , 37 ] , indicating that the application of these drugs does not represent a contraindication for spls . in patients undergoing restorative proctocolectomy for medically refractory ulcerative colitis , a three - stage spls procedure was advocated when patients received more than 20 mg of prednisolone or anti - tnf- agents such as infliximab or adalimumab . in some studies , benefits of spls in colorectal procedures such as shorter hospital stays [ 11 , 15 ] , reduction of estimated blood loss , reduced time to flatus and bowel movement , or better cosmetic results were claimed , but results from these studies appear to be limited by inhomogeneous cohorts , small sample size with low statistical power , or possible selection bias . a small randomized prospective study including 16 spls patients and 16 patients with standard laparoscopic surgery in colon cancer found no differences in terms of morbidity and operation time . in the available literature on spls in ibd , the present review of the literature shows the feasibility of spls in patients with ibd in selected cases . the patient selection however depends on the surgeon 's experience and the patient 's condition . currently , the literature on spls techniques in ibd is shifting from case reports on single applications to reports on larger series . at present evidence from prospectively randomized trials is required to clarify whether there is a true benefit compared to standard laparoscopic techniques .

background . single port laparoscopic surgery ( spls ) is being increasingly employed in colorectal surgery for benign and malignant diseases . the particular role for spls in inflammatory bowel disease ( ibd ) has not been determined yet . in this review article we summarize technical aspects and short term results of spls resections in patients with crohn 's disease or ulcerative colitis . methods . a systematic review of the literature until january 2012 was performed . publications were assessed for operative techniques , equipment , surgical results , hospital stay , and readmissions . results . 34 articles , published between 2010 and 2012 , were identified reporting on 301 patients with ibd that underwent surgical treatment in spls technique . surgical procedures included ileocolic resections , sigmoid resections , colectomies with end ileostomy or ileorectal anastomosis , and restorative proctocolectomies with ileum - pouch reconstruction . there was a wide variety in the surgical technique and the employed equipment . the overall complication profile was similar to reports on standard laparoscopic surgery in ibd . conclusions . in experienced hands , single port laparoscopic surgery appears to be feasible and safe for the surgical treatment of selected patients with ibd . however , evidence from prospective randomized trials is required in order to clarify whether there is a further benefit apart from the avoidance of additional trocar incisions .

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

spinal and bulbar muscular atrophy ( sbma ) is a slowly progressive adult onset neurodegenerative disease that affects men in mid - life . the disease causes degeneration and atrophy of the muscles of the proximal limb girdle and the bulbar muscles of the jaw ( kennedy et al . , 1968 ) , as well as the accumulation of nuclear aggregates of mutant androgen receptor ( ar ) protein , otherwise known as neuronal intranuclear inclusions ( niis ) ( li et al . , 1998 ) . sbma results from a cag expansion in the first exon of the ar gene , located on the proximal long arm of the x chromosome ( la spada et al . , 1991 ) . in the rare instances when female carriers manifest disease , their symptoms are mild ( ferlini et al . , 1995 ) . initially , it was unclear whether female carriers were protected from disease symptoms owing solely to x - inactivation . however , the finding of homozygous females with very mild symptoms ( schmidt et al . , 2002 ) and evidence from several cell and animal models of sbma indicate that testosterone plays a crucial and causative role in disease pathogenesis . cellular models of disease show an increase in nuclear inclusion formation upon hormone treatment ( stenoien et al . , 1999 ; walcott and merry , 2002 ) , whereas fly models demonstrate that consumption of testosterone induces disease as well as nuclear inclusion formation ( takeyama et al . , 2002 ) . transgenic mouse models have indicated that both surgical and chemical castration of sbma males alleviates or prevents disease progression , depending on the stage of disease at the time of intervention ( katsuno et al . , 2002 ; furthermore , administration of testosterone to transgenic female sbma mice exacerbates disease phenotype ( katsuno et al . , 2002 ) , as does giving testosterone to male sbma mice treated with leuprorelin , a lutenizing hormone - releasing hormone ( lhrh ) analog ( katsuno et al . , 2003 ) . for years , many patients with sbma received testosterone treatment . the rationale for this treatment was complex , but a partial loss of ar function coupled with the known anabolic effects on muscle suggested that testosterone might prove beneficial ( sheffield - moore , 2000 ) . despite the evidence indicating that testosterone contributes to the progression of sbma , these patients have not reported any negative effects as a result of androgen administration ( goldenberg and bradley , 1996 ; neuschmid - kaspar et al . , 1996 ) . however , one case study reported on the substantial decline of a patient with sbma , with subsequent recovery upon cessation of testosterone treatment ( kinirons and rouleau , 2008 ) . however , the fact that this patient was taking paroxetine , a selective serotonin reuptake inhibitor ( ssri ) , might complicate the interpretation of this case . paroxetine has been shown to enhance the conversion of dihydrotestosterone ( dht ) to androstanediol ( griffin and mellon , 1999 ) , and thus there might have been relatively low relative levels of dht within his spinal motor neurons . treatment of this patient with testosterone might have restored his dht levels and exacerbated disease . previously , we created and characterized mice that were transgenic for the full - length expanded human ar gene , containing 112 cag repeats , driven by the prion protein ( prp ) promoter ( ar112q mice ) ( chevalier - larsen et al . , 2004 ) . the disease in this model progresses slowly , with ar112q males beginning to exhibit mild motor dysfunction at 3 months of age and developing late - stage disease by 10 months of age ( chevalier - larsen et al . , 2004 ) . , we treated a cohort of these ar112q male mice with testosterone , beginning at 2 months of age , for a period of 6 months . behavioral and histopathological measures of disease indicate no change in severity or age of onset of disease with testosterone treatment ( t - treatment ) . these data have important clinical implications for patients and suggest that there is a point at which the role of hormone in the pathogenic mechanism of sbma becomes saturated . ( a ) testosterone treatment resulted in elevated serum testosterone levels in transgenic ( tg ) t - treated ( n=8 ) and non - transgenic ( ntg ) t - treated ( n=10 ) animals when compared with tg sham - treated ( n=6 ) mice ( p<0.05 ) , with the exception of months three and six . although , testosterone levels dropped at the end of pellet life , testosterone levels were still significantly elevated in ntg t - treated mice in month six and there was a trend towards elevated testosterone in tg t - treated animals . ( b ) ability to remain on a rotarod was impaired in ar112q t - treated ( n=10 ) and ar112q sham - treated ( n=12 ) males when compared with t - treated non - transgenics ( n=14 ) ( p<0.05 ) , but rotarod performance was similar between t - treated ar112q males and sham - treated ar112q males ( p>0.05 ) . ( c e ) grip strength [ using forepaws ( c ) or all paws ( d ) ] and vertical activity ( e ) of all ar112q males was reduced in comparison to t - treated non - transgenic males ( p<0.05 ) , but no significant decrease in any of these measures was observed upon t - treatment of ar112q males ( p>0.05 ) . ( f ) clasping behavior increased with age in all ar112q males and was not affected by t - treatment ; non - transgenic males rarely showed clasping behavior . transgenic ar112q and non - transgenic mice were implanted with timed - release pellets designed to deliver 46 ng / ml of testosterone for 90 days . over the course of the experiment , implantation of testosterone pellets increased circulating testosterone an average of threefold , from 0.400.23 ng / ml ( ar112q sham - treated ; n=10 ) to 1.260.48 ng / ml [ non - transgenic ( ntg ) t - treated ; n=8 ] and 1.170.35 ng / ml [ transgenic ( tg ) t - treated ; n=6 ] ; this is a significant elevation of testosterone levels in treated animals ( ntg t - treated and tg t - treated ) over those implanted with placebo pellets ( tg sham - treated ) ( p<0.001 ) . although 90-day testosterone pellets were used , we observed a decline in potency of the pellets by the end of the 90-day period ( months three and six ) ( fig . implantation of new pellets at the end of 90 days restored circulating testosterone levels ( fig . although circulating testosterone was elevated in treated mice , motor function assays did not reveal any effect of testosterone treatment on phenotype . motor function assays were performed monthly for 6 months ; results were consistent for all 6 months of treatment . beginning at 3 months of age ( 1 month following treatment ) , ar112q males showed decreased rotarod performance , regardless of t - treatment , when compared with non - transgenic t - treated males ( fig . 1b ) . decreased grip strength was apparent in all ar112q animals , regardless of t - treatment , when animals gripped with either forepaws only ( fig . 2004 ) , weakness was more pronounced when assessing all paws , implying greater weakness in the hindlimbs . vertical activity , or rearing behavior , was also reduced in ar112q males ( fig . 1e ) , although no deficits were seen in t - treated ar112q males when compared with sham - treated ar112q males . clasping behavior was greater in both t - treated and sham - treated ar112q males when compared with t - treated non - transgenic mice and increased steadily with time ( fig . t - treatment had no effect on weight or survival and no gross alterations in muscle fiber morphology were observed ( data not shown ) . cellular markers of disease were also examined in spinal cord and brain tissue to determine whether t - treatment expedited cellular dysfunction that might not be detected by behavioral changes . as with the behavioral analyses , no changes were detected between the t - treated and sham - treated ar112q males . niis were present in both t - treated and sham - treated males ; nii size and frequency were similar between these two groups ( fig . a total of 49.66.5% of motor neurons from lumbar spinal cord contained niis in tg sham - treated animals , whereas niis were seen in 48.510.5% of motor neurons in tg t - treated mice ( fig . similarly , the level of unphosphorylated neurofilament heavy chain ( nf - h ) was reduced in the neuronal soma of all ar112q animals when compared with non - transgenic mice ( fig . 2a ) , and the extent to which unphosphorylated nf - h immunoreactivity was reduced was similar between all ar112q animals , regardless of t - treatment ( fig . 2c ) . in summary , signs of cellular pathogenesis were consistent with those observed during characterization of this disease model and were unchanged by long - term treatment with testosterone . although it is clear that testosterone plays a key role in sbma pathogenesis , the mechanism leading to neurodegeneration remains unknown , although an increased understanding of the specific aspects of ar metabolism that contribute to its misfolding , aggregation and toxicity have come to light in recent years ( montie et al . the data from this study suggest that , whatever the mechanism that leads to neuronal dysfunction , there is a threshold at which the effect of testosterone on this pathway plateaus . it is therefore likely that testosterone would have a greater capacity to exert its pathogenic effects in ar112q mice than in sbma patients expressing normal levels of ar . despite their increased capability to bind ligand , testosterone in excess of endogenous levels niis were detected with ar - h280 antibody in spinal cord of all ar112q males ( a , top ) ; no differences in nii frequency were detected upon t - treatment ( b ; n=2 ) . nuclei of non - transgenic ( ntg ) t - treated males show diffuse nuclear ar and no niis ( a ) . unphosphorylated nf - h , as detected by smi32 immunostaining ( a , bottom ) , was abundant in neuronal soma of the non - transgenic spinal cord but was reduced in ar112q tissue . ( c ) quantification of smi32 intensity did not reveal any differences between transgenic ( tg ) t - treated and tg sham - treated males ( n=2 ) . panels showing niis ( a , top ) and smi32 ( a , bottom ) are not from the same fields . insets in a represent the location within the anterior horn from which higher - magnification images were taken ( yellow box ) . the red oval outlines the central canal , with the dorsal aspect to the left and the ventral aspect to the right . several previous studies have demonstrated that hormone withdrawal can ameliorate disease onset and/or progression ( katsuno et al . , 2002 ; katsuno et al . , 2003 ; chevalier - larsen et al . it is possible that the brief decline in elevated testosterone levels seen at the end of month three of these studies might alleviate any adverse effects of hormone treatment . this seems unlikely , however , because behavioral improvement due to androgen ablation was not observed in mice of a comparable age until 3 months following castration ( chevalier - larsen et al . , 2004 ) and a caveat to the studies reported here is that our transgenic mice express the mutant ar primarily in the nervous system , through the use of the prp promoter , and thus the evaluation of the mutant protein ( and added testosterone ) in muscle , which plays an important role in disease pathogenesis ( yu et al . , expression of the prp - promoter - driven transgene in muscle was observed , albeit at levels lower than those in neural tissue ( chevalier - larsen et al . , 2004 ) , and nuclear inclusions in muscle were also noted ( data not shown ) . whether testosterone treatment would exacerbate disease through effects on muscle is unknown , although we find the possibility of different pathogenic processes in motor neurons and muscle unlikely . mutant ar protein might exert its toxic effects on neurons through multiple pathways , including disruption of protein folding homeostasis , interference with protein degradation , transcriptional dysregulation and/or by inhibition of axonal transport . it is possible that a threshold level of mutant ar present in the nucleus would lead to maximal disruption of neuronal function . in motor neurons , testosterone and dht ; thus , an excess of these ligands might actually moderate the prevalence of the mutant ar . it is likely that dht , not testosterone , is the ligand responsible for neuronal pathology in sbma , given the high motor neuron levels of the testosterone - to - dht converting enzyme 5-reductase ( pozzi et al . , 2003 ) . in this case , the conversion of testosterone to dht via 5-reductase could represent a point of saturation in the pathological pathway . this hypothesis is particularly attractive because it would suggest a mechanism for cell - type specificity in sbma . however , manipulation of 5-reductase activity with the inhibitor dutasteride failed to modulate disease in sbma patients ( fernandez - rhodes et al . , 2011 ) or in a transgenic mouse model of sbma regardless of the molecular mechanism by which this saturation occurs , our data are encouraging in that sbma patients who had previously taken supplemental testosterone are unlikely to have exacerbated their disease . genotyping was performed as described previously ( chevalier - larsen et al . , 2004 ) . the area between the shoulder blades was shaved and the surgical area sterilized with alcohol . a small ( 12 cm ) dorsal midline incision was made . a pellet containing a 90-day - release 12.5 mg testosterone pellet or a placebo pellet ( both from innovative research of america ) was placed into the pocket and the incision was closed with a wound clip . mice were re - implanted with hormone or sham pellet once during the course of this study . all animal surgeries were performed according to the guidelines of the office of laboratory animal welfare ( olaw ) and the institutional animal care and use committee ( iacuc ) at thomas jefferson university . sera were assessed for circulating testosterone levels using a colorimetric eia kit ( caymen chemical ) according to the manufacturer s instructions . testing was carried out as previously described ( chevalier - larsen et al . , 2004 ) . immunofluorescence of brain and spinal cord tissue was carried out as previously described ( chevalier - larsen et al . , 2004 ) . antibodies used include ar h280 ( 1:100 ; santa cruz ) and smi32 ( 1:1000 ; sternberger monoclonal ) . motor neurons of the lumbar spinal cord were scored as inclusion - positive whether they had several punctate or single , large inclusions . unphosphorylated neurofilament immunoreactivity was quantified using imagej software ; integrated density values were acquired for regions of interest delineated around motor neuron soma of the lumbar spinal cord . spinal and bulbar muscular atrophy ( sbma ) is a neurodegenerative disorder that is caused by a polyglutamine expansion in the androgen receptor ( ar ) and affects approximately 1 in every 40,000 men . affected men typically develop muscle weakness of the proximal limbs and bulbar muscle , tremors and fasciculations in mid - life . signs of androgen insensitivity , such as gynecomastia and reduced fertility , often accompany muscle deficits . female carriers of the disease are protected both by x - inactivation of the gene encoding the mutant ar and by lower levels of circulating ar ligands ( testosterone and dihydrotestosterone ) . prior to the discovery that disease pathogenesis is androgen - dependent , many individuals with sbma received androgens therapeutically , based on the assumption at the time that a loss of ar function contributed to disease symptoms . this study examines the impact of exogenous testosterone on disease progression in a mouse model of sbma to determine whether testosterone treatment of the patient population might in fact have exacerbated the disease . the data indicate that the addition of exogenous testosterone does not exacerbate disease progression in intact male mice that are transgenic for a mutant ar . behavioral measures of disease in testosterone - treated transgenic males are indistinguishable from transgenic males that are sham treated . additionally , cellular markers of disease in testosterone - treated transgenic males are comparable to those in sham - treated transgenic males . implications and future directions these data suggest that it is unlikely that androgen therapy has hastened the progress of disease in sbma patients . they also suggest that there is a threshold at which the contribution of testosterone to disease pathogenesis becomes saturated .

summaryevidence from multiple animal models demonstrates that testosterone plays a crucial role in the progression of symptoms in spinal and bulbar muscular atrophy ( sbma ) , a condition that results in neurodegeneration and muscle atrophy in affected men . mice bearing a transgene encoding a human androgen receptor ( ar ) that contains a stretch of 112 glutamines ( expanded polyglutamine tract ; ar112q mice ) reproduce several aspects of the human disease . we treated transgenic male ar112q mice with testosterone for 6 months . surprisingly , testosterone treatment of ar112q males did not exacerbate the disease . although transgenic ar112q males exhibited functional deficits when compared with non - transgenics , long - term testosterone treatment had no effect on motor function . testosterone treatment also failed to affect cellular markers of disease , including inclusion formation ( the accumulation of large nuclear aggregates of mutant ar protein ) and levels of unphosphorylated neurofilament heavy chain . these data suggest that the mechanism of disease in sbma saturates at close to endogenous hormone levels and that individuals with sbma who take , or have taken , testosterone for its putative therapeutic properties are unlikely to suffer adverse effects .

INTRODUCTION RESULTS DISCUSSION METHODS

sturge - weber - krabe syndrome ( sws ) , also known as encephalotrigeminalangiomatosis and named the forthfacomatosis , recall the names of the authors who first described it in its basic clinical , radiological , and anatomopathologicalaspects . sturge - weber syndrome is a neurocuta - neoussyndrome , characterized by the association between facial port - wine hemangiomas in the trigeminal nerve distribution area and vascular malformation(s ) of the brain ( leptomeningealangioma ) with or without glaucoma . although sws is a congenital disorder , which usually presents in infancy , occasionally neurological symptoms first present in adulthood . most affected individuals do survive into adulthood with varying degrees of neurological impairment , including epilepsy , hemiparesis , visual field deficits , and cognitive impairments ranging from mild learning disabilities to severe deficits . sws is a multisystem disorder that requires the neurologist to be aware of the possible endocrine , psychiatric , ophthalmological , and other medical issues that can arise and impact the neurological status of these patients . seizures , stroke - like episodes , glaucoma , headache , and developmental delay are frequent associated features . this study describes 14 subjects with sws who express various characteristics of the clinical spectrum of the syndrome . our study reports 14 cases of sturge - weber disease , 6 male and 8 female patients , studied in follow up . we observed 10 cases since they presented seizures , and 4 cases for the presence of a severe picture of psychomotorial retardation . in 5 of 10 cases , present an extension of the angioma in other body parts , and , particularly in one case , the nevus extends itself on the whole left lower lateral aspect up to the foot , in another case it extends itself on the breech and on both lower limbs , in 2 other cases it extends itself on the dorsum and in the omphalic area . in table 1 we report the main features of the syndrome both described in literature and those observed and/or detected also by neurodiagnostic exams in our cases , i.e. brain computed axial tomography ( cat ) and nuclear magnetic resonance ( nmr ) in our cases . each case with epilepsy has reached a good level of control of the crises without using any invasive therapy , and this has certainly slowed down the progressive course of the syndrome . of course in these cases it is necessary , on the basis of the long survival of patients , to first give pharmacological and neuropsychomotor therapies , but also a psychotherapeutic support to patients . it should be noted that the literature does not take psychotherapeutic support into consideration . in most cases , relationship difficultiesare highlightedon the basisofalow self - esteemthat can be related to anexperience of illnessthattends toisolatethesesubjects . facialangiomacertainly hasled tothe emergence of aself - image characterized by depressive elements with features of self - depreciation that interfere also in one patient s work at school . sturge weber syndrome ( sws ) , also called encephalotrigeminalangiomatosis , is a sporadically occurring neurocutaneoussyndrome , characterized by vascular malformation with capillary venous angiomas that involve face , choroid of eye and leptomeninges with resulting neurological and orbital manifestations . as far as the nature of angiopathy is concerned , the vascular alterations is not an angioblastoma . the prevalent orientation is to consider the alteration as a malformation that starts prematurely during intrauterine life . the subarachnoid and choroid angiodysplasia , like the cutaneous one , is made by mature vessels , without any note of endothelial proliferation ; that explains why it is so resistant to the ionogetic radiation treatments with antiblastic chemotherapies , withglycoactive steroids . the nevus of the face , evident already at the birth , has the color of port - wine , it is located in the orbital area , extending itself generally in the frontal area and also on the cheek in a distribution corresponding to the first and the second branch of trigeminus , exceptionally to the whole hemicorpo . it can also be located in the oral cavum , in the pharynx , in the throat , in the tongue , inside the nose , on the eyelid conjunctiva , in the episclera , in the retrobulbar orbit causing proctosis with hipomobility of the bulb and through the orbital fissures ; and it can have endocranial development in the extradural site . the lesion is flat , just palpable and it decolorizes onvitropression . over the years , a progressive development of the anomalous vascularity has been noted and the nevus becomes prominent , thicker , darker , mamelonated , and sometimes hypertrophic . the leptomeningealangiomatosis is located mainly in the occipital areas and in occipital parietal areas of a cerebral hemisphere . its structural and developing characteristics recall those of the red nevus ( it is also a progressive developing ectasiant capillary venous angiodysplasy ) . under the vascular mass the cerebral tissue meets a gradual process of atrophy with a dilatation of the homolateralventriculum and increase insubarachnoid spaces . the subintimal calcification of meningeal arteries is associated to the cerebral atrophy ; it extends gradually at the intima and at the media giving to the vessels the appearance of petrified tubes . the calcification process takes place mainly in the intravascular parenchyma with accumulation , in the external layers of the cortex , of pulverized granules of calcium salts that form heaps which progressively increase and replace the cerebral tissue . the calcium accumulations , absent at birth and exceptional in the newborn , are formed later . they translate themselves into a peculiar radiological picture of the syndrome : the ring - shape calcifications , appearing with parallel spires , are 2 - 3 mm apart . severe calcification in the affected hemisphere is related to severely decreased perfusion in underlying white matter and is associated with more severe epilepsy in sws patients . the presence of cerebral calcifications , evident at cat and at mr has a negative prognostic meaning since it stresses the unavoidable and irreversible deterioration of the child s condition . the direct visualization of the leptomeningealangioma is very inconstant with tomodensitometry or with arteriography ; the encephalon nmr reveals an atrophy and involvement of white mass and of the deep venous dilatations on sequences in t2 . recently sequences have also been used in t1 after an injection of gadolinium in order to visualize the leptomeningeal angioma . a congenital and hypsilateralangioma of the choroid can be present : this angioma can determine a light blue staining of the iris ( even in the case of the dark color of the other eye ) . it is usually located at the posterior pole in the inter papillary macular area where a diskshapeunpigmented white - gray stain turns to red . the structural and evolutive characteristics of this vascular anomaly recall those of the letangiectatic nevus the area of which is related to it . the hypertension , acting in a phase of the development in which the structures of containment of the globe ( sclera and cornea ) are more pliable , causes exhaustion and then dilatation . the glaucoma can be present at birth with megophthalmo , megalocornea and other anomalies such as coloboma of iris , deformity of the lens , or it ninety percent of cases present focal or generalized convulsions that often represent the onset of neurological symptoms and quite often these start during the first year of life . the seizures , which at first can be of the jacksonian type , controlateral and later generalized , later become more and more frequent , and show little sensitivity to pharmacological treatment . the seizures are associated to controlateral hemiparesis . the first attacks are temporybut later become persistent with spastic alterations , disturbances of sensibility , deficit of limb growth of the hemiparetic side . moreover , they are associated to homonymous hemianopsia , a consequence of the angiomatosis location of the occipital lobe . the outcome of hemispherectomy in pediatric patients is good for those with refractory epilepsies , such as west syndrome , lennox - gastaut syndrome , epileptic encephalopathy with continuous spikes and waves during slow sleep , and startle epilepsy arising from a hemispheric lesion associated with hemiplegia . the aphasia is frequent and follows the seizural symptomatology revealing itself just the first one of them . it has been noted in 80% of cases , sometimes in early infancy , other times after normal psychological development after the early infancy ; for this reason it has been called nevus amentia . a significant quadratic relationship was found between iq and extent of severe ( but not total ) hypometabolism . results suggest that intermediate size of severe hemispheric hypometabolism is associated with the worst cognitive outcomes , and small or absent lesions , with the best cognitive outcomes ; children in whom a very large extent of the hemisphere is severely affected are likely to have relatively preserved cognitive function . nevertheless cognitive and fine motor functions are related to diffusion abnormalities in specific ipsilateral , mostly frontal , wm regions . quaintness in behavior , hyperactivity , irritability , muddle , suicide and homicidal trends are frequent . the most frequent diagnoses were mood disorder ( 31% ) , disruptive behavior disorder ( 25% ) , and adjustment disorder ( 25% ) . a significant association was found between disruptive behavior disorder not otherwise specified and more left frontal and left parietal involvement . a trend toward significant association of having a seizure in the past three months with disruptive behavior disorder not otherwise specified was observed . problems with mood , attention , sleep , learning , and substance use were common . there are also symptoms related to infundibulum - hypophyseal deranged functioning ( growth disorder , acromegaly , insipid diabetes , adipositas , genital adipicdystrophia ) . sturge - weber - krabe syndrome is a multisystem disorder that requires the neurologist to be aware of the possible endocrine , psychiatric , ophthalmological , and other medical issues that can arise and impact the neurological status of these patients . two elements are interesting : the exceptional diffusion to the whole hemicorpo of the red nevous and the evaluation of the living patterns related to the disease that is not considered in literature . on the contrary , this is an important factor since new pharmacological therapies can obtaina good control of the epilepsy and consequently an extension of life , and less deterioration of the patient s condition .

sturge - weber - krabe syndrome ( sws ) , also known as encephalotrigeminalangiomatosis and named the forthfacomatosis , recall the names of the authors who first describedit in its basic clinical , radiological andanatomopathological aspects . we report here 14 cases of sturge - weber disease . in 6 of these , despite what had been previously described in literature , an extension of the angioma has been noted in other parts of the body . the study of these subjects stresses not only the need for a pharmacological / neuropsychomotor intervention , but alsothe need of a psychotherapeutic approach , for the emotional and affective implications thatcould derive from this syndrome . the reported cases are similar to those presented in literature for their main features . in particular , two elements are interesting : i ) the exceptional diffusion of the red nevousto the whole hemicorpo ; and ii ) the evaluation of the way the patients live the disease , which has not beenpreviously considered in literature . we can conclude that sws is a multisystem disorder that requires the neurologist to be aware of the possible endocrine , psychiatric , ophthalmological , and other medical issues that can arise and impact on the neurological status of the patients .

Introduction Case Report Discussion Conclusions

the sunlight - driven generation of the energy carrier h2 from water using earth - abundant materials is considered one of the key processes to generate more sustainable fuels in a postfossil age . synthetic first - row transition - metal complexes containing co , fe , and ni are under intense development as scalable alternatives to the benchmark h2 evolution catalysts platinum and hydrogenases . complexes with a catalytic bis(1,5-r-diphospha-3,7-r-diazacyclooctane)nickel(ii ) core , [ ni(p2n2)2 ] , developed by dubois and co - workers , emerged in the past decade as probably the most active synthetic 3d transition - metal electrocatalysts for proton reduction . the catalytic cycle has been studied experimentally and computationally , and the catalyst mimics important features of the hydrogenase active site . photocatalytic h2 generation requires the efficient coupling of an efficient proton reduction electrocatalyst with a light - harvesting component . in particular , the development and investigation of molecular h2 evolution catalysts made of abundant elements that show stability and activity in water are of major interest . many reports on homogeneous photocatalytic systems for water reduction using fe or co complexes are available , but there are relatively few using ni - based catalysts . heterogenization of well - defined molecular catalysts on semiconductor surfaces is an emerging approach for photocatalytic h2 generation . systems that have been shown to have notable h2 production efficiencies include cobaloximes immobilized on tio2 nanoparticles or cdse / zns quantum dots , and [ fefe]-hydrogenase mimics on cdte or zns quantum dots . efforts have also been made to integrate molecular catalysts on photoelectrodes , such as an [ fefe ] complex on inp , a cobaloxime catalyst on p - type gap , and fe(dithiolato)(diphosphine ) or cobaloxime catalysts on dye - sensitized nio films . however , the frequent use of organic solvents , poor light - to - h2 conversion efficiencies , and photoinstabilities are drawbacks in these systems . despite the promising properties of dubois - type catalysts , they were previously only applied in homogeneous photocatalytic schemes in the presence of organic solvents or a biological matrix . surface immobilization of a [ ni(p2n2)2 ] derivative on carbon nanotubes yielded highly active electrodes for h2 generation under strongly acidic conditions . when a [ ni(p2n2)2 ] derivative was immobilized on a p - type silicon photoelectrode , no ( photo)activity for h2 production was reported . water - soluble [ ni(p2n2)2 ] derivatives were previously not shown to operate in aqueous homogeneous or heterogeneous environments for photocatalytic h2 generation . in this work , we report on a novel dubois - type catalyst ( nip ; figure 1 ) , which not only contains a highly electroactive [ ni(p2n2)2 ] core , but also is surrounded by an outer sphere containing four dangling phosphonic acid moieties . this outer - sphere feature provides the catalyst with good solubility in aqueous solutions and anchors for immobilization onto metal oxide surfaces . electrochemical studies of nip in an aqueous electrolyte solution demonstrated sustained electrocatalytic activity in the absence of organic solvents . subsequently , we studied nip in aqueous photochemical systems with a phosphonated bipyridine - based ru(ii ) photosensitizer ( rup ; figure 1 ) in both a homogeneous system and heterogeneous photocatalytic environments where rup and nip were immobilized on different metal oxide semiconductors . chemical structures of the doubly charged cationic complexes used in this study : the electrocatalyst nip and dye rup . the kinetics of charge separation and recombination were studied by time - correlated single photon counting ( tc - spc ) and transient absorbance spectroscopy ( tas ) . this spectroscopic study reveals that distinct reaction mechanisms are possible in these remarkably versatile photochemical systems . in particular , we consider whether this coimmobilization strategy on metal oxide surfaces reduces the rapid electron / hole recombination losses typically observed when such photosensitizers and catalysts are covalently attached via molecular linker groups . the visible - light - driven h2 generation of the different systems was also studied in bulk experiments and revealed that nip can operate with a high photocatalytic turnover frequency for a ni - based molecular proton reduction catalyst in purely aqueous systems . all synthetic procedures involving air- or moisture - sensitive materials were carried out under n2 by using either a glovebox or schlenk techniques . chemicals for the synthetic part of this work were purchased from commercial suppliers and used without further purification . tio2 nanoparticles ( aeroxide tio2 p25 particles ; anatase / rutile ( 8/2 ) mixture , average particle size 21 nm ) were a gift from evonik industries , and zro2 nanoparticles ( 99.9% , 2030 nm ) were obtained from skyspring nanomaterials inc . nanostructured anatase tio2 and zro2 films were prepared by the doctor blading technique from colloidal pastes as reported previously . the films were annealed at 450 c for 30 min prior to use , and the anatase phase for tio2 was confirmed by x - ray diffraction studies after heating . the resulting film thicknesses , determined by profilometry ( tencor instruments ) , were 4 m . [ ni(p2n2)2](bf4)2 ( nip ) ( figure s1 , supporting information ) and rup(24 ) were prepared according to published procedures . h nmr spectra were referenced to the solvent residual peaks as an internal reference , and p nmr spectra were referenced to an external standard ( 85% h3po4 in d2o ) . vis spectra were recorded on a varian cary 50 uv vis spectrophotometer using quartz glass cuvettes . high - resolution electrospray ionization mass spectra ( hr - esi - ms ) were recorded on a quattro lc spectrometer , and the theoretical and experimental isotope distributions were compared . elemental analysis was carried out by the microanalytical laboratory in the department of chemistry of the university of cambridge . bromotrimethylsilane ( 0.25 ml , 1.86 mmol ) was added dropwise to a deep red solution of nip ( 250 mg , 0.143 mmol ) in degassed dichloromethane ( 15 ml ) under a n2 atmosphere at room temperature . the color changed to deep purple , and the solution was stirred for 2 days at room temperature . the solvent was removed , followed by addition of deoxygenated meoh ( hplc grade , 15 ml ) to the dark solid residue and stirring under n2 for 1 day at room temperature . the volume of the solvent was reduced to approximately one - third , and the product was precipitated with et2o ( 25 ml ) . after the mixture was stirred for 30 min , the deep purple precipitate was filtered off under n2 , washed with et2o , and dried in vacuo . the solid was redissolved in meoh , the solution was stirred for several minutes , and the solvent was evaporated under high vacuum to give a purple solid . h nmr ( 400 mhz , cd3od , ) : very broad signals , 8.236.70 ( m , 36h , ph ) , 3.604.70 ( m , 16h , nch2p ) , 2.973.22 ( m , 8h , phch2po(oh)2 ) ppm . p{h } nmr ( 162 mhz , cd3od , ) : 25.8 ( po(oh)2 ) , 21.0 ( nch2p ) , 13.9 ( nch2p ) ppm . hr - esi - ms ( meoh , negative ) : m / z calcd for [ m 2h + br ] 1419.1121 , found 1419.1154 ; m / z calcd for [ m 3h ] , 1339.1895 , found 1339.1903 . calcd for c60h68br2n4nio12p8hbr2h2o : c , 44.47 ; h , 4.54 ; n , 3.46 ; p , 15.29 ; br , 14.79 . found : c , 44.45 ; h , 4.70 ; n , 3.24 ; p , 14.61 ; br , 15.08 . vis ( meoh ) : max , nm ( ) 510 ( 1100 ) . electrochemical measurements were performed on an iviumstat or ivium compactstat potentiostat under an argon atmosphere using a three - electrode configuration . a glassy - carbon disk ( 3 mm diameter ) working electrode , a platinum - wire counter electrode , and a ag / agcl / kcl(sat ) reference electrode were used for cyclic voltammetry ( cv ) . all potentials were converted to the normal hydrogen electrode ( nhe ) by addition of + 0.197 v. controlled - potential electrolysis ( cpe ) was carried out in a three - necked flask with a three - electrode setup using glassy - carbon rods as working and counter electrodes ( surface area in solution 2 cm ) and a ag / agcl / kcl(sat ) reference electrode . a solution of nip ( 0.18 mm ) in ascorbic acid ( aa , 0.1 m , ph 4.5 ) was purged with n2 containing ch4 ( 2% ) as the internal gas chromatography ( gc ) standard , and a potential of 0.5 v vs nhe was applied during cpe for 2 h. the faradaic yield was calculated from the amount of h2 accumulated in the headspace , as measured by gc . cpe of catalyst - free electrolyte at the same potential showed no production of h2 . to prepare tio2 and zro2 films sensitized with rup , an aqueous solution of rup ( 10 l of 4 m ) was spread onto the films ( geometrical surface area 1.5 cm ) , and the solvent was dried in air for 30 min . for tio2 and zro2 films cofunctionalized with rup and nip , first a solution of nip ( 10 l of 8 m ) in meoh was spread onto the surface of the film , followed by drying for 30 min in air . then , an aqueous rup solution ( 10 l of a 4 m ) was spread onto the surface of the film and was dried for an additional 30 min . spectroscopic measurements on homogeneous solutions and functionalized tio2 and zro2 films were carried out in water or in aqueous aa solution ( 0.1 m ) carefully adjusted to ph 4.5 with naoh ( 0.1 m ) , unless otherwise stated . the aqueous solutions were purged with n2 for 15 min prior to the measurements . the uv vis and fluorescence spectra of the solutions and films were recorded using a quartz cuvette ( 1 cm path length ) on a perkin elmer lambda 35 uv vis spectrophotometer and a horiba jobin yvon fluorolog luminescence spectrophotometer , respectively . tc - spc measurements were performed by using a horiba jobin yvon tbx fluorocube system . as the excitation source , a pulsed laser with 467 nm nominal wavelength at a repetition rate of 100 khz was used . the photoluminescence intensity of rup at em 650 nm was measured as a function of time after the excitation pulse , for a fixed data collection period to ensure matched densities of absorbed photons between samples ( 600 s ) . the instrument response was measured at the full width at half - maximum and showed typically a 200250 ps value . all tc - spc experiments undertaken on tio2 and zro2 films were measured under aqueous conditions or a 0.1 m aa solution at ph 4.5 in air . the microsecond to second transient absorption decays were measured using a nd : yag laser ( big sky laser technologies ultra cfr nd : yag laser system , 6 ns pulse width ) . the second and third harmonics of the laser ( corresponding to 532 and 355 nm , respectively ) were used to excite rup . the laser intensity was adjusted using neutral density filters as appropriate , with experiments typically employing 350 j cm , and the frequency of the laser pulse was fixed to 1 hz . a liquid light guide with a diameter of 0.5 cm was used to transmit the laser pulse to the sample . the probe light source was a 100 w bentham il1 tungsten lamp , and the probing wavelength was selected by using a monochromator ( obb-2001 , photon technology international ) placed prior to the sample . several high - pass , low - pass and neutral - density filters ( comar optics ) were used to decrease the light arriving to the detector . the information was passed through an amplifier box ( costronics ) and recorded using a tektronics tds 2012c oscilloscope ( microsecond to millisecond time scale ) and a national instruments ( ni usb-6211 ) daq card ( millisecond to second time scale ) . all photocatalytic experiments were carried out using a solar light simulator ( newport oriel , 100 mw cm ) equipped with an air mass 1.5 global filter ( am 1.5 g ) . uv irradiation was filtered using a 420 nm cutoff filter ( uqg optics ) , and ir irradiation was filtered by a water filter ( path length 10 cm ) . the photoreactor was held at a constant temperature of 25 c in all experiments . samples were generally prepared in air protected from light by an al foil . the reaction vessel was sealed with a rubber septum , and air was replaced by n2 containing 2% ch4 ( internal gc standard ) . the irradiated cross section of the solution in the vials was approximately 3.3 cm . h2 evolution was monitored by gc measurements with an agilent 7890a series gc equipped with a 5 molecular sieve column . the gc oven temperature was kept constant at 45 c , n2 was used as a carrier gas at an approximate flow rate of 3 ml min , and a thermal conductivity detector ( tcd ) was used . samples ( 1520 l ) for headspace gas analysis were taken from the reaction vessel headspace in 30 min intervals for 2 h in the screening experiments . for long - term measurements the response factor of the thermal conductivity detector for h2 compared to ch4 was 1.91 . in a typical experiment , a freshly prepared aa solution was titrated to the desired ph using naoh ( 0.1 m ) or hbf4 ( 0.1 m ) and diluted to a final aa concentration of 0.1 m ( the final ph was confirmed ) . stock solutions of rup ( 1.0 or 2.0 mm in h2o ) and nip ( 2.0 mm in meoh ) were added in the desired ratio to the aqueous aa solution ( 0.1 m ) to reach a final volume of 2.25 ml , leaving 5.59 ml of gas headspace in the vial . control experiments in the absence of meoh were carried out and confirmed a comparable photocatalytic activity . zro2 or tio2 nanoparticles ( 2.5 mg ) were placed in a vial containing an aa solution and dispersed by sonication for 5 min . to load the particles , first nip was added and after stirring for several min rup was added , resulting in a final volume of 2.25 ml , leaving 5.59 ml of gas headspace in the vial . the adsorption of nip on tio2 and zro2 nanoparticles was quantified by recording the difference uv vis spectrum of a solution of nip in aa ( 0.1 m , ph 4.5 , 2.25 ml ) before and after exposure to tio2 or zro2 nanoparticles ( 2.5 mg ) . the solution was stirred with the nanoparticles for 30 min , followed by centrifugation ( 7000 rpm , 5 min ) between the measurements . adsorption of rup on nanoparticles after coadsorption with nip was estimated by adding rup ( 0.05 mol ) to a suspension of nip - sensitized tio2 or zro2 particles ( 0.02 or 0.1 mol nip loading on 2.5 mg nanoparticles ) . quantitative adsorption of 0.05 mol rup and 0.02 mol nip would result in an estimated surface coverage of approximately 400 rup and 150 nip molecules per nanoparticle and approximately 45% coverage of the tio2 surface ( see the supporting information ) . the external quantum ( photon to h2 conversion ) efficiency ( eqe ) was determined by an led light source ( modulight , ivium ) using blue light ( 460 nm , 5 mw cm ) . the light intensity was measured with a newport thermopile detector ( 818p-020 - 12 ) coupled with an optical power meter ( 1916-r ) . samples of rup ( 0.3 mol ) and nip ( 0.1 mol ) in solution were used . the efficiency was determined from the amount of h2 produced after 2 h of irradiation using the equation all analytical measurements were repeated at least three times . the obtained data were treated as follows : for a sample of n observations xi , the unweighted mean value x0 and the standard deviation were calculated using the equationsa minimum of 10% was assumed in all experiments . the light sources and gas chromatographs were calibrated regularly to ensure reproducibility throughout all experiments . the catalyst nip was synthesized through hydrolysis of the octaethyl phosphonate ester analogue nip by dealkylation of the ethyl ester groups with trimethylsilyl bromide in dichloromethane ( figure s1 , supporting information ) . nip was obtained in 81% yield and characterized by h and p nmr spectroscopy , high - resolution mass spectrometry , and elemental analysis for c , h , n , p , and br . nip is soluble in water ( 0.3 mg ml ) and dissolves well in an aqueous ascorbic acid solution ( 0.1 m , ph 4.5 ) with a solubility of more than 5 mg ml . nip therefore is an example of a dubois - type ni catalyst that is soluble in aqueous solutions and can be employed homogeneously in organic solvent free aqueous catalytic systems . before studying nip in a purely aqueous electrolyte solution , we compared nip with the previously studied complex nip ( figure s1 ) in acetonitrile / water mixtures and in the presence of added triflic acid . on a glassy - carbon working electrode under ar at a scan rate of 100 mv s , cv measurements of nip ( 0.30.5 mm ) show two quasi - reversible waves at e1/2 = 0.38 and 0.54 v vs nhe in h2o ( 0.1 m na citrate ph 5)/acetonitrile ( 0.1 m tbabf4 ) ( 1/1 ) . the two waves are assigned to the ni and ni redox couples , respectively ( figure 2a and figures s2 and s3 ( supporting information ) ) . addition of triflic acid induced a catalytic current attributed to proton reduction below wsph 4.9 following the reduction of ni to ni with an onset potential of 0.3 v vs nhe ( figure 2a ) . the small cathodic shift in potential of the reduction wave upon acidification is presumably due to protonation of the dangling phosphonate moieties . thus , the [ ni(p2n2)2 ] core and electrocatalytic proton reduction activity are intact in nip and the electrochemical response is indeed comparable to that of nip in the presence of an organic acid ( figures s2s4 , supporting information ) . ( a ) cv of nip ( 0.5 mm , black trace ) in h2o ( 0.1 m na citrate ph 5)/acetonitrile ( 0.1 m tbabf4 ) ( 1/1 ) , followed by titration with increasing amounts of triflic acid ( 0.678 m in h2o ) . wsph is the concentration of protons in a mixed solvent ( water / organic solvent ) where the ph was measured in water against an aqueous reference . ( b ) cv of nip ( 0.3 mm , solid red trace ) in an aqueous aa solution ( 0.1 m , ph 4.5 ) . wave ( a ) shows the first reduction wave of nip , and wave ( b ) indicates the onset of catalytic proton reduction . reduction potentials of possible reactive intermediates of rup in a photocatalytic process and the conduction band potential of tio2 ( ecb(tio2 ) are also shown . a glassy - carbon working electrode , a ag / agcl / kcl(sat . ) reference electrode , and a platinum - wire counter electrode were employed at room temperature with a scan rate of 100 mv s in ( a ) and ( b ) . electrocatalytic proton reduction by nip was also observed in the absence of organic solvent in an aqueous solution buffered with ascorbic acid ( aa ) or na citrate ( 0.1 m , ph 4.5 ) . a single , irreversible reduction wave ( a ) was observed at ep = 0.35 v vs. nhe , followed by the onset of a catalytic wave ( b ) at approximately 0.48 v vs nhe ( figure 2b and figure s5 ( supporting information ) ) . therefore , the catalyst operates with a small overpotential requirement of approximately 0.2 v in comparison to the thermodynamic potential for proton reduction of 0.27 v vs nhe at ph 4.5 . the reduction potentials of intermediates of rup , which are likely to be formed during the quenching process after photoexcitation ( see below ) are also indicated in figure 2b . the position of these potentials and the conduction band edge of tio2 suggest that enough driving force for photocatalytic proton reduction with nip would be available when using rup and rup - sensitized tio2 as a light harvester , as described in detail in the kinetic and photocatalytic sections . controlled - potential electrolysis with nip ( 0.18 mm ) in an aqueous ascorbic acid solution ( 0.1 m , ph 4.5 ) on a glassy - carbon - rod working electrode ( surface area 2 cm ) at 0.5 v vs nhe for 2 h confirmed the generation of h2 gas with a faradaic yield of 85 4% ( h2 in the headspace quantified by gc ) . rinsing the electrode with h2o after cpe and immersing it in a fresh electrolyte solution ( in the absence of nip catalyst ) did not result in the formation of h2 when continuing with cpe at the same potential . thus , h2 production originates from a dissolved catalyst and not from electrodeposited decomposition products on the electrode . thus , the water - soluble nip displays electroactivity for the reduction of aqueous protons with a high faradaic yield in the absence of organic solvents , a prerequisite for the use of nip in photocatalytic schemes in water . the suitability of nip in photocatalytic h2 generation was studied with rup , aa ( 0.1 m ) as a sacrificial electron donor , and buffer ( pka 4.17 ) in aqueous solution . photoexcitation of rup to rup * ( max 455 nm , mlct ) can result in either oxidative or reductive quenching of the photoexcited state ( figures 3 and 4 ) . first , the kinetics and mechanisms of et between rup and nip in homogeneous aqueous solution ( rup - nip system ) and anchored onto metal oxide surfaces ( rup - zro2-nip and rup - tio2-nip systems ) were studied by tc - spc and tas . the three photocatalytic systems are illustrated in figure 3 , and the mechanistic and kinetic details are summarized in figure 4 and described below . the rup - nip and rup - zro2-nip systems generate h2 through a reductive quenching mechanism of rup in solution or on the particle , respectively . rup - tio2-nip undergoes oxidative quenching and electrons are transferred through the particle . summary of et kinetics for the three photocatalytic systems studied as determined by tc - spc and tas ( defined as t50% times ) . tio2 was observed to cause oxidative quenching of the immobilized rup * , resulting in the oxidized intermediate rup ( e(rup / rup * ) = 0.95 v vs nhe ) by electron injection into the conduction band of tio2 ( ecb = 0.55 v vs nhe at ph 4.5 ) . these kinetics were measured by tc - spc to take place in approximately 180 ps , with an injection efficiency of > 95% ( figure 5 ) . the transient absorption spectrum of a photoexcited rup - tio2 film shows a maximum transient absorption peak at 700 nm , corresponding to the absorption spectrum of the oxidized rup ( figure s6 , supporting information ) . in the absence of aa , the tio2 conduction band electrons recombine with rup within approximately 1 ms . upon reduction of rup by aa ( t50% 50 s ) , corresponding to the regeneration of rup , the resulting photoinjected tio2 electrons exhibit a lifetime of 0.5 s. following the codeposition of nip ( molecular ratio rup / nip of 1/2 ) , the decay of these electrons is accelerated to approximately 1 ms , assigned to et to nip ( figure s7 , supporting information ) . the oxidative et mechanism from rup to nip through the semiconductor ( through particle system , figure 3c ) , is possible with tio2 but not with zro2 , due to the energetic mismatch between rup * and the conduction band of zro2 ( ecb = 1.26 v vs nhe at ph 4.5 ) . zro2 is therefore unable to accept electrons from rup * and can only be used as a matrix to immobilize the compounds in close proximity on the particle ( on particle system , figure 3b ) . the reductive quenching through intermolecular et from aa ( e = 1.17 v vs nhe ) to rup * ( e(rup*/rup ) = 1.08 v vs nhe ) was measured by tc - spc to take place in approximately 250 ns with an estimated efficiency of 70% in homogeneous rup - nip and heterogenized on particle rup - zro2-nip systems ( figure 5 ) . photoexcitation of an aqueous rup solution ( 4 m ) containing aa ( 0.1 m , ph 4.5 ) resulted in the appearance of a transient absorption peak at 500 nm ( figure s8 , supporting information ) assigned to the formation of the reactive intermediate rup . this reduced state of the dye , rup , is a strong reducing agent and has a large driving force for the reduction of nip ( e(rup / rup ) = 1.09 v vs nhe , figure 2 ) . time - resolved luminescence measurements of rup with and without nip in water and in the presence of aa ( 0.1 m ) at ph 4.5 : ( a ) anchored onto the surface of a tio2 and zro2 film ( 10 l of 4 m rup , 10 l of 8 m nip ) ; ( b ) in a homogeneous solution ( [ rup ] = 4 m , [ nip ] = 8 m ) . the addition of increasing amounts of nip ( from 0 to 16 m ) to a solution containing rup ( 4 m ) and aa ( 0.1 m , ph 4.5 ) results in the linear decrease of the lifetime of rup from 700 to 29 s , following first - order kinetics with respect to nip concentration for the intermolecular et between rup and nip ( figure 6 ) . the second - order rate constant of this et is ket = 1.4 10 m s , indicating that the et kinetics are diffusion limited ( figure 6b and table s1 ( supporting information ) ) . in addition , the presence of nip resulted in the appearance of a long - lived ( t50% 0.1 s ) bleaching signal assigned to the reduction of ni species ( figure s9 ( supporting information ) ) upon reduction by rup . further experiments are ongoing to monitor the nickel catalytic species involved in the h reduction reaction . ( a ) transient absorption decays of rup ( 4 m ) in an aqueous aa solution ( 0.1 m , ph 4.5 ) after addition of different concentrations of nip ( 0 , 2 , 4 , 8 , and 16 m ) . the excitation wavelength was ex 532 nm , and the decays were probed at probe 500 nm . ( b ) calculated pseudo - first - order rate constant ( ket[nip ] ) for the et from rup to nip as a function of the concentration of nip catalyst ( table s1 ( supporting information ) ) . the reductive quenching of rup * also occurs through intermolecular et from aa to rup * in rup - zro2-nip . the photoluminescence intensity and lifetime of rup * on nanostructured zro2 films decrease upon the addition of an aqueous aa solution ( 0.1 m , ph 4.5 ) , and tc - spc measurements reveal that reductive quenching of rup * to form rup occurs in approximately 250 ns with an estimated et efficiency of 70% ( figure 5b ) . the cofunctionalization of zro2 with nip ( 8 m ) and rup ( 4 m ) in water does not affect the luminescence of rup , indicating that et between the two molecules requires the formation of rup in the presence of a sacrificial electron donor . a transient absorption peak at 500 nm corresponding to the signal of rup was also observed in the rup - zro2 system in the presence of aa ( 0.1 m at ph 4.5 ) , with the decay of rup accelerating following the codeposition of nip ( figure s10 ( supporting information ) ) . it can be concluded that the same reductive et mechanism and similar kinetics ( dependent upon component loadings / concentrations ) take place both in homogeneous media and on anchoring of the molecules onto the surface of a material that does not allow for dye electron injection . although the reductive et mechanism is the same for both homogeneous and the on - particle zro2 systems , charge separation is achieved through two different processes : while in homogeneous media charge separation occurs through the diffusion of molecules into the solution , when the dye and catalyst are immobilized in rup - zro2-nip charge separation can take place through the intermolecular et between neighboring molecules . the spectroscopic studies revealed that all three photocatalytic systems drive the efficient photoreduction of nip . all three systems were indeed efficient in producing h2 during irradiation with visible light . consequently , the systems were studied and optimized by varying the ph value of the aa solution and concentrations of rup and nip ( table 1 and tables s2s4 ( supporting information ) ) . the photocatalytic activity and longevity of h2 production was dependent on the conditions and type of system employed . the photocatalytic performance of nip generally varied in all three environments , and the rate of photogenerated h2 was constant over at least 1 h in all experiments ( figure 7a ) . the turnover frequencies based on nip ( tofnip ) were calculated from the amount of h2 accumulated in a photoreactor after 1 h irradiation ( tables s3 and s4 ) . all samples were irradiated with visible light ( am 1.5 g filter , 100 mw cm , > 420 nm , 25 c ) under an n2 ( 2% ch4 ) atmosphere and a standard solvent volume of 2.25 ml , leaving a gas headspace volume of 5.59 ml . standard screening samples were irradiated for 2 h in aa ( 0.1 m , ph 4.5 ) , and the tof was determined after 1 h irradiation . homogeneous system with nip ( 0.1 mol ) , rup ( 0.3 mol ) , and different ph values . rup ( 0.05 mol ) ; in heterogenized systems tio2 or zro2 ( 2.5 mg per sample ) nanoparticles were used as dispersions . tofnip after 15 min visible light irradiation reported due to rapid bleaching of eosin y. no h2 detected in gc measurements ( limit of detection < 0.01% ) . ( a ) visible - light - driven generation of h2 in a homogeneous aqueous system ( 0.1 m aa , ph 4.5 ) comprised of ( a ) nip ( 0.1 mol ) with different amounts of rup and ( b ) rup ( 0.05 mol ) with low ( 0.02 mol , squares ) and high loadings of nip ( 0.1 mol , circles ) . in a homogeneous rup - nip system , a tofnip value of 460 60 h was observed when rup ( 0.05 mol ) was used with a low amount of nip ( 0.02 mol ) in aqueous aa ( 0.1 m , 2.25 ml ) . this rup - nip system was photoactive for 2 h with a final tonnip value of 723 171 ( 15 3 mol of h2 ) . an increasing amount of nip ( 0.1 mol ) resulted in a decreased initial tofnip concomitant with an increased system lifetime and a comparable overall tonnip value of 651 30 ( 65 3 mol of h2 ) after 30 h irradiation ( table 1 and figure 7b ) . this observation might be explained by the less likely double reduction of a single molecular catalyst at high nip concentrations . addition of rup or aa to a photodegraded system did not result in reactivation , suggesting that decomposition of nip occurred after approximately 700 tons . addition of nip to a deactivated homogeneous system did result in reactivation , but full photoactivity was not restored , indicating that photodegradation affected not only nip but also other system components such as rup . indeed , photobleaching of rup became evident after 1 h irradiation by recording an electronic absorption spectrum of the homogeneous solution after irradiation in a gastight quartz cuvette under standard conditions ( figure s11 ( supporting information ) ) . several sets of control experiments were carried out , which showed that no or only negligible amounts of h2 were produced in the absence of rup , nip , aa or light . the presence of additional buffers such as citrate and acetate did not impede the photocatalytic activity of the original system . replacement of nip by different ni salts such as nicl2 and nibr2 in combination with 4 equiv of a water - soluble phosphine ( [ 2-(dicyclohexylphosphino)ethyl]trimethylammonium chloride ) resulted only in negligible amounts of h2 . homogeneous systems using commercially available dyes such as [ ru(bipy)3]cl2 ( tofnip = 220 20 h ) or the organic , noble - metal - free dye eosin y ( used as the disodium salt ; tofnip = 189 31 h ) also resulted in efficient photocatalytic h2 production in an aqueous aa solution ( 0.1 m , ph 4.5 ) ( table 1 and table s6 ( supporting information ) ) . the external quantum efficiency ( eqe ) of the homogeneous photocatalytic system with rup ( 0.3 mol ) and nip ( 0.1 mol ) in aa ( 0.1 m , ph 4.5 ) was measured using an led light source ( 460 nm , 5 mw cm ) . an eqe value of 9.7 1.2% was determined after 2 h irradiation . we note that this external yield assumes that all photons emitted by the light source were absorbed by rup and therefore represents a lower estimate of the quantum efficiency of the system . the photon - to - h2 quantum yield observed for our homogeneous rup - nip system is remarkable in comparison to other photocatalytic systems with molecular 3d transition - metal catalysts operating homogeneously or immobilized in aqueous solution . recently reported quantum yields for homogeneous photocatalytic systems ranged from 0.23 to 0.6% for a co - pentapyridine catalyst with [ ru(bpy)3 ] as photosensitizer in an aqueous system at neutral ph to 4.6% for a cobaloxime - based h2 generation system with an al - porphyrin dye in a water / acetone solvent system . in systems with a molecular catalyst immobilized on a solid - state material , eqes of approximately 11.5% for a cobaloxime immobilized on rup - sensitized tio2 and a ni(teoa)3 ( teoa = triethanolamine ) complex on graphitic carbon nitride , respectively , were reported . eqes for self - assembled photocatalytic systems using ni salts and 2-mercaptoethanol ( 24.5% ) or dihydrolipoic acid ( 36% qe ) ligands are still benchmarks ; however , a defined catalytically active species has not been reported for these systems . various homogeneous photocatalytic schemes have been reported for the reduction of aqueous protons with molecular catalysts . examples of efficient photo - h2 generating systems include a molecular dye combined with [ ni(p2n2)2]-type and fe - based molecular catalysts , but in these systems water / organic solvent mixtures were employed . photocatalytic schemes for h2 generation which operate in pure aqueous systems are highly desirable . very recently , the water - insoluble [ ni(p2n2)2](bf4)2 was incorporated into the photosynthetic protein photosystem i ( psi ) for photocatalytic h2 production in aqueous solution , but only a very limited amount of ni catalyst can be loaded onto dilute enzyme systems and the photostability of the hybrid assembly remained an issue ( more than half of the photoactivity ceased after 30 min of irradiation ) . nickel - thiolate and cobalt - dithiolene catalysts have been reported to achieve several thousand turnovers in organic solvent / water mixtures . cobaloximes have been reported to evolve h2 in a homogeneous system and on rup - sensitized tio2 in water , but the tofco value never exceeded 20 h and the photon - to - h2 efficiency was only 1% . the higher photocatalytic activity and eqe of nip in comparison to those of cobaloxime catalysts may be related to the similar reduction potentials required for both reduction steps in nip . we have previously shown that the photocatalytic activity of a cobaloxime complex is significantly limited by its relatively slow second reduction kinetics , attributed at least in part to the relatively unfavorable energetics for this second reduction . the use of a co - pentapyridine catalyst and a [ fefe]-hydrogenase mimic in photocatalytic schemes in an aqueous solution resulted in tofs of approximately 20 and 50 h , respectively . thus , our rup - nip system compares favorably with previously reported systems in organic solvent free aqueous solution . the immobilization of a molecular catalyst on a photoelectrode is a prerequisite for fuel generation in a photoelectrochemical device , and the attachment on a semiconductor material is particularly desirable . therefore , we extended our studies using nip for h2 generation in heterogeneous photocatalytic assemblies with nanoparticle suspensions as a first step towards an electrode assembly . we first determined the maximum loading capacity of rup(6c ) and nip on the metal oxide particles by spectrophotometry . approximately 0.05 mol of rup or nip can be immobilized per milligram of tio2 or zro2 when adding an excess of phosphonated catalyst ( see the supporting information ) . the metal oxide nanoparticles were loaded by the following procedure : nip was added to a suspension of tio2 or zro2 in aqueous aa solution ( 2.5 mg in 2.25 ml ) , and then rup was added . nip was loaded first due to the optimized geometry of adsorption of phosphonated bipyridine ligands of rup . the photoactivity of the suspensions was studied under irradiation with visible light ( am1.5 g , 100 mw cm , > 420 nm ) . upon investigating the heterogeneous photocatalytic systems , notably different trends in performance were observed in comparison to the homogeneous system at lower nip loadings ( figure 8) . visible - light - driven h2 evolution rate with different amounts of nip and rup ( 0.05 mol ) : ( triangle ) rup - nip ; ( square ) rup - tio2-nip ; ( circle ) rup - zro2-nip . in a rup - zro2-nip system , a low amount of nip ( 0.02 mol ) with rup ( 0.05 mol ) on 2.5 mg of zro2 resulted in a tofnip value of 27 3 h. the results obtained in tas measurements suggest that direct interaction of the quenched dye ( rup ) with nip is required to drive the reaction . et between rup and nip on the particle as observed by the spectroscopic studies above becomes difficult under such dilute conditions due to the spatial separation of the compounds on the zro2 surface . when the amount of nip added to zro2 is increased to 0.1 mol , a significant enhancement in tofnip to 92 26 h was observed ( figure 8 and table s4 ( supporting information ) ) . we note that , at low concentrations of compounds carrying phosphonic acid groups , attachment on zro2 or tio2 is not quantitative , presumably due to competitive binding of aa to metal oxides . spectrophotometric studies indicate that nip is almost quantitatively ( > 80% ) adsorbed on zro2 , whereas only approximately 10% of rup adsorbs on nip - modified zro2 ( table s7 ( supporting information ) ) . when the zro2 nanoparticles were loaded with nip ( 0.02 mol ) and rup ( 0.05 mol ) , separated by centrifugation , and redispersed in fresh aa ( 0.1 m , ph 4.5 ) , the amount of h2 produced was 0.16 mol after 1 h irradiation ( in comparison to 0.54 mol of h2 before centrifugation ) . thus , the photodriven h2 production in bulk experiments can best be described as a mixture of et between surface - immobilized catalysts through an on particle mechanism and from solubilized rup to surface - bound nip . a rup - tio2-nip system displays a tofnip value of 51 7 h at a low nip loading of 0.02 mol on 2.5 mg of tio2 ( table 1 ) . the rate of h2 production reached a maximum with a nip loading of 0.1 mol , whereupon a tofnip value of 72 5 h and an overall tonnip value of 278 19 h ( after 30 h ) was obtained ( table s5 ( supporting information ) ) . under these conditions , at least 80% of nip and more than 20% of rup are attached on tio2 , as measured by spectrophotometry ( figures s12 and s13 ( supporting information ) ) . once nip or rup is bound to the tio2 surface , it can not easily be removed from the solid - state material . redispersion of loaded particles in a fresh aa solution did not result in the detection of significant amounts of rup or nip in solution . loading the tio2 nanoparticles with nip ( 0.02 mol ) and rup ( 0.05 mol ) , centrifugation , and resuspension in fresh aa ( 0.1 m , ph 4.5 ) resulted in a tofnip value of 24 h during irradiation . thus , 50% of the photocatalytic activity remained , thereby establishing the importance of the role of the conduction band and the through particle this experiment and the significantly higher rate of photocatalytic h2 production for rup - tio2-nip in comparison to rup - zro2-nip at very low loading of the particles with nip ( table 1 ) support a preferential through particle et mechanism for tio2 : a mechanism which does not require the direct electronic communication of rup and nip as needed on zro2 . these results show that photocatalytic h2 production can be achieved with a dubois - type catalyst attached on a solid - state semiconductor . however , to this point limitations by the loading capacity of materials used and by competitive binding of electron donor or electrolyte are still being faced when investigating such hybrid materials . work is in progress to overcome these limitations by investigating the binding modes of the molecular components and photocatalytic activity of rup - nip systems on thin films and electrode materials . in summary , we describe a novel [ ni(p2n2)2]-type h2 evolution catalyst ( nip ) , which is soluble in water and can be immobilized on metal oxide surfaces . nip is electrocatalytically active in organic solvent free aqueous solution and evolves h2 with an onset potential of only 0.48 v vs nhe under mild conditions ( ph 4.5 ) . photocatalytic and spectroscopic studies were performed with nip in three different systems in a purely aqueous solution containing aa . a homogeneous rup - nip system operates through reductive quenching of rup * in solution . the heterogeneous rup - zro2-nip system shows the same et mechanism , and zro2 acts merely as a matrix to retain the attached molecules closely together , hence facilitating et on the particle . in rup - tio2-nip mechanism , where rup * is oxidatively quenched upon injection of an electron into the conduction band of tio2 , which can subsequently be harvested by nip . a high tof value of 460 60 h for light - driven h2 evolution with a molecular 3d transition metal catalyst in pure aqueous solution was obtained , with tons of approximately 700 for nip . advanced spectroscopic methods ( tc - spc and tas ) confirmed that directed et from rup to nip occurs efficiently in all systems on the nano- to microsecond time scale . losses due to charge recombination are minimized , as et occurs efficiently within the lifetimes of the excited species . the highly efficient et from dye to proton reduction catalyst is also reflected in the high photon to h2 quantum yield of the homogeneous system of almost 10% in the presence of the sacrificial electron donor aa . work is in progress to assemble a photoelectrode with nip for use in a photoelectrochemical water splitting cell .

the generation of renewable h2 through an efficient photochemical route requires photoinduced electron transfer ( et ) from a light harvester to an efficient electrocatalyst in water . here , we report on a molecular h2 evolution catalyst ( nip ) with a dubois - type [ ni(p2rn2r)2]2 + core ( p2rn2r = bis(1,5-r-diphospha-3,7-r-diazacyclooctane ) , which contains an outer coordination sphere with phosphonic acid groups . the latter functionality allows for good solubility in water and immobilization on metal oxide semiconductors . electrochemical studies confirm that nip is a highly active electrocatalyst in aqueous electrolyte solution ( overpotential of approximately 200 mv at ph 4.5 with a faradaic yield of 85 4% ) . photocatalytic experiments and investigations on the et kinetics were carried out in combination with a phosphonated ru(ii ) tris(bipyridine ) dye ( rup ) in homogeneous and heterogeneous environments . time - resolved luminescence and transient absorption spectroscopy studies confirmed that directed et from rup to nip occurs efficiently in all systems on the nano- to microsecond time scale , through three distinct routes : reductive quenching of rup in solution or on the surface of zro2 ( on particle system ) or oxidative quenching of rup when the compounds were immobilized on tio2 ( through particle system ) . our studies show that nip can be used in a purely aqueous solution and on a semiconductor surface with a high degree of versatility . a high tof of 460 60 h1 with a ton of 723 171 for photocatalytic h2 generation with a molecular ni catalyst in water and a photon - to - h2 quantum yield of approximately 10% were achieved for the homogeneous system .

Introduction Experimental Section Results and Discussion Conclusions

the role of hla compatibility as a parameter for allocation of kidneys from deceased donors to patients on the kidney waiting list remains a matter of debate . it is well accepted that the best possible organ for an end stage renal disease patient is a fully hla compatible kidney . both graft and patient survival in post mortal kidney transplantation are significantly better in compatible groups compared to kidneys transplanted with incompatibilities ( doxiadis et al . , 2004 ; kaneku and terasaki , 2006 ; opelz and dhler , 2007 ; johnson et al . , 2010 ) . furthermore , hla incompatible organs are the source of hla antigens toward which the patient can , even under immunosuppressive therapy , form alloantibodies . following the rules of immunology this step these alloantibodies are currently suggested to be the main cause of graft destruction ( sijpkens et al . , 1999 ) . furthermore , incompatibilities render the patient ( highly ) sensitized and after primary graft loss more difficult to be offered a suitable donor kidney for retransplantation . alloantibodies have been related not only to acute but especially to chronic allograft dysfunction , better discussed as chronic rejection . intervening therapies are used to treat patients with alloantibodies such as intravenous immunoglobulines ( glotz et al . , 1993 ; gebel and halloran , 2010 ) , anti - cd20 monoclonal antibodies recognizing the b cells and proteasome inhibitors as bortezomib ( trivedi et al . , 2010 ) prevention of alloantibody formation would be a good alternative to the unsatisfactory treatment , which in the case of transplantation results often into secondary problems like degenerative diseases such as cancer ( opelz and dhler , 2010 ) . many mostly european organ exchange organizations allocate kidneys on the basis of hla matching by giving more points to better matched patients than those with a lower matching degree ( doxiadis et al . , 2004 ; johnson et al . , a selection of a specific locus , e.g. , hla - dr , is not yet the rule . earlier it has been shown that hla - dr compatibility leads to an increased graft survival for patients receiving a primary graft . the effect of hla - a , b compatibility disappears when patients receive a hla - dr incompatible organ ( doxiadis et al . , 2007 ) . these results have been obtained using a large number of transplant ( n = 35,205 ) in the transplantation period of 19852005 . in a follow - up the recipients were 18 years old while the donors were over 5 years old . meier product limit method was used to estimate survival rates . for the estimation of relative risks ( hazard ratios ) for different variables all analyses were performed with the spss statistical package , version 14 ( spss inc . , the course of post transplantation follow - up was analyzed in the three separate phases post transplantation : initial ( < 1 year ) , intermediate ( 1 to < 5 years ) , and late phase ( 5 years follow - up ) . the results of these analyses are similar to the ones obtained for the primary transplants . the effect of hla - a , b compatibility exists as expected over time and mimics the results already shown for primary transplants . by introducing an hla - dr incompatibility the effect of hla - a the risk factors in multivariate analysis showed that donor age already reported for the primary grafting and also in many other studies and sensitization degree of the patient at transplantation are deleterious for the patient . finally , hla - a , b incompatibility is an independent factor resulting in a higher graft loss in the first phase ( 1 year ) post transplantation , presumably because of alloantibodies or unreported earlier sensitization of the patients . these results are based on a seroequivalent hla - dr typing ( dr1dr18 ) or a molecular typing of the first field , according to the new nomenclature . in a simulation study we could show that even on a local basis it is possible to offer to almost all patients on the waiting list a fully hla - dr compatible organ even including patients of different genetic background , allowing an equitable allocation to all patients on the waiting list . the production of alloantibodies toward incompatibilities of the donor is related to the mhc class ii compatibility . in case patients receive incompatible hla - dr organs they have a significantly higher chance to produce alloantibodies against the donor than in the case of compatible grafts ( doxiadis et al . , 2007 ) . many studies have been reported since the seminal publication of terasaki ( 2003 ) regarding the production of alloantibodies toward the graft and its relation to transplantation . if one admits that these alloantibodies affect allograft survival then prevention of their formation might be the answer since selective elimination is impossible to date . the alloantibody prevention scenario for future transplant might look as follows : as a first step patients are offered a fully hla - dr ( drb1 ) compatible graft . epitopes leading to alloantibody formation should be avoided , e.g. , bw4 in hla - dr1 or dr3 recipients ( fuller and fuller , 1999 ) . after this initial phase full compatibility also for other than hla - drb1 specificities should be aimed . at present no conclusive data for any effect of hla - drb3,4,5 in large registries are available , the same holds true for hla - dq . the very strong associations between most of the hla - drb1 alleles to hla - dq specificities make this type of analyses difficult and require large data sets , which are currently not available . the hla phenotype of the patient will lead to the selection of organs with a low chance of alloantibody formation . the patients should be closely monitored with respect to alloantibody production , e.g. , monthly at the initial phase of up to 6 months , and then at intervals of about 6 months , for two reasons : first , for possible treatment and second for data collection in order to fully understand alloantibody formation under transplantation circumstances . the final step in this scenario would be to offer every patient a graft with the least chances for hla alloantibody formation ( duquesnoy et al . , 2003 ) . the proposed scenario would offer the following benefits : increased graft and patient survival . a significant decrease of the sensitization toward donor hla incompatibilities during the transplantation period and , in case of organ loss , more , and better possibilities for retransplantation . decrease of waiting time for the retransplant . reduced incidence of side effects , normally associated with the additional immunosuppressive regiments necessary to treat rejection episodes . this proposal is only the first step needed to reduce allosensitization of patients receiving a kidney allograft . it is our intention to introduce on the long term additional steps such as compatibility of epitopes leading to alloantibodies . it is essential to reproduce the data mentioned at the beginning of this review using local or regional transplantation outcome , since controversial results have been reported ( e.g. , gritsch et al . , 2008 ) . all patients should receive a fully hla - dr compatible graft , resulting in an increased graft survival and reduced allosensitization . until immunosuppressive regiments are directed toward the humoral component of graft rejection simple prevention should be aimed in the field of post mortal kidney transplantation . alloantibodies are not only a relevant factor before transplantation but are also a cause of allograft dysfunction after transplantation . full compatibility for mhc class ii together with intelligent introduction of incompatibilities could be seen as a first step in a series of possibilities to diminish allosensitization without the need of additional immunosuppressive treatments . in kidney transplantation it is important to note that by eliminating the one component of the immune system other components can take over . until now the t cells were quoted as the bad guys . in the present period b cells took over . recently , we showed that nk cells ( van bergen et al . , 2011 ) are associated with graft loss in the later period of transplantation ( gritsch et al . , 2008 ) . the author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .

long lasting debates in the past questioned the relevance of any sort of compatibility in post mortal kidney transplantation . it is for no say that fully compatible transplants have the highest chances for a long patient and graft survival . in the present report the use of hla - dr as a representative of the major histocompatibility complex class ii genes in the allocation of organs is discussed . the major arguments are the easiness to offer to patients a compatible graft in a relatively short waiting time , an increase in graft survival , the less sensitization during the transplantation period , and the lower waiting time for a retransplant . even if the number of organ donors remains the same a lowering of the mean waiting time is expected because of the longer period of graft survival .

Introduction HLA-DR Compatibility Conclusion Summary Author Note Conflict of Interest Statement

de van stated , meet the mind of the patient before meeting the mouth of the patient . thus , understanding the mental attitude of the patient is very helpful for the clinician to anticipate the patient 's response during the clinical procedures . krochak stated that many patients with favorable anatomy can not tolerate a well - fabricated denture , and yet other patients with unfavorable anatomy willingly endure dentures that may be ill - fitting . patients seeking prosthodontic care arrive with an accumulation of experiences and resulting attitudes . in 1950 , house devised a classification system in which he classified the patient 's psychological responses to becoming edentulous and adapting to dentures into following four types : philosophical mind , exacting mind , hysterical mind , and indifferent mind . philosophical patients anticipate the need for treatment with complete dentures and are willing to rely on the dentist 's advice for diagnosis and treatment . these patients follow the dentist 's advice when advised to replace their dentures . the best mental attitude for denture acceptance exacting mind patients usually have poor health and show resistance to accommodate suggestions given by the clinician . they also doubt the dentist 's ability and even try to direct them regarding the treatment procedure . often , the exacting mind patient demands extra - ordinary efforts and guarantees of treatment outcome at no additional cost . the prognosis is often unfavorable , and additional professional help ( psychiatric ) is required before and during treatment . the patient must be made aware that his / her problem is primarily systemic and that many of his symptoms are not the result of dentures . they pay no attention to instructions , will not cooperate and are prone to blame the dentist for poor dental health . truly stated by jamieson , fitting the personality of the aged patient is often more difficult than fitting the denture to the mouth . it has also been said that the patient 's attitude is probably influenced by a series of social factors ( the patient 's sex , age , education , vocation , social factors , upbringing , home environment , etc . , ) and by the state of general health . on the other hand , it has also been reported that little correlation exists between satisfaction with dentures and the patient 's personality , social adaptation , and appreciation of the dentist . no correlation was seen between the patient 's intelligence and his satisfaction with the dentures . careful observation and listening in a structured interview and questionnaire provide ample information to the dentist regarding the patient 's mental attitude . to supplement this , the additional aid of graphoanalysis has also been advised to identify the behavior of the patients . knowledge of patient 's mental attitude may provide insight into patient behavior , which may help in the overall improvement of the treatment rendered . the aim of this study was , to find any relationship , if exists , between the patient 's mental attitude with age , sex , or educational qualification . philosophical patients anticipate the need for treatment with complete dentures and are willing to rely on the dentist 's advice for diagnosis and treatment . these patients follow the dentist 's advice when advised to replace their dentures . the best mental attitude for denture acceptance exacting mind patients usually have poor health and show resistance to accommodate suggestions given by the clinician . they also doubt the dentist 's ability and even try to direct them regarding the treatment procedure . often , the exacting mind patient demands extra - ordinary efforts and guarantees of treatment outcome at no additional cost . the prognosis is often unfavorable , and additional professional help ( psychiatric ) is required before and during treatment . the patient must be made aware that his / her problem is primarily systemic and that many of his symptoms are not the result of dentures . they pay no attention to instructions , will not cooperate and are prone to blame the dentist for poor dental health . truly stated by jamieson , fitting the personality of the aged patient is often more difficult than fitting the denture to the mouth . it has also been said that the patient 's attitude is probably influenced by a series of social factors ( the patient 's sex , age , education , vocation , social factors , upbringing , home environment , etc . , ) and by the state of general health . on the other hand , it has also been reported that little correlation exists between satisfaction with dentures and the patient 's personality , social adaptation , and appreciation of the dentist . no correlation was seen between the patient 's intelligence and his satisfaction with the dentures . careful observation and listening in a structured interview and questionnaire provide ample information to the dentist regarding the patient 's mental attitude . to supplement this , the additional aid of graphoanalysis has also been advised to identify the behavior of the patients . knowledge of patient 's mental attitude may provide insight into patient behavior , which may help in the overall improvement of the treatment rendered . the aim of this study was , to find any relationship , if exists , between the patient 's mental attitude with age , sex , or educational qualification . a total of 200 patients who attended the outpatient department of post graduate institute of dental sciences , university of health sciences , rohtak , from september 2009 to october 2010 , for fabrication of new complete dentures were selected . the eligibility criteria for the study included : subjects above 45 years of age , ability to provide informed consent , and completely edentulous subjects with or without prior denture wearing experience . patients were given a verbal explanation of the study , and an informed consent was obtained . during the same appointment , was then filled by the clinician as answered by the patient [ table 1 ] . questionnaire form ( 22 questions under four categories ) after completion of the questionnaire , the required prosthodontic treatment procedures were completed for all the participants as per routine . selected subjects were evaluated and categorized based on questionnaire and clinical experience during treatment according to classification by house . participants were categorized for data processing as follows : on the basis of sex : group m : male , group f : female;on the basis of age : group 1 : 4554 years , group 2 : 5564 years , group 3 : 65 years and above;on the basis of educational status : group a : less than high school , group b : high school and group c : more than high school attended any college ( participants were asked about the highest qualification ) . group m : male , group f : female ; group 1 : 4554 years , group 2 : 5564 years , group 3 : 65 years and above ; on the basis of educational status : group a : less than high school , group b : high school and group c : more than high school attended any college ( participants were asked about the highest qualification ) . a total of 200 participants were recruited for the study , of which 83 were male and 117 female . many participants found to have a hysterical mindset was the lowest , a group which comprised 4 participants only . based on the statistical analysis , no significant differences were found between males and females in philosophic and hysterical mind category . the numbers of females were significantly higher than males in the exacting group ( p = 0.007 ) . interestingly , their number declined significantly in the indifferent category ( p = 0.002 ) . thus , on this basis , females were found to be more exacting and males more indifferent [ table 2 and figure 1a ] . distribution of mental attitude according to gender it shows graph ( a ) correlation of mental attitude with : sex graph ( b ) : correlation of mental attitude with : age graph ( c ) : correlation of mental attitude with : educational status table 3 and figure 1b shows the participants categorized according to age into three groups : 4554 years , 5564 years , and 65 years and above . the youngest age group ( 4554 years ) showed significantly more number of subjects with exacting mindset compared to the older ones ( p < 0.001 ) . besides , only four patients of more than 65 years old group were found in hysterical category . there was no significant correlation found in philosophic and indifferent attitude patients when correlated with age . distribution of mental attitude according to age when these patients were enquired about highest education attained , it was found that out of 200 subjects , 54 were found in group a ( never went to school or less than high school ) , 106 in group b ( high school level ) and 40 in group c ( higher than high school ) [ table 4 and figure 1c ] . when this data were correlated with the mental attitude of the patients , no significant values were found in philosophical and hysterical types . group b subjects were found significantly higher with exacting attitude ( p < 0.001 ) , and group a patients were found to be significantly inclined toward indifferent attitude ( p < 0.001 ) . the aim of this study was to find the mental attitudes of patients presenting for prosthodontic treatment and to determine if any relationship existed between the patient 's mental attitude with age , sex , or educational qualification . stated that the house classification is based on extensive clinical experience , and it has stood up well to the test of time . the results of this study show that females were found to be more exacting in nature and while males , on the other hand , revealed more indifferent attitude . brunello and mandikos in a study found a higher dental anxiety scale scores among female subjects than males . this can also be correlated with myers opinion , who studied the relationship of the women irritable personality , that this related to psychosomatic pressures , and he believed that happiness associated with social support , marriage , career , and religious faith , can play an effective role in general and mental health . shah et al . in a study stated women have a better health seeking behaviors and more conscious for their appearance , and this could be one of the main reasons that females visit dental clinic more than males . polsani et al . stated that the changes influencing one 's appearance , such as loss of hair and face height , wrinkling of skin , changes in tooth appearance , and loss of natural teeth , seem to affect women more than men . women frequently voice their concern while men may be more dramatic but concealed . as far as the relationship of age with mental attitude is concerned , according to this study , the younger edentulous age groups in their late 40s and early 50s were more demanding and presented exacting behavior throughout the treatment procedure as compared to other age groups . shrivastava et al . in a study found no correlation between dental anxiety and denture satisfaction with age and period of edentulousness . hgglin et al . stated that persons in the age group of 5564 years were mature and wise mentally than other ages . women in this age group may be in postmenopausal phase , particularly those who have been admired for their beauty , may be quite demanding about the cosmetic arrangement of their artificial teeth . the loss or decline of the senses of vision , hearing , and taste has psychological implications . the elderly population has a high prevalence of psychiatric complications associated with medical illnesses . about 1520% seeing the effect of education status on mental attitude , with the decline in the education level , patients were more indifferent and needed motivation for wearing and maintaining dentures . interestingly , a significantly high number of high school pass outs had an exacting mindset when compared to college graduates . concluded that the level of education had relation with the satisfaction and acception of the complete denture , because the ignore person could not assess his condition in a suitable manner . the clinical significance of this study lies in anticipating the mental attitude of the patient sitting in the dental office by correlating it with sex , age , and education level . though , we found statistically significant results in indifferent and exacting categories , but this study has certain limitations . the study had a small sample size to generalize the facts in a huge population . the other limitation was that only one method was used to judge the mental attitude of the patient . to conclude , females were found to be more exacting in nature while males presented with indifferent attitude in context to the fabrication of complete denture procedure . patients in the age groups of 4554 years revealed exacting nature where 65 years showed hysterical attitude when compared with other age groups . an illiterate or minimally educated group outnumbered in the indifferent type and were in the exacting category . within limitations , it could be said that the mental attitudes of patients could vary according to gender , age , and educational status , which could affect patient cooperation and satisfaction with oral rehabilitation , eventually manipulating the overall success of the treatment rendered . this study encourages further studies to evaluate such correlations in large sample size , in different regions and using different methodology .

objective : the aim of this study was to determine any relationship , if exists , between the patient 's mental attitude with age , sex , or educational qualification.methods:a total of 200 patients who attended the outpatient department during a span of 1 year , for the fabrication of new complete dentures , were chosen for the study . after completing a routine case history and examination , a questionnaire was filled by the clinician as answered by the patient . participants were evaluated and categorized based on the questionnaire and clinical experience during treatment according to a predefined classification of determining mental attitudes . outcomes from the survey were correlated with participant gender , age , and educational status.results:the male to female ratio was 83:117 , out of the 200 enrolled participants . the results from the questionnaire showed that females were found to be more exacting ( p = 0.007 ) in nature , while males , on the other hand , revealed more indifferent attitude ( p = 0.02 ) ; both differences being statistically significant . of the three age group categories : participants in the age group of 4554 years revealed a significant inclination toward an exacting attitude when compared with other age groups ( p < 0.001 ) . in regards to educational status , an illiterate or minimally educated group significantly outnumbered the college graduates in the indifferent attitude group ( p < 0.001).conclusions : within the limitations of this study , it could be said that the mental attitudes of patients could vary according to gender , age , and educational status , which could affect patient cooperation and satisfaction with oral rehabilitation , eventually manipulating the overall success of the treatment rendered .

INTRODUCTION Philosophical mind Exacting mind Hysterical Indifferent mind MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION Financial support and sponsorship Conflicts of interest

the interaction between genetic and environmental factors lies behind the neuronal representation of sensory stimuli in the nervous system . sensory experience actually drives the refinement of immature neural circuitries into organized patterns of synaptic connectivity that subserve adult brain functions . environmental influences play a key role in sculpting the central nervous system architecture during early life , when neural circuitries are highly sensitive to experience ( reviewed in [ 2 , 3 ] ) . this seems to be a period of time ( so called critical period ) in which an individual acquires an indelible memory of relevant stimuli in the environment , which ensures proper development of sensory functions and/or behaviours . an emerging view in the field of plasticity is that the effects caused by early developmental experience in the remodeling of neural networks seem to be actively preserved by the late appearance of structural and functional factors that restrict plasticity over the time course . this feature seems to be of relevance in terms of adaptive functions but determines diminished plasticity in the adult brain , which in turn severely restricts the functional reorganization of the nervous system thus posing a limit for feasible clinical interventions after brain injury or disease in humans ( for review see [ 4 , 5 ] ) . the capacity of neural circuitries to change in response to sensory experience is of high relevance in fields of neuronal rehabilitation and brain repair . this is clear , for instance , in the case of stroke , which is a major cause of long - term disability for which there is currently no clinical treatment . reactivating juvenile - like plasticity in the adult brain would be beneficial in poststroke patients , whose recovery depends on a reorganization of neuronal networks in adult life ( reviewed in ) . experience - dependent modifications of brain functions depend , at least partially , on gene expression patterns that have evolved to meet specific environmental demands . the structure and function of the bdnf gene are a compelling example of physiological mechanisms by which experience - dependent plasticity is achieved . since promoter areas of the bdnf gene are differentially regulated by distinct neurotransmitter systems , the levels of which vary in response to environmental influences ( for review see ) , bdnf protein synthesis in the brain is regulated by experience in a spatiotemporal - dependent manner [ 8 , 9 ] . this neurotrophin drives different forms of synaptic plasticity and therefore epitomizes how the nervous system mediates fast adaptive responses to changing environmental conditions . a hot spot in the neuroscience field is the identification of physiological mechanisms associated with experience that trigger alterations in the pattern of dna methylation and/or posttranslational modifications of histones that in turn control the expression of genes underlying phenomena of plasticity in the brain ( reviewed by [ 10 , 11 ] ) . indeed , epigenetic mechanisms that exert a long - lasting control of gene expression by modifying chromatin structure rather than changing the dna sequence itself have been recognized as experience - dependent mechanisms that regulate the occurrence of brain plasticity ( [ 1215 ] , reviewed in [ 1618 ] ) . transcriptional mechanisms that are mediated by immediate early genes ( iegs ) and lie behind the occurrence of plasticity in the nervous system have also been subject of recent studies ( , for review see [ 20 , 21 ] ) . it is becoming increasingly clear that experience - dependent plasticity is achieved when neuronal activity triggers intracellular signal pathways that promote the induction of iegs ( e.g. , c - fos , c - jun , creb , and zif268 ) that in turn control the expression of downstream targets , the products of which then work via the activation of structural and functional mechanisms that eventually modify the strength of synaptic connections so as to change the computational properties of neural networks in the brain ( reviewed in [ 2022 ] ) . in this review , i shall focus on the role of the recently discovered neuronal - specific transcription factor npas4 as a key regulator of brain plasticity and cognition . it has been suggested that npas4 may be involved in phenomena of plasticity after local [ 23 , 24 ] and global [ 24 , 25 ] cerebral ischemia , seizures [ 26 , 27 ] , and brain injury [ 25 , 27 , 28 ] . more recently , it has been reported that the npas4 transcription factor plays a key role in mediating a transcriptional program underlying amygdala - dependent and hippocampal - dependent processes of memory , social , and cognitive functions . the upregulation of npas4 in the striate nucleus after chronic amphetamine administration , which is a pharmacological model of plasticity with high relevance for mechanisms of addiction [ 33 , 34 ] , has also been described . moreover , impairments of neurogenesis and deficits in fear and spatial memories by social isolation and chronic stress seem to be associated with the transcriptional suppression of the npas4 gene , suggesting a central role for this transcription factor as a mediator of plasticity . here , i will highlight recent advances that have brought to light some of the structural and functional mechanisms underlying the action of npas4 in experience - dependent plasticity . i shall also cover novel findings on npas4-mediated gene programs that lie behind phenomena of cortical plasticity caused by either pharmacological treatments or experimental strategies based on the enhancement of environmental stimulation levels in adult life . studies aimed at the identification of genes that mediate the activity - dependent regulation of inhibitory synapses formation during development , revealed that npas4 is an ieg induced by neuronal activity that seems to lie behind homeostatic mechanisms that keep neuronal firing in response to sensory experience within normal levels . the exposure of primary neuronal cultures to high levels of potassium chloride leads to membrane depolarization and calcium influx through l - type voltage - sensitive calcium channels . the resulting increase in intracellular calcium levels then triggers calcium - dependent signaling pathways that eventually mediate changes in gene transcription . the analysis of dna microarrays upon this experimental design , in cortical neurons of young mice when development of inhibitory synapses is underway , revealed that npas4 is a transcription factor regulated by neuronal activity , whose expression parallels the development of inhibitory synaptic contacts . npas4 is selectively induced by calcium influx only in neurons but not in other cell types . the expression of npas4 , unlike other activity - dependent transcription factors such as creb and c - fos , is triggered selectively by excitatory synaptic transmission but not by neurotrophic factors . as observed in the cortex , npas4 expression in primary hippocampal neurons increases with the formation and maturation of synaptic contacts that occur during development , presumably , because of enhanced endogenous spontaneous levels of activity . of note , npas4 expression in response to stimulation of primary sensory areas has been reported ; visual experience in mice after one week of dark exposure actually increases mrna and protein levels of npas4 in visual cortex pyramidal cells . studies in hippocampal cell cultures , using shrna interference ( shrnai ) against npas4 and immunohistochemistry for both the gaba synthetizing enzyme gad65 and the gabaa - receptor 2 subunit as pre- and postsynaptic markers , respectively , revealed that the downregulation of npas4 expression markedly reduces inhibitory synaptic contacts formation on perisomatic and dendritic regions of excitatory neurons , suggesting that this transcription factor positively regulates the number of inhibitory synapses that form during early life . these findings were confirmed by recordings of miniature inhibitory postsynaptic currents ( mipscs ) in ca1 pyramidal cells , which decrease in amplitude after npas4 downregulation by npas4-shrnai infection . furthermore , experiments performed in conditional knockout mice ( npas4 ) in which the npas4 gene is selectively deleted by cre - mediated recombination revealed that cre expression leads to a significant increase of interevent intervals of mipscs , thus showing that ca1 pyramidal neurons lacking npas4 receive fewer inhibitory synaptic inputs . in contrast , increasing npas4 levels in cultured hippocampal neurons enhances the formation of inhibitory synapses , as suggested by a marked increase in the number of the gabaa - receptor 2 subunits . in line with this , expression of npas4 in ca1 pyramidal neurons increases the amplitude of mipscs while decreasing mipscs interevent intervals , consistent with an enhanced inhibitory synaptic signaling . notably , modifications of excitatory synaptic transmission also seem to occur after alterations of npas4 expression . in addition to the induction of genes that control the development of inhibition , npas4 also seems to regulate a gene program that includes a wide variety of transcription factors , genes encoding channel proteins , g - protein signaling molecules , protein kinases and phosphatases , and genes involved in membrane receptors trafficking and synaptic transmission . moreover , it has been reported that npas4 mediates bdnf expression in primary cortical neurons [ 30 , 38 , 40 , 41 ] . bdnf is reduced in neurons with decreased levels of npas4 after lentiviral npas4-shrnai infection and primary cell cultures from npas4 knockout mice consistently show a similar reduction of depolarization - induced bdnf expression . chromatin immunoprecipitation ( chip ) studies have shown that npas4 binds to the bdnf promoters i and iv in membrane - depolarized neurons , indicating that npas4 directly mediates the activity - dependent bdnf transcription . this phenomenon seems to underlie , at least partially , the effect of npas4 in increasing the formation of inhibitory synapses , as the number of inhibitory synaptic contacts induced by npas4 is moderately attenuated in cells in which bdnf has been knocked down by bdnf - shrnai infection . accordingly , the enhancement of inhibition caused by npas4 in ca1 neurons is partially but not totally attenuated by knocking down bdnf levels . in summary , npas4 induction in response to excitatory transmission appears to mediate a reduction of neuronal activity levels and therefore may function as a homeostatic mechanism during phases of enhanced excitability . to what extent npas4 mediates , directly or indirectly , the development of inhibitory synaptic contacts formed by different types of gabaergic interneurons on excitatory cells is an open question that remains to be explored . further studies of npas4 physiological functions may shed light on mechanisms by which experience - dependent neuronal activity regulates the balance between inhibition and excitation in the brain and how alterations in such a balance may contribute to pathological conditions such as down syndrome , autism , and rett syndrome in which inhibitory transmission seems to be altered [ 4244 ] . the formation and storage of memories are a classical example of experience - dependent plasticity mechanisms that allow an individual to modify behaviour by learning . what structural and functional changes occur in the brain as we learn ? it is well established that there are stages in memory that are encoded as modifications in the strength of synapses that correlate with behavioural phases of short- and long - term memory . pioneering studies from molluscs to flies , and mammals revealed highly conserved signal transduction pathways that are critical for the occurrence of synaptic plasticity underlying the establishment of long - term memories . these conserved pathways involve calcium - mediated activation of intracellular protein kinases , translocation of these proteins to the nucleus , and subsequent activation of transcription factors that mediate gene transcription ( for review see ) . activation of glutamate n - methyl - d - aspartate ( nmda)-receptors , for instance , seems to induce phosphorylation of creb , which causes alterations of chromatin structure that allow for the induction of gene programs and de novo synthesis of proteins that eventually mediate long - term changes of synaptic transmission during learning ( figure 1 ) . in rodents , the hippocampus is involved in the formation of memory for new environments or contexts ( reviewed by ) , this phenomenon being dependent on the activation of the ca3 hippocampal area [ 4951 ] . contextual memory formation can be examined using the contextual fear conditioning ( cfc ) task ( for review see ) , which consists of exposure of an animal to a given context in which an electric shock , that may or may not be accompanied by a tone , occurs . after training in this protocol , wild - type animals normally remember and associate the context with the aversive shock experience , which can be later evaluated in terms of freezing behaviour ; 1 hour or 24 hours after training , the animals are exposed to the same aversive context to explore either short- or long - term contextual memory , respectively . using this experimental paradigm , a novel role for npas4 in the regulation of contextual memory formation has been recently uncovered . these studies initially evaluated the expression of the iegs c - fos , arc , and npas4 in the dorsal hippocampus of mice that were exposed to the cfc task and sacrificed at different time points . notably , npas4 expression was found to peak much before that of c - fos and arc ; npas4 mrna reached its peak after 5 min of training , returning to basal levels of expression after 4.5 hours . instead , c - fos and arc reached their peak levels of expression after 30 min of training . these findings highlight a hierarchical genetic program in which npas4 is upstream of several other iegs in the dorsal hippocampal area . this notion was later confirmed by the observation that conditional deletion of the npas4 gene by cre recombination in hippocampal neurons of npas4 transgenic mice results in a marked loss of c - fos , arc , and zif268 expression . after 5 min of training in the cfc test , robust freezing behaviour was observed in both wild - type and npas4 littermates , indicating that learning capabilities were normal in npas4 animals . in contrast , freezing behaviour was significantly reduced 1 hour and 24 hours after cfc training , showing that both short - term and long - term memory formation is impaired in npas4 mice . after cfc training , npas4 expression was localized mainly in the ca3 area of the hippocampus . the selective deletion of npas4 in ca3 but not in ca1 impaired long - term contextual memory formation ; 24 hours after cfc training , npas4 mice injected in ca3 with a virus expressing the cre recombinase showed attenuated freezing responses as compared with wild - type or npas4 animals injected in ca1 , thus demonstrating that deleting npas4 specifically in ca3 replicates the memory deficits seen in the npas4 knockout . the issue of whether npas4 expression in the ca3 area of the npas4 background leads to the expression of the npas4-mediated gene program and rescues memory formation was also investigated . remarkably , the expression of npas4 in ca3 completely reversed the short - term and long - term contextual memory deficits previously observed in the npas4 background ; npas4-expressing mice in ca3 but not in ca1 showed similar freezing behaviour as wild - type control animals after either 1 hour or 24 hours of training in the cfc behavioural task . in summary , this elegant set of experiments demonstrates that the activity - dependent transcription factor npas4 is a key mediator of plastic phenomena that underlie hippocampal - dependent contextual memory formation . on the one hand , acute deletion of the npas4 gene in ca3 results in a dramatic diminishment of iegs expression and impaired contextual memory formation . on the other hand , expression of npas4 mrna in npas4 knockout animals the first model to provide a specific mechanism for modifications of synaptic transmission involved in associative learning was advanced by donald hebb in 1949 ; it was proposed that modifications in the strength of synapses might occur only if the use of those synapses was associated with and contributes to the generation of action potentials in the postsynaptic neuron ( reviewed by ) . hebb 's principle has been summarized as follows : neurons that fire together wire together whereas neurons that fire out of synchrony lose their connection . thus , an essential feature of this postulate is that modifications of synaptic transmission depend on coincidence activity of the presynaptic and the postsynaptic neuron . nmda - receptors actually function as coincidence detectors in synaptic plasticity , as they open and mediate excitatory synaptic transmission only when the presynaptic release of glutamate is coupled to the postsynaptic depolarization ( [ 54 , 55 ] , for review see ) , thus fulfilling hebb 's rule at molecular level . although hebbian mechanisms provided an initial and important framework for the interpretation of neuronal network alterations , it has become clear that there are mechanisms of metaplasticity controlling changes of synaptic plasticity ( reviewed by ) . indeed , due to positive feedback , hebbian plasticity could lead to a saturation of the synaptic strength in the absence of proper constraints . there is now a general consensus that homeostatic mechanisms are regulatory adjustments that work to maintain the stability and functionality of neuronal networks when modifications of synaptic transmission are underway ( reviewed in ) . a classical form of homeostatic plasticity is epitomized by the bienenstock - cooper - munro ( bcm ) model , which states that synaptic inputs driving postsynaptic firing to high levels result in an increase in synaptic strength , whereas inputs that trigger low levels of postsynaptic firing result in a decrement of synaptic transmission . the threshold for neuronal activation in the bcm model is not fixed but changes itself as a function of postsynaptic activity , the threshold slides as to make potentiation more likely whenever average activity is low , and less likely when average activity is high ( reviewed by ) . this is thought to maintain the stability of synapses in neuronal circuitries upon changes of synaptic transmission . mechanisms of homeostatic plasticity described so far ( for review see [ 57 , 58 ] ) include ( i ) synaptic scaling ( i.e. , scaling of the strength of excitatory synapses depending on the average activity of the postsynaptic neuron ) and ( ii ) the regulation of intrinsic excitability ( i.e. , changing the way in which postsynaptic neurons integrate synaptic inputs and fire action potentials ) . the identification of molecular substrates underlying these forms of homeostatic plasticity , however , still needs further research . hence , the discovery that the activity - dependent expression of npas4 is implicated in a transcriptional program that regulates neuronal firing responses to excitatory transmission by enhancing inhibition is of high relevance for homeostatic plasticity research . it will be interesting to evaluate mechanisms of metaplasticity in npas4 knockout animals or in conditional npas4 mice after deletion of the npas4 gene by selective cre recombination . experience - dependent functional modifications of neuronal circuitries in the brain are accompanied by structural rearrangements of neuronal connectivity . excitatory synaptic structures such as dendritic spines , for instance , are particularly sensitive to experience during development . a total lack of visual experience in early life ( dark rearing ) actually modifies spines morphology and density in the visual system , these two phenomena being partially reversible by subsequent light exposure . in agreement with this notion , monocular deprivation during the critical period influences motility , turnover , number , and morphology of dendritic spines in the visual cortex [ 6266 ] . these findings highlight a correlation between the structural remodeling of single synapses and functional modifications of neural circuitries in response to changing environmental conditions . this question has been recently addressed by signal optical imaging of functional responses to visual stimulation and by longitudinal two - photon imaging experiments , showing that dendritic spine dynamics of pyramidal neurons in the mouse neocortex is maximal during early stages of development but decreases thereafter , in parallel to the decline of functional plasticity that occurs over development ( , for review see ) . although most studies on structural plasticity have focused on modifications in excitatory cells , there is also evidence that structural plasticity occurs in inhibitory neurons . it has been demonstrated that gabaergic interneurons in superficial layers of the visual cortex exhibit dendritic arbor growth and remodeling in adult life . moreover , structural modifications of inhibitory synapses onto pyramidal excitatory cells seem to be a major component of plasticity in the adult mouse neocortex [ 6971 ] . the dynamic turnover of dendritic spines on pyramidal neurons and the remodeling of interneurons dendritic arbors actually appear to be a common feature among primary sensory areas . in summary , cortical plasticity seems to be associated with a structural rearrangement of excitatory connections during early development whereas structural modifications of dendritic arbors in gabaergic interneurons seem to correlate with adult cortical plasticity . an unresolved and interesting question in the field is whether npas4 activates downstream targets associated with structural plasticity in the nervous system . very recent studies suggest that npas4 may be involved , at least in part , in some forms of structural plasticity . there is evidence that differentiation - induced neurite outgrowth in cell cultures is inhibited if npas4 expression is knocked down , whereas overexpression of npas4 appears to accelerate neurite outgrowth . moreover , depolarization - induced neurite outgrowth is impaired in the hippocampus of npas4 knockout animals . this phenomenon appears to depend on phosphorylation of the protein synapsin - i by the cyclin - dependent protein kinase cdk5 and npas4 seems to mediate cdk5 expression by binding to the cdk5 gene promoter area . whether these findings bear any physiological significance in naturally occurring processes of neuronal plasticity . it may be interesting to examine whether dendritic spines in excitatory neurons and dendritic arbors in gabaergic cells are fewer and/or lessened in the visual cortex of npas4 mice or in conditional npas4 mice after deletion of the npas4 gene by selective cre recombination . since the expression of npas4 seems to take place predominantly in excitatory neurons , another interesting consideration would be to investigate whether npas4 influences dendritic spines turnover and density in pyramidal cells in the visual cortex . this issue is of particular relevance for the process of plasticity reactivation late in life as there is evidence that the maturation of the extracellular matrix composition during development stabilizes neuronal connectivity patterns while inhibiting structural and functional plasticity of dendritic spines . the degradation of extracellular matrix components known as chondroitin sulphate proteoglycans ( cspgs ) by exogenous administration of the bacterial enzyme chondroitinase actually reinstates ocular dominance plasticity in adulthood , probably by modifying dendritic spines dynamics and associated neuronal connectivity changes in the visual cortex . the extent to which environmental influences modify brain structure and function has been extensively studied in the developing visual system . an experience - dependent reorganization of eye - specific inputs during early life is actually the major mechanism by which neuronal connectivity is established in the primary visual cortex . the monocular deprivation paradigm has been a classical model to assess neuronal plasticity in the visual system . early electrophysiological and anatomical studies in cats and monkeys revealed that short periods of sensory deprivation by unilateral eye closure during early life cause major structural and functional modifications of visual cortical circuitries . visual cortex responsiveness markedly shifts in favour of the non deprived eye after monocular deprivation during the critical period ( [ 7579 ] ) . in addition , the deprived eye becomes amblyopic ; its visual acuity ( spatial resolution ) and contrast sensitivity are severely impaired [ 77 , 7981 ] . is the activity - dependent npas4 expression involved in the occurrence of critical period plasticity ? before addressing this question , a brief overview of the developmental functional organization of the visual system should be considered . sensory experience during early life signals the time course of the critical period by promoting the transfer of the protein otx2 from the retina to the visual cortex , where otx2 appears to drive the maturation of parvalbumin - positive gabaergic interneurons ( for review see [ 83 , 84 ] ) . the experience - dependent developmental maturation of gaba - mediated inhibition then establishes the threshold for both the start and the end of the critical period for visual cortical plasticity [ 8587 ] . indeed , transgenic mice with reduced levels of intracortical inhibition due to the lack of the gaba - synthetizing enzyme gad65 exhibit no modifications of visual cortex responsiveness after monocular deprivation in early life , whereas enhancing inhibition by exogenous administration of gabaa - receptor agonists in the knockout background rescues the impairment of plasticity [ 86 , 87 ] . on the other hand , transgenic animals that show an accelerated maturation of intracortical inhibition due to bdnf overexpression in forebrain regions display a precocious development of the visual system and an accelerated end of the critical period for ocular dominance plasticity . in summary , an initial threshold of inhibition triggers a sensitive period in which neuronal networks in the visual system are highly susceptible to sensory experience , whereas a second inhibitory threshold signals the end of this phase of enhanced plasticity ( for review see ) . since the transcriptional program activated by npas4 enhances inhibition by promoting the expression of genes that direct the formation of inhibitory synaptic contacts , it emerges clearly that npas4 expression is likely involved in the regulation of the critical period for visual cortex plasticity . it will be interesting to evaluate whether npas4 animals with reduced levels of inhibition show impairments of ocular dominance plasticity in response to monocular deprivation during early development . this could be complemented by studies of plasticity in wild - type animals after npas4 downregulation by selective npas4-shrnai infection or in npas4 mice with a selective deletion of the npas4 gene by cre recombination in the developing visual cortex . moreover , the induction of the npas4-mediated gene program by infection with an npas4 expressing virus in the visual cortex of either wild - type or npas4 animals may be a feasible strategy to evaluate whether npas4 overexpression accelerates visual system development and the time course of the critical period for plasticity . another interesting issue is whether the activation of the retinogeniculocortical transfer of the protein otx2 in the visual pathway correlates with the activity - dependent expression of npas4 in pyramidal neurons of the primary visual cortex . an alternative approach to assess the impact of npas4 expression in visual cortical plasticity may be rearing animals in total darkness from birth . experiments that combine dark rearing and electrophysiology as a functional readout have demonstrated that the absence of visual inputs during development leads to a delayed maturation of the visual cortex . it will be exciting to evaluate whether dark rearing decreases the expression of npas4 in the visual cortex and whether the effects of dark rearing in the visual system of wild - type animals are prevented by selective npas4 expression . converging lines of evidence attribute the decline of plasticity that occurs with age to the maturation of intracortical inhibitory circuitries [ 8587 ] . consistently , it is possible to restore a high degree of plasticity in adult life by reducing levels of inhibition . this is in line with the observation that experimental paradigms , such as dark exposure [ 9193 ] , environmental enrichment [ 9496 ] , food restriction , long - term fluoxetine treatment [ 15 , 98 , 99 ] , exogenous igf-1 administration , and genetic manipulations [ 101 , 102 ] , all promote plasticity in adult life by shifting the intracortical inhibitory / excitatory ratio in favour of excitation . recent studies in rodents and cats have revealed that the process of plasticity reactivation appears to be a multifactorial event that comprises the action of different structural and functional mechanisms , working in parallel or in series , the sum of which results in the activation of intracellular signal pathways regulating the expression of plasticity genes ( reviewed by [ 18 , 104 ] ) . indeed , experience - dependent modifications of chromatin structure that control gene transcription are recruited as targets of plasticity - associated processes in adulthood [ 14 , 15 , 97 , 101 ] . there is evidence that npas4 mediates the activity - dependent expression of bdnf [ 30 , 38 , 40 , 41 ] , a neurotrophin that has been clearly linked to multiple forms of synaptic plasticity in diverse brain areas ( [ 105110 ] , for review see ) . of note , chronic infusion of bdnf into the visual cortex restores susceptibility to monocular deprivation in adulthood , whereas the impairment of bdnf - trkb signaling effectively prevents the process of plasticity reactivation caused by fluoxetine in the adult visual system . these findings portray the activity - dependent npas4 transcription factor as an appealing candidate for the regulation of visual cortical plasticity in adulthood . the following is an overview of the potential role of npas4 as a mediator of plasticity induced by different noninvasive experimental approaches in the adult visual system . compelling experimental evidence for npas4-mediated transcriptional mechanisms that lie behind phenomena of visual cortex plasticity in adulthood has been recently obtained using the monocular deprivation paradigm and chronic treatment with fluoxetine as a pharmacological strategy for the induction of plasticity . there is evidence that the plastic outcome caused by fluoxetine in adulthood is accompanied by increased levels of serotonin , reduced levels of gabaergic inhibition , and increased bdnf expression in the visual cortex . more recently , it was demonstrated that the reinstatement of plasticity caused by fluoxetine is paralleled by epigenetic modifications of chromatin structure that promote gene transcription . on the one hand , an increased histone acetylation status at bdnf promoter regions occurs in concomitance with bdnf expression . on the other , a reduction in the methylation status at the npas4 promoter area parallels an enhanced npas4 expression after pharmacological treatment . of note , the impairment of serotonergic signaling prevents the remodeling of chromatin structure caused by the pharmacological treatment in gene promoter areas . this points toward a hierarchical model in which serotonin seems to be the primum movens in a series of signal transduction pathways leading to epigenetic modifications of chromatin structure and subsequent expression of plasticity genes in the adult visual cortex ( for review see ) . in this context , npas4 is upstream bdnf expression and seems to direct the gene program mediating this plastic phenomenon . electrophysiological experiments in combination with gene delivery by lentiviral infection have actually shown that npas4 expression in the visual cortex of nave animals restores susceptibility to monocular deprivation in adulthood . consistently , npas4 downregulation by npas4-shrnai in the adult visual system effectively prevents plastic events caused by fluoxetine treatment . how does npas4 fit into a model of enhanced plasticity , which correlates with a decrease of inhibition in adulthood ? given that npas4 expression increases the formation of inhibitory synaptic contacts , one might expect npas4 to be inversely correlated with the occurrence of plasticity ; that is , npas4 expression should occlude plasticity whereas npas4 knockdown should enhance it . this scenario , however , seems to be unlikely as there is evidence that knocking down npas4 expression by npas4-shrnai infection in the visual cortex of nave animals does not restore visual cortex susceptibility to monocular deprivation in adult life . instead , based on extensive data in the hippocampus , it seems reasonable to hypothesize that the activity - dependent npas4 expression caused by serotonin ( fluoxetine ) in the adult visual cortex may turn on a transcriptional program that upregulates the expression of plasticity genes while facilitating , in parallel or in series , a functional reorganization of inhibitory circuitries that might contribute to the homeostasis of cortical excitability during this phase of enhanced plasticity . this is in line with two recent observations : ( i ) npas4 interacts with a wide variety of neuronal activity - regulated gene expression enhancers and promoters in the nervous system and ( ii ) different homeostatic mechanisms assist to keep neuronal activity within normal levels as synaptic modifications of neuronal circuitries are underway in the visual cortex . the available experimental evidence for a combined action of fluoxetine - induced serotonergic signaling , reduced levels of inhibition , and npas4 expression in driving adult visual cortex plasticity is consistent with a model ( figure 2 ) in which the serotonin - mediated shift of the intracortical inhibitory / excitatory balance that occurs in favour of excitation [ 15 , 98 ] may induce the activity - dependent expression of npas4 . this in turn could mediate the expression of plasticity genes and subsequently promote the formation of inhibitory synaptic contacts on excitatory neurons as a compensatory mechanism for the reduction of the inhibitory tone after fluoxetine treatment . in line with this notion , there is evidence that the fluoxetine - induced mechanism of disinhibition in the visual cortex of adult animals after a brief period of monocular deprivation is accompanied by an increase in elongations of gabaergic interneuron dendritic branch tips in superficial cortical layers . hence , this points toward a compensatory mechanism for the reduction of inhibition that involves the formation and/or strengthening of inhibitory contacts on neighbouring excitatory neurons , that is , a mechanism in which npas4 is likely to be involved . understanding how npas4 expression regulates inhibitory synapse density and function in the adult visual system is important to interpret these findings . it may be interesting to evaluate the time course of expression of gabaergic markers in the adult visual cortex ( e.g. , gad65 , gad67 , vgat , gabaa - receptor 2 subunits , and parvalbumin ) by means of immunohistochemistry in a nave background versus a background of npas4 knockdown or npas4 mice with a selective deletion of the npas4 gene by cre recombination . this could also be assessed in a nave background before and after long - term fluoxetine treatment and may be complemented by electrophysiological analysis of spontaneous and evoked ipscs to examine functional connectivity . the use of dna microarrays in this context might be of relevance for the identification of npas4 downstream targets associated with structural and functional modifications in the adult visual system after fluoxetine treatment . the enhancement of environmental stimulation levels has recently proved to be a powerful and noninvasive strategy to promote juvenile - like plasticity in the adult visual system . environmental enrichment ( ee ) is an experimental paradigm characterized by enhanced sensory - motor activity and social stimulation that has a profound impact on brain structure and function ( reviewed by ) . in rodents , it has been demonstrated that short period of ee in adult life reactivates ocular dominance plasticity [ 94 , 116 ] and there is evidence that resetting adult visual cortex circuitries to a more plastic stage by ee favours the rescue of sensory functions after long - term deprivation [ 95 , 96 , 117 , 118 ] . in humans , enriching the environment in terms of body massage triggers plastic phenomena that accelerate the maturation of visual functions during development . does npas4 play a role in the effects caused by ee in visual cortical plasticity ? this is a likely scenario as the reinstatement of plasticity caused by ee in adulthood is accompanied by an increment in serotonin signaling , reduced levels of inhibition , and enhanced bdnf expression , much as in the case of long - term fluoxetine treatment . it is worth noting that decreasing bdnf signaling by exogenous administration of antisense oligonucleotides in the visual cortex of adult animals exposed to enriched environmental conditions prevents partially but not totally the shift of ocular dominance in response to monocular deprivation . considering that npas4 directly promotes bdnf expression , this suggests that the effects caused by ee in visual cortical plasticity could be only partially dependent on npas4 expression . another possibility is that npas4 drives phenomena of plasticity even in the absence of bdnf signaling and therefore could lie behind the plasticizing effects of ee in adult life . . it will be interesting to assess the effects caused by ee in adult visual cortex plasticity in npas4 knockout animals or in npas4 mice with a selective deletion of the npas4 gene by cre recombination . brief periods of visual deprivation by dark exposure and food restriction have also proved to be effective approaches to reactivate plasticity in the adult visual system . juvenile - like ocular dominance plasticity can actually be restored in adult animals if monocular deprivation is preceded by visual deprivation or by a reduction of the caloric intake . it may be interesting to investigate the effects caused by brief periods of dark exposure and food restriction in the npas4 knockout versus a nave background . long regarded as a rather static and unchanging structure , the adult brain has increasingly been recognized as a system that retains a degree of plasticity that allows for structural and functional modifications of neuronal networks if exposed to certain experimental conditions . in animal models , it has been demonstrated that both pharmacological treatments and experimental paradigms based upon manipulation of environmental stimulation levels effectively promote a rewiring of visual cortical circuitries in adulthood . it seems reasonable to speculate that these noninvasive approaches , when combined with appropriate instructive environmental stimuli , could be exploited for clinical applications in humans . since the decline of plasticity that occurs over the life course severely restricts the functional reorganization of neuronal circuitries , these studies are beginning to elucidate physiological processes that lie behind modifications of neuronal networks ' connectivity in adulthood in which the activity - dependent transcription factor npas4 seems to play a critical role . in light of this , structural and functional mechanisms leading to the activity - dependent npas4 expression arise as potential therapeutic targets for future development of drugs that could be used in a variety of pathological conditions in which a reorganization of neuronal circuitries is needed late in life . long - term fluoxetine administration , indeed , not only induces npas4 expression , but also has been proved successful in promoting the functional recovery from stroke in humans , which is a major cause of long - term disability for which there is currently no clinical treatment .

the capability of the brain to change functionally in response to sensory experience is most active during early stages of development but it decreases later in life when major alterations of neuronal network structures no longer take place in response to experience . this view has been recently challenged by experimental strategies based on the enhancement of environmental stimulation levels , genetic manipulations , and pharmacological treatments , which all have demonstrated that the adult brain retains a degree of plasticity that allows for a rewiring of neuronal circuitries over the entire life course . a hot spot in the field of neuronal plasticity centres on gene programs that underlie plastic phenomena in adulthood . here , i discuss the role of the recently discovered neuronal - specific and activity - dependent transcription factor npas4 as a critical mediator of plasticity in the nervous system . a better understanding of how modifications in the connectivity of neuronal networks occur may shed light on the treatment of pathological conditions such as brain damage or disease in adult life , some of which were once considered untreatable .

1. Introduction 2. Neuronal Activity and 3. NPAS4 Upregulates a Gene Program That Underlies Memory Formation 4. Role of NPAS4 in the Regulation of Homeostatic Plasticity 5. NPAS4 and Structural Plasticity in the Nervous System 6. 7. 8. Conclusion and Implications

the ecls - b includes data from a cohort of ~10,200 children born in the us in 2001 . note that all sample sizes reported in this paper have been rounded to the nearest 50 , as specified by ecls - b data confidentiality requirements ( which are available at the ecls - b website : http://nces.ed.gov/ecls/birthdatainformation.asp ) . the ecls - b sample was selected from birth certificate records and includes oversamples of asian and pacific islanders , native americans and alaska natives , low birthweight ( 1,5002,500 g ) and very low birthweight ( < 1,500 g ) children , and multiple births . sample weights are provided in the ecls - b dataset to allow for calculation of nationally - representative estimates . the ecls - b study protocol includes direct developmental assessments and interviews with family members when children are 9 months , 24 months , and 48 months of age . data used in the present analyses were gathered at the 24 and 48 month assessments . in this study , we restricted the analytic sample to the approximately 7,200 children having cognitive assessment data from the 24 and 48 month developmental assessments , as well as data for each of the other variables included in the multivariate models . thus , the analytic sample excluded some children who participated in the ecls - b study but had missing data on variables of interest . however , those children who were excluded were generally similar to those included in the analytic sample on most observed socioeconomic characteristics . data for the 48 month developmental assessments were slightly more likely to be missing among males and among black children , resulting in analytic sample sizes for these groups that were smaller than their representation in the full sample by 1.2 percent and 3 percent , respectively . the outcome of interest , delayed cognitive functioning , was quantified based on standardized cognitive assessments administered to children at the 24 and 48 month survey waves . at 24 months , the mental scale of the bayley short form - research edition ( bsf - r ) was used . this is a modified version of the bayley scales of infant development , second edition ( bsid - ii ) , which is designed for use in children from birth to 36 months of age . in both the bsid - ii and the bsf - r , the mental score is based on trained interviewer assessments of age - appropriate cognitive development as manifested in tasks demonstrating memory , habituation , preverbal communication , problem - solving , and concept attainment . the shortened bsf - r was extensively tested to ensure that the psychometric properties of the bsid - ii were maintained and that it accurately measured children s performance over the entire ability distribution . a mental scale score is provided in the ecls - b , along with the child s age at the time of administration . for children born preterm , age at administration a dichotomous variable was created having a value of 1 for children scoring in the lowest 10% of the bsf - r scale distribution and 0 otherwise . this 10th percentile cut - point has been used to identify cognitive delay in previous research by our team and by others [ 2628 ] . the patterns of results using these alternative cut points were consistent with the results we report using the 10% cut point . administration of the bayley assessment was no longer age - appropriate by the 48 month time point . instead , a standardized assessment battery measuring literacy , thus , the 48 months battery consisted of items relating more directly to preschool - aged children s academic school readiness . the battery incorporated items from a number of standardized assessments developed for use in other large studies of child development such as the head start impact study , and included elements of the peabody picture vocabulary test , the preschool comprehensive test of phonological and print processing , the prelas 2000 , and the test of early mathematics ability-3 . we converted children s scores on the measures of literacy , math concepts , color knowledge , and receptive vocabulary into z - scores and summed them to produce a summary cognitive score . similar to the procedure used for the bsf - r scores , we created a dichotomous variable that was given a value of 1 for those scoring in the lowest 10% of the distribution , and 0 otherwise . socio - demographic data were collected in parent interviews and from birth certificates , and variables were included in the analyses capturing children s race / ethnic origin ( non - hispanic white , non - hispanic black , hispanic , asian , native american , and other ) , maternal education ( less than 9th grade , 9th12th grade , high school graduate , some college or other training after high school , and 4-year college degree and above ) , family income ( less than $ 10,000 , $ 10,00120,000 , $ 20,001$40,000 , $ 40,001$75,000 , or over $ 75,000 ) , maternal age , and marital status . birth certificate records provided data on medical risk factors during pregnancy ( quantified as a count of problems present including incompetent cervix , acute or chronic lung disease , chronic hypertension , pregnancy - induced hypertension , eclampsia , diabetes , hemoglobinopathy , cardiac disease , anemia , renal disease , genital herpes , oligohydramnios , uterine bleeding , rh sensitization , previous birth weighing 4,000+g , or previous preterm birth ) , behavioral risk factors during pregnancy ( alcohol and tobacco use during pregnancy , coded as 1 if present and summed to form a scale that ranged from 0 to 2 ) , obstetrical procedures ( measured as a count of procedures including induction of labor , stimulation of labor , tocolysis , amniocentesis , and cesarean section ) , labor complications ( measured as a count of complications including abruption placenta , anesthetic complications , dysfunctional labor , breech / malpresentation , cephalopelvic disproportion , cord prolapsed , fetal distress , excessive bleeding , fever of > 100f , moderate / heavy meconium , precipitous labor ( <3 h ) , prolonged labor ( > 24 h ) , placental previa , or seizures during labor ) , multiple birth , preterm delivery ( represented by two dichotomous indicator variables : ( 1 ) very preterm ( 32 weeks ) and ( 2 ) moderately preterm ( 3336 weeks ) ) , low birthweight ( represented by 2 dichotomous indicator variables : ( 1 ) very low birthweight ( 1,500 g ) and ( 2 ) moderately low birthweight ( 1,5012,500 g ) , and presence of any congenital anomaly . child age was included to control for variations in actual age at administration of the 24 and 48 month assessments . descriptive analyses were conducted for study variables , as well as cross - tabulation of cognitive delay at 48 months by delay status at 24 months to assess the relative stability of cognitive delay over the two time points . two nested multiple logistic regression models were estimated for each of these study time points to examine associations between low cognitive functioning and socio - demographic characteristics and gestational and birth - related factors . to identify potential differences between children with persistent and emerging delay over the study period , additional sets of multiple logistic models predicting cognitive delay at 48 months were estimated separately for children with and without delay at 24 months . analyses were weighted to appropriately account for the oversampling of some population groups and the stratified cluster design of the ecls - b . the ecls - b includes data from a cohort of ~10,200 children born in the us in 2001 . note that all sample sizes reported in this paper have been rounded to the nearest 50 , as specified by ecls - b data confidentiality requirements ( which are available at the ecls - b website : http://nces.ed.gov/ecls/birthdatainformation.asp ) . the ecls - b sample was selected from birth certificate records and includes oversamples of asian and pacific islanders , native americans and alaska natives , low birthweight ( 1,5002,500 g ) and very low birthweight ( < 1,500 g ) children , and multiple births . sample weights are provided in the ecls - b dataset to allow for calculation of nationally - representative estimates . the ecls - b study protocol includes direct developmental assessments and interviews with family members when children are 9 months , 24 months , and 48 months of age . data used in the present analyses were gathered at the 24 and 48 month assessments . in this study , we restricted the analytic sample to the approximately 7,200 children having cognitive assessment data from the 24 and 48 month developmental assessments , as well as data for each of the other variables included in the multivariate models . thus , the analytic sample excluded some children who participated in the ecls - b study but had missing data on variables of interest . however , those children who were excluded were generally similar to those included in the analytic sample on most observed socioeconomic characteristics . data for the 48 month developmental assessments were slightly more likely to be missing among males and among black children , resulting in analytic sample sizes for these groups that were smaller than their representation in the full sample by 1.2 percent and 3 percent , respectively . the outcome of interest , delayed cognitive functioning , was quantified based on standardized cognitive assessments administered to children at the 24 and 48 month survey waves . at 24 months , the mental scale of the bayley short form - research edition ( bsf - r ) was used . this is a modified version of the bayley scales of infant development , second edition ( bsid - ii ) , which is designed for use in children from birth to 36 months of age . in both the bsid - ii and the bsf - r , the mental score is based on trained interviewer assessments of age - appropriate cognitive development as manifested in tasks demonstrating memory , habituation , preverbal communication , problem - solving , and concept attainment . the shortened bsf - r was extensively tested to ensure that the psychometric properties of the bsid - ii were maintained and that it accurately measured children s performance over the entire ability distribution . a mental scale score is provided in the ecls - b , along with the child s age at the time of administration . for children born preterm , age at administration a dichotomous variable was created having a value of 1 for children scoring in the lowest 10% of the bsf - r scale distribution and 0 otherwise . this 10th percentile cut - point has been used to identify cognitive delay in previous research by our team and by others [ 2628 ] . the patterns of results using these alternative cut points were consistent with the results we report using the 10% cut point . administration of the bayley assessment was no longer age - appropriate by the 48 month time point . instead , a standardized assessment battery measuring literacy , math concepts , color knowledge , and receptive vocabulary skills was administered . thus , the 48 months battery consisted of items relating more directly to preschool - aged children s academic school readiness . the battery incorporated items from a number of standardized assessments developed for use in other large studies of child development such as the head start impact study , and included elements of the peabody picture vocabulary test , the preschool comprehensive test of phonological and print processing , the prelas 2000 , and the test of early mathematics ability-3 . we converted children s scores on the measures of literacy , math concepts , color knowledge , and receptive vocabulary into z - scores and summed them to produce a summary cognitive score . similar to the procedure used for the bsf - r scores , we created a dichotomous variable that was given a value of 1 for those scoring in the lowest 10% of the distribution , and 0 otherwise . socio - demographic data were collected in parent interviews and from birth certificates , and variables were included in the analyses capturing children s race / ethnic origin ( non - hispanic white , non - hispanic black , hispanic , asian , native american , and other ) , maternal education ( less than 9th grade , 9th12th grade , high school graduate , some college or other training after high school , and 4-year college degree and above ) , family income ( less than $ 10,000 , $ 10,00120,000 , $ 20,001$40,000 , $ 40,001$75,000 , or over $ 75,000 ) , maternal age , and marital status . birth certificate records provided data on medical risk factors during pregnancy ( quantified as a count of problems present including incompetent cervix , acute or chronic lung disease , chronic hypertension , pregnancy - induced hypertension , eclampsia , diabetes , hemoglobinopathy , cardiac disease , anemia , renal disease , genital herpes , oligohydramnios , uterine bleeding , rh sensitization , previous birth weighing 4,000+g , or previous preterm birth ) , behavioral risk factors during pregnancy ( alcohol and tobacco use during pregnancy , coded as 1 if present and summed to form a scale that ranged from 0 to 2 ) , obstetrical procedures ( measured as a count of procedures including induction of labor , stimulation of labor , tocolysis , amniocentesis , and cesarean section ) , labor complications ( measured as a count of complications including abruption placenta , anesthetic complications , dysfunctional labor , breech / malpresentation , cephalopelvic disproportion , cord prolapsed , fetal distress , excessive bleeding , fever of > 100f , moderate / heavy meconium , precipitous labor ( <3 h ) , prolonged labor ( > 24 h ) , placental previa , or seizures during labor ) , multiple birth , preterm delivery ( represented by two dichotomous indicator variables : ( 1 ) very preterm ( 32 weeks ) and ( 2 ) moderately preterm ( 3336 weeks ) ) , low birthweight ( represented by 2 dichotomous indicator variables : ( 1 ) very low birthweight ( 1,500 g ) and ( 2 ) moderately low birthweight ( 1,5012,500 g ) , and presence of any congenital anomaly . child age was included to control for variations in actual age at administration of the 24 and 48 month assessments . the outcome of interest , delayed cognitive functioning , was quantified based on standardized cognitive assessments administered to children at the 24 and 48 month survey waves . at 24 months , the mental scale of the bayley short form - research edition ( bsf - r ) was used . this is a modified version of the bayley scales of infant development , second edition ( bsid - ii ) , which is designed for use in children from birth to 36 months of age . in both the bsid - ii and the bsf - r , the mental score is based on trained interviewer assessments of age - appropriate cognitive development as manifested in tasks demonstrating memory , habituation , preverbal communication , problem - solving , and concept attainment . the shortened bsf - r was extensively tested to ensure that the psychometric properties of the bsid - ii were maintained and that it accurately measured children s performance over the entire ability distribution . a mental scale score is provided in the ecls - b , along with the child s age at the time of administration . for children born preterm , age at administration a dichotomous variable was created having a value of 1 for children scoring in the lowest 10% of the bsf - r scale distribution and 0 otherwise . this 10th percentile cut - point has been used to identify cognitive delay in previous research by our team and by others [ 2628 ] . the patterns of results using these alternative cut points were consistent with the results we report using the 10% cut point . administration of the bayley assessment was no longer age - appropriate by the 48 month time point . instead , a standardized assessment battery measuring literacy , math concepts , color knowledge , and receptive vocabulary skills was administered . thus , the 48 months battery consisted of items relating more directly to preschool - aged children s academic school readiness . the battery incorporated items from a number of standardized assessments developed for use in other large studies of child development such as the head start impact study , and included elements of the peabody picture vocabulary test , the preschool comprehensive test of phonological and print processing , the prelas 2000 , and the test of early mathematics ability-3 . we converted children s scores on the measures of literacy , math concepts , color knowledge , and receptive vocabulary into z - scores and summed them to produce a summary cognitive score . similar to the procedure used for the bsf - r scores , we created a dichotomous variable that was given a value of 1 for those scoring in the lowest 10% of the distribution , and 0 otherwise . socio - demographic data were collected in parent interviews and from birth certificates , and variables were included in the analyses capturing children s race / ethnic origin ( non - hispanic white , non - hispanic black , hispanic , asian , native american , and other ) , maternal education ( less than 9th grade , 9th12th grade , high school graduate , some college or other training after high school , and 4-year college degree and above ) , family income ( less than $ 10,000 , $ 10,00120,000 , $ 20,001$40,000 , $ 40,001$75,000 , or over $ 75,000 ) , maternal age , and marital status . birth certificate records provided data on medical risk factors during pregnancy ( quantified as a count of problems present including incompetent cervix , acute or chronic lung disease , chronic hypertension , pregnancy - induced hypertension , eclampsia , diabetes , hemoglobinopathy , cardiac disease , anemia , renal disease , genital herpes , oligohydramnios , uterine bleeding , rh sensitization , previous birth weighing 4,000+g , or previous preterm birth ) , behavioral risk factors during pregnancy ( alcohol and tobacco use during pregnancy , coded as 1 if present and summed to form a scale that ranged from 0 to 2 ) , obstetrical procedures ( measured as a count of procedures including induction of labor , stimulation of labor , tocolysis , amniocentesis , and cesarean section ) , labor complications ( measured as a count of complications including abruption placenta , anesthetic complications , dysfunctional labor , breech / malpresentation , cephalopelvic disproportion , cord prolapsed , fetal distress , excessive bleeding , fever of > 100f , moderate / heavy meconium , precipitous labor ( <3 h ) , prolonged labor ( > 24 h ) , placental previa , or seizures during labor ) , multiple birth , preterm delivery ( represented by two dichotomous indicator variables : ( 1 ) very preterm ( 32 weeks ) and ( 2 ) moderately preterm ( 3336 weeks ) ) , low birthweight ( represented by 2 dichotomous indicator variables : ( 1 ) very low birthweight ( 1,500 g ) and ( 2 ) moderately low birthweight ( 1,5012,500 g ) , and presence of any congenital anomaly . child age was included to control for variations in actual age at administration of the 24 and 48 month assessments . descriptive analyses were conducted for study variables , as well as cross - tabulation of cognitive delay at 48 months by delay status at 24 months to assess the relative stability of cognitive delay over the two time points . two nested multiple logistic regression models were estimated for each of these study time points to examine associations between low cognitive functioning and socio - demographic characteristics and gestational and birth - related factors . to identify potential differences between children with persistent and emerging delay over the study period , additional sets of multiple logistic models predicting cognitive delay at 48 months were estimated separately for children with and without delay at 24 months . analyses were weighted to appropriately account for the oversampling of some population groups and the stratified cluster design of the ecls - b . table 1 provides descriptive statistics for the study sample , including gestational and birth factors , demographic characteristics , and mean cognitive scores at the 24 and 48 month assessments . of the 11% of children born preterm , 9 percentage points were due to those born between 33 and 36 weeks , and 2 percentage points were due to those born very preterm at 32 weeks gestation or fewer . similarly , a small percentage ( 1% ) of the sample were born at very low birthweight ( 1,500 g ) , while 6% had moderately low birthweight ( 1,5012,500 g ) . the next largest ethnic groups , hispanic and black children , made up 22 and 14% of the sample , respectively.table 1demographic characteristics , gestational and birth factors , and cognitive test scores , early childhood longitudinal study - birth cohort ( ecls - b ) study sample ( n = 7,200)mean or percentstandard deviationmale50%child age ( months ) , ecls - b wave 224.391.16child age ( months ) , ecls - b wave 352.544.10ethnic origin white , non - hispanic57% black14% hispanic22% asian3% native american0.4% other4%mother s education , 24 month assessment less than 9th grade3% from 9th to 12th grade12% high school graduate31% some training / college after high school27% four year college degree and above26%family income , 24 month assessment less than $ 10,0009% between $ 10,001 and $ 20,00014% between $ 20,001 and $ 40,00027% between $ 40,001 and $ 75,00026% at or above $ 75,00124%mother s education , 48 month assessment less than 9th grade3% from 9th to 12th grade10% high school graduate30% some training / college after high school30% four year college degree and above27%family income , 48 month assessment less than $ 10,0008% between $ 10,001 and $ 20,00012% between $ 20,001 and $ 40,00026% between $ 40,001 and $ 75,00025% at or above $ 75,00128%maternal age = 35 or older14%marital status = unmarried , 48 month assessment32%medical risk factor(s)0.180.52behavioral risk factor(s)0.120.33obstetric procedure(s)0.590.68labor complication(s)0.360.67multiple birth3%gestation very preterm 32 1,500 g1% moderately low birthweight 1,5012,500 grams6%congenital anomaly5%bayley mental score , 24 month assessment127.7510.66literacy score , 48 month assessment13.117.14math score , 48 month assessment22.647.42color knowledge score , 48 month assessment8.772.28receptive vocabulary score , 48 month assessment8.601.9248 month assessment score53.1216.00the bayley mental score was used as the cognitive score at 24 months ; at 48 months , literacy , math , color knowledge , and receptive vocabulary scores were converted into z - scores and summedsample size rounded to nearest 50 in accordance with ecls - b data confidentiality requirements ( available at http://nces.ed.gov/ecls/birthdatainformation.asp)estimates are weighted to take the complex sampling design into accountany congenital abnormality identified on the birth certificate demographic characteristics , gestational and birth factors , and cognitive test scores , early childhood longitudinal study - birth cohort ( ecls - b ) study sample ( n = 7,200 ) the bayley mental score was used as the cognitive score at 24 months ; at 48 months , literacy , math , color knowledge , and receptive vocabulary scores were converted into z - scores and summed sample size rounded to nearest 50 in accordance with ecls - b data confidentiality requirements ( available at http://nces.ed.gov/ecls/birthdatainformation.asp ) estimates are weighted to take the complex sampling design into account any congenital abnormality identified on the birth certificate table 2 displays information for two groups of children . data for the nearly 1,000 children who scored in the lowest 10% of the weighted cognitive score distribution at 24 months are shown in the top panel . the bottom panel contains data for children not in the low range at 24 months . of the children classified as cognitively delayed at the 24 month assessment , conversely , 7.9% of children who were not delayed at 24 months fell into the low range by 48 months . this indicates that the majority of children displaying cognitive delay at 24 months of age do not display atypically low levels of school readiness at 48 months of age.table 2patterning of cognitive scores over time in early childhood , ecls - b unweighted study sample ( n = 7,200)number notnumberpercent ( 95% ci)delayed at 48 modelayed at 48 modelayed at 48 moi . all children cognitively delayed at 24 mo ( n = 1,000)75025024.2 ( 21.6 , 27.0)ii . all children not cognitively delayed at 24 mo ( n = 6,250)5,7505007.9 ( 7.2 , 8.6)the bayley mental score was used as the cognitive score at 24 months ; at 48 months , literacy , math , color knowledge , and receptive vocabulary scores were converted into z - scores and summed . at both 24 and 48 months , cognitive delay was defined as falling in the lowest 10% of scorescutoffs for the bottom 10% of scores at 24 and 48 months were determined from the weighted , full available sample of cases at each time point . ( this , plus oversampling of low birth weight and multiple birth children accounts for the fact that the analysis sample contains more than 10% delayed children at 24 months.)all sample sizes in this table are rounded to the nearest 50 in accordance with ecls - b data confidentiality requirements ( available at http://nces.ed.gov/ecls/birthdatainformation.asp)percentages and confidence intervals are calculated from actual sample sizes and have not been rounded patterning of cognitive scores over time in early childhood , ecls - b unweighted study sample ( n = 7,200 ) the bayley mental score was used as the cognitive score at 24 months ; at 48 months , literacy , math , color knowledge , and receptive vocabulary scores were converted into z - scores and summed . at both 24 and 48 months , cognitive delay was defined as falling in the lowest 10% of scores cutoffs for the bottom 10% of scores at 24 and 48 months were determined from the weighted , full available sample of cases at each time point . ( this , plus oversampling of low birth weight and multiple birth children accounts for the fact that the analysis sample contains more than 10% delayed children at 24 months . ) all sample sizes in this table are rounded to the nearest 50 in accordance with ecls - b data confidentiality requirements ( available at http://nces.ed.gov/ecls/birthdatainformation.asp ) percentages and confidence intervals are calculated from actual sample sizes and have not been rounded table 3 shows logistic regression models predicting the child s cognitive delay status separately at 24 and 48 months . the first model includes demographic information including sex , age , and race / ethnic origin . the second model also adds socioeconomic variables and characteristics of gestation and infant status at birth . the numbers shown are the effects of each predictor on the odds of a child displaying a low cognitive score , after statistically controlling for all of the other variables in the model.table 3logistic regression estimates ( odds ratios ) of displaying low cognitive scores at the 24 and 48 month assessments ( n = 7,200)24 mo assessment48 mo assessmentmodel 1model 2model 1model 2male1.93***2.00***1.55***1.66***child age ( months)0.66***0.64***0.86***0.83***ethnic origin black3.00***1.92***4.47***1.97 * * * hispanic2.89***2.04***5.69***2.61 * * * asian2.45***2.74***1.68 * 1.61 native american1.961.464.45***2.37 * * * other1.85 * 1.492.40***1.70 * white , non - hispanic1.00 ( ref)1.00 ( ref)1.00 ( ref)1.00 ( ref)mother s education at corresponding assessment less than 9th grade2.33**13.65 * * * from 9th to 12th grade2.45***6.88 * * * high school graduate1.97***3.13 * * * some training / college after high school1.81**1.83 * four year college degree and above1.00 ( ref)1.00 ( ref)family income at corresponding assessment less than $ 10,0001.437.01 * * * between $ 10,001 and $ 20,0001.565.13 * * * between $ 20,001 and $ 40,0001.313.92 * * * between $ 40,001 and $ 75,0001.102.52 * * at or above $ 75,0011.00 ( ref)1.00 ( ref)maternal age = 35 or older0.900.96marital status = unmarried , 48 month assessment1.100.99medical risk factor(s)0.980.89behavioral risk factor(s)0.790.87obstetric procedure(s)0.850.83*labor complication(s)1.041.03multiple birth1.52**1.52*gestation very preterm 32 weeks1.521.86 moderately preterm 3336 weeks1.071.10birthweight very low birthweight4.38***1.10 moderately low birthweight1.70***1.17congenital anomaly1.191.27 * p 0.05 , * * p 0.01 , * * * p 0.001literacy , math , color knowledge , and receptive vocabulary scores were converted into z - scores and summed to produce cognitive score at 48 months ; lowest 10% were defined as lowbayley mental score was used as cognitive score at 24 months ; lowest 10% were defined as lowsample size rounded to the nearest 50 in accordance with ecls - b data confidentiality requirements ( available at http://nces.ed.gov/ecls/birthdatainformation.asp ) logistic regression estimates ( odds ratios ) of displaying low cognitive scores at the 24 and 48 month assessments ( n = 7,200 ) * p 0.05 , * * p 0.01 , * * * p 0.001 literacy , math , color knowledge , and receptive vocabulary scores were converted into z - scores and summed to produce cognitive score at 48 months ; lowest 10% were defined as low bayley mental score was used as cognitive score at 24 months ; lowest 10% were defined as low sample size rounded to the nearest 50 in accordance with ecls - b data confidentiality requirements ( available at http://nces.ed.gov/ecls/birthdatainformation.asp ) model 1 , displaying findings from the 24 month assessment , indicates that male children were approximately twice as likely as female children to exhibit low cognitive functioning . those children who were comparatively older at the time of the assessment were less likely to have lower scores . compared to non - hispanic white children , black , hispanic , and asian children , as well as those of other race / ethnic origins ( with the exception of native americans ) had significantly elevated odds of displaying cognitive delay . children of mothers in the lowest education category experiencing over twice the risk of low cognitive scores compared to children of highly educated mothers . other significant predictors of cognitive delay at 24 months include multiple birth ( or = 1.52 ) and being born at very low or moderately low birthweight ( or = 4.38 and or = 1.70 , respectively ) . the addition of socioeconomic and birth - related variables in model 2 reduces the effects associated with black and hispanic race / ethnicity , but does not completely account for those effects as race / ethnicity remains a statistically significant risk factor . the effects of low maternal education become even stronger at 48 months . at this time point , and controlling for many other variables , children of mothers with the lowest educational attainment are more than 13 times more likely to be cognitively delayed than children of the most highly educated mothers . this indicates that income and education each exert independent effects on children s risk of displaying delayed cognitive functioning at 48 months . by 48 months , birthweight is no longer predictive of cognitive delay . however , gender and multiple birth remain significant , and having had obstetrical procedures at birth is associated with lower risk . similar to the findings at 24 months , the estimated effects of race / ethnicity are generally reduced in magnitude but not eliminated after accounting for additional social and birth - related factors . table 4 shows estimated effects of risk factors for cognitive delay at 48 months , separately for children who were , and who were not cognitively delayed at 24 months . looking first at those who were delayed at 24 months , the full model ( model 2 ) indicates that maternal education is an extremely strong risk factor for continued delay at 48 months ( or = 30.49 for lowest maternal education category , and 10.66 for the second - lowest category ) , with family income also shown to be important ( or = 7.58 for the lowest income category and 4.17 for the second - lowest category ) . very low birthweight is significant in model 2 , however race / ethnicity is not.table 4logistic regression estimates ( odds ratios ) of displaying low cognitive scores at 48 months , shown separately for children with and without cognitive delay at 24 monthschildren with low cognitive score at the 24 month assessment ( n = 1,000)children with non - low cognitive score at the 24 month assessment ( n = 6,250)model 1model 2model 1model 2male0.881.061.57***1.68***child age ( months)0.85***0.82***0.85***0.83***ethnic origin black2.30**1.604.39***1.89 * * * hispanic2.32**1.366.18***2.87 * * * asian0.881.101.731.54 native american0.860.385.66***3.31 * * * other3.36 * 2.162.00 * 1.40 white , non - hispanic1.00 ( ref)1.00 ( ref)1.00 ( ref)1.00 ( ref)mother s education at corresponding assessment less than 9th grade30.49***11.46 * * * from 9th to 12th grade10.66***5.93 * * * high school graduate5.61**2.62 * * some training / college after high school3.031.56 four year college degree and above1.00 ( ref)1.00 ( ref)family income at corresponding assessment less than $ 10,0007.58***6.20 * * * between $ 10,001 and $ 20,0004.17**4.85 * * * between $ 20,001 and $ 40,0003.19 * 3.86 * * * between $ 40,001 and $ 75,0003.17**2.31 * at or above $ 75,0011.00 ( ref)1.00 ( ref)maternal age = 35 or older1.220.89marital status = unmarried , 48 month assessment0.781.02medical risk factor(s)1.360.77behavioral risk factor(s)1.170.83obstetric procedure(s)0.910.81*labor complication(s)0.981.04multiple birth1.251.48*gestation very preterm 32 weeks0.333.07 * * moderately preterm 3336 weeks0.821.17birthweight very low birthweight3.42 * 0.56 moderately low birthweight1.261.13congenital anomaly1.071.26 * p 0.05 , * * p 0.01 , * * * p 0.001literacy , math , color knowledge , and receptive vocabulary scores were converted into z - scores and summed to produce cognitive score at 48 months ; lowest 10% were defined as lowbayley mental score was used as cognitive score at 24 months ; lowest 10% were defined as lowall sample sizes in this table are rounded to the nearest 50 in accordance with ecls - b data confidentiality requirements ( available at http://nces.ed.gov/ecls/birthdatainformation.asp ) logistic regression estimates ( odds ratios ) of displaying low cognitive scores at 48 months , shown separately for children with and without cognitive delay at 24 months * p 0.05 , * * p 0.01 , * * * p 0.001 literacy , math , color knowledge , and receptive vocabulary scores were converted into z - scores and summed to produce cognitive score at 48 months ; lowest 10% were defined as low bayley mental score was used as cognitive score at 24 months ; lowest 10% were defined as low all sample sizes in this table are rounded to the nearest 50 in accordance with ecls - b data confidentiality requirements ( available at http://nces.ed.gov/ecls/birthdatainformation.asp ) among children who were not delayed at 24 months , the results of model 2 show both similarities and differences with those who were delayed . one difference is that for those not delayed at 24 months , race / ethnic origin effects are larger and more likely to be statistically significant than for those who were delayed . these occur for blacks ( or = 1.89 ) , hispanics ( or = 2.87 ) , and native americans ( or = 3.31 ) . mother s education and family income effects are now smaller in magnitude than for the previously delayed , but still strikingly large overall . the effect for the lowest category of mother s education has an or = 11.46 , while the lowest category of family income has an or = 6.20 . very preterm birth ( or = 3.07 ) and multiple birth ( or = 1.48 ) are also associated with elevated risk for low cognitive scores at 48 months in this group of children , and a history of obstetric procedures is associated with lower risk ( or = 0.81 ) . collectively , these analyses indicate that particular socioeconomic factors ( i.e. , low maternal education , low family income ) strongly elevate a child s risk for cognitive delay . this is the case for each time period separately , and , when separate analyses are run according to cognitive delay status at 24 months , for both persistent and emerging delay . such is not the case for the gestational and birth characteristics , where effects become much weaker when predicting cognitive delay at 48 months . the importance and regularity of the effects of the socio - economic factors are also underscored by the fact that they display a monotonic increasing pattern , in that each increase in either the mother s education level or the family s income corresponds to a decrease in a child s risk of persistent or variable cognitive delay . the largest of these effects occurs at the very lowest level of mother s education , when predicting child outcomes at 48 months . the goal of this study was to identify patterns of cognitive delay in the us child population between 24 and 48 months of age , and quantify the factors associated with increased risk of experiencing such delay . the findings provide evidence for a high likelihood of recovery among children who exhibit cognitive delay at 24 months , with 76% testing in the non - delayed range by 48 months . the analyses also identified a group of children who repeatedly display cognitive delay during the toddler to preschool period . the estimates indicate that about one - quarter of those delayed at 24 months will also experience delay at 48 months ( i.e. , persistently delayed children ) . these children will likely require early intervention services if they are to successfully meet the increasing demands of primary school classrooms . multivariate analyses involving children delayed at 24 months indicate that low levels of maternal education and family income are by far the most salient risk factors for continued delay at 48 months . these factors are associated with dramatic increases in risk of 30-fold ( mother s education ) and sevenfold ( family income ) , respectively . in contrast , very low birthweight status is associated with a more modest but still significant threefold increase in risk . among children without delay at 24 months , socio - demographic factors including low maternal education and low family income also had the strongest effects on the risk of delay at 48 months , while perinatal characteristics including prematurity and multiple gestation were associated with lesser but still significantly elevated odds of delay . the analyses presented here provide important new evidence about the patterning and relative importance of socio - demographic and perinatal factors in determining the risk of cognitive delay over time within the general population of young children . much previous research about the adverse effects of perinatal factors on subsequent child development has been based on follow - up of clinical samples of low and very low birthweight children [ 5 , 9 , 3437 ] , who are often compared with similar numbers of normal birthweight children matched on demographic characteristics or selected from regional samples . consistent with the present analyses , those prior studies identify persistent negative effects of low birthweight and prematurity on cognitive development [ 3 , 6 ] . although these studies provide valuable information , they do not directly contrast the relative magnitude of these gestational and birth factors with the risks of cognitive delay associated with socioeconomic disadvantage among young children who are seen in general pediatric practice . population - level research on developmental outcomes has generally been limited to secondary analysis of datasets such as the national health and nutrition examination survey , the national health interview survey , and the panel study of income dynamics , in which small sample sizes necessitate pooling data from children of widely varying ages , often over both child and adolescent age ranges . the present analyses underscore the strong effects of lower socioeconomic status , including lower maternal education and family income , on children s developmental risk . in particular , children with cognitive delay at 24 months who have less educated mothers are at markedly elevated risk of persistent delay through 48 months . lower socioeconomic status is known to be associated with a range of exposures that can adversely influence cognitive development . these exposures include suboptimal nutritional status , both in overall caloric intake and with regard to specific nutrients needed for central nervous system functioning including iron , iodine and essential fatty acids [ 3841 ] , as well as increased incidence of chronic health conditions such as asthma . economically disadvantaged families are also more likely to live in neighborhoods that contain sources of lead , pcbs , and other toxins that can impair cognitive functioning . lower socioeconomic status is also associated with less optimal home and childcare environments that tend to provide less cognitive stimulation . for example , mothers with lower education levels typically use both lower quantity and quality vocabularies when interacting with their children . children in low - income families tend to watch tv more frequently , and often lack access to storybooks and other types of print material [ 49 , 50 ] . the finding in this study that black , hispanic , and native american children are at increased risk of emerging delay after taking family income and education into account is consistent with findings that minority families are differentially more likely to experience adverse health - related and environmental exposures than white families of similar socioeconomic status [ 51 , 52 ] . culturally diverse and non - english - speaking families also tend to have reduced access to and utilization of high quality pediatric health care that could counteract or buffer at least some of the effects of adverse physical and environmental exposures [ 53 , 54 ] . the study findings have a number of practical implications for providers of pediatric health care . first , universal and comprehensive developmental screening in pediatric practice is critical for detecting early developmental delays . a recent survey of pediatricians , however , indicates that only about a quarter adhere to current screening guidelines , which call for standardized developmental screening tests at 9 , 18 , and 24 or 30 months of age , and screening for school readiness at age 4 . moreover , findings of the present study suggest that this recommended timetable may be insufficient for timely identification of cognitive delay in socioeconomically disadvantaged populations . in addition to standardized testing , evidence suggests that systematically eliciting and evaluating parental concerns about their children s developmental progress at each well - child care visit is a highly beneficial component of surveillance . results of the present study also underscore the importance of strategies to facilitate cognitive development . in reviewing the evidence base for primary health care services to promote early child development , regalado and halfon found support for the efficacy of anticipatory guidance encouraging positive parent - child contact through education about children s normal social development and emphasizing the importance of responsive reciprocal interactions . the incorporation in pediatric practices of structured programs such as reach out and read that facilitate interactive reading among parents and children have also been found to increase children s cognitive and language test scores [ 58 , 59 ] . pediatric practitioners can also stress the importance of high quality child care in promoting child development , and provide information to parents about the components of quality programs . gestational and birth information comes from birth certificates , which although widely used may contain erroneous information , especially concerning complications of pregnancy and delivery , prenatal substance use , and congenital abnormalities ( some of which may not be recognized until after the newborn period ) . this was because the standardized cognitive assessment included in the ecls - b at 24 months , the short form research version of the bayley assessment , was not age - appropriate for administration to 48 month olds . in its place , the designers of the ecls - b administered age - appropriate tests of early literacy , mathematics , color knowledge , and receptive vocabulary at 48 months . although we can not directly assess how use of different assessments at the two time points may have influenced the results , our strategy of specifying a cutoff of the 10th percentile of standardized score to identify delay has been used in other research , and it is reassuring that tests of alternative 5 and 15 percentile cutoff values yielded similar patterns of results . in conclusion , this study underscores the dynamic nature of cognitive developmental progress from 24 to 48 months of age . although low birthweight increases the risk of cognitive delay , especially at 24 months , socioeconomic disadvantage is more common in the pediatric population and is by far a much stronger predictor of persistent as well as emerging delay . comprehensive developmental screening in pediatric practice is needed , especially in low - income subpopulations , to identify children who are experiencing delays and initiate ameliorative action .

the objective of this paper is to examine patterns of cognitive delay at 24 and 48 months and quantify the effects of perinatal and sociodemographic risk factors on persistent and variable cognitive delay . using data from 7,200 children in the early childhood longitudinal study , birth cohort ( ecls - b ) , multiple logistic regression models identified significant predictors of low cognitive functioning at 24 and 48 months . additional multiple logistic models predicting cognitive delay at 48 months were estimated separately for children with and without delay at 24 months . of the nearly 1,000 children delayed at 24 months , 24.2% remained delayed by 48 months ; 7.9% of the children not delayed at 24 months exhibited delay at 48 months . low and very low birthweight increased cognitive delay risk at 24 , but not 48 months . low maternal education had a strongly increasing effect ( or = 2.3 at 24 months , or = 13.7 at 48 months ) , as did low family income ( or = 1.4 at 24 months , or = 7.0 at 48 months ) . among children delayed at 24 months , low maternal education predicted delay even more strongly at 48 months ( or = 30.5 ) . low cognitive functioning is highly dynamic from 24 to 48 months . although gestational factors including low birthweight increase children s risk of cognitive delay at 24 months , low maternal education and family income are more prevalent in the pediatric population and are much stronger predictors of both persistent and emerging delay between ages 24 and 48 months .

Methods Data and Sample Measures Low Cognitive Functioning Predictor Variables of Interest Analyses Results Discussion

it is estimated that 6685 % of the world s population depends directly on plants as medicines [ 13 ] . since the existence of human civilization , plants and their by - products have been used by a large proportion of the population living in rural and urban areas for various purposes such as medicine , healthcare , food , clothing , shelter , agriculture , agrochemicals , pharmaceuticals , narcotics , etc . medicinal plants are defined as plants having one or more organs containing substances that can be used for therapeutic purposes or which are precursors for the synthesis of useful drugs . thus , medicinal plants represent , for the local population , a possibility of simple and cheap treatment . in addition , they represent sources of potentially important new pharmaceutical substances since all parts of a plant , from roots to seed heads and flowers , are employed in traditional remedies and can therefore act as sources of lead compounds . more than 80 % of the world s populations , especially in developing countries , depend on traditional systems of medicine for the treatment of a variety of diseases . this observation could be attributed to two main factors ; inaccessibility of modern drugs and the low purchasing power within the populations living in the rural areas . moreover , some of the local remedies work and so the populations have no need for anything different . from antiquity , mankind has been developing a traditional medicinal system , based on the knowledge of medicinal plants throughout the world . africans , in particular , have used medicinal plants and animal - derived remedies in their struggle for survival and in their quest for religious experiences . the world health organization ( who ) defines traditional medicine as practices , knowledge and belief systems which use minerals , plants and animal based remedies , spiritual therapies and exercises to prevent , treat and this knowledge became enriched over numerous generations due to experimentation but also through observations of animal behaviour . most often , knowledge of traditional medicine is only inherited orally , thereby facing the danger of being lost in favor of western medicine ( wm ) . many africans believe in the manifestation of life forces or spirits in every creation , and that these spirits constitute the heart of all life forms , natural events or non - living things . this gives herbal medicine a vital role in health care delivery systems especially in remote areas where clinics and hospitals are sparsely located [ 8 , 11 ] . despite the advances in wm , african traditional medicine ( atm ) this could probably be due to the rapidly increasing awareness of the potential and curative abilities of alternative medicines , especially from the use of medicinal plants , as well as the inadequate access to wm and physicians and the high cost for western drugs . the argument for the local african populations resorting to traditional remedies could also be partly justified by the fact that natural product inspired molecules represented about 80 % of drugs that had been put into the market [ 1 , 13 ] . previous ethnobotanical studies of medicinal plants confirm the rational use of recipes by different people or groups from different communities . numerous varieties of medicinal plants growing in africa are widely used against many diseases ranging from endemic tropical diseases like malaria and leishmaniasis to complex illnesses such as asthma , psychosis , hepatitis and even cancer . searching for new lead compounds to be developed as drugs or as templates for analogue synthesis and the evaluation of traditional medicine and herbal medicinal products , are the two basic reasons for the advancement of work on medicinal plants . the use of plant preparations can be supported if it is safe and if their biological activity can be scientifically confirmed . this calls for quality control and standardization . if the activity can not be confirmed , and certainly if there is a risk of toxicity , the use of herbal medicinal products should be discouraged . the study of plants used traditionally as medicines is therefore an interesting discipline because of the possibility to find new drugs and also because of the strong adhesion of local populations , for the aforementioned reasons [ 1113 , 22 , 23 ] . this has prompted many research teams to carry out studies on plants used in africa by traditional healers against diseases . the united nations definition of western africa includes the following 16 countries : benin , burkina faso , cape verde , ivory coast , the gambia , ghana , guinea , guinea - bissau , liberia , mali , mauritania , niger , nigeria , senegal , sierra leone and togo . these countries occupy an area of over 6,140,000 km and the natural environment in this area consists of subtropical and tropical regions with semi - arid and humid climates . in these communities , traditional herbalists operate closer to the people , taking advantage of the biodiversity of plant species in such areas to cure various diseases and ailments . nigeria is located in west africa on the gulf of guinea and shares borders with cameroon ( 1,690 km ) in the east , chad ( 87 km ) in the northeast , niger ( 1,497 km ) in the north , benin ( 773 km ) in the west and the atlantic ocean in the south ( fig . 1 ) . the country is divided administratively into the federal capital territory ( abuja ) and 36 states . as a result of the large surface area occupied by nigeria these include about 20,000 species of insects , almost 1,000 species of birds , 247 species of mammals , 123 species of reptiles , about 1,000 species of fish and about 7,895 species of plants . consequently atm is a common and acceptable practice in nigeria as in many countries in africa and asia . many plants from the nigerian flora which provide very useful clues for potential therapeutic compounds have been investigated by mostly university research groups . some of the plants investigated are commonly used in the treatment of several microbial infections , a range of neglected tropical diseases including malaria , trypanosomiasis , as well as diabetes and diverse infections . it is therefore very important to scientifically document the available knowledge for its exploitation towards the enhancement of human health .fig . 1map showing nigeria and her neighbours map showing nigeria and her neighbours several attempts towards the documenting of ethnobotanical and ethnopharmacological knowledge for medicinal plants in nigeria have been carried out . raphael has provided a useful work on traditional medicine in nigeria , about its current status and the future , while soladoye et al . provided a list of 143 plant species belonging to 58 families which were found to be useful for the treatment of haemorrhoids in south western nigeria . . also carried out an ethnobotanical survey around ogun state , south western nigeria , in which 38 plant species belonging to 24 families were identified to be used in herbal anti - malarial recipes , while ajibesin et al . identified a total of 114 medicinal plant species representing 102 genera and 54 families employed in the traditional medical practice of the people of akwa ibom state , nigeria . . carried out a similar survey of wound - healing plants , while sonibare and gbile , listed plants that are being used in the treatment of asthma in south western nigeria . gbolade made an inventory of plants used in the treatment of diabetes in lagos state and plants used in treating hypertension in edo state of nigeria [ 36 , 37 ] . idu et al . conducted an ethnobotanical survey of plants among the esan tribe of edo state , and identified 32 plant species belonging to 31 genera and 23 families which have been used for oral healthcare . one of the goals of our research centre is to document and establish knowledge bases for chemical substances , including those derived from plant use in atm . our previous studies have been focused on central africa [ 13 , 3941 ] , including the pharmacokinetics profiling of natural products ( nps ) derived from plant materials [ 4244 ] . it has been our aim to extend the study to different regions and eventually to cover the entire continent of africa . due to the different works carried out by different research teams on the nigerian flora , the aim of this survey is to identify those plants used traditionally for the treatment of various ailments in nigeria that show promising lead - like activity . our focus has been on the nps whose measured biological activities correlate with the use of the plant in atm . in this survey , the ethnobotanical uses of the plant species from nigeria versus the bioactivities of the derived nps are presented by plant family and in alphabetical order . the ethnobotanical uses of plants in the above families are shown in table 1 , along with the biological activities of the most remarkable isolated nps . the biological activities which correlate with the enthnobotanical uses of the plants have been highlighted in bold . from the alliaceae family , ajoene ( 1 ) and allicin ( 2 ) , were isolated from the bulb of allium sativum ( commonly known as garlic ) , a plant traditionally used in nigeria for the treatment of malaria , among diverse uses . ajeone was active against plasmodium berghei in mice , thus validating the ethnobotanical use of the plant , meanwhile allicin was reported as a p. falciparum cysteine protease inhibitor . it should be mentioned that rodent malaria is a well - known animal model for testing new compounds and plant extracts . however , trial in humans is decisive to identify a hit as a real hit ; and this is a good way to assess toxicity and safety . crinumglaucum is used in the treatment of cough , asthma , and convulsions in nigeria [ 48 , 49 ] , while crinum jagus is a plant used in traditional medicine , either singly or in a combination with chromoleana odorata and emilia prateramisa ( both of asteraceae family ) in the treatment of all forms of convulsion . ( amaryllidaceae ) , which exhibit acetylcholinesterase inhibition , the most active alkaloids isolated being hamayne ( 7 , ic50 = 250 m ) and lycorine ( 5 , ic50 = 450 m ) , while the other alkaloids were comparatively inactive , with haemanthamane ( 6 ) giving 3 % inhibition and crinamine ( 8) giving 4.4 % inhibition at 50 mg ml ( 174 m ) . these results contrast with the positive control physostigmine which gave ic50 of 0.25 m . thus , cholinesterase activity appears to be associated with the presence of two free hydroxy groups in this structural type of amaryllidaceae alkaloids . crinamine has also been isolated from the aerial parts of the asian subspecies crinum asiaticum var . the compound showed potent dose dependent inhibition ( ic50 = 2.7 m ) of hif-1 in a cell - based reporter gene assay . the other components of the asian subspecies ( from korea ) showed no significant inhibition of hif-1 induced transcriptional activity.table 1summary of ethnobotanical uses versus measured biological activities of isolated secondary metabolites from ; alliaceae , amaryllidaceae , annonaceae , apocynaceae , asteraceae and bignoniaceae plant familiesplant familyplant nameuse in traditional medicinepart of plant studiedactive principlemeasured activityauthor and referencesalliaceae allium sativum treatment of malaria , seasoning food , cleansing of blood , prevention and fighting of common cold , boost testosterone levels , regulation of blood sugar levels and as an antisepticbulb 1 and 2 antiplasmodial activity adebayo et al . amaryllidaceae crinum glaucum used in the treatment of cough , asthma , and convulsions . the plant extracts have also exhibited analgesic , anti - inflammatory and antianaphylactic propertieswhole plant 3 , 4 and 5 acetylcholinesterase inhibitionokpo and adeyemi , houghton et al . [ 50 ] crinum jagus treatment of all forms of convulsions and some infectious diseaseswhole plant 5 , 6 , 7 and 8 acetylcholinesterase inhibition , hif-1 inhibitionazikiwe et al . , kim et al . annonaceae enantia chlorantha treatment of malaria , jaundice , dysentery , hypertension , skin , gastric and duodenal ulcers , inflammation , and liver - related diseases and to make unpainted furniture and veneersstem bark 9 and 10 antiplasmodial and antiviral activitiesadebayo et al . , bhadra and kumar , bidla et al apocynaceae picralima nitida treatment of malaria , diarrhea and as a painkiller stem bark , seed 11 , 12 , 13 , 14 , 15 , 16 and 17 antiplasmodial activity , antipsychotic and anxiolytic properties and known potent -opioid agonists adebayo et al . , , ezeamuzie et al . , okokon et al . , elisabetsky and costa - campos asteraceae struchium sparganophora treatment of malaria and measles , cutaneous , subcutaneous parasitic infection , rheumatic pains , diarrhea , dysentery as well as venereal diseases , as an abortifacient , and in the treatment of inflammatory and tumor - related ailments . also used in the preparation of soup in the south western part of nigerialeaf 18 , 19 and 20 antimicrobial and antitumour activitieskasim et al . bignoniaceae spathodea campanulata extracts of bark , leaves and flowers are used to treat malaria , hiv , diabetes mellitus , oedema , dysentery , constipation , gastrointestinal disorders , ulcers , skin diseases , wounds , fever , urethral inflammation , liver complaints and as a poison antidotestem bark 21 antiplasmodial activity , cardioprotective and aromatase inhibitoradebayo et al . treatment of diseases ( ulcers , dysentery , oedemas , skin eruptions , scabies , wound healing and urethral discharge ) and veterinary application have been attributed to the plant in different culturesflowers , fruits , leaf and stem bark 22 , 23 , 24 , 25 , 26 and 27 antioxidant activity elusiyan et al . 2active principles from allium sativum , crinum glaucum , crinum jagus , enantia chlorantha , picralima nitida , struchium sparganophora , spathodea campanulata , guiera senegalensis and morinda lucida summary of ethnobotanical uses versus measured biological activities of isolated secondary metabolites from ; alliaceae , amaryllidaceae , annonaceae , apocynaceae , asteraceae and bignoniaceae plant families active principles from allium sativum , crinum glaucum , crinum jagus , enantia chlorantha , picralima nitida , struchium sparganophora , spathodea campanulata , guiera senegalensis and morinda lucida enantia chlorantha ( annonaceae ) is an ornamental tree , of up to 30 meters in height , with dense foliage and spreading crown . it s stem bark is used against fever / malaria by traditional medicine practitioners in the forest regions , in addition to its use in the treatment of jaundice , dysentery , hypertension , inflammation , and liver - related diseases . the isolated compounds palmatine ( 9 ) and jatrorrhizine ( 10 ) are known to exhibit anti - malarial activity , while palmatine also has weak in vitro activity against flavivirus . seven compounds with anti - malarial properties ; akuammicine ( 11 ) , akuammine ( 12 ) , alstonine ( 13 ) , picraline ( 14 ) , picratidine ( 15 ) picranitidine ( 16 ) and -akuammigine ( 17 ) have been isolated from the stem bark and seeds of picralima nitida ( apocynaceae ) , a plant used in the treatment of malaria and in the management of pains and other ailments [ 55 , 56 ] . based on these results , an enterprising ghanaian hospital started manufacturing standardised 250 mg capsules of the powdered p. nitida seed , and sold around the country where they became widely accepted as a safe and effective pain relief product . the extract showed potent and dose - dependent anti - inflammatory , anti - pyretic and anti - malarial activities . given intraperitoneally , this extract inhibited carrageenan - induced rat paw oedema with ic50 of 102 mg kg , and with the highest dose tested ( 300 mg kg ) producing 72.2 % inhibition . on the lps - induced pyrexia in rabbits , 50 mg kg of the extract produced a mean percentage antipyrexia of ( 38.7 % ) compared with ( 29.0 % ) by 200 mg kg of aspirin . in a 4-day in vivo schizontocidal test in mice infected with p. berghei , up to 300 mg kg daily for 4 days was ineffective in preventing the development of parasitaemia or the consequent mortality . however , marked inhibitory activity was obtained on multi - drug resistant human p. falciparium parasites cultured in vitro . the dose causing 50 % inhibition of parasite growth was 1.75 g ml , compared with 0.14 g ml for chloroquine . the results justify the use of this plant by natives of west africa in the treatment of malaria . akuammidine and -akuammigine , are known to be potent -opioid agonists , although not particularly selective . investigated the leaves of struchium sparganophora ( asteraceae ) , a plant used traditionally to treat malaria and measles . the isolated compounds ( 1820 ) demonstrated antimicrobial activities against the bacteria staphylococcus aureus ( nctc 6571 ) , klebsiella aerogenes ( welcome res . lab.cn 345 ) , escherichia coli ( nctc 9001 ) and proteus vulgaris ( nctc 8313 ) , as well as against the fungal strains candida albicans ( atcc10231 ) and aspergilus niger ( ncpf3149 ) . vernodalin ( 19 ) has been also isolated from vernonia amygdalina , along with vernomygdin , and shown to demonstrate antitumour activities . the anti - malarial activity of ursolic acid ( 21 ) , derived from spathodea campanulata ( bignoniaceae ) , popularly known as african tulip tree , has been used to endorse the use of the plant in southwestern nigeria for malaria treatment by drinking the decoction of its stem bark . ursolic acid also has potential use as a cardioprotective compound and was found to be a weak aromatase inhibitor ( ic50 = 32 m ) . additionally , the antioxidant activities of compounds 2227 isolated from the flowers , fruits , leaf and stem bark of the same plant have been investigated by elusiyan et al . . the results show that the antioxidant principles isolated from the various parts of the plant are verminoside ( 24 ) , from the leaves , stem bark and flowers ( ec50 = 2.04 g ml ) , specioside ( 23 ) , from the flowers ( ec50 = 17.44 g ml ) , kampeferol diglucoside ( 22 ) , from the leaves ( ec50 = 8.87 g ml ) and caffeic acid ( 26 ) , from the leaves and fruits . verminoside has also been isolated from kigelia africana , a plant used in africa for anti - inflammatory , anti - microbial , and anti - skin - aging effects . the anti - inflammatory activity of the compound has been evaluated by cutaneous irritation in cell cultures and reconstituted human epidermis and revealed significant anti - inflammatory activity . the non antioxidant components isolated in the study include ajugol ( 25 ) , from the stem bark and fruits and phytol ( 27 ) , from the leaves . a summary of the biological activities of the compounds that mark the ethnobotanical uses of the plants of the above families is given in table 2 . guiera senegalensis ( combretaceae ) is often used in nigeria for the treatment of malaria . the leaf extract of the plant harvested in nigeria showed positive anti - malarial activity in vitro in plasmodium yoelii nigeriensis . the alkaloids harman ( 28 ) and tetrahydroharman ( 29 ) and the methoxylated naphthalene derivative guieranone a ( 30 ) , fig . 3 , were shown to be the active principles from this plant species harvested in mali and so tom [ 65 , 66 ] in addition to its antifungal activity . the leaves of tithonia diversifola ( compositae ) have also been extensively used in nigerian traditional medicine to treat malaria and fevers . oral decoction of the leaves of tithonia diversifolia are used for treatment of hepatitis , diabetes , malaria , pain , chemoprevention and anti - helicobacter pylori [ 6870 ] , external application of dried leaves on wounds and infusion of leaves for the treatment of measles . the anti - plasmodial compound 31 has been proven to be the active principle responsible for the use of the plant leaves in malaria treatment . another species from the compositae family , laggera pterodonta , is often used against insect attack , athlete s foot , skin infections , pediatric malaria and wounds . the plant is also used in the treatment of hepatitis , arthritis , bronchitis and nephritis . five sesquiterpenes ( 3235 ) , exhibiting antimicrobial activities , have been identified from the species growing in china .table 2summary of ethnobotanical uses versus measured biological activities of isolated secondary metabolites from ; combretaceae , compositae , connaraceae , crassulaceae and ebenaceae plant familiesplant familyplant nameuse in traditional medicinepart of plant studiedactive principlemeasured activityauthor and referencecombretaceae guiera senegalensis treatment of malaria , diarrhea , dysentery , venereal diseases and microbial infections leaves 28 , 29 and 30 antiplasmodial and antifungal activitiesiwalewa et al . , silva and gomez compositae tithonia diversifola treatment of malaria , hepatitis , diabetes , malaria , pain , measles , chemoprevention and anti - helicobacter pylori leaves 31 antiplasmodial and anti - ulcer activitieskuroda et al . , castillo - jurez et al . , adebayo et al . , goffin et al . , snchez - mendoza et al . laggera pterodonta against insect attack , athlete s foot , skin infections , pediatric malaria and wounds . also used in treatment of hepatitis , arthritis , bronchitis and nephritisair - dried aerial part 32 , 33 , 34 , 35 and 36 antimicrobial activity egharevba et al . connaraceae byrsocarpus coccineus leaf decoction is used for the treatment of venereal diseases and as antidote to arrow poisoning and as remedy for pile , while the decoction of the whole plant is applied to swelling and tumours and also to arrest bleeding , the plant has also been reported as a remedy for diarrheaair - dried leaves 37 , 38 and 39 not testedahmadu et al . crassulaceae bryophyllum pinnatum treatment of ulcers , allergic inflammation and epilepsydried whole plant 18 , 40 and 41 antibacterial activity ogungbamila et al . ebenaceae diospyros mespiliformis leaf decoction used for whooping cough treatment and root extracts as worm expellantsroot 42 cytotoxicityadeniyi et al . 3active principles from tithonia diversifola , laggera pterodonta , byrsocarpus coccineus , bryophyllum pinnatum and diospyros mespiliformis summary of ethnobotanical uses versus measured biological activities of isolated secondary metabolites from ; combretaceae , compositae , connaraceae , crassulaceae and ebenaceae plant families active principles from tithonia diversifola , laggera pterodonta , byrsocarpus coccineus , bryophyllum pinnatum and diospyros mespiliformis the leaf extract of byrsocarpus coccineus ( connaraceae ) has been used for the treatment of several venereal diseases and as an antidote to arrow poisoning . it also acts as a remedy for piles , while the decoction of the whole plant is applied to swellings and tumours and to arrest bleeding . the isolated flavonoid and flavonoid glycosides ( 3739 ) , have been respectively identified as quercetin 3-o--arabinoside , quercetin and quercetin 3--d - glucoside . the flavonoid luteolin ( 18 ) , epigallocatechin 3-o - syringate ( 40 ) and gallic acid ( 41 ) have been identified as the active principles responsible for the antibacterial activity of this plant , which explains why it is used in many west african traditional medicinal recipes for the treatment of ulcers . the main antibacterial constituent was found to be free gallic acid , which accounted for about 0.014 % w / w of the fresh aerial part . however , luteolin and a new acylated flavan-3-ol , epigallocatechin-3-o - syringate , were isolated as minor constituents in the active fraction . luteolin exhibits a wide range of biological activities , including antioxidant activity promotion of carbohydrate metabolism , and immune system modulation . other in vitro studies suggest luteolin has antiinflammatory activity [ 77 , 78 ] , and that it acts as a monoamine transporter activator , a phosphodiesterase inhibitor , and an interleukin 6 inhibitor . in vivo studies show that luteolin affects xylazine / ketamine - induced anesthesia in mice . in vitro and in vivo experiments also suggest the compound may inhibit the development of skin cancer [ 81 , 82 ] . diosquinone ( 42 ) , derived from the roots of diospyros mespiliformis ( ebenaceae ) , has shown very good activity against all the cell lines tested with ed50 values ranging between 0.18 g ml against human glioblastoma ( u373 ) and 4.5 g ml against hormone dependent human prostate cancer ( lncap ) . the relationship with the ethnobotanical use of this plant has not however been established ( the leaf decoction has been used for the treatment of whooping cough and root extracts used as worm expellants ) . amongst the plants of the euphorbiaceae family , tor - anyiin et al . have reported the use of alchornea cordifolia in the treatment of malaria / fevers in nigeria ( table 3 ) . . identified ellagic acid ( 43 ) , fig . 4 , to be the active ingredient from the stem of this plant , responsible for its anti - malarial activity . the leaves of the same plant are used as a remedy for arthritis , muscle pain and other inflammatory disorders . carried out an analysis of the volatile oil extracted from the fresh leaves of a. cordifolia and revealed the presence of high concentrations of eugenol ( 44 : 41.7 % ) , cadinol ( 45 : 2.46 % ) , caryophylene ( 56 : 1.04 % ) , linalool ( 57 : 30.59 % ) and ( e)--bergamotene ( 59 : 4.54 % ) . these compounds could be contributing to the topical anti - inflammatory effects of a. floribunda and a. cordifolia leaf extracts . okoye and osadebe had also carried out a bioactivity - guided fractionation of the ethyl acetate fraction of the methanol leaf extract of the plant material , leading to the isolation of a new flavonol glycoside , 3,5,7,3-tetrahydroxyflavone-3-o--l - rhamnoside ( 61 ) . the anti - inflammatory activity ( 50 mg kg ) of this compound was higher than that of the standard anti - inflammatory drug , aspirin ( 100 mg kg ) . the compound also significantly ( p < 0.001 ) inhibited heat - induced haemolysis of human erythrocytes in vitro . these results demonstrated that the anti - inflammatory activity of a. floribunda leaves may be , in part , a result of the flavonol glycoside , compound 61.table 3summary of ethnobotanical uses versus measured biological activities of isolated secondary metabolites from euphorbiaceae plant familyplant familyplant nameuse in traditional medicinepart of plant studiedactive principlemeasured activityreferenceeuphorbiaceae alchornea cordifolia the leaves or leafy stems , as an infusion or chewed fresh , are taken for their sedative , antimalarial and antispasmodic activities to treat a variety of respiratory problems including sore throat , cough and bronchitis , genital urinary problems including venereal diseases and female sterility , and intestinal problems including gastric ulcers , diarrhoea , amoebic dysentery and wormsstem 43 antiplasmodial activity tor - anyiin et al . remedy for arthritis , muscle pain and other acute and chronic inflammatory disorders leaves 4451 , 5360 and 62 anti - inflammatory activity okoye et al . alchornea floribunda leaves are traditionally used as a remedy for arthritis , muscle pain and other inflammatory disorders leaves 6175 anti - inflammatory activity okoye et al . , okoye and osadebe jatropha gossypifolia treatment of various disease conditions such as cough , tuberculosis , bacterial infections and cancerous growths . the leaves of the plant are traditionally being applied to boils , carbuncles , eczema , itches , and veneral diseases and also used as febrifuge , while its bark is used as emmenagogue . the leaves and seeds are considered as a purgative and are widely used to treat obstinate constipation . roots are used to treat leprosy , and stem latex possess coagulant activityseeds 76 antifungal activityfalodun et al . i summary of ethnobotanical uses versus measured biological activities of isolated secondary metabolites from euphorbiaceae plant family active principles from alchornea cordifolia i from this same family , jatropha gossypifolia has been used to treat various disease conditions such as cough , tuberculosis , bacterial infections and cancerous growths . the phytochemical studies revealed the presence of some secondary metabolites such as alkaloids , saponins and tannins . there was no activity against the tested bacteria ( gram positive and gram negative organisms at 2.5100 mg ml ) . the seed extract however showed significant antifungal activity . from the seeds of this plant falodun et al . carried out a spectroscopic analysis ( 1d and 2d nmr ) of the colourless oil , giving 9-acetoxynerolidol ( 76 ) , which they suggested was the active ingredient figs . 59.fig . 6active principles from jatropha gossypifolia , cajanus cajan , ganoderma colossum , hyptis suaveolens , cassia alata , berlina grandiflora , abrus precatorius and cassia siamea fig . 9active principles from ixora coccinea , fagara zanthoxyloides , quassia amara , quassia indica and aframomum danielli active principles from alchornea floribunda ii active principles from jatropha gossypifolia , cajanus cajan , ganoderma colossum , hyptis suaveolens , cassia alata , berlina grandiflora , abrus precatorius and cassia siamea active principles from loranthus micranthus , sida acuta and ixora coccinea active principles from azadiracta indica , khaya grandifoliola and pavetta crassipes active principles from ixora coccinea , fagara zanthoxyloides , quassia amara , quassia indica and aframomum danielli our survey also involved five species from the fabaceae or leguminoceae family , table 4 . these are cajanus cajan used in cancer treatment , cassia alata used in the treatment of skin diseases such as ringworm , eczema , pruritis , itching , scabies , ulcers and other related diseases , berlina grandiflora used in the treatment of gastrointestinal disorders , as well as abrus precatorius and cassia siamea , both used in the treatment of malaria . a dichloromethane ( ch2cl2 ) fraction of the leaves of cajanus cajan had ic50 value 510 g ml , with the two constituent stilbenes , longistylins a ( 78 ) and c ( 79 ) , being primarily responsible , with ic50 values of 0.714.7 m against the range of cancer cell lines [ 8991].table 4summary of ethnobotanical uses versus measured biological activities of isolated secondary metabolites from ; fabaceae , ganodermataceae , lamiaceae and loranthaceae plant familiesplant familyplant nameuse in traditional medicinepart of plant studiedactive principlemeasured activityauthor and referencefabaceae cajanus cajan cancer treatment , for the treatment diabetes and as an energy stimulantleaves 7779 cytotoxicity ashidi et al . ganodermataceae ( fungus ) ganoderma colossum mushrooms of this genus are known to possess anti - tumour , anti - cancer , immunomodulatory and immunotherapeutic qualities whole fungus 8082 antimicrobial activity , activity against hiv-1 protease ofodile et al . , lamiaceae hyptis suaveolens treatment of respiratory tract infections , colds , pain , fever , cramps and skin diseasesleaves 83 antiplasmodial activity chukwujekwu et al . leguminosae cassia alata treatment of skin diseases such as ringworm , eczema , pruritis , itching , scabies , ulcers and other related diseasewhole plant 84 antibacterial activity okwu and nnamdi berlina grandiflora treatment of gastrointestinal disorders whole plant 85 anthelmitic activity enwerem et al . abrus precatorius treatment of malaria stem bark 86 antiplasmodial activity muhammad et al . stem bark is used as a mild , pleasant , safe purgative ; to treat diabetes ; a paste is used as a dressing for ringworm and chilblains ; the roots are used as an antipyretic ; and the leaves are used for the treatment of constipation , hypertension , and insomnialeaves 8789 antiplasmodial activity , vasodialator effect ajaiyeoba et al . , loranthaceae loranthus micranthus treatment of diarrhea , epilepsy , hypertension and rheumatismleafy twigs 9095 antioxidant activityagbo et al . treatment of several diseases including immune - modifying diseases leaves 96 and 97 immunostimulatory activity omeje et al . summary of ethnobotanical uses versus measured biological activities of isolated secondary metabolites from ; fabaceae , ganodermataceae , lamiaceae and loranthaceae plant families ofodile et al . have isolated and characterized three colossolactones ; colossolactone e ( 80 ) , colossolactone b ( 81 ) and 23-hydroxycolossolactone e ( 82 ) , from an n - hexane : dichloromethane ( 2:7 ) extract of ganoderma colossum . the antimicrobial screening of these compounds revealed that colossolactone e and 23-hydroxycolossolactone e were active against bacillus subtilis and pseudomonas syringae . potency of the compounds against the tested bacteria supports the use of this mushroom in therapeutic medicine , since this genus of mushrooms is known to possess anti - tumor , anti - cancer , immunomodulatory and immunotherapeutic qualities . other colossolactones ( v viii and g ) and schisanlactone a have also been isolated from the mushroom , ganoderma colossum , harvested from vietnam , together with colossolactone e ( 80 ) . the isolated compounds were evaluated for inhibition of hiv-1 protease , with ic50 values for the most potent compounds ranging from 5 to 13 g / ml . colossolactone g showed the most promising anti - viral activity , thus justifying the immunomodulatory and immunotherapeutic qualities of this mushroom . hyptis suaveolens ( lamiaceae ) , used in the treatment of respiratory tract infections , colds , pain , fever , cramps and skin diseases has been investigated by chukwujekwu et al . . a bioactivity - guided fractionation of the petroleum ether extract of the leaves of this plant led to the isolation of the abietane - type diterpenoid endoperoxide , 13-epi - dioxiabiet-8(14)-en-18-ol ( 83 ) , a molecule with high anti - plasmodial activity ( ic50 = 0.1 g ml ) . this might explain the usefulness of the plant for treating fever , which might be due to malaria . among the other plants of the leguminoceae family , the antibacterial activity of 4-butylamine-10-methyl-6-hydroxy cannabinoid dronabinol ( 84 ) , isolated from cassia alata , could explain the use of this plant in the treatment of ulcers , amongst other skin diseases . the anthelmintic activity of betulinic acid ( 85 ) was also used to validate the use of berlina grandiflora for treating gastrointestinal disorders . the investigations of muhammad and amusa have reported the use of the stem bark of abrus precatorius ( leguminosae - caesalpinioideae ) in the treatment of malaria . isolated abruquinone b ( 86 ) , which showed anti - plasmodial activity , with ic50 = 1.5 g ml against the k1 strain of p. falciparum , from the stem bark of this plant . also reported the use of the leaves and stem bark of cassia siamea in the treatment of malaria . investigation of the leaves of this plant led to the isolation of emodin ( 87 ) , lupeol ( 88 ) and cassiarin a ( 89 ) , with ic50 values of 5.0 g ml against the k1 strain for both compounds 87 and 88 and an ic50 value of 0.02 m for compound 90 [ 101 , 102 ] . in asian traditional folk medicine , the stem bark of cassia siamea is used as a mild , pleasant , safe purgative ; a decoction of the bark is given to treat diabetes ; a paste is used as a dressing for ringworm and chilblains ; the roots are used as an antipyretic ; and the leaves are used for the treatment of constipation , hypertension , and insomnia . the vasodilator effect of cassiarin a , could explain the use of this plant in the treatment of hypertension , amongst other ailments . from the leafy twigs of loranthus micranthus ( loranthaceae ) the antioxidant activities of the isolated compounds were evaluated using the 2,2-diphenyl-1-picrylhydrazyl ( dpph ) assay , giving ic50 values varying from 23.8 and 50.1 m , all being more active than the reference drug chlorogenic acid ( with ic50 = 67.9 m ) . the immunostimulatory activities of 2,3-dimethoxy - benzo[a , b]cyclopentenyl-3,3,5-trimethyl pyran-4-carboxylic acid ( 96 ) and lupinine ( 97 ) have been used to validate the use of the leaves of this same plant in the treatment of several diseases including immune - modulating diseases . sida acuta ( malvaceae ) is widely used for the treatment of malaria in west africa , among other diseases . the ethanol , aqueous and chloroform extracts are known to display antiplasmodial activity at 3.90 , 0.92 and 0.87 g ml respectively against the p. falciparum fcm29 strain [ 106108 ] . identified cryptolepine ( 98 ) as the active ingredient from samples collected in ivory coast , west africa . cryptolepine analogues have also been isolated from cryptolepis sanguinolenta ( periplocaceae ) , harvested from diverse locations in west africa and exhibiting antiplasmodial activity [ 109114 ] . different parts of azadiracta indica and khaya grandifoliola ( meliaceae ) are also used in west africa for traditional preparations to treat malaria [ 115117 ] . the limonoid gedunin ( 99 ) , isolated from the leaves of a. indica , inhibits p. falciparum at ic50 of 1.25 g ml [ 115 , 118 ] . gedunin was also isolated from k. grandifoliola , along with the other limonoids 2,6-dihydroxyfissinolide ( 100 ) , methylangolensate ( 101 ) , 7-deacetylkhivorin ( 102 ) , 1-deacetylkhivorin ( 103 ) and 6-acetylswietenolide ( 104 ) , exhibiting significant antiplasmodial activities . additionally , gedunin has displayed anticancer activities and hsp90 inhibition . among the surveyed plants of the rubiaceae family , pavetta crassipes has been used in handling respiratory infections and abdominal disorders , while the leaves and stems of morinda lucida are used in treating malaria [ 45 , 84 ] and ixora coccinea is used to treat a variety of infections , including hypertension , menstrual irregularities , sprains , chronic ulcers and skin diseases . a bioactive flavonoid ( quercetin-3-o - rutinoside , 105 ) has been isolated from the aqueous extract of p. crassipes leaves , which showed activity against some pathogenic microorganisms , including streptococcus pyogenes , corynebacterium ulcerans , klebsiella pneumoniae , neisseria gonorrhoeae , pseudomonas aeruginosa , and escherichia coli at a concentration < 50 mg ml . the compound had minimum inhibitory concentration ( mic ) ranging from 6.25 to 12.5 mg ml and minimum bactericidal concentration ( mbc ) ranging from 12.5 to 25 mg ml . this supports the ethnomedicinal use of the plant in the treatment of respiratory infections and abdominal disorders . the ethanol , ch2cl2 and petroleum ether extracts of the leaves and stems of morinda lucida exhibited respective ic50 values of 5.70 , 5.20 and 3.90 g ml . . identified ursolic acid ( 21 ) as the active ingredient , which exhibited antiplasmodial activity at ic50 of 3.10 g ml against the chloroquine - sensitive strain of p. falciparum . identified a doubly linked , a - type proanthocyanidin trimer and other constituents of ixora coccinea leaves . the antioxidant and antibacterial properties of the identified compounds ( 93 , 106111 ) were also investigated . all tested compounds inhibited the growth of b. subtilis , while only epicatechin ( 93 ) and quercetin-3-o--l - rhamnopyranoside ( 110 ) inhibited the growth of e. coli . antioxidant evaluation of isolated compounds revealed that ixoratannin a-2 ( 107 ) and cinnamtannin b-1 ( 108 ) were the most active compounds in dpph , inhibition of lipid peroxidation and nitric oxide radical scavenging assays . the presence and antiplasmodial property of the alkaloid fagaronine ( 112 ) , with an ic50 of 0.018 m against the 3d7 strain of p. falciparum , in fagara zanthoxyloides ( rutaceae ) , could explain why the roots of this plant are used in preparations against malaria , amongst other applications in atm . the leaves and stems of plants from the quassia species ( simaroubaceae ) are generally employed in anti - malarial preparations , q. amara ( also called bitterwood tree ) having the highest anti - malarial reputation for curative and preventive purposes in the simaroubaceae family [ 45 , 126 ] . simalikalactone d ( 113 ) , samaderine x ( 114 ) , samaderine z ( 115 ) , samaderine b ( 116 ) and samaderine e ( 117 ) have been isolated from q. amara and q. indica , displaying anti - plasmodial properties in the range 0.010 and 0.210 g ml [ 127 , 128 ] . the compound displayed anti - malarial activity and was shown to be less toxic than simalikalactone d ( 113 ) . have shown that the alcohol and petrol extracts of seeds of aframomum danielli ( zingiberaceae ) , used traditionally as a food spice and also as an anti - inflammatory agent , inhibit the soya 5-lipoxygenase enzyme and thus may show anti - inflammatory activity . compounds isolated from the active extract were shown to be long chain polypropenyl benzoquinone derivatives . phytyl plastoquinone ( 119 ) and heptaplastoquinone ( 120 ) were respectively isolated from the petrol and alcohol extracts , both compounds inhibiting 5-lipoxygenase at 6.25 and 18.5 m , respectively , compared with the standard drug fisetin , which inhibited the enzyme at 0.92 m , table 5.table 5summary of ethnobotanical uses versus measured biological activities of isolated secondary metabolites from ; malvaceae , meliaceae , rubiaceae , rutaceae and simaroubaceae plant familiesplant familyplant nameuse in traditional medicinepart of plant studiedactive principlemeasured activityauthor and referencemalvaceae sida acuta treatment of malaria , ulcer , fever , gonorrhea , abortion , breast cancer following inflammation , wound infectionsstem 98 antiplasmodial activityobute , bertani et al . it is also known for anthelmintic , antifungal , antidiabetic , antibacterial , antiviral , contraceptive and sedative propertiesleaves 99 antiplasmodial activityisah et al . extracts also show anti - inflammatory and toxic effectsbark , roots and seeds 99104 antiplasmodial and anticancer activities and hsp90 inhibitionagbenanusi et al . rubiaceae pavetta crassipes management of respiratory infections and abdominal disordersfresh plant 105 antimicrobial activitybello et al . morinda lucida treatment of malaria , diabetes , leaves , stem bark 20 antiplasmodial activityawe and makinde , cimanga et al . ixora coccinea treatment of a variety of infections ; hypertension , menstrual irregularities , sprains , chronic ulcers and skin diseasesleaves 93 , 106111 antioxidant activitiesidowu et al . the stem and the root of the plant are both used as chewing stick in nigeria particularly among the yoruba ethnic group in the south - western part of the countryroot 112 antiplasmodial activity odebiyi and sofowora simaroubaceae quassia amara treatment of malaria , as a digestive , treat fever , against hair parasites ( lice , fleas ) , and mosquito larvae in ponds ( and do not harm the fishes ) . extracts of quassia wood or bark act as a natural insecticideleaves , stem 113 antiplasmodial activity ajaiyeoba et al . quassia indica treatment of malaria , rheumatism , asthma , skin diseases and as an insecticide , especially against white antsleaves , stem 114118 antiplasmodial activity bertani et al . zingiberaceae aframomum danielli used as traditional food spice , as an anti - inflammatory agent and also for crop protectionseeds 119 and 120 lipoxygenase inhibitionodukoya et al . summary of ethnobotanical uses versus measured biological activities of isolated secondary metabolites from ; malvaceae , meliaceae , rubiaceae , rutaceae and simaroubaceae plant families the results presented in this review represent an overview of the biological activities of selected nps isolated from plants used in traditional medicine in nigeria . our intention has been to focus on those plants whose ethnobotanical uses correlate with the biological activities of the derived nps . even though this report does not claim to be exhaustive , the goal of documenting the baseline knowledge , from which further investigations could be carried out , has been achieved . this work has also led to the collection of a chemical database for plants used in traditional medicine in nigeria ( naijaplant ) , to be made available separately . the plant sources , geographical collection sites , chemical structures of pure compounds as well as their spectroscopic data , were retrieved from literature sources comprising data collected from 176 articles from 68 peer reviewed journals , spanning the period 1971 to 2013 . our survey consisted in collecting data from the literature sources , mainly from msc and phd theses from nigerian university libraries and also using the author queries in major natural product and medicinal chemistry journals . the collected data includes plant sources , uses of plant material in traditional medicine , plant families , region of collection of plant material , isolated metabolites and type ( e.g. flavonoid , terpenoid , etc . ) , measured biological activities of isolated compounds , and any comments on significance of isolated metabolites on the chemotaxonomic classification of the plant species ( as commented in the literature ) . this survey suggests that information gathered from compounds derived from african medicinal plants , in particular from nigeria , could be a suitable starting point for extensive drug discovery projects , especially for some of the most frequently mentioned diseases like malaria , since malaria is the most endemic disease in africa and plants used in the treatment of malaria have been the most investigated on the continent [ 39 , 40 , 45 , 131134 ] .

traditional medicinal practices play a key role in health care systems in countries with developing economies . the aim of this survey was to validate the use of traditional medicine within local nigerian communities . in this review , we examine the ethnobotanical uses of selected plant species from the nigerian flora and attempt to correlate the activities of the isolated bioactive principles with known uses of the plant species in african traditional medicine . thirty - three ( 33 ) plant species were identified and about 100 out of the 120 compounds identified with these plants matched with the ethnobotanical uses of the plants .

Introduction Alliaceae, Amaryllidaceae, Annonaceae, Apocynaceae, Asteraceae and Bignoniaceae Combretaceae, Compositae, Connaraceae, Crassulaceae and Ebenaceae Euphorbiaceae Fabaceae, Ganodermataceae, Lamiaceae and Loranthaceae Malvaceae, Meliaceae, Rubiaceae, Rutaceae and Simaroubaceae Conclusions

a 16-year - old female patient came to department of oral medicine and radiology with chief complaints of bleeding from gums . on examination , she was thin built . extraoral examination of face and skull revealed hypoplasia of the malar eminences and zygomata , hypoplasia of maxilla and underdevelopment of the mandible [ fig . 1 ] . ears appeared low - set and rotated with presence of skin tags [ fig . 2 ] . intraoral examination revealed inflamed gingiva due to poor oral hygiene . radiological examination supported the clinical findings . patient was then referred to ophthalmology department where ocular examination showed downward slant of palpebral fissures , ptosis of upper lids and limbal dermoid in right eye measuring around 1 1 cm in diameter [ fig . the patient was uncooperative and refused any other treatment except for oral prophylaxis which we performed to relieve her from bleeding gums . extraoral examination of face and skull showing hypoplasia of the malar eminences and zygomata , hypoplasia of maxilla and underdevelopment of the mandible low - set ears along with presence of skin tags ocular examination showing limbal dermoid in right eye the most ordinary type of acrofacial dysostosis is the nager type ( nafd ) of the so - called pre - axial acrofacial dysostosis . this disorder is categorized by the office of rare diseases in the united states as a rare illness ; thus , it has only affected less than 200,000 of the country 's population . some studies have shown that the gene which causes the illness might be located around 9q32 and it is possible that the zfp-37 gene could also be involved . the major symptoms of nager syndrome often include the underdevelopment of the jaw and cheek areas , a jaw that may appear smaller than the usual or normal jaw , hearing problems ( and some defects on the external or internal ear area ) , cleft palate ( which is a congenital defect of the mouth or the lip ) , the absence or underdevelopment of the thumb or thumbs , forearms that appear short ( the forearms could altogether be absent of the radius bone ) , fusing of the bones of the forearms , poor elbow movements ( or limited extension of this body part ) , ears that appear to be backward rotated , the ears manifesting some skin tags ( or they could be low - set ) , and feeding and breathing problems ( especially among infants who are afflicted with this syndrome ) . our patient showed underdevelopment of jaws , hypoplasia of the malar eminences , zygomata and hypoplasia of maxilla . ocular manifestations of this syndrome are rare and include downward slant of palpebral fissures , ptosis of upper lids , coloboma of lower lids , and deficiency of eyelashes of the medial one - third of the lower eyelids . our patient showed downward slant of palpebral fissures , ptosis of upper lids and limbal dermoid in right eye . traditionally , the differential diagnosis of nager syndrome is done with treacher - collins , pierre - robin and goldenhar syndrome . treacher - collins syndrome is characterized by maxilla and mandible hypoplasia , downslanting palpebral fissures , colobomas of inferior eyelid and auricular malformation . pierre - robin syndrome presents with micrognathia , cleft palate and glossoptosis . in goldenhar syndrome , we find hemifacial microsomy , flattening of maxilla , temporal and zygomatic bone , microtia , ocular and vertebral alterations , and in 50% of cases , fallot tetrad . the echographic findings of these malformations become evident usually in the third trimester , therefore a systematic ultrasound exploration including visualization of fetal face line is essential for its detection . if the patient suffers from speech retardation , then a speech therapist could help the patient . they may not totally cure the condition , but they can help in managing the symptoms . treatment of limbal dermoids may consist of periodic removal of irritating cilia , topical lubrication to prevent foreign body sensation , or excision of the lesion if it is causing significant cosmetic disfigurement or interfering with vision . our patient refused to take any ocular treatment and insisted on taking only the dental treatment for which she visited our hospital . correct understanding of the underlying pathophysiologic pattern of this disease is necessary for optimal diagnosis and treatment planning . ophthalmological examination of these patients is a must as ocular manifestations are not uncommon in these patients .

nager syndrome , also called preaxial acrofacial dysostosis , comprises two groups of defects involving the limbs and craniofacial region , respectively . this syndrome is rare and only 70 cases have been reported in the literature . the exact cause of this syndrome is unknown , but there is indication that it is genetically based . ocular manifestations of this syndrome include widely separated downward slanting eyes , absence of eyelashes , ptosis of upper eyelids and colobomas on the inner aspect of lower eyelids . we report limbal dermoid in a patient with nager syndrome . we did not find such an association of limbal dermoid in nager acrofacial dysostosis syndrome on pubmed using nager acrofacial dysostosis , limbal dermoid and ocular manifestations as the keywords .

Case Report Discussion Conclusion

growth prediction is defined as the estimation of alterations in speed and direction of future growth . the growth patterns of the mandible , maxilla , and other craniofacial structures should be taken into consideration as essential components to determine the time of occurrence , duration , and prognosis of malocclusions . development of craniofacial bones takes place in relation to one another and is influenced by various factors . the frontal sinus , one of the paranasal sinuses located in the skull , is formed following pneumatization of the frontal bone , which is directly influenced by the interactions of the respiratory epithelium and activity of adjacent osteoclasts . since the left and right sinuses are developed independently , asymmetrical sinuses may also be found . variations in the extent of pneumatization individualize frontal sinus morphology and diversity in shape and capacity and symmetry of the frontal sinus . frontal sinus anatomy can also help in identification of the deceased by comparing pre- and post - mortem radiographs . evidence shows that frontal sinus dimensions are multifactorial and related to genetic factors and weather conditions . in 1990 , blaney stated that craniofacial structures affect the morphology of the paranasal sinuses . an association between hypertrophic frontal sinus and overgrowth of the mandible exists in acromegaly ( production of excessive amounts of growth hormone ) . the correlation of frontal sinus anatomy and its dimensions with the mandible in patients with normal systemic conditions has been previously evaluated . suggested the area of frontal sinus as a predictor of mandibular growth pattern and concluded that a class iii patient with a larger frontal sinus would more likely need an orthognathic surgery besides orthodontic appliances in the future . they reported that larger frontal sinuses were associated with skeletal class iii malocclusion , longer condyles , and wider symphysis . another study on adults with skeletal class i , ii and iii relations measured the area of frontal sinus on their lateral cephalograms ( lcs ) and indicated that large frontal sinuses were associated with large mandibles irrespective of their positional relationship ; however , there was evidence that class iii skeletal malocclusions were more likely associated with larger frontal sinus areas . others have investigated the relationship between the development of the frontal sinus and the person 's height and concluded that frontal sinus can serve as an indicator for assessment of developmental maturity . frontal sinus can be evaluated in the sagittal plane using lateral cephalometry and in the coronal plane using posterior - anterior ( pa ) cephalometry , which are routinely ordered for orthodontics patients . this study was designed to assess the possible associations between the frontal sinus dimensions and craniofacial indices both sagittal and vertical , using pretreatment lc and pa radiographs in a group of the iranian population . this retrospective study was approved by the research institute of dental sciences , shahid beheshti university of medical sciences , tehran , iran . it was performed on patients presenting to the department of orthodontics , dental school , shahid beheshti university of medical sciences from 2011 to 2013 . the included patients were 12 years old and had true scale lc and pa radiographs . all these radiographs were taken using a cranex d x - ray unit ( soredex , helsinki , finland ) with exposure settings of 6670 kvp , 10 ma , and 14.2 s , with patient in centric occlusion and natural head position . the exclusion criteria were as follows : patients with no records of their age or genderpatients with signs of infection or sinus pathology based on radiographscongenital syndromes involving craniofacial bones , palatal clefts , hemifacial microsomia , or hypertrophyhistory of trauma to the nasomaxillary complexhistory of orthognathic surgerypoor quality of radiographs . patients with no records of their age or gender patients with signs of infection or sinus pathology based on radiographs congenital syndromes involving craniofacial bones , palatal clefts , hemifacial microsomia , or hypertrophy history of trauma to the nasomaxillary complex history of orthognathic surgery poor quality of radiographs . sinus dimensions were measured on pa radiographs and lc of patients before orthodontic treatment by dolphin imaging software ( dolphin imaging , chatsworth , ca , usa ) figure 1a and b. briefly , sinus borders were marked excluding the crista galli by an experienced orthodontist . extension of the superior orbital rim was considered as the lower limit of sinus , in cases in whom the inferior sinus border was not detectable . measurements of the maximum height , width , and area of the frontal sinuses were made by autocad 2007 software ( autodesk inc . , san rafael , ca , usa ) with 2.5% error in both sagittal and frontal planes by two examiners [ figure 1c and d ] . the mean values of measurements made by the two examiners were reported for each patient . ( d ) measurement of sinus dimensions on the coronal plane anatomical landmarks were identified through dolphin software , and their accuracy was ensured by an orthodontist [ figure 1a ] . fifteen cephalometric variables representing maxillofacial growth pattern in both vertical and anterior - posterior dimensions were calculated [ tables 1 and 2 ] . definition of the cephalometric landmarks used in the study definition of assessed cephalometric indices descriptive values ( mean and standard deviation ) were reported for each parameter . student 's t - test for independent samples was used to compare sinus dimensions between males and females . the relationship between sinus dimensions and cephalometric indices was assessed by the pearson 's correlation coefficient . to control for the effect of age and gender on this relationship , a linear regression model was applied . this retrospective study was approved by the research institute of dental sciences , shahid beheshti university of medical sciences , tehran , iran . it was performed on patients presenting to the department of orthodontics , dental school , shahid beheshti university of medical sciences from 2011 to 2013 . the included patients were 12 years old and had true scale lc and pa radiographs . all these radiographs were taken using a cranex d x - ray unit ( soredex , helsinki , finland ) with exposure settings of 6670 kvp , 10 ma , and 14.2 s , with patient in centric occlusion and natural head position . the exclusion criteria were as follows : patients with no records of their age or genderpatients with signs of infection or sinus pathology based on radiographscongenital syndromes involving craniofacial bones , palatal clefts , hemifacial microsomia , or hypertrophyhistory of trauma to the nasomaxillary complexhistory of orthognathic surgerypoor quality of radiographs . patients with no records of their age or gender patients with signs of infection or sinus pathology based on radiographs congenital syndromes involving craniofacial bones , palatal clefts , hemifacial microsomia , or hypertrophy history of trauma to the nasomaxillary complex history of orthognathic surgery poor quality of radiographs . sinus dimensions were measured on pa radiographs and lc of patients before orthodontic treatment by dolphin imaging software ( dolphin imaging , chatsworth , ca , usa ) figure 1a and b. briefly , sinus borders were marked excluding the crista galli by an experienced orthodontist . extension of the superior orbital rim was considered as the lower limit of sinus , in cases in whom the inferior sinus border was not detectable . measurements of the maximum height , width , and area of the frontal sinuses were made by autocad 2007 software ( autodesk inc . , san rafael , ca , usa ) with 2.5% error in both sagittal and frontal planes by two examiners [ figure 1c and d ] . the mean values of measurements made by the two examiners were reported for each patient . intraclass correlation coefficient was measured to assess the interexaminer reliability . a sample case of frontal sinus measurement and cephalometric analysis . anatomical landmarks were identified through dolphin software , and their accuracy was ensured by an orthodontist [ figure 1a ] . fifteen cephalometric variables representing maxillofacial growth pattern in both vertical and anterior - posterior dimensions were calculated [ tables 1 and 2 ] . student 's t - test for independent samples was used to compare sinus dimensions between males and females . the relationship between sinus dimensions and cephalometric indices was assessed by the pearson 's correlation coefficient . to control for the effect of age and gender on this relationship , a linear regression model was applied . a total of 144 subjects were enrolled ; of which , 78 were female and 66 were male . the mean age of patients was 19.26 4.66 years ( range : 1331 years ) . the average dimensions of the frontal sinus in both sagittal and frontal planes in males were greater than those in females . however , according to the independent t - test , this difference was only significant in the transverse dimension in the frontal plane ( p = 0.032 ) . the average dimensions of the frontal sinus in two planes ( width and height in cm and area in cm . sample size : 144 ) the mean values of cephalometric indices are presented in table 4 . the average measurements on lc were not significantly different between males and females except for the anb angle , which was 0.56 4.93 in males and 3.32 3.96 in females ( p = 0.048 ) . average cephalometric indices ( all indices in degrees except for wit 's [ mm ] and jarabak index [ ratio ] . sample size : 144 ) assessment of the relationship between the dimensions of the frontal sinus and cephalometric indices revealed a significant association of sn - fh and sna angles with frontal sinus size [ table 5 ] . in males , the sn - fh , sum of posterior angles , pal - sn , and jarabak index had significant associations with the size of the frontal sinus [ table 6 ] ; while in females , only sn - fh and gonial angles were significantly correlated with frontal sinus dimensions [ table 7 ] . significant correlations between the dimensions of the frontal sinus and cephalometric indices in total significant correlations between the dimensions of the frontal sinus and cephalometric indices in males significant correlations between the dimensions of the frontal sinus and cephalometric indices in females a regression model was applied adjusted for age and gender . the results showed a significant correlation between sinus dimensions and sn - fh , sum of posterior angles , and jarabak index [ table 8 ] . neither age nor gender affected the correlation between frontal sinus dimensions and cephalometric indices . linear regression model adjusted for age and gender , showing significant correlations between the dimensions of the frontal sinus and cephalometric indices the average dimensions of the frontal sinus in both sagittal and frontal planes in males were greater than those in females . however , according to the independent t - test , this difference was only significant in the transverse dimension in the frontal plane ( p = 0.032 ) . the average dimensions of the frontal sinus in two planes ( width and height in cm and area in cm . the average measurements on lc were not significantly different between males and females except for the anb angle , which was 0.56 4.93 in males and 3.32 3.96 in females ( p = 0.048 ) . average cephalometric indices ( all indices in degrees except for wit 's [ mm ] and jarabak index [ ratio ] . assessment of the relationship between the dimensions of the frontal sinus and cephalometric indices revealed a significant association of sn - fh and sna angles with frontal sinus size [ table 5 ] . in males , the sn - fh , sum of posterior angles , pal - sn , and jarabak index had significant associations with the size of the frontal sinus [ table 6 ] ; while in females , only sn - fh and gonial angles were significantly correlated with frontal sinus dimensions [ table 7 ] . significant correlations between the dimensions of the frontal sinus and cephalometric indices in total significant correlations between the dimensions of the frontal sinus and cephalometric indices in males significant correlations between the dimensions of the frontal sinus and cephalometric indices in females a regression model was applied adjusted for age and gender . the results showed a significant correlation between sinus dimensions and sn - fh , sum of posterior angles , and jarabak index [ table 8 ] . neither age nor gender affected the correlation between frontal sinus dimensions and cephalometric indices . linear regression model adjusted for age and gender , showing significant correlations between the dimensions of the frontal sinus and cephalometric indices growth of the maxilla and nasal cavity is closely related to the development of paranasal sinuses and all these structures ultimately determine the final morphology of the face . studies conducted on twins have shown that the shape of sinuses is mainly determined by genetics ; however , environmental factors , trauma , allergies , acquired conditions , nutrition , and drug use can also affect the development of sinuses . genotype and growth of maxillofacial structures are two major factors that affect frontal sinus dimensions . considering the multifactorial size of the frontal sinus , the current study assessed the relationship between the cephalometric indices and frontal sinus dimensions in a group of iranian patients . the results revealed good correlations between some cephalometric indices and frontal sinus size . in the current study , lcs provide an optimal view to examine the pa walls of the frontal sinus , and the sinus area is visible on pa radiographs . the previous studies have shown that physiological changes in the size of the frontal sinus after the age of 12 are minimal ; thus , only patients over 12 years of age were enrolled in this study . a noteworthy result was that the dimensions of the frontal sinus were larger in males compared to females ; however , this difference was not statistically significant . similarly , previous studies reported larger frontal sinus in men compared to women , and this difference was not significant in some of them . in total population of the study , a significant inverse correlation was found between sn - fh angle and the dimensions of the frontal sinus , especially its height in sagittal and frontal planes . the increase in the size of frontal sinus resulted in the lower inclination of the anterior cranial base from the horizontal plane . it could be justified that the nasion point , which is the anterior landmark of the sn plane , is influenced by the dimensions of the frontal sinus . an inverse correlation between the sn - fh angle and sinus dimensions was also observed when analyzing females and males separately . this provides strong evidence for the influence of frontal sinus on the inclination of anterior cranial base . only one correlation was found between the anterior - posterior growth of skeletal structures and dimensions of the frontal sinus . this correlation was found between the sna and height of the sinus in the frontal plane and might indicate a tendency for maxillary retrognathism in individuals with increased height of frontal sinus . on the other hand , the anb and wits indices showed no significant relations with the dimensions of the frontal sinus . therefore , according to the results presented here , the skeletal relation of the maxilla and mandible in the sagittal plane was not influenced by the dimensions of the frontal sinus . the results did not show any correlation between the size of the frontal sinus and angle classification in the studied population . in both females and males , after controlling for the confounding factors of age and gender , a significant correlation was found between frontal sinus size and sn - fh , sum of posterior angles and jarabak index . in males , the sum of posterior angles showed a positive correlation with the sinus area . in cases with larger sinus area the negative correlation between the jarabak index and sinus dimensions in the lateral view supports this finding . a poor inverse correlation was also observed in males between the pal - sn angle and the sinus area . this finding could again be justified by the influence of frontal sinus on the location of nasion and the inclination of cranial base as well as clockwise rotation of the maxilla , which could result in increased anterior facial height . similar to males , a correlation was found in females between the sinus area and the gonial angle , which supports increased steepness of the mandible . as previously described , there were no significant associations between the position of the maxilla and mandible in the sagittal plane with frontal sinus dimensions , other than the weak tendency for maxillary retrognathism in the present study , while in other studies , larger frontal sinus was correlated with excessive mandibular growth and class iii skeletal malocclusion . assessed the association between the area of the frontal sinus and excessive mandibular overgrowth by comparing fifty skeletal class i and 53 skeletal class iii patients . the only radiograph used in their study was the lc , and the area of the sinus was measured using a digitizer connected to a microcomputer . they claimed that sinus area can serve as a predictor of abnormal mandibular growth according to the measurements of mandibular length , symphysis width , porion location , ramus position , and condylar axis . among the anteroposterior angles , anb was the common variable measured in both studies ; however , a negative correlation between anb and sinus area was reported by rossouw et al . no correlations were reported in their study between the frontal sinus size and the facial angle , indicating that a large frontal sinus may be present in vertical growers . while , there was a significant association between increased vertical dimension and sinus size in the current study . prashar et al . also demonstrated that larger sinus areas were strongly associated with larger mandibles , with a tendency for class iii skeletal malocclusion . the inherent limitation of conventional two - dimensional radiographs for measuring the volume of complex three - dimensional anatomical structures is considered as a limitation of the present study . a computed tomography scan of the frontal sinus would enable more accurate analysis of sinus dimensions ; however , the aim of this study was to identify expected associations between the frontal sinus anatomy and craniofacial structures on lc and pa radiographs routinely ordered by orthodontists . furthermore , patient malpositioning or slight movements when taking radiographs could cause inaccuracies in the measurement of frontal sinus size and cephalometric indices . in this study , radiographs with any sign of distortion were excluded . to generalize the results to the clinical setting , it should be noted that prediction of growth of craniofacial structures in the diagnosis phase can help achieve stable and favorable outcomes in orthodontic patients , and clinicians should use all the tools available for this purpose . dimensions of the frontal sinus , which are visible on routine orthodontic radiographs , may be used as an indicator to assess the vertical growth . however , the current study was a cross - sectional study and growth prediction could not be concluded . the results showed an association between frontal sinus size and some cephalometric indices in adult patients . based on these results , the authors could only suggest that in young adults , in whom the frontal sinus has reached its maximum size ( while the vertical growth continues ) , larger frontal sinus might be associated with future vertical growth . to confirm this , considering the limitations of the present study , greater dimensions of the frontal sinus might be associated with decreased inclination of the anterior cranial base ( sn ) . there was also a correlation between frontal sinus dimensions and increased anterior facial height ( sum of posterior angles , pal - sn , and jarabak index ) in males and increased gonial angle in females .

objective : growth prediction plays a significant role in accurate diagnosis and treatment planning of orthodontics patients . it was hypothesized that the unique pattern of pneumatization of the frontal sinus as a component of craniofacial structure would influence the skeletal growth pattern and may be used as a growth predictor.materials and methods : a total of 144 subjects ( 78 females and 66 males ) with a mean age of 19.26 4.66 years were included in this retrospective study . posterior - anterior and lateral cephalograms ( lcs ) were used to measure the frontal sinus dimensions . the skeletal growth pattern and relations of craniofacial structures were analyzed on lc using variables for sagittal and vertical analyses . correlation between the frontal sinus dimensions and cephalometric indices was assessed by the pearson 's correlation coefficient.results:the sn - fh and sna angles had significant associations with frontal sinus dimensions in all enrolled subjects ( p < 0.05 ) . in males , the sn - fh , sum of posterior angles , pal - sn , and jarabak index were significantly associated with the size of frontal sinus ( p < 0.05 ) . in females , the associations of sn - fh and gonial angles with frontal sinus dimensions were significant ( p < 0.05).conclusion : the results show that larger size of frontal sinus was associated with reduced inclination of the anterior cranial base , increased anterior facial height ( in males ) , and increased gonial angle ( in females ) in the study population .

INTRODUCTION MATERIALS AND METHODS Study population Measuring sinus dimensions Cephalometric analysis Statistical analysis RESULTS Frontal sinus dimensions Cephalometric indices Correlation between the frontal sinus size and maxillofacial dimensions DISCUSSION CONCLUSION Financial support and sponsorship Conflicts of interest

the introduction of highly active antiretroviral therapy ( haart ) has led to significant improvement in the morbidity , mortality , and quality of life of people living with human immunodeficiency virus ( hiv ) . however , this improved outcome is being mitigated by increase in deaths from liver disease , cancer and cardiovascular disease ( cvd ) . the increase in cvd in people living with hiv ( plwhiv ) has been attributed to increased prevalence of traditional cardiovascular risk factors ( cvrfs ) , induction of potentially atherogenic dyslipidemia and vascular and immune changes by the virus and some anti - retroviral agents . reports suggest that there is excess risk of cardiovascular diseases ( cvds ) among plwhiv . these reports have come mainly from high- and middle - income countries with very few reports from low - income countries , particularly sub - saharan africa ( ssa ) . although reports from ssa indicated that tuberculosis , sepsis , advanced hiv disease and pulmonary infections are the leading causes of death , the opportunity to nip in the bud the emergence of cvd as an important cause of death in plwhiv in ssa will be missed if attention is not given to the surveillance and control of cvrfs in this population . nigeria , with an estimated population of plwhiv of approximately 3.5 million , has the second largest population of plwhiv in the world , second only to south africa , and is responsible for 10% of the global burden of hiv . due to the differences in the prevalence of cvd and the socio - demographic characteristics of plwhiv ( which are known to influence to cvd ) in different economic regions of the world , reports from high- and middle - income countries also , failure to appreciate possible emerging problems of cvd in plwhiv in ssa may mitigate the gains from haart in this region . we , therefore , embarked on this study to determine the prevalence of traditional cvrfs in our patients with no previously known cvrfs or cvd . also , we determined the 10-year cardiovascular risk in our cohort using the framingham , the world health organization / international society of hypertension ( who / ish ) and the systematic coronary risk evaluation ( score ) equations . the patients involved gave both verbal and written consent after the study was duly explained to them . this cross - sectional study involved 265 consecutive patients with documented hiv infection aged 18 years seen at the outpatient clinic dedicated to plwhiv in our facility over a 4 month period . we excluded participants with the following features : acute illness necessitating medical or surgical intervention ; clinical and biochemical profile indicative of liver disease ; clinical thyroid disease ; pregnancy ; known personal history of hypertension , diabetes mellitus ( dm ) , ischemic heart disease ( ihd ) , stroke ; patients on lipid - lowering medication ; and those that declined to be part of the study . we used the step - wise approach to surveillance ( steps ) questionnaire developed by the who for non - communicable disease . the questionnaire was used to obtain information such as age ; gender ; educational status ; use of cigarette ; alcohol intake ; physical activity ; dietary habit ; and family history of hypertension , dm , ihd , and stroke . intake of haart was noted and duration of haart was calculated from the time of commencement of haart to the time of inclusion of the patient in the study . haart was defined as the use of 2 nucleoside reverse transcriptase inhibitors ( nrtis ) and at least one non - nucleoside reverse transcriptase inhibitor ( nnrti ) ; or 2 nrtis and at least one protease inhibitor ( pi ) or one nrti in combination with one pi and at least one nnrti . the weight ( kilogram ) and height ( metres ) of the participants were measured in light clothing and with the shoes off . the body mass index ( bmi ) of the participants was calculated from weight ( kg)/height ( m ) . the waist circumference ( wc ) in ( cm ) and the hip circumference ( hc ) in ( cm ) were measured without clothing with a tape measure in light contact with but not compressing the skin midpoint between the lowest rib and the iliac crest and at the levels of the greater trochanters , respectively . blood pressure ( bp ) was taken in a sitting position using the a and d ua 767 automated digital manometer which had been validated by the british hypertension society . three bp and pulse rate ( pr ) readings were obtained for each participants and the average of the last two readings were used for current bp and pr readings for analysis . blood samples were drawn after an 8- to 12-h fast to determine the serum total cholesterol ( tc ) , high density lipoprotein cholesterol ( hdl - c ) , and triglycerides ( tg ) . the tc and the different cholesterol fractions were determined using commercially available reagents ( randox laboratories ltd , uk ) . the concentration of the low - density lipoprotein cholesterol ( ldl - c ) was determined using the friedwald equation for participants with a tg the presence of various traditional cvrfs was determined using cut - off values based on guidelines of the national cholesterol education program ( ncep atp iii ) , the joint national committee on prevention , detection , evaluation and treatment of high blood pressure ( jnc7 ) and the american diabetes association and are as shown in table 1 . framingham , the who / ish risk prediction chart for africa , sub - region d ( which included nigeria ) and the score equations . in view of the relatively low incidence of chd in nigeria we classified the subjects as having low , moderate or high cardiovascular risk using framingham and who / ish ( < 10% , 10 - 20% , and > 20% , respectively ) and score ( < 3% , 3 - 4% and 5% , respectively ) equations . patients with markedly raised levels of single risk factors i.e. tc 8 mmol / l ( score and who / ish equations ) , ldl - c 6 mmol / l ( score ) , bp 180/110 mmhg ( score ) , bp 160/100 mmhg ( who / ish ) , and fpg 7 mmol / l [ all risk equations ] were defined as having coronary risk equivalents and classified as having high risk . differences between two means were assessed using the student 's t - test while the chi - square was used to assess the degree of association of categorical variables with fishers test applied as appropriately . cohen 's kappa ( ) coefficient was used to assess agreements between two risk scores . the level of agreement was considered poor if = 0.20 , fair if = 0.21 - 0.40 , moderate if = 0.41 - 0.60 , substantial if = 0.61 - 0.80 , and very good if > 0.80 . all p values were two - tailed and p < 0.05 was considered statistically significant . all statistical analyses were done using the statistical package for social sciences software , version 15 ( spss , chicago , il ) . differences between two means were assessed using the student 's t - test while the chi - square was used to assess the degree of association of categorical variables with fishers test applied as appropriately . cohen 's kappa ( ) coefficient was used to assess agreements between two risk scores . the level of agreement was considered poor if = 0.20 , fair if = 0.21 - 0.40 , moderate if = 0.41 - 0.60 , substantial if = 0.61 - 0.80 , and very good if > 0.80 . all p values were two - tailed and p < 0.05 was considered statistically significant . all statistical analyses were done using the statistical package for social sciences software , version 15 ( spss , chicago , il ) . the study population consisted of 179 ( 67.5% ) females and 86 ( 32.5% ) males . table 2 shows the demographic and social characteristics of the study population according to gender and haart use . the mean age of the study population was 38.7 8.7 years and the males were significantly older than the females . current cigarette smoking and alcohol intake were found in 5 ( 1.9% ) and 19 ( 7.2% ) participants , respectively , and were commoner in the males . two hundred and fourteen ( 80.8% ) participants were on haart with 194 ( 90.7% ) on zidovudine / lamivudine / nevirapine ) , 7 ( 3.3% ) on stavudine / lamivudine / nevirapine , 6 ( 2.8% ) on tenofovir / lamivudine / efavirenz , 6 ( 2.8% ) on tenofovir / emtricitabine / efavirenz , and 1 ( 0.4% ) on zidovudine / lamivudine / efavirenz combinations . participants on haart were older than haart - nave patients and females had significantly higher cd4 count when compared to males [ table 2 ] . demographic and social characteristics of the study population according to gender and haart use males had significantly higher sbp and whr than the females while the females had significantly wider hc [ table 3 ] . there was no statistically significant gender difference in the mean values of the wc , fpg , tc , hdl - c , ldl - c , and tg [ table 3 ] . participants on haart had higher sbp and dbp when compared to those not on haart [ table 3 ] . although those on haart had higher mean values of tc , hdl - c , and tg when compared to haart - nave participants , the differences were not statistically significant [ table 3 ] . anthropometric , clinical and laboratory characteristics of the study population according to gender and haart use tables 4 and 5 show respectively the prevalence of the traditional cvrfs and cardiovascular risk scores in the study population and their comparison by gender and haart intake . low physical activity was present in 175 ( 66% ) of the study participants with no significant gender difference . overweight , generalized obesity , abdominal obesity , and increased whr were present in 69 ( 26% ) , 18 ( 6.8% ) , 48 ( 13.1% ) , and 108 ( 40.8% ) participants , respectively , and these variables were significantly higher in females compared to males . hypertension and diabetes were found in 30 ( 11.3% ) and 28 ( 10.6% ) participants respectively with no significant gender difference . mmol / l ) , hypertriglyceridemia , and elevated ldl - c were found in 89 ( 33.6% ) , 34 ( 12.8% ) and 75 ( 28.3% ) participants respectively with no significant gender difference . low hdl - c was present in 130 ( 49.1% ) participants and was significantly higher in females compared to males ( 55.3% vs. 36.0% , p = 0.003 ) . there was no statistically significant difference in the prevalence of hypertension , diabetes , hypercholesterolemia and cholesterol fractions in participants on haart when compared with haart - nave participants [ table 4 ] . traditional cardiovascular risk factors in the study population according to gender and haart use cardiovascular risk assessment using framingham , who / ish and score risk equations according to gender and haart use the 10 year - cardiovascular risk was low in 234 ( 88.3% ) , 231 ( 87.2% ) , and 231 ( 87.2% ) participants according to the framingham , who and score risk equations , respectively [ table 5 ] . the prevalence of moderate to high 10-year coronary risk in the present study was 11.7% , 12.8% and 12.8% using the framingham , who and score risk equations , respectively . the prevalence of high cardiovascular risk according to the three risk scores was insignificantly higher in males when compared with females and in those on haart when compared to haart - nave participants . the observed agreement between framingham and who risk equations was substantial ( = 0.670 ) ; very good between framingham and score ( = 0.809 ) and substantial between who and score risk equations ( = 0.732 ) . the traditional cvrfs observed in our study population in decreasing order of frequency were low physical activity ( 66.0% ) , low hdl - c ( 49.1% ) , increased whr ( 40.8% ) , overweight and obesity ( 32.8% ) , hypercholesterolemia ( tc 5.0 mmol / l ) ( 33.6% ) , elevated ldl - c ( 28.3% ) , older age ( 14.3% ) , abdominal obesity ( 13.1% ) , elevated tg ( 12.8% ) , hypertension ( 11.3% ) , diabetes ( 10.6% ) , alcohol consumption ( 7.2% ) , and smoking ( 1.9% ) . the prevalence of low hdl - c in our cohort of 49.1% is comparable to 49.5% by cahn et al . , in latin - american cohort receiving haart but higher than 44.9% and 36.3% reported by saves et al . , and edwards - jackson et al . , respectively . the prevalence of bmi 25kg / m in our cohort of 32.8% is higher than 30.2% and 19.0% reported in simone study and malawians , respectively . many published reports have drawn attention to the increasing prevalence of overweight and obesity in plwhiv . this is due to the fact that being overweight removes / reduces the suspicion of hiv positivity and stigma , and allows acceptability and integration into the society . hypertriglyceridaemia was present in 12.8% of our cohort and this is much less than 30 - 60% reported in non - african cohorts . blacks and people of african descent tend to have low prevalence of elevated tg , even in the presence of low hdl - c , the so - called triglyceride paradox . the explanations adduced to this low prevalence of elevated tg in people of african descent include higher activity of lipoprotein lipase ( lpl ) , lpl activity uninhibited by insulin resistance and lower levels of apolipoprotein c iii activity which inhibits lpl activity in blacks compared to caucasians . the prevalence of hypertension in our cohort of 11.3% falls within the reported prevalence of 5.2 - 31.5% from published studies . the higher sbp and dbp seen in patients receiving haart when compared with haart - nave patients may be explained by the older age and slightly higher prevalence of overweight / obesity of patients on haart . the prevalence of elevated blood sugar in our cohort of 10.1% is higher than 2.0 - 7.3% reported by various workers in plwhiv . the higher prevalence reported in our cohort may not be unconnected with the fact fpg was done once in our cohort . smoking is estimated to cause 10% of cvd and its prevalence is estimated to be lowest in the who african region . unlike reports from middle- and high - income countries and certain parts of west africa ( mali , cote d ivoire ) where the prevalence of smoking is reported to be between 13.2 - 66.8% , the prevalence of smoking in our cohort is remarkably low at 1.9% . reports from high - income countries have shown that tobacco smoking is higher in plwhiv than in the general population and this is particularly related to the presence of a specific subpopulation of multidrug users . the low prevalence of smoking in our cohort can be explained partly by the low prevalence of smoking in our population , the higher prevalence of females ( which reflects of our patient population ) in our study population unlike reports from middle- and high - income countries with higher male population , the continuous counselling of plwhiv by health care workers of the danger of smoking and the fact that our patients do not share the same addiction characteristics common in plwhiv in certain high - income countries . patients on haart , particularly those on protease inhibitors ( pis ) present more metabolic alterations such as dyslipidaemia and metabolic syndrome than do haart - nave patients . also , these alterations tend to be prevalent with increased duration of treatment . the absolute values of tc , hdl - c , tg and fpg were higher in people on haart compared to haart - nave patients similar to other published studies . our findings can partly be explained by the fact that the haart regimens in our cohort were different from that in high - income countries in that none of our patients was on pis and the duration of treatment in our cohort was relatively shorter when compared with other studies , with the mean duration of treatment of 17.3 months . in this cohort of nigerians living with hiv / aids , we found low overall cardiovascular risk as predicted by the framingham , who and score risk equations . this study showed a high prevalence of nigerians living with hiv / aids at low cardiovascular risk regardless of the coronary risk equation used . previous reports from nigeria have documented a low prevalence of coronary heart disease though there are suggestions that the incidence of chd will increase as nigerians continue to adapt western lifestyle . the low prevalence of cvd risk factors in our population can be explained by the relatively young age of the study cohort and the low incidence of chd in our population . the 10-year coronary risk greater than 10% according to framingham score of 11.7% obtained in this study is slightly higher than 9.0% and 9.9% reported in the data collection on adverse events of anti - hiv drugs ( d : a : d ) and by edwards - jackson et al . , in thais patients , respectively . however , our value is much lower than 23.4% reported by knobel et al . , in spain , 24.1% by moreira guimaraes et al . however , which of the three risk equations will best predict cardiovascular risk in hiv - infected nigerians can only be determined by a longitudinal study . studies have shown that how well the predicted outcomes agree with the actual outcome ( i.e. the calibration ) using the various risk systems may vary widely . reasons adduced to this poor calibration include differences in the baseline rates of cvd in the different geographic regions which in turn may lead to overestimation or underestimation of risk ; change in the secular trend of the baseline prevalence of cvd ; assumption of constant effects of the risk factors at different ages and levels of the other risk factors by risk estimation systems ; and variation in the different end - point definition of various risk systems . despite the shortcomings in the risk equation systems , clinicians must be encouraged to regularly evaluate and manage patients for cvd risk in order to improve cvd outcomes . our study has some limitations which should be taken into consideration when interpreting our results . first , our cohort consisted of consecutive patients with no overt cardiovascular disease attending a sub - urban hiv care center . thus , our sample may not be representative of the whole population of nigerians living with hiv / aids . however , we are unaware of any other study in our population that has attempted to assess cardiovascular risk using three cvd risk scores as done in this study . our study therefore provides a useful comparison with other published studies from middle- and high - income countries . second , the exclusion of patients with known personal history of hypertension , diabetes and use of lipid - lowering medications which are important cvrfs could explain the low prevalence of medium cardiovascular risk in this study cohort . third , we did not include controls of hiv - negative people from the population . this is because our goal was to show the current status of cvd risk in nigerians living with hiv / aids . fourth , we did not assess for altered body compositions such as lipodystrophy and lipoatrophy which had been shown to be associated with increased cvd risk in some published studies . fifth , the risk equations were not developed in the nigerian population and an overestimation of cvd risk in our population with lower incidence of chd is possible . lastly , we can not make causal relationships between the cvrfs , hiv infection , and treatment with haart since our study is cross - sectional . most of the study participants had low overall cardiovascular risk according to the three risk equations . whilst the frequencies of certain cvrfs such as low physical activity , low hdl - c , overweight and hypercholesterolemia were high , others such as smoking was remarkably low in our cohort . as more nigerians have access to haart with consequent improvement in longevity , the emergence of cvds as an important health challenge can not be ruled out . thus , clinicians involved in taking care of plwhiv must not only pre - occupy themselves with achieving and maintaining virological control , but also assess and control for cvrfs .

background : reports from middle- and high - income countries suggest that the improved health outcome from highly active antiretroviral therapy ( haart ) in people living with human immunodeficiency virus ( plwhiv ) is being mitigated by increase in deaths from cardiovascular disease ( cvd).aims : this study was to determine the prevalence of traditional cardiovascular risk factors ( cvrfs ) and the 10-year cardiovascular risk using three risk equations in plwhiv with no overt vascular disease.materials and methods : this cross - sectional study involved 265 plwhiv . we classified the subjects as having low , moderate or high cardiovascular risk using the framingham , world health organization / international society of hypertension ( who / ish ) and systematic coronary risk evaluation ( score ) equations.results:the mean age of the cohort was 38.7 8.7 years ; 179 ( 67.5% ) were females and 214 ( 80.8% ) were on haart . the prevalent traditional cvrfs in our cohort were low physical activity ( 66% ) , low hdl - c ( 49.1% ) , hypercholesterolaemia ( 33.6% ) , bmi 25 kg / m2 ( 32.8% ) and elevated ldl - c ( 28.3% ) . the prevalence of smoking was very low ( 1.9% ) . the prevalence of moderate to high 10-year coronary risk was 11.7 , 12.8 , and 12.8% according to the framingham , who / ish and score risk equations , respectively.conclusion:most of our patients had low overall cardiovascular risk according to the three risk equations .

Introduction Materials and Methods Statistical analysis Results Discussion Conclusion

data synthesized from existing randomised controlled trials show that pyloric drainage procedures reduce the occurrence of early post - operative gastric outlet obstruction after oesophagectomy with gastric reconstruction ( 1,2 ) . pyloromyotomy and pyloroplasty are generally considered simple procedures , however , they may be complicated by stricture , leak , and even death ( 3 ) . because of these complications some advocate not performing an emptying procedure during oesophagectomy ( 3 ) . a 61-year - old gentleman presented in january 2010 with dysphagia for solids and regurgitation . this arose from a long segment of multifocal dysplastic barrett s metaplasia ( 22 - 39 cm ) . full pre - operative staging investigations diagnosed a poorly differentiated mid to distal oesophageal adenocarcinoma ( t3 , n1 , m0 ) . the patient was recruited to the oeo5 trial in april 2010 and underwent neo - adjuvant chemotherapy with 5-fluorouracil and cisplatin . in may 2010 he underwent 3-stage mckeown oesophagectomy with a formal heineke - mikulicz pyloroplasty ( 2-layer , interrupted closure with omental patch ) . final histology of the resected specimen revealed t3 , n1 ( 2/22 ) , m0 , poorly differentiated adenocarcinoma with microscopically involved margins ( circumferential ) with vascular and lymphatic invasion . on post - operative day 7 a routine post - operative contrast swallow was performed . this identified a spill of contrast due to a 5 mm anterior defect at the level of the oesophago - gastric anastomosis suggestive of focal ischaemia . a further contrast swallow revealed contrast aggregating around the drain ( figure 1 ) and that the covered oesophageal stent was appropriately positioned with no evidence of on - going leak ( figure 2 ) . a contrast swallow demonstrating contrast aggregating around the drain & a covered oesophageal stent appropriately positioned with no evidence of leak on post - operative day 11 , at routine dressing change on the ward , a full thickness abdominal wall dehiscence was identified . the patient was managed with washout , drainage and re - suturing of the abdominal wall over a vicryl mesh . the original intra - abdominal tube drain was left undisturbed . on post - operative day 16 the defect at the pyloroplasty suture line was now visible endoscopically and a covered pyloric stent was located appropriately across the defect . due to proximal stent migration this was unsuccessful in controlling the leak ( figure 4 ) . a late pyloroplasty leak diagnosed on contrast swallow & a contrast swallow demonstrating proximal stent migration and evidence of on - going leak the patient was now stable and he requested to eat and drink . a surgical approach to the management of fistula after pyloroplasty was deemed inappropriate because of the recent surgery for intra - abdominal sepsis and the presence of an abdominal wall mesh . a novel approach to the management of a fistula after pyloroplasty was employed . a combined endoscopic and fluoroscopic procedure was performed where a radiologically placed snare was inserted through the abdominal drain and manipulated into the gastric tube through the defect in the pyloroplasty . a gastroscope was used to introduce a t - tube into the gastric tube and into the snare . the abdominal drain and snare were withdrawn and the t - tube placed appropriately , plugging the hole in the pyloroplasty , under direct vision . a further guide wire was left in place along the tract of the tube drain and was used to deploy a pigtail drain alongside the pyloroplasty ( figures 5 and 6 ) . an intra - operative image demonstrating the combined endoscopic and fluoroscopic procedure a pictorial representation of the result of the procedure follow up ct , with contrast both orally and through the t - tube , showed no evidence of ongoing leak from the pyloroplasty . tube satisfactorily plugged the hole through the anterior wall of the pyloroplasty , best demonstrated in the lateral decubitus position ( figures 7 and 8) . the patient was discharged with a capped off t - tube in situ and made a good recovery . a follow - up ct demonstrating no evidence of on - going leak from the pyloroplasty & a follow - up ct demonstrating the flanged t - tube satisfactorily plugging the hole through the anterior wall of the pyloroplasty treatment of leaks by direct suture repair , revision , patching , and application of fibrin glue to leaks have failed to gain widespread acceptance owing to a high failure rate ( 4 ) . alternative methods of controlling leaks such as the placement of covered stents are feasible but can be complicated by stent migration ( 4 ) and inadequate drainage of associated collections . to our knowledge this is the only reported case of management of late pyloroplasty leak following oesophagectomy with a combined endoscopic and radiological placement of a t - tube . the t - tube provided an ideal plug at the site of leak which enabled the patient to consume a normal diet and return home safely .

management of upper gastrointestinal anastomotic leaks is an inter - disciplinary challenge . we present a case of late pyloroplasty leak following 3-stage oesophagectomy . we describe a novel , combined endoscopic and fluroscopic procedure to introduce a t - tube into the anastomotic leak proving an ideal plug at the site of leak which enabled the patient to consume a normal diet and return home safely .

INTRODUCTION CASE REPORT DISCUSSION

the goal of treatment in phenylketonuria ( pku ) is to control hyperphenylalaninemia by phenylalanine dietary restriction . patients with persistent plasma levels of phenylalanine > 6 mg / dl ( 360mole / l ) should be treated . continuation of this dietary restriction during the whole life is necessary for optimal outcomes [ 3 , 4 ] and families should be trained , as most of the treatment is done by themselves . mothers of the pku children have the most important role in helping children take responsibility for their own . a study recommended that illiteracy of the mothers decreased the rate of compliance with the rigid and long - term dietary treatment . socioeconomic , psychosocial and emotional factors and health care system can be barriers to dietary compliance in pku patients too . there are a lot of differences in health services for pku patients between iran and other countries such as providing free supplements for patients . as there are not accurate data on family social status of these patients , this study is performed to evaluate family social status and dietary adherence of iranian pku patients . we hope by this research , we were able to realize some barriers of dietary compliance in iran . in this cross - sectional study , we studied all 105 treated pku patients ( 46 males , 59 females , born between 1984 to 2010 ) diagnosed by newborn screening , or later , and followed at the metabolism clinic of mofid children 's hospital , which is a referral children 's hospital in tehran , iran . we considered a patient to have pku if plasma phenylalanine concentrations were more than 6 mg / dl in the untreated newborn infant/ child . social status was defined by number of children in family , number of affected children in family , maternal and paternal education , marital status and employment status of the parents . the educational level of parents was classified into four groups of illiterate , primary school , high school , and higher education ( which includes university education or passing any courses after graduating from high school ) . the marital status was classified into divorced or non divorced , and employment status into employed or unemployed . dietary phenylalanine intake , was adjusted to achieve blood phenylalanine levels between 2 - 6 mg / dl in children less than 12 years old and between 2 - 10 mg / dl in patients more than 12 years old . this level of control was considered as appropriate control , and blood phenylalanine less than 2 mg / dl was considered as overtreatment . mean plasma phenylalanine level was considered as a sign of dietary adherence in pku patients and understanding the factors that might have an effect on it . the mean plasma phenylalanine for each patient was calculated , considering the phenylalanine measurements throughout at least one year.all the information was collected via interviewing by the same blinded interviewer after informed parents consented orally with the aim of this study . anova t - test was done to understand the association between educational levels of parents and blood phenylalanine levels . independent t - test was done to assess the association between employment status of parents and phenylalanine levels . mann - whitney u test was done to understand the association between marital status of parents and phenylalanine levels . pearson correlation test was performed to assess the correlation between number of children in family , affected children in family , age at diagnosis , treatment duration and phenylalanine levels . the mean plasma phenylalanine concentration was 8.064.9 mg / dl ( range 1.13 to 23.60 mg / dl ) . it was 5.93.6 mg / dl in patients < 12 y / o and , 13.13.9 mg / dl in patients > 12 y / o . 50 ( 47.6% ) patients kept their mean plasma phenylalanine concentrations in normal range . the rest of the patients had mean plasma phenylalanine concentrations above age related reference range ( 44.8% ) , or under the normal range ( < 2 mg / dl , 8% ) as overtreatment . comparison of dietary control between two age groups we could n't find any significant association between four levels of maternal or paternal education and mean blood phenylalanine levels ( p=0.34 and p=0.33 respectively ) . table 1 displays mean phenylalanine concentrations in each group of parental educational level.we had only 3 divorced parents . the mean rank of phenylalanine in their children was 92 and it was 51.8 in the rest of cases ( mean plasma phenylalanine concentration in patients with divorced and not divorced parents was 14.56 mg / dl and 7.86 mg / dl respectively ) . there was a significant increase in mean phenylalanine rank of patients whose parents were divorced ( p=0.02 ) . 17 of 105 patients had at least one unemployed parent ( the one who was responsible for the family income ) . the mean phenylalanine in the unemployed and employed group was 10.36mg / dl and 7.61mg / dl respectively . this increase in the mean blood phenylalanine concentration of unemployed group was significant ( p=0.03 ) . parental educational level and mean phenylalanine concentrations in each group there was a significant positive correlation between the age at diagnosis and the mean blood phenylalanine ( r=0.2 , p=0.03 ) of the patients . also , there was a significant positive correlation between treatment duration and mean blood phenylalanine ( r=0.7 , p<0.001 ) . there was a positive correlation between number of children in the family and patients ' mean blood phenylalanine , although not significant ( r=0.1 , p=0.08 ) . there was a significant positive correlation between the number of affected patients in a family and mean blood phenylalanine ( r=0.43 , p<0.001 ) . mean ( sd ) , median and range of age of diagnosis , treatment duration , number of children in family and affected children in family of 105 cases with phenylketonuria it seems that younger patients ( < 12 y / o ) in our study , had a better dietary control . in contrast , averagely 12 year - old patients did not control their plasma phenylalanine concentrations properly . gokmen ozel et al also found the blood phenylalanine concentration higher in school - age children than in preschool children . in a study of crone et al , mean phenylalanine was kept on constant concentrations until 13 years old , but increased after 13 . in another study during the first year of life , average blood phenylalanine levels were < 5 mg / dl in 45% of cases . however , some of children with good metabolic control moved to poor control section during the mentioned study . of societal barriers in dietary adherence , especially in adolescents , we could mention to " peer pressure " as stated in nevins ' study . therefore it is much easier to control children , in the preschool ages than in elder ages , when the children are in open area of school and are going to be autonomous . there was an association between higher knowledge in parents and lower phenylalanine concentrations in a study of bekhof et al . also , patient 's age and parental educational level were effectively related to blood phenylalanine concentrations . mothers ' illiteracy was a negative factor on dietary compliance in ozalp et al study . macdonald et al , believe that dietary knowledge is an essential factor on dietary compliance . in contrast , gokmen ozel et al found that there was a non significant negative correlation between maternal total knowledge score ( r=0.140 , p=0.093 ) , and median blood phenylalanine concentration . olsson et al found parents ' educational level was not statistically significant related to blood phenylalanine concentrations . macdonald et al found was a positive but not significant correlation between higher knowledge of mothers and lower blood phenylalanine levels in their children ( r= 0.27 , p=0.06 ) . we also , found no relation between the four groups of maternal or paternal educational level and patients ' plasma phenylalanine concentrations . this may be due to nutrition specialists who train families and increase parents ' knowledge independently from their education . we suppose , in comparison with educational level , having precise dietary knowledge is a more vital demand of families and patients . in spite of very slight number of divorced parents ( 3 of 105 ) in our study , dietary adherence in these cases also , olsson et al found divorced parents had more difficulty in controlling their children 's phenylalanine level . this result apparently leads to the consequence that child neglect happens more common in divorced families . phenylalanine levels were not affected by parental employment status in a study of macdonald et al . in contrary , gokme ozel et al realized that currently employed mothers had more knowledge than unemployed ones . in our study unemployment of the parents , who were responsible for family income , significantly impressed the patients ' phenylalanine levels . although the specific " phenylalanine free " formulas are given at no cost to the families in our referral hospitals , these unemployed families are under pressure to afford many other requirements of their children . we found , blood phenylalanine concentrations did not raise significantly by increase in family members . as well , a study found weakening in children 's care giving in thickly populated families , also they found maternal knowledge was lower in large families . but macdonald et al found that phenylalanine levels were not affected by family size . quite the opposite , a study accepted that large families could improve the dietary compliance by food shopping , cooking and supporting each other . by increasing number of affected children in the family it seems that by increasing affected children in the family there will be more financial pressure on them , because in contrast with many other countries , secondary services like special foods or supplements are not available free of charge for them . since there was a positive correlation between age at diagnosis and phenylalanine levels in our study , it looks that by a late diagnosis there is an increase in plasma phenylalanine levels . besides , late diagnosis causes more complications including physical and mental retardation that could be another barrier for dietary compliance . therefore , we did not insert the data concerning family income to the study , because most of the families have various sources of income and their report might not be valid . the interesting finding in our study it looks as if growing up does not lead to better dietary compliance , but it may be due to some kind of refractory behavior in these children . although , increasing in blood phenylalanine levels in elder patients could have many explanations , spending more time on training families and patients themselves , might lead to understanding the importance of dietary control and its continuation during whole life . consulting the families to have better contraception and have less affected children in family , financial and emotional support especially for divorced parents can help them to have a better dietary adherence . finally sending children to special pku schools could help them to avoid peer pressure.consequently , finding the level of dietary knowledge in patients and their caregivers could be the next step .

objectivethere are several problems associated to the management of patients with phenylketonuria ( pku ) . social status could be one of the affecting factors on dietary adherence in these patients . the aim of this study was to evaluate family social status and dietary adherence of pku patients in iranian population.methodsin a cross - sectional study , we studied 105 iranian pku patients ( born 1984 to 2010 ) , treated and followed at mofid children 's hospital , tehran . social status was defined by number of children in family , number of affected children in family , maternal and paternal education , marital and employment status of the parents . age at diagnosis and duration of treatment were also recorded . mean plasma phenylalanine level was considered as a sign of dietary adherence in pku patients and was calculated considering the phenylalanine measurements throughout at least one year.findingsmean plasma phenylalanine concentration was 5.93.6 mg / dl in patients < 12 years old and 13.13.9 mg / dl in patients > 12 years old . blood phenylalanine concentrations in 47.6% of patients were in normal age - related reference range . there was a significant association between divorced and unemployed parents , and higher levels of blood phenylalanine concentration ( p=0.02 and p=0.03 respectively ) . there was a significant positive correlation between number of affected children in the family ( r=0.43 , p<0.001 ) , age at diagnosis ( r=0.2 , p=0.03 ) , treatment duration ( r=0.7 , p=<0.001 ) and blood phenylalanine concentrations . there was no significant relation between parental education , family size and dietary adherence.conclusionsocial status affects dietary adherence to some extent . we suggest exploring care - givers dietary knowledge as the next step to improve dietary compliance in these patients .

Introduction Subjects and Methods Findings Discussion Conclusion Conflict of Interest

this disorder is relatively frequent on the beard of adult men and on the scalp of children . a 33-year - old man was referred for evaluation of an itchy sensation on his back that had been present for more than 10 years . physical examination revealed a large patch with short and irregular hairs that involved his entire back [ figure 1a ] . dermoscopic evaluation showed that hair follicles contained several short hair shafts with different lengths [ figure 1b ] . histological examination revealed that the follicular structures consisted of different hair shafts , which were enclosed in a common outer root sheath [ figure 2 ] . these findings suggest the diagnosis of pili multigemini . each hair had its own inner root sheath . ( a ) physical examination revealed an extensive and well demarcated patch with short and irregular hairs that involved the entire back ( b ) dermocopic image shows that hair follicles contained several short hair shafts with different lengths histologic image . follicular structures include different hair shafts ( yellow arrows ) , which were enclosed in a common outer root sheath pili multigemini is a hair disorder characterized by clusters of shafts that emerge from a single follicle . a common bulb encloses a dermal papilla that splits into different - sized hair shafts with separate cuticles . normal hair grows from a single hair follicle and exits from one hair canal to the surface of the skin . the cause of pili multigemini is unknown , although there may be a genetic link . it has been suggested that a subdivided papilla produced divided hairs or that multiple hairs may be due to the partial merging of several papillae . an extensive presentation of pili multigemini over a large area has been previously reported in three patients . they showed a wide patch consisting of irregular and coarse thicker hairs over the entire back . the authors propose that this extensive condition could be underdiagnosed because the nevoid multiple hairs have no specific symptoms . trichoscopy enhances the evaluation of hair disorders and facilitates the diagnosis of this rare hair shaft dysplasia . differential diagnosis includes mainly pili bifurcate , which is characterized by bifurcation of the hair shaft . each bifurcation produces two separate parallel branches , which fuse again to form a single shaft , and each branch of the successive bifurcations is covered with its own cuticle . however , in pili gemini , a kinetic papilla splits at the upper end from single to double - tipped during the anagen phase and consequently the same follicular matrix produces two different - sized hair shafts having separate cuticles that emerge through a single pilary canal . pili gemini maintains the double tipped papilla and consequently the hair shaft does not fuse again . in conclusion , we present an extensive case of pili multigemini over the back . this condition has been described previously as the nevoid pili multigemini . despite the rarity of this entity , probably it could be a commonly recognizable condition with an exhaustive exploration . the large areas implicated in the patients described and the normal hair appearance in other locations suggests a genetic link .

pili multigemini is a rare disorder where more than one hair exists in a single hair follicle . papillar tips that divide into several tips will produce several hair shafts , so that characteristically do not fuse again . this disorder is relatively frequent on the beard of adult men and on the scalp of children . however , extensive areas of pili multigemini in other locations have rarely been described .

INTRODUCTION CASE REPORT DISCUSSION

his development of alternative continent bladder access through a continent stoma has relieved the necessity for the patient to either void efficiently or to catheterize through the urethra . there have been other alternatives to mitrofanoff 's original use of appendix for extra - anatomic access to the bladder . other options for continent bladder access include ureter , fallopian tube , bladder tubes , and tubularized bowel.[13 ] many surgeons believe that catheterizable abdominal stomas promote continence by avoiding manipulation of the urethra or in some cases a reconstructed bladder neck . in some cases they encourage patient compliance with intermittent catheterization because catheterizing through an abdominal stoma is easier and less time consuming than urethral catheterization for many patients . regular catheterization keeps bladder pressures low and minimizes stress on the urethra or reconstruction done at the bladder neck . stomas allow the surgeon to be more aggressive in creating resistance at the bladder outlet because the urethra will not be needed as the primary access for catheterization . the combination of continent stomas and augmentation has allowed patients who have suffered multiple failed continence surgeries to be salvaged with good results . the mitrofanoff procedure has traditionally been performed using an open surgical technique using either a lower midline or pfannenstiel incision . there have been a few reports in the literature where laparoscopic techniques have been applied to this procedure in an attempt to minimize morbidity and improve cosmesis . jordan and winslow , and van savage and slaughenhoupt described a laparoscopic assisted appendicovesicostomy where the appendix and right hemicolon were mobilized laparoscopically and the appendicovesical anastomosis was completed through a pfannenstiel incision . intracorporeal suturing is a technically demanding task that requires a significant amount of experience and surgical skill . we report our experience with a complete laparoscopic mitrofanoff appendicovesicostomy to promote a catheterizable abdominal stoma . a 10 mm laparoscopic camera port was placed through the umbilicus and 2 , 5 mm laparoscopic ports were placed in the left lower quadrant and right midaxillary line at the level of the umbilicus . a fourth port was placed in the left midaxillary line , also at the level of the umbilicus . the appendix was ligated and divided at its base and the stump was buried into the caecum using a 3-zero silk purse - string suture . the appendix was assessed to determine whether the length was adequate to reach the anterior abdominal wall ( approximately 5 to 6 cm ) and whether the diameter was of sufficient caliber to accommodate at least a 12 fr catheter . this maneuver was done without desufflating the abdomen to allow more room for error in appropriating an adequate distance . ligation of the base of the appendix appendix mobilized on an adequate length of mesoappendix the bladder was filled with saline and a 5 cm vertical seromuscular incision was made in the right posterior wall of the bladder down to the mucosa [ figure 3 ] . after incision of the mucosa the appendix was anastomosed to the bladder using 4 zero polyglactin interrupted sutures . the seromuscular layer of the bladder was closed using interrupted 3 zero polyglactin sutures , [ figure 4 ] creating a tunnel for the appendix . the appendix was then brought up to the umbilicus and a catheterizable stoma was created [ figure 5 ] . smooth catheterization through the appendix with full pneumoperitoneum and with the pneumoperitoneum released was performed . a 16 fr suprapubic catheter was inserted into the bladder and a 10 fr stenting catheter was left through the appendix . pouting bladder mucosa after dissection of detrusor completed appendico - vesical anastomosis and detrussoraphy distal end of the appendix being brought out to the skin via a 10 mm port the child was started on oral liquid feeds within 6 - -8 hours . the catheter was left indwelling for 3 weeks and was removed after ensuring that the patient or family could catheterize without difficulty . during the period january 2004 and december 2009 , six children with a mean age of 12.8 years ( range 9 - -16 years ) underwent laparoscopic mitrofanoff appendicovesicostomy . three of these children had neurogenic bladder secondary to spina bifida , two had non - neurogenic neurogenic ( hinman ) bladder , and one child was a case of urethral stricture following posterior urethral injury and had undergone multiple surgeries for the same . the reasons for failed urethral cic in these children included urethral strictures in three , down 's syndrome in two and painful cic in the remaining child . overall the mean operative time was 139.6 min ( range 125 - -175 min ) . all children had liquid diet in the evening of surgery or on the postoperative day 1 . two children developed urinary incontinence via the mitrofanoff ; however both responded well to anticholinergic therapy . one of these children has been off anticholinergic therapy , while the other child still needs low dose anticholinergic therapy . one child had a severe episode of urinary infection in the follow - up period , but responded well to antibiotics . the mitrofanoff principle was originally described as a method to provide an alternative means to access the bladder . it creates a conduit to the bladder through which patients with a sensitive , absent , or traumatized urethra can perform clean intermittent catheterization ( cic ) easily . fashioning this continent , catheterizable channel using the appendix the use of laparoscopic and robot - assisted approaches to reconstruct the lower urinary tract has been slowly introduced in the past several years . individual cases of laparoscopic and robot - assisted mitrofanoff appendicovesicostomy have appeared in the literature . the adoption of a purely laparoscopic or robot - assisted approach to lower urinary tract surgery in pediatric patients has been slow , given the steep learning curve and relative paucity of cases . congruent with the limited published experience , the surgical outcomes have compared favorably with those of an open approach in these selected patients . parents of children undergoing this minimally invasive procedure were satisfied with the short - term outcomes and postoperative recovery , particularly early resumption of physical activity and the avoidance of a prolonged absence from school . in children approaching adolescence and adulthood , the perceived benefits of a minimally invasive approach such as lower analgesic requirements , improved recovery time and improved cosmesis might be more apparent than in younger children . some surgeons have reported that prolonged operative time was a potential disadvantage associated with laparoscopic approach . the operative time should improve significantly in subsequent cases with better handling of technical issues . our reduced operative time has been due to our experience in performing laparoscopic pyeloplasty in children . in our experience the use of the da vinci robotic technology has added advantages for the pediatric population . it has allowed for the more complex laparoscopic procedures to be performed with greater dexterity . pedraza et al found that by incorporating the da vinci robotic system they were able to create efficiently a circumferential watertight appendicovesical anastomosis and decrease the overall operative time of the procedure . nguyen et al reported on 10 patients who underwent the robot - assisted laparoscopic mitrofanoff procedure using the da vinci surgical system . they similarly believe that the robot - assisted laparoscopic mitrofanoff procedure is feasible to perform , is associated with satisfactory outcomes and minimal complications , and has the benefits of a minimally invasive approach . pure laparoscopic mitrofanoff appendicovesicostomy is feasible and is associated with reasonable outcome with early recovery , resumption of normal activities , and excellent cosmesis .

introduction : the mitrofanoff principle was originally described as a method to provide an alternative means to access the bladder . it creates a conduit to the bladder through which patients with a sensitive , absent , or traumatized urethra can perform clean intermittent catheterization ( cic ) easily . we report our experience with complete laparoscopic mitrofanoff appendicovesicostomy to promote a catheterizable abdominal stoma.materials and methods : a 4-port transperitoneal approach was used to create a complete laparoscopic mitrofanoff appendicovesicostomy.results:six children with a mean age of 12.8 years ( range 9 - 16 years ) underwent laparoscopic mitrofanoff appendicovesicostomy . mean operative time was 139.6 min and mean estimated blood loss was 46 cc . no cases of urinary leaks were noted . there have been no cases of either stomal stenosis or appendicovesical stenosis noted.conclusions:pure laparoscopic mitrofanoff appendicovesicostomy is feasible and is associated with reasonable outcome with early recovery , resumption of normal activities and excellent cosmesis .

INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS

the transsphenoidal approach is the most commonly utilized operation for the surgical treatment of sellar lesions and is a relatively safe operation in experienced centers.1 following resection of pituitary adenomas and other sellar tumors , many surgeons utilize absorbable plates to reconstruct the bony sellar floor to serve as a buttress for the sellar contents and repair construct . although usually safe , vascular injury in conjunction with insertion of rigid plates following sellar tumor resection has been described once before.2 common complications of transsphenoidal operations include endocrine abnormalities and cerebrospinal fluid ( csf ) leaks.3 vascular injury is a rare but serious complication of transsphenoidal surgery encountered in 0.8 to 1.1% of cases , with an associated mortality of nearly 30%.456 the majority of vascular injuries are identified at the time of surgery , usually resulting from direct injury to the internal carotid artery during resection of tumor within the cavernous sinus or upon opening of the dura , often resulting in profuse arterial hemorrhage.6789 other described vascular complications include vasospasm , carotid thrombosis , cavernous sinus thrombosis , embolism , caroticocavernous fistula , or pseudoaneurysm.237810111213141516171819 postoperative carotid pseudoaneurysm , though rare , represents a grave risk to the patient if unrecognized . it may lead to delayed hemorrhagic or embolic complications when the patient is no longer in a monitored hospital setting . we present a rare case of delayed pseudoaneurysm and embolic stroke following erosion of a rigid plate into the cavernous internal carotid artery . a 57-year - old man with a nonfunctional pituitary macroadenoma causing vision loss underwent a gross total , endoscopic transsphenoidal resection ( fig . 1 ) . the tumor was invading the right cavernous sinus wall . during the procedure to resect the tumor from this region , there was some venous bleeding that was easily controlled by temporarily packing the area using gelfoam ( pfizer , new york , new york , usa ) with thrombin . following resection , a custom - fit bioabsorbable plate was placed extradurally to reconstruct the sellar floor . four weeks later , he experienced epistaxis for which he was treated at an outside emergency department . six weeks following the operation , he presented to clinic with dysarthria as well as left hand and facial weakness and was admitted for further work - up . neuroimaging revealed subacute infarcts in right middle cerebral artery distribution . computed tomography ( ct ) angiogram showed a 6 4 mm pseudoaneurysm located on the medial wall of the right cavernous internal carotid artery . a hypodensity likely representing the implanted absorbable plate was noted to be compressing the right carotid artery in this region ( fig . stent coiling with placement of eight detachable coils was used to successfully obliterate the pseudoaneurysm ( fig . 3 ) . follow - up digital subtraction angiogram and ct angiogram demonstrated durable obliteration of the pseudoaneurysm with preserved flow through the carotid artery . on follow - up , the patient 's neurological deficits have resolved . ct - angiogram of the brain showing hypodense absorbable plate compressing the right cavernous carotid artery . digital subtraction angiogram showing right cavernous carotid pseudoaneurysm before and after stent - assisted coil embolization . iatrogenic carotid pseudoaneurysm is an extremely rare complication of transsphenoidal surgery , with only 24 reported cases found in our review of the literature . only one previous case was associated with sellar floor reconstruction.2 a pseudoaneurysm is caused by injury to the carotid artery wall leading to an encapsulated hematoma in communication with the ruptured artery . in general , an aneurysmal rupture of the cavernous carotid will result in a caroticocavernous fistula . however , severe epistaxis and pituitary apoplexy have been reported in patients whose medial cavernous sinus wall has been eroded by tumor or violated during surgery.4 thus , the consequences of aneurysmal rupture support urgent treatment of even incidentally diagnosed lesions , as they represent a grave risk to the postoperative patient . because of the extremely low reported incidence of pseudoaneurysms after transsphenoidal surgery , postoperative angiography is not routinely performed.6 although in cases where there is significant arterial bleeding or concern for carotid injury , many experts recommend immediate postoperative angiography.615 based on the uneventful operative course and features observed on ct angiogram , the pseudoaneurysm was likely caused by erosion of the absorbable plate into the cavernous carotid artery . this has been previously reported by crowley et al and was also associated with delayed severe epistaxis followed by transient ischemic deficits.2 despite its low complication rates , these cases highlight the risks associated with sellar floor reconstruction.20 both patients were treated via endovascular means with good results . in a majority of cases , the absorbable plates used to reconstruct the bony sellar floor can be custom fit to approximate the size of the defect . the surgeon must take extra care to ensure that the cut edges are as round and smooth as possible , and that the size of the plate is just large enough to be inserted beneath the bony edges of the lateral sellar floor , and in an extradural fashion . perhaps more importantly , the authors now make every attempt to only use a rigid buttress when necessary that is , only in cases where intraoperative csf leaks are observed in the setting of a large bony defect that can not be repaired or adequately buttressed with routine techniques , including cellulose sponge , duragen ( integra lifesciences corporation , plainsboro , new jersey , usa ) , and fibrin glue . according to our search of the literature , 18 publications reporting 24 cases of iatrogenic carotid pseudoaneurysm following transsphenoidal surgery were identified ( table 1 ) . among published cases , the mean patient age was 45.6 ( range 22 to 74 ) , with a slight female predominance ( 13/22 patients ) . intraoperative arterial hemorrhage requiring packing was described in 17 of 24 cases ; 21% of cases reported the operation as uneventful . we recommend additional delayed imaging in patients with high suspicion for arterial injury , considering multiple reports in which the immediate postoperative angiogram was negative , only to have the patient present with a pseudoaneurysm in a delayed fashion.161721 common presentations prior to diagnosis included severe epistaxis,10 postoperative high - suspicion angiogram,10 routine follow - up imaging,2 and cranial neuropathy.1 the interval to diagnosis following surgery varied between 0 days and 10 years . delay to diagnosis may lead to aneurysmal rupture or embolic stroke outside of a monitored setting . in fact , in nine cases reported in the literature , the patient was discharged from the hospital prior to the diagnosis being made . abbreviations : acth , adrenocorticotropic hormone ; bto , balloon test occlusion ; csf , cerebrospinal fluid ; ct , computed tomography ; gh , growth hormone ; ica , internal carotid artery ; mri , magnetic resonance imaging ; nr , not reported , pod , postoperative day ; ptca , percutaneous trasnluminal coronary angioplasty ; ptfe , polytetrafluoroethylene . although conservative management may be an option , one report in which a patient refused surgical management resulted in death at 2 years secondary to uncontrollable epistaxis.15 despite reports that conservatively managed postprocedure femoral pseudoaneurysms spontaneously close in 89% of patients , we recommend treatment even in incidental false aneurysms of the carotid because of the higher morbidity associated with carotid rupture.22 additional surgical options for cavernous carotid pseudoaneurysms include extracranial to intracranial ( ec - ic ) bypass with carotid occlusion , craniotomy with direct repair of the carotid artery within the cavernous sinus , or trapping the aneurysm via cervical carotid ligation and supraclinoid carotid clip ligation.613152324 endovascular options may include balloon occlusion of the carotid artery , coil embolization of the pseudoaneurysm or carotid artery , and/or placement of a flow - diverting stent.378121518 with the advancement in endovascular techniques , open surgery has become less common . successful carotid artery occlusion , aneurysm trapping , stenting , or coiling can be achieved without subjecting the patient to the risks associated with open craniotomy and manipulation of a cavernous carotid pseudoaneurysm . some authors prefer carotid occlusion , when feasible , to avoid the risks associated with manipulation of the false aneurysm . struffert described two cases requiring repeat transsphenoidal surgery to retrieve coils that had migrated from a pseudoaneurysm into the patients ' nostril.17 the use of a flow - iverting stent may obviate the need for an intra - aneurysmal embolization.1825 however , flow - diverting stents surrender future endovascular access to the pseudoaneurysm , because once deployed a microcatheter can not be passed through it . if the patient experiences breakthrough bleeding , he or she could not be treated with further embolic agents although rare , erosion of an absorbable plate used to reconstruct the bony sellar floor may cause a carotid artery pseudoaneurysm , potentially resulting in a subacute presentation of epistaxis or embolic stroke . the authors ' preference is to avoid the use of rigid plating for sellar floor reconstruction in the absence of intraoperative csf leaks , unless required to buttress a large skull base defect and csf leak repair construct . short - segment embolization with stent coiling is the preferred option for the treatment of iatrogenic carotid pseudoaneurysms following endonasal skull base operations .

background bioabsorbable plates are frequently utilized in the repair of skull base defects following transsphenoidal operations . traumatic intracranial pseudoaneurysms are a rare complication of transsphenoidal surgery . to date , iatrogenic carotid pseudoaneurysm associated with the use of an absorbable plate has been reported once.results a 57-year - old man with a large nonfunctional pituitary macroadenoma underwent an endoscopic transsphenoidal operation with gross total resection . an absorbable plate was placed extradurally to reconstruct the sellar floor . he experienced delayed repeated epistaxis , followed by a right middle cerebral artery distribution embolic stroke . computed tomorgraphy ( ct ) angiogram 6 weeks postoperatively revealed a 6 4 mm pseudoaneurysm located on the medial wall of the right cavernous internal carotid artery . stent coiling was used to successfully obliterate the pseudoaneurysm , and the patient fully recovered.conclusion delayed erosion of the carotid artery wall caused by a plate used to reconstruct the sellar floor may manifest with epistaxis or embolic stroke . the authors ' preference is to avoid insertion of a rigid plate for sellar floor reconstruction in the absence of intraoperative cerebrospinal fluid ( csf ) leaks , unless it is required to buttress a large skull base defect . short - segment embolization with stent coiling is the preferred treatment option for carotid pseudoaneurysms following transsphenoidal operations .

Introduction Case Report Discussion Conclusion

the health protection agency centre for infections has coordinated national surveillance of listeriosis in england and wales since 1990 . cases are included in the system by voluntary referral of cultures to the national reference laboratory or by electronic reporting of confirmed cases from local laboratories . clinical data , including details of patients concurrent conditions , are subsequently sought from the consultant clinical microbiologist involved in the care of the case - patient . microbiologic data from local and reference laboratories and clinical and risk factor data are linked for each case , deduplicated as necessary , and stored in a bespoke microsoft access database ( microsoft , redmond , wa , usa ) access database . a case of listeriosis is defined as a person with clinically compatible illness and from whom l. monocytogenes was isolated from a normally sterile site . cases are subsequently classified as either non pregnancy - associated ( persons > 1 month of age ) or pregnancy - associated ( a maternal fetal or maternal neonatal pair ; such pairs were considered a single case ) . in this study , we included non pregnancy - associated cases reported from laboratories in england for which a clinical questionnaire was available and showed that at least 1 reported concurrent condition was present . we included cases reported during april 1 , 1999march 31 , 2009 because denominator data were arranged by fiscal years . these cases included sporadic cases and cases that were identified as being part of common source foodborne outbreaks . authors ( p.m. and i.a.g . ) reviewed each reported concurrent condition and assigned an international classification of diseases , 10th revision ( icd-10 ) ( 8) , code when appropriate . rules for assigning codes were developed at the outset to ensure standardized coding throughout the study ( technical appendix ) . these rules were validated by a third author ( s.j.o . ) , a clinically qualified investigator , who also reviewed any coding disparities . counts were calculated of all persons and those > 60 years of age for each icd-10 chapter ( icd-10 codes are aggregated into 22 chapters ) and subgroup ( within each chapter ) . hospital episode statistics finished consultant episodes ( fce ) data , which were aggregated by icd-10 code , age group ( 014 years , 1559 years , 6074 years , and > 75 years ) , and fiscal year , were obtained from the health and social care information centre ( 9 ) and used as denominator data . these data describe episodes of continuous admitted patient care under a specific consultant for national health service hospital inpatients in england , and a primary diagnosis is assigned to each episode by using icd-10 coding . to ensure reliable confidence intervals ( cis ) , we calculated incidence rates / million fces and 95% cis for each icd-10 chapter and subgroup in which there were > 10 cases . two icd-10 chapters not used by hospital episodes statistics to code primary diagnoses , external causes of morbidity and mortality ( v01y98 ) and codes for special purposes ( u00u99 ) , were not considered . relative risks ( rrs ) and corresponding 95% cis were calculated as appropriate when > 10 cases were reported for a concurrent condition subgroup or chapter . data were stored , manipulated , and summarized by using microsoft access , and incidence rates and rrs were calculated by using microsoft excel . differences in proportions and changes in proportions over strata were assessed by using the test and the test for trend , respectively . a total of 1,239 icd-10coded concurrent conditions were reported by 1,413 case - patients with non pregnancy - associated listeriosis in england during april 1 , 1999march 31 , 2009 ( figure ) . of those patients who reported > 1 underlying condition , 21 ( 2.2% ) were identified as being part of a common source outbreak . characteristics of case - patients with and without a completed clinical questionnaire are shown in table 1 . overall , 9.1 cases of listeriosis / million fces were reported over the study period ( 95% ci 8.69.6 ) ( table a1 ) . compared with all other reported conditions , higher rates of disease were reported for the following chapters ( in order of highest to lowest rr ) : endocrine , nutritional , and metabolic diseases ( rr 5.3 , 95% ci 4.26.6 ) ; neoplasms ( rr 4.9 , 95% ci 4.45.5 ) ; mental and behavior disorders ( rr 3.1 , 95% ci 2.44.1 ) ; diseases of the circulatory system ( rr 1.4 , 95% ci 1.21.6 ) ; diseases of the digestive system ( rr 1.3 , 95% ci 1.11.5 ) ; and diseases of the musculoskeletal system and connective tissue ( rr 1.3 , 95% ci 1.11.6 ) ( table 2 ) . study population and reported international classification of diseases , 10th revision ( icd-10)coded concurrent conditions for 1,413 case - patients with non pregnancy - associated listeriosis , england , april 1 , 1999march 31 , 2009 . * * icd-10 , international classification of diseases , 10th revision ; ci , confidence interval ; nc , not calculated ( for conditions with < 10 cases ) ; , data not available . within these chapters , only certain subgroups showed increased rates : diabetes mellitus ; malignant neoplasms of the lymphoid , hematopoietic , and related tissues ; eye , brain , and other parts of the central nervous system ( cns ) ; respiratory and intrathoracic organs ; digestive organs ; breast ; male and female genital organs ; thyroid and other endocrine glands ; mental and behavior disorders caused by psychoactive substances ( alcohol - related in 96% of reports ) ; hypertensive diseases , other forms of heart disease , and diseases of arteries , arterioles , and capillaries ; diseases of the liver and noninfective enteritis and colitis ; and systemic connective tissue disorders ( table 2 ) . in addition , several subgroups were associated with increased risk even when the corresponding chapter was not : renal failure , diseases of blood and blood - forming organs , and chronic lower respiratory diseases ( table 2 ) . concurrent conditions were disproportionately reported for persons > 60 years of age ( p<0.001 ) , and the rate of listeriosis for this age group ( 16.8/million ; 95% ci 15.817.9 ) was significantly higher than that for younger persons ( rr 4.6 , 95% ci 4.15.3 ) ( table 2 ) . when the rr for each chapter for persons > 60 years of age ( using persons < 60 years of age as the reference population ) was calculated , the following were associated with increased risk : endocrine , nutritional and metabolic diseases ; genitourinary system diseases ; diseases of the musculoskeletal system and connective tissue ; neoplasms ; certain infectious and parasitic diseases ; diseases of the digestive system ; and mental and behavior disorders ( table 2 ) . in instances where the risk for each subgroup in persons > 60 years of age could be calculated and compared with that for persons < 60 years of age , all subgroups of previously identified chapters were associated with increased risk . we analyzed surveillance data that included detailed denominator data by using an internationally recognized diagnostic classification system and found that a wide variety of conditions seem to increase the risk for serious infection with l. monocytogenes . malignancies accounted for more than one third of conditions , and cancer patients had a 5-fold increased risk for development of listeriosis . other high - risk conditions included diabetes mellitus ; alcoholism ; certain diseases of the circulatory system and the musculoskeletal system and connective tissue ; noninfective enteritis and colitis ; and diseases of the liver and kidney . for most high - risk conditions , the risk for infection was higher among older patients . case identified by the national surveillance program in england are laboratory confirmed , and most cases result in serious illness requiring hospitalization or death . given this finding , a hospitalized population better represents the population at risk than a community population , which was used in previous studies ( 10,11 ) . the response rate to the clinical questionnaire that captured information on concurrent conditions was high and not influenced by age or sex of the case - patient , which minimized differential ascertainment of clinical data . however , we could not assess concurrent conditions for which completed clinical questionnaires were not returned . this issue indicates that the role of some conditions might be underestimated if clinicians were unwilling to return questionnaires and disclose information for certain case - patients ( e.g. , those with aids ) . similarly , but less likely , reporting bias might exist if the propensity to report certain concurrent conditions were affected by the presence or absence of others conditions , or if only concurrent conditions considered relevant to l. monocytogenes infection were reported . concurrent conditions were reported by the clinical microbiologist rather than by the consultants responsible for the care of the patients with concurrent conditions . these consultants might be better informed of existing concurrent conditions . however , hospital microbiologists need to be aware of such conditions to provide treatment accordingly , and questioning several consultants for each case - patient may have a negative effect on questionnaire response because questionnaires might be lost if passed between multiple consultants . although we acknowledge that such grouping might mask high - risk conditions apparent at the 4-character icd-10 code level , routine surveillance data were not specific enough to further discriminate among conditions . in some instances , in which treatments were reported in the absence of relevant conditions ( e.g. , chemotherapy , dialysis , splenectomy ) , we made assumptions about the conditions requiring such treatment and coded accordingly ( table a1 ) . although these assumptions could inflate the incidence rates for certain conditions , they occurred relatively infrequently and were not used for treatments that could be prescribed for a range of conditions ( e.g. , broad - spectrum antimicrobial drugs ) . because only single - variable analysis could be performed , we could not assess the extent to which concurrent conditions were correlated , which led to the potential for uncontrolled confounding . such method limitations might explain the high incidence associated with both diabetes and kidney disease and reinforce the need to consider these findings as highly refined hypotheses to be tested by other methods ( 12 ) . to our knowledge , few studies have attempted to quantify the risk for listeriosis by patient concurrent conditions . as part of a risk assessment of l. monocytogenes in ready - to - eat foods , researchers from the world health organization ( who ) and the food and agriculture organization ( fao ) calculated the relative susceptibility to listeriosis for certain conditions ( 10 ) . furthermore , risk levels for listeriosis by predisposing condition in denmark have also been estimated ( 11 ) . despite differences in methods between those studies and our study , several high - risk conditions were also identified in those studies : malignancies ( most notably those of the blood ) , kidney disease ( recorded as dialysis and renal transplant ) , diabetes , alcoholism , and increased age in all 3 studies ; liver disease and pulmonary cancer in the who / fao study and our study ; and systemic lupus erythematosus in the study in denmark and our study ( as systemic connective tissue disorders ) . the absence of aids as a high - risk condition in our study and its presence in both previous studies ( 10,11 ) , might reflect improved treatment for hiv infection that prevents aids and , consequently , l. monocytogenes infection ( 13 ) or highlight a reporting bias by the consultant microbiologist . a general transplantation status , identified as a condition leading to the highest relative susceptibility in the who / fao study , was not coded in our study because it is a treatment . noninfective enteritis and colitis and certain diseases of the circulatory system were identified as additional high - risk conditions in our study but not in the previous studies . these additional conditions might be the result of improved accuracy , use of icd-10 coding and a hospitalized reference population instead of the general population , different susceptibility calculations , or changes in the prevalence of certain conditions in the interim period ( the previous studies used data from 1992 and 19891990 ) . however , we acknowledge that links between these conditions and listeriosis have been reported ( 1418 ) . with these caveats in mind , the number and diversity of conditions that appear to increase the risk for listeriosis imply that physicians working in all specialties should consider listeriosis when treating patients with concurrent conditions and provide appropriate food safety advice . similarly , current uk government food safety advice on avoidance of listeriosis , which is delivered passively and is specific mainly for pregnant women ( 19,20 ) , should be communicated actively to all high - risk groups . in prioritizing advice , policy makers should consider not only the associated risk but also the prevalence of the concurrent condition . cancer patients accounted for more than one third of listeriosis cases , and high risks were observed for most cancer subgroups . because we are not aware of any appropriate food safety advice that is tailored specifically for cancer patients in the uk , emphasis on this group might help to prevent further cases .

the epidemiology of listeriosis in england and wales changed during 20012008 ; more patients > 60 years of age had bacteremia than in previous years . to investigate these changes , we calculated risk for listeriosis by concurrent condition for non pregnancy - associated listeriosis cases reported to the national surveillance system in england during 19992009 . conditions occurring with l. monocytogenes infection were coded according to the international classification of diseases , 10th revision , and compared with appropriate hospital episode statistics inpatient denominator data to calculate incidence rates / million consultations . malignancies ( especially of the blood ) , kidney disease , liver disease , diabetes , alcoholism , and age > 60 years were associated with an increased risk for listeriosis . physicians should consider a diagnosis of listeriosis when treating patients who have concurrent conditions . providing cancer patients , who accounted for one third of cases , with food safety information might help limit additional cases .

Methods Results Discussion Supplementary Material

abdominal aortic aneurysms ( aaa ) can be repaired with an aortic bifemoral bypass graft , which is considered an ideal procedure for aortic aneurysms , aorto - iliac disease or aortic atherosclerosis . though rare , infections involving the graft can happen , typically , two to five years following implantation . anaerobic , non - sporulating , gram - positive bacilli from the normal human gut flora can occasionally be implicated in invasive diseases . notably , eggerthella lenta , formerly known as eubacterium lentum , often causes clinically significant bacteremia with a significant mortality rate . herein we report a case of an aortobifemoral bypass graft with an aortoenteric fistula , giving rise to a very rare bacteremia caused by the species eggerthella lenta . a 75-year - old male with a past medical history significant for type 2diabetes mellitus , hypertension , chronic thrombocytopenia and chronic kidney injury underwent resection of a ruptured aaa and placement of an aortobifemoral bypass graft . his postoperative course was eventful for renal failure , which required four cycles of dialysis with improvement . twenty - nine months later , the patient was seen in the emergency department with a two - day history of rigors , dysuria , polyuria and burning micturition complicated by fever , weakness and lethargy observed by his family on the day of admission . blood pressure upon examination was 70/35 mmhg , rising to 90/48 mmhg after a fluid bolus ; temperature was 101 degrees fahrenheit ; heart rate 90/min ; and respiratory rate was 20/min . blood work was significant for a white blood cell count of 18,000 ; lactic acid of 4.1 , 20 percent bands and platelet count of 20,000.urinalysis was remarkable for white blood cells , red blood cells and moderate leukocyte esterase . physical examination revealed diffuse abdominal tenderness , and peri - umbilical tenderness with deep palpation without peritoneal signs.the patient was admitted to the intensive care unit with a diagnosis of severe sepsis secondary to urinary tract infection with high anion gap metabolic acidosis secondary to lactic acidosis . blood cultures and urine cultures were drawn , which demonstrated eggerthella lenta septicemia and escherichia coli extended spectrum beta - lactamase ( esbl)-positive urinary tract infection . a subsequent abdominal computed tomography ( ct ) with contrast demonstrated induration with inflammatory changes around the aortobifemoral bypass graft . furthermore , the graft showed stranding around the aorta near the iliac components with presence of an air bubble between these components [ figure 1 ] . this finding raised the possibility of gas producing infection or an aortoenteric fistula communicating with an adjacent loop of bowel , either of which could have feasibly contributed to the aortic graft harboring eggerthella lenta . the patient was recommended to have graft replacement surgery with repair of the fistula in order to remove the source of infection ; however , he refused , preferring to be treated with iv antibiotics instead , despite the considerable risk . repeated blood cultures after treatment with antibiotics were negative and the patient was discharged home on antibiotics as an outpatient with the possibility of requiring chronic suppression with antibiotics . ct scan of the abdomen and pelvis showing an induration with inflammatory changes around the aorto - iliac graft and an small air bubble ( yellow arrow ) between the two legs of the graft , possibly caused by a fistulous communication with the bowel aortoenteric fistulas can involve any part of the small or large bowel , but it is more common to find them in the third and fourth portions of the duodenum . mucosal bleeding can be one of the signs as erosions of the graft into the intestines can precede massive gastrointestinal bleeding from an established aortoenteric fistula . staphylococcus aureus and gram - negative bacilli , accompanied by fever , signs of infection and leukocytosis , typically cause early infections ( less than four months after repair ) . the presentation of late - onset infections can be subtle without any specific signs or symptoms with an absence of fever being common . in fact , in few studies , the absence of fever and stay in the intensive care unit ( icu ) , was associated with an increased risk of mortality . they can also present with complications of the graft infection , such as a pseudoaneursym , sepsis or bleeding . our patient did have fever , leukocytosis and hypotension , but thiscould have been caused by his concurrent urinary tract infection with e. coli . it is possible that had this patient not had symptoms from his urinary tract infection , his bacteremia may have been missed . the use of graft devices for repair of an aaamay require a complex , open - air procedure with exposure of the compromised vessel . this , of course , increases the risk of an infectionfrom the device as well as the wound . the incidence of aaa graft infection is low ( 0.4% to 3% ) , with one - third of those infections manifesting as aortoenteric erosions / fistula . the management of an aortoenteric fistula has typically required replacement of the affected graft and repair of the fistulae , a surgery with a mortality risk of 20 to 30 percent . bacteremia associated with the aortoenteric fistula has also been reported to have a high mortality risk . in our case , the ct scan of the patient raised the possibility of an aortoenteric fistula that was likely to be the source of the patient 's infection with eggerthella lenta . after being explained the risk of the surgery , as well as the possibility of not being able to resolve the infection without surgery , the patient declined to move forward with the procedure . although lactobacillus , clostridium and propionibacterium species are more commonly encountered , many other lesser known anaerobic gram - positive bacilli can be involved in clinical infections as well . members of this genus are anaerobic , non - sporulating , non - motile , gram - positive bacilli that grow singly , as pairs or in short chains . classification and identification of this bacterium is reliably achieved through specialized cultured blood samples identifying the gram - positive bacilli as eggerthella lenta with 94% and 99% probability and confirmed by 16srna pcr gene sequencing . eggerthella lenta can be found in many different parts of the body , most commonly in the colon and feces . bacteremia associated with this bacterium carries a high mortality rate ( 20 - 40% ) especially if it is found to be the only pathogen in the blood . eggerthella lenta has been linked with many conditions such as ulcerative colitis , crohn 's disease , sinusitis , abscesses , pelvic inflammatory disease , malignancy and pancreatic abscesses among others . nearly all infections with anaerobic bacteria originate from the patient 's own microflora ; therefore , we can conclude that this patient 's bacteremia was from his own flora , with the likely source to be the aortoenteric fistula . due to the fastidious nature of these organisms , it can be exceedingly difficult to isolate these anaerobes from the site of an infection and easy to miss . isolating them requires the appropriate method of collection , transportation and cultivation of specimens , which requires a laboratory capable of performing 16s rrna gene analysis . , it took three days in order to get a positive blood culture and another three days to identify the organism and get antibiotic susceptibility . treatment with etrapenem proved to be successful as repeat cultures were negative . though bacteremia caused by eggerthella lenta is rare , if it is present , it is almost always clinically significant and requires prompt diagnosis and treatment . we believe that patients , who have had repair of an aaa and present with abdominal pain with or without fever , should have their blood appropriately cultured for anaerobic bacteria . it should be noted that patients who undergo this procedure are recommended to have yearly ct scans performedto rule out infections or fistulas that can lead to dangerous bacteremia .

the presence of an aortoenteric fistula following aortobifemoral graft repair of an abdominal aortic aneurysm is associated with a high probability of infection leading to clinically significant bacteremia . we report a case of an aortoenteric fistula that developed two years after initial aortic grafting resulting in colonization with the anaerobe , eggerthella lenta . this dangerous bacterium is difficult to culture , associated with high mortality and the patient may have mild symptoms on presentation .

INTRODUCTION CASE REPORT DISCUSSION Financial support and sponsorship Conflicts of interest

dyslipidemia is a significant morbidity associated with diabetes and cardiovascular disorders . the present study was undertaken to assess the lipid profile of type 2 diabetic and age - gender matched healthy subjects and its association , if any , with fasting plasma glucose . clinically diagnosed diabetic subjects were recruited for the study . the fasting plasma glucose and lipid profiles were analyzed for 99 diabetic and 101 healthy volunteers . the blood samples were analyzed for fasting plasma glucose , total cholesterol , triglycerides , high density lipoprotein - cholesterol , low density lipoprotein - cholesterol and very low density lipoprotein - cholesterol . correlation analysis of lipid profile with fasting plasma glucose and calculation of risk ratio was done . the levels of high density lipoprotein - cholesterol and low density lipoprotein - cholesterol were found to be significantly low in diabetics and subjects with lower low density lipoprotein - cholesterol were on statins . inspite of lower lipid values , the risk ratio for diabetics was significantly higher . the correlation analysis indicated significant difference in relationship between fasting plasma glucose , lipid parameters and risk ratios in the two groups . diabetics with lower high density lipoprotein - cholesterol and higher total cholesterol present with a higher risk ratio pointing to need of non - statin high density lipoprotein - raising medications decreasing their predisposition to cardiovascular disorders . the study highlights the altered pattern of correlation of lipid profile with fasting plasma glucose in diabetics and their increased risk of cardiovascular disorders . the dyslipidemia in the form of triglyceridemia and significantly low high density lipoprotein - cholesterol in diabetics point towards the need of non - statin high density lipoprotein - raising medications .

Financial support and sponsorship: Conflicts of interest:

from the time of the earliest descriptions of growing pains ( gp ) to the most recent reviews,13 the nature of the prevalent disorder of childhood , gp ( benign nocturnal limb pains of childhood ) , has remained enigmatic . early etiological theories of gp addressed growth,4 relative hyperactivity and fatigue,58 anatomical / biomechanical factors,9,10 and psychogenic factors.4,11 however , none of these factors have been shown to be more than potential contributors . the current study was designed to shed light on the question put forward by naish and apley , what is this malady called gp?4 evidence that gp may be arising from somatic tissues , particularly musculoskeletal structures , in the limbs has not been convincing.1,3 the possibility that gp is mediated by peripheral nerve dysfunction has not been definitively tested . evidence of disordered somatosensory processing in the form of widespread deep pressure allodynia ( pain due to a stimulus which does not normally provoke pain)/hyperalgesia ( an increased response to a stimulus which is normally painful ) has been demonstrated by pain threshold testing by hashkes et al.12 this is the only published study of somatosensory testing in gp and did not include responses to cutaneous stimuli which are less sensitively elicited in clinical conditions characterized by a widespread deep hyperalgesic state . the hypothesis of this paper is based on haschkes et al s evidence for deep hyperalgesia , with the aim of confirming this hypothesis and extending the evidence to cutaneous somatosensory responses . arising from this background , the hypothesis that gp is characterized by widespread disorder of somatosensory processing was tested . alternatively , it is a peripheral neuropathic pain syndrome ( pain initiated or caused by a primary lesion or dysfunction in the ( peripheral ) nervous system).43 thirty - three children ( 14 males , 19 females ) aged 512 years were recruited by advertisement ( newspaper , radio , school newsletter , hospital notice board ) , conscious that the peak age of point prevalence for gp is 46 years.8 however , symptoms may develop later and continue into adolescence,35 and somatosensory test responses are less reliable under 5 years.23 the written advertisements were headlined : volunteers , children aged 512 years , are required for an ethically approved study into the nature of nocturnal limb pain syndrome ( gp ) . the content included : this study will be conducted by personnel from the sydney children s hospital ( pain medicine unit ) and the university of new south wales . if your child has aches and pains in the limbs , especially legs and particularly at night , participation in this study would be greatly appreciated . we shall also be asking for a volunteer , such as a friend , of similar age and same sex as your child to come along and participate in what will be an interesting study . those who responded were mailed a package containing further information about the study including the planned sensory testing , consent forms , and a gp screening questionnaire,13 based on petersen s gp criteria ( table 1).14 with there being no gold standard for the diagnosis of gp , the petersen criteria have been widely applied in published studies to a point of reasonable consensus.15 a case selection flowchart is presented as figure 1 . each participant ( cases and controls ) was also reviewed by a pediatrician ( ay ) to consider differential diagnoses of lower limb pain . the control group comprised 29 children ( 12 males , 17 females ) who did not have limb pain . in view of the difficulties in recruiting volunteers to undergo somatosensory testing , 19 controls were siblings of cases without gp , eight of whom were unaffected twins ( three monozygotic ; five dizygotic ) . the high number of twins was the result of advertisements that , for purposes external to the current study , indicated a particular interest in twins . a set of clinical somatosensory test procedures was developed based on the standard neurological clinical examination and selected somatosensory tests applicable to children in an office practice to obtain evidence about peripheral neuropathic disorders and/or abnormal central sensory processing . these methods were derived from published protocols.1620 the selection of multimodal - stimulus response measures has been further supported recently by neziri et al,21 who showed that responses to different modalities represent different specific dimensions and should be assessed in combination . the current study however did not use any noxious stimuli ( pain provocation in children with a normally functioning nervous system ) on request from the south eastern sydney area health service ethics committee , who approved the final protocol . the procedures were demonstrated by the researcher ( sp ) , who was blinded as to case or control status . the process was organized by the participating pediatrician so that the blinding was strictly applied . taking into consideration the avoidance of quantitative sensory testing apparatus requiring a laboratory base , the related portability and time availability for office / bedside application , and the avoidance of pain thresholds for heat , cold , and mechanical stimuli , the following test stimuli ( and responses ) were selected and applied to specified sites . additionally , the cases and controls were assessed for abnormal responses in peripheral nerve or dermatomal distribution . the procedures are summarized in table 2.2225 with focus on the distribution of quantitative or qualitative abnormalities , static light touch was assessed by nonstroking touch of obliquely applied soft brush fibers to lower legs , forearms , and abdomen . this was the first assessment of a - beta peripheral sensory channel , central lemniscal pathway , and a large array of cortical regions . calibrated von frey monof ilament stimuli ( vf1 optihair-2 [ set of 12 optical glass filaments with force range 0.25512 mn ] ; marstock , marburg , germany ) enabled fixed graded stimuli to assess static light touch . applied to lower legs , forearms , and abdomen , it provided mechanical detection threshold for touch , pain threshold in states of cutaneous hyperalgesia . for touch threshold , the method of limits was used with the average of threshold estimations from two tests commencing with a subthreshold force ( 0.25 mn ) and two tests commencing with a suprathreshold force ( 64 mn ) . mean stimulus - response determinations from the same sites to von frey filament number 9 were determined using the color analog scale ( 010 ) with upper anchor very strong touch . repetitive dynamic brush stimuli were applied to the limbs and abdomen ( as control site ) to determine the presence of cutaneous hyperesthesia or dynamic mechanical allodynia . the dynamic repetitive stimuli test for temporal summation effects which correlate better with neuropathic pain states than single static stimuli . a standardized camel hair brush ( sense lab brush-05 ; somedic , horby , sweden ) exerting a force of approximately 200400 mn was applied with a single stroke of approximately 2 cm in length over the skin in runs of ten per test site . rydel - seiffer graduated tuning fork ( 64 hz , 8/8 scale ; us neurologicals , poulsbo , wa ) was applied to bony prominences , tibial tuberosities , olecranons , and skin over soft tissues ( lower legs , forearms , and abdomen ) . vibration threshold was determined as a disappearance threshold ( method of limits ) . a mean stimulus - response determination for the maximal vibration intensity was determined on a 010 scale using an adapted color analog scale with upper anchor very strong buzzing . the assessment of somatosensory responses to cool stimuli was determined by a metal thermoroll ( marstock ) . cool/ cold stimuli activate a - beta and a - delta peripheral sensory channels and spinothalamic central pathways . the upper anchor of the color analog scale ( 010 ) was ice cold . responses to blunt deep pressure stimuli were assessed by application of the fischer pressure algometer ( fdk 10 ; pdt inc , ny ) to consistent sites over deep somatic tissues ( middle lower legs posterolaterally , middle forearms posterolaterally , abdomen , tibial tuberosities , and olecranons ) . this device has been used in children.12,26 blunt deep ( muscle ) pressure , as it increases , activates intramuscular afferents type iii and iv and the spinothalamic central pathway . responses to deep force reaching 2 kg / cm over 3 seconds , a level comfortably below the mean pain threshold in healthy children , were obtained using the color analog scale ( 010 ) with adapted anchors , the upper being very strong push . the color analog scale27 was applied with anchors modified in accordance with the type of stimulus . the color analog scale has been shown to be reliable and valid in pain contexts in children as young as 5 years.2830 generally , visual analog scales have also been used in diverse nonpain applications . children were assessed for abnormal mechanosensitivity of peripheral nerves with a stretch by straight leg raising31 and brachial plexus provocation tests.32 student s t - tests for independent samples were performed to compare cases and controls for all sensory testing measures . for each sensory modality , each tested site was analyzed as well as the mean across all tested sites . with focus on the distribution of quantitative or qualitative abnormalities , static light touch was assessed by nonstroking touch of obliquely applied soft brush fibers to lower legs , forearms , and abdomen . this was the first assessment of a - beta peripheral sensory channel , central lemniscal pathway , and a large array of cortical regions . calibrated von frey monof ilament stimuli ( vf1 optihair-2 [ set of 12 optical glass filaments with force range 0.25512 mn ] ; marstock , marburg , germany ) enabled fixed graded stimuli to assess static light touch . applied to lower legs , forearms , and abdomen , it provided mechanical detection threshold for touch , pain threshold in states of cutaneous hyperalgesia . for touch threshold , the method of limits was used with the average of threshold estimations from two tests commencing with a subthreshold force ( 0.25 mn ) and two tests commencing with a suprathreshold force ( 64 mn ) . mean stimulus - response determinations from the same sites to von frey filament number 9 were determined using the color analog scale ( 010 ) with upper anchor very strong touch . repetitive dynamic brush stimuli were applied to the limbs and abdomen ( as control site ) to determine the presence of cutaneous hyperesthesia or dynamic mechanical allodynia . the dynamic repetitive stimuli test for temporal summation effects which correlate better with neuropathic pain states than single static stimuli . a standardized camel hair brush ( sense lab brush-05 ; somedic , horby , sweden ) exerting a force of approximately 200400 mn was applied with a single stroke of approximately 2 cm in length over the skin in runs of ten per test site . rydel - seiffer graduated tuning fork ( 64 hz , 8/8 scale ; us neurologicals , poulsbo , wa ) was applied to bony prominences , tibial tuberosities , olecranons , and skin over soft tissues ( lower legs , forearms , and abdomen ) . vibration threshold was determined as a disappearance threshold ( method of limits ) . a mean stimulus - response determination for the maximal vibration intensity was determined on a 010 scale using an adapted color analog scale with upper anchor very strong buzzing . the assessment of somatosensory responses to cool stimuli was determined by a metal thermoroll ( marstock ) . cool/ cold stimuli activate a - beta and a - delta peripheral sensory channels and spinothalamic central pathways . the upper anchor of the color analog scale ( 010 ) was ice cold . responses to blunt deep pressure stimuli were assessed by application of the fischer pressure algometer ( fdk 10 ; pdt inc , ny ) to consistent sites over deep somatic tissues ( middle lower legs posterolaterally , middle forearms posterolaterally , abdomen , tibial tuberosities , and olecranons ) . this device has been used in children.12,26 blunt deep ( muscle ) pressure , as it increases , activates intramuscular afferents type iii and iv and the spinothalamic central pathway . responses to deep force reaching 2 kg / cm over 3 seconds , a level comfortably below the mean pain threshold in healthy children , were obtained using the color analog scale ( 010 ) with adapted anchors , the upper being very strong push . the color analog scale27 was applied with anchors modified in accordance with the type of stimulus . the color analog scale has been shown to be reliable and valid in pain contexts in children as young as 5 years.2830 generally , visual analog scales have also been used in diverse nonpain applications . children were assessed for abnormal mechanosensitivity of peripheral nerves with a stretch by straight leg raising31 and brachial plexus provocation tests.32 student s t - tests for independent samples were performed to compare cases and controls for all sensory testing measures . for each sensory modality , each tested site was analyzed as well as the mean across all tested sites . there were no significant demographic or anthropometric differences between cases and controls ( table 3 ) . in the gp case sample ( n = 33 ) , pain was present in the knee region and lower leg in all cases , additionally in the upper leg in eight cases ( 24% ) , and in the arms in three cases ( 9% ) . pain was described as aching in 30 cases ( 94% ) , deep in 13 ( 39% ) , and there were accompanying pins and needles in nine ( 27% ) . episodes occurred daily in six cases ( 19% ) , weekly in nine ( 28% ) , monthly in eleven ( 34% ) , and less frequently in six ( 19% ) . a total of 24 parents ( 72% ) of children with gp reported that their child experienced pain of sufficient severity to wake the child and 22 ( 66% ) reported that the pain induced crying . there was no clinical evidence of a peripheral neuropathic pain syndrome with there being no indication of sensory impairment in peripheral nerve or dermatome distribution , no neurological motor deficits , and no abnormal mechanosensitivity of peripheral nerves according to sciatic and brachial plexus stretch maneuvers . there were no significant differences in somatosensory test responses between cases and controls in the lower leg sites ( except for pressure response magnitude at the lateral calf [ p = 0.04 ] ) , nor in the upper limbs or abdominal site . response magnitudes to cold , vibration , and deep pressure stimulation were significantly greater in cases than controls while for static punctate tactile magnitude the apparent greater sensitivity in the cases did not reach statistical significance . there were no significant differences between cases and controls in touch threshold and vibration threshold . there was a nonsignificant trend to prolonged brush - induced after - sensations in cases . the study was designed to investigate the nature of the pain , specifically to determine whether there was regional ( lower limb ) or widespread disorder of somatosensory test responses , in children with gp . the diagnosis of gp is based on history , examination , and the exclusion of identifiable musculoskeletal / orthopedic , neurological , or other causes of limb ( especially leg ) pains . the features are characteristic of , and are reflected in , the criteria derived from petersen.14 criteria have been determined by gradually acquired consensus but with there being no gold standard for diagnosis , specificity has not been adequately validated . because patients were accrued by advertisement , the extent to which their features would be similar to a random sample can not be determined and thus extrapolation to the population of children with gp requires caution . consistent with previous studies and reviews,13 and consistent with the criterion - based , widely - applied gp definition,14 there was no evidence for a musculoskeletal / somatic tissue disorder . further , the essentially negative responses to the neuropathic pain questionnaire , the absence of sensory impairment in peripheral nerve or dermatomal distribution , and the lack of abnormal mechanosensitivity to brachial plexus and lumbosacral nerve root ( sciatic ) provocation provided no support for the interpretation of a peripheral neuropathic pain syndrome.33,34 the investigator - blind somatosensory testing covered a range of stimulus modalities . although the lower leg cutaneous testing showed consistent trends to more sensitive responses in cases than controls , these differences did not reach statistical significance . however , the greater response to deep pressure in the soft tissues of the lower legs in children with gp compared with responses in control children was statistically significant , consistent with previous evidence of deep pressure allodynia / hyperalgesia in children with gp.12,35 the overall greater sensitivity of responses averaged across all sites ( statistically significant for response magnitude to cutaneous cold , vibration , and deep pressure ) suggests a mild widespread disorder of somatosensory processing . the reference values for children in the relevant age range for face , hand , and foot sites , applying the german research network on neuropathic pain protocol,20 were reported in detail by blankenburg et al.23 the present somatosensory test protocol was in accord with procedures in the blankenburg et al study23 and in studies which the authors cited , however , it was simpler , avoided pain threshold , and added response magnitude tests . these investigators found that the younger children ( 68 years ) were generally less sensitive to thermal and mechanical detection stimuli but more sensitive to pain stimuli than older children ( 912 years ) , who in turn were similar to adolescents ( 1317 years ) . these punctuate pressure ( blunt , static ) , vibration intensity , and deep ( blunt , static ) stimulus procedures were used in general by those investigators , but not in the stimulus response manner the present study applied to avoid pain threshold testing . based on published evidence and results from this present study , gp does not appear to be a somatic / nociceptive ( ie , musculoskeletal pain ) syndrome , nor is it a peripheral neuropathic pain syndrome . the widespread distribution of sensory disturbances within this sample suggests the likelihood that underlying spontaneous pain is central sensitization of nociception36 and/or disordered descending pain modulation in the central nervous system.37 the authors suggest that , on the basis of this and the authors previous studies , and other published studies , gp can be provisionally allocated to the group of idiopathic pain disorders as defined by diatchenko et al38 and to the functional pain syndromes as defined by mayer and bushnell.39 these disorders are characterized by an absence of definable somatic disease , by comorbid associations with other idiopathic pain syndromes , by indicators of disordered central processing of nociception , genetic influence , often neuroendocrine dysfunction , and by psychosocial associations . as a group they are prevalent , eg , 1-year prevalence of any syndrome in 57 year old children of 23.2%.40 given the modest sample size , the multiple statistical comparisons should be acknowledged as a point of caution regarding extrapolation . the diagnosis of gp was based on consensus criteria and the exclusion of other conditions . the controls included siblings , of whom a disproportionate number were twins ; however , the reasonable expectation is that such selection would tend to reduce contrast in somatosensory responses in cases and controls . the somatosensory test methods used in this study are relatively novel and have only recently been applied to children . the somatosensory test procedures were conservative in that they involved the quantification of responses to subpain threshold stimuli whereas quantitative sensory testing has been used most extensively in the assessment of pain thresholds or the quantification of responses to suprathreshold stimuli.41 suprathreshold measures ( tolerance ) are believed to be more clinically relevant than threshold measures.42 temporal summation , a dynamic psychophysical measure , is thought to better capture the pain modulatory ability of the central nervous system as compared to state measures , such as threshold and tolerance that only measure a single point in the pain processing continuum.43 thus , for most of the measures , the contrast in responses between cases and controls were relatively minor although the direction consistently favored greater sensitivity of cases for all modalities . a greater contrast between cases and controls would be expected from deep repetitive pressure stimuli testing for temporal summation , radiation , and persistent after - sensations . the current study has shown that the prevalent pain syndrome of childhood , gp , is characterized by widespread mildly disordered ( relatively sensitive ) cutaneous and deep somatosensory responses to several stimulus modalities . the perspective of the authors is that although gp has been considered an end - organ

purposeto further the understanding of growing pains ( gp ) , in particular , the nature of this pain disorder.methodsthis study included 33 children aged 512 years who met criteria for gp ( cases ) and 29 children without gp of similar age and sex ( controls ) . nineteen controls were siblings of cases . gp was diagnosed by standard consensus questionnaires . a questionnaire addressed characteristics of the pain and family history of gp . evidence for peripheral neuropathic disorder was tested by somatosensory testing and provocation tests of peripheral nerves . somatosensory testing by a blinded researcher involved threshold determination and/or response magnitude to nonpainful stimuli including touch , dynamic brush , cold , vibration , and deep pressure applied to limb and abdominal sites.resultsdistributional , temporal , and quality characteristics of the pain were in accordance with published descriptions . there was no indication of primary musculoskeletal disorder . no evidence was found that gp is a peripheral neuropathic pain syndrome . there were minor but statistically significantly increased responses to cutaneous cold , vibration , and to deep pressure stimuli in cases compared to controls , evident in a wider distribution than the symptomatic lower limbs.conclusiongp is a regional pain syndrome with evidence in this study of mild widespread disorder of somatosensory processing .

Introduction Materials and methods Test stimuli Results Discussion Conclusion

in this paper , we report on a general synthetic strategy for the assembly of glycopolymers that capitalizes on the intrinsic reactivity of reducing glycans toward hydrazides to form stable cyclic n - glycosides . we developed a poly(acryloyl hydrazide ) ( pah ) scaffold to which we conjugated a variety of reducing glycans ranging in structure from simple mono- and disaccharides to considerably more complex human milk and blood oligosaccharides . the conjugation proceeds under mild conditions with excellent ligation efficiencies and in a stereoselective manner , providing glycopolymers with pendant glycans accommodated mostly in their cyclic -glycosidic form . utilizing a biotin - terminated pah scaffold prepared via raft polymerization , we quickly assembled a panel of glycopolymers that we microarrayed on streptavidin - coated glass . we then demonstrated that in these microarrays , the glycopolymer ligands bind lectins according to the structures of their pendant glycans . importantly , glycopolymers containing biologically relevant branched oligosaccharides , such as sialyl lewisx , as well as sulfated glycosaminoglycan - like epitopes can be readily prepared using our methodology .

Supplementary Material

computer - aided diagnosis ( cad ) has been the subject of a lot of research as a tool to help health professionals in medical decision making . as a result , many cad systems integrate image processing , computer vision , and intelligent and statistical machine learning methods to aid radiologists in the interpretation of medical images and ultimately help improve diagnostic accuracy . these systems have been employed to analyze and classify various types of digitized biomedical images , including retina [ 1 , 2 ] , mammograms [ 35 ] , brain magnetic resonance images [ 68 ] , skin cancer images [ 9 , 10 ] , lung images [ 11 , 12 ] , and ulcer detection in endoscopy images [ 13 , 14 ] , just to name a few . the typical cad process starts with a segmentation stage to identify one or more regions of interest ( roi ) in the image of interest . then , the roi(s ) is processed for image enhancement and/or feature extraction before classification . because the segmentation step requires prior knowledge of discriminant image features and its implementation these approaches realize feature space reduction by applying one or more transforms to the whole image and extracting the feature vector to classify from one or more of the obtained components [ 3 , 5 , 714 ] . texture analysis methods can be categorized as statistical , geometrical , and signal processing types . statistical methods are mainly based on the spatial distribution of pixel gray values , while geometrical approaches depend on the geometric properties of texture primitives . as for signal processing methods , they use texture filtering in the spatial or frequency domain to extract relevant features . multiresolution analysis is the most widely employed signal processing technique for characterizing biomedical images due to its capability to obtain high time - frequency resolutions . the basic wavelet transform [ 15 , 16 ] starts with a basis function , the mother wavelet , and decomposes a signal into components of different time and frequency scales ; longer time intervals are used to obtain low - frequency information and shorter intervals are used to obtain high - frequency information . the most commonly used wavelet transform in biomedical image processing is the discrete wavelet transform ( dwt ) whose discrete time shifting and stretching variables lead to a sparse and efficient representation . the dwt takes an input image and decomposes into four subimage components that characterize it for different orientations in the horizontal and vertical frequency axes . more precisely , the dwt decomposition yields the approximation subband ( ll ) , the horizontal detail subband ( lh ) , the vertical detail subband ( hl ) , and the diagonal detail subband ( hh ) . these describe , respectively , the low - frequency components in the horizontal and vertical directions , the low - frequency components in the horizontal direction and high - frequency components in the vertical direction , the high - frequency components in the horizontal direction and low - frequency components in the vertical direction , and the high - frequency components in both directions . thus , in essence , the standard dwt algorithm yields horizontal , vertical , and diagonal directional information about the frequency spectrum of an image . however , these three directions may not be sufficient to express all the directional information in digital images , particularly biomedical images [ 4 , 14 ] . in an attempt to express the directional features more efficiently , these include the gabor wavelets , the dual - tree complex wavelet transform ( dt - cwt ) , the ridgelet , the curvelet , and the contourlet . there exist also reports on biomedical applications of gabor filter banks , dt - cwt , ridgelets , curvelets , and contourlets . the two - dimensional ( 2d ) gabor filter decomposes an image into components corresponding to different scales and orientations . as a result , it captures visual properties such as spatial localization , orientation selectivity , and spatial frequency . the 2d gabor filter has real and imaginary parts and is highly flexible in its representation as its parameters can be adapted to the structure of the patterns that one wants to analyze in the image . it is however difficult to find the optimal set of parameters to characterize a given image . in comparison , the dt - cwt transform provides directional selectivity , shift invariant features , and complex images . the ridgelet transform is appropriate to capture radial directional details in the frequency domain ; in particular it is optimal for representing straight - line singularities . however , those structures are not dominant in medical images and are rarely observed in real world images . this limits the suitability of the ridgelet transform to characterize the texture of real images . the curvelet transform is an extension of the ridgelet transform for detecting image edges and singularities along curves while analyzing images at multiple scales , locations , and desired orientations . unfortunately , the curvelet transform is highly redundant and only few choices of mother functions are available for the curvelets as opposed to the many choices available for the standard wavelet transform . finally , the contourlet transform can capture directional details and smooth contours in a given image . in particular , it is suitable in the analysis of images containing textures and oscillatory patterns . its main drawback is the high degree of information redundancy and occurrence of artefacts [ 30 , 31 ] . in past works , we proposed several transform - based approaches to account for directional features in classifying biomedical images . for instance , in the case of brain magnetic resonance images , we proposed a simple methodology in [ 32 , 33 ] where features are extracted from the lh and hl components of the dwt instead of the more common ll , or image approximation , component . we found that the lh and hl coefficients are efficient at characterizing changes in the biological tissue and help distinguish normal and abnormal image textures . for mammograms , we investigated in a hybrid processing system that sequentially uses the discrete cosine transform ( dct ) to obtain the high - frequency component of the mammogram and then applies the radon transform ( rt ) to the result in order to extract its directional features . the validation results showed that the rt helps improve the recognition rate of the detection system . in subsequent work the approach targeted the hh component of the dwt decomposition and improved classification accuracy when compared to using the dwt or rt alone or the dct - rt used in . in addition , the dwt - rt detection system was more efficient for classifying normal and abnormal images than the dct - rt , possibly because of the multiresolution capability of the dwt and the fact that it leads to a sparser signal representation than the dct . still , the rt can not capture spatial frequency , a potential feature to improve further the classification accuracy . in this paper , we describe a hybrid biomedical image processing and classification system that uses both the dwt and gabor filter as directional transforms and statistical features derived from them for the classification task which is accomplished by support vector machines ( svms ) . as stated before , however , except for the lh and hl subbands whose coefficients depend on image row and column information , respectively ( an effect of the subband coding used by the algorithm ) , the standard dwt is essentially an image compression tool and it can not perform directional analysis at arbitrary directions . on the other hand , the gabor filter can process images in terms of preferred orientations at arbitrary spatial frequencies thus , combining dwt and gabor filter banks in sequence may lead to improved feature extraction from biomedical images and better classification of normal versus abnormal images in comparison to using dwt or gabor filter banks alone . in this hybrid processing scheme , the dwt acts both as high - frequency filter to extract abrupt changes in image texture and image compression engine to reduce image dimensionality and a gabor filter bank extracts the directional information . in a preliminary work , the previously mentioned dwt - gabor hybrid system was successfully applied to mammograms to extract features that allow discriminating normal and cancer images . more specifically , the goal was to detect the presence of malign microcalcifications ( specs of calcium in the breast tissue that appear in the mammogram as small bright spots that are scattered or grouped in clusters ) , whose early detection is important for cancer screening [ 38 , 39 ] . the results showed the superiority of the approach over simply using the dwt alone . in the present work , we widen our study to retina digital images and brain magnetic resonance images to investigate the effectiveness of the dwt - gabor approach across application domains with similar image features . indeed , the images of some pathologies related to brain , retina , and breast present similar contrast features characterized by abrupt changes in image texture with directional properties ( see examples in figures 1 , 2 , and 3 ) . for instance , breast cancer is characterized by dense concentration of contrast cells in the biological tissue , cancer in brain magnetic resonance images is often characterized by large cells with high contrast , and many forms of retinopathy are characterized by the presence of spots on the retina or covering the macula . as a result , the dwt - gabor hybrid system we have used in our previous work to detect cancer in mammograms could potentially also be applied to brain magnetic resonance images and retina digital images with similar properties . circinate retinopathy is a retinal degeneration characterized by a circle of white spots encircling the macula that causes complete foveal blindness ; retinal microaneurysms are due to a swelling of the capillaries caused by a weakening of the vessel wall and are considered to be the earliest sign of diabetic retinopathy , among others . magnetic resonance imaging ( mri ) is a noninvasive imaging modality largely used for brain imaging to detect diseases such as alzheimer 's and multiple sclerosis [ 6 , 8 ] . alzheimer 's disease is the most frequent cause of age - related dementia and multiple sclerosis is a progressive neurological disorder that can result in various dysfunctions . additional brain pathologies that can be detected from mr images and that are investigated in this work include glioma , herpes encephalitis , and metastatic bronchogenic carcinoma ( figure 1 ) . all of these are characterized by large cells with high contrast , hence the interest in being able to detect them with the same algorithm . first , we propose a relatively simple and fast approach to biomedical image characterization that relies on the directional properties of high - frequency components . the dwt is applied first to extract high - frequency components that characterize abrupt changes in the biological tissue and , then , the gabor filter is applied to the obtained hh subimage to extract directional features . second , the statistical features extracted from the hybrid dwt - gabor transform are processed by an svm for classification . this statistical binary classifier has proven its efficiency [ 46 , 3235 , 37 ] and ease of tuning in comparison to alternatives such as artificial neural networks . third , contrary to alternatives that focus on rois or specific image details , the proposed methodology is of more general reach as three different types of images used for validation show . section 2 reviews previous works related to the automatic classification of normal versus abnormal images in the context of brain magnetic ( mr ) resonance imaging , mammograms , and retina digital images . section 3 describes our proposed approach for directional features extraction from biomedical images using discrete wavelet transform followed by gabor filter banks and support vector machines classifier . mammograms , retina , and mr images are the subject of many research efforts on feature extraction and subsequent classification . next is a summary of some recent works related to dwts and/or gabor filters . in the problem of automatic classification of mammograms , the authors in used gabor filter banks to process images and k nearest neighbour ( k - nn ) algorithm as classifier . the obtained classification rate was 80% . in the dual - tree complex wavelet transform ( dt - cwt ) and support vector machine ( svm ) were employed to classify benign and malignant images . the authors in employed the contourlet transform and successive enhancement learning ( sel ) weighted svm to obtain 96.6% correct classification rate . the previous studies all used images of size 1024 1024 pixels . in the problem of retina digital image classification , the authors in employed the belkyns 's shift - invariant dwt to classify normal against abnormal retina images of size 700 605 pixels . the pathologies of the abnormal images included exudates , large drusen , fine drusen , choroidal neovascularization , central vein and artery occlusion , histoplasmosis , arteriosclerotic retinopathy , hemicentral retinal vein occlusion and more . in order to capture texture directional features , they employed normalized gray level cooccurrence matrices ( glcms ) . the authors in employed the probabilistic boosting algorithm and morphological scale space analysis and glcm to extract texture features . the detection accuracy of normal images varied between 81.3% and 92.2% , and that of abnormal images varied between 71.7% and 85.2% depending on texture complexity ( grade of pathology ) . the authors in used four approaches to extract features from retina digital images of size 300 300 pixels to automatically classify glaucoma images . the first set of features is obtained by taking the pixel intensities as input to principal component analysis . the third set of features is computed from the coefficients of the fast fourier transform and the fourth set of features is obtained from the histogram of the intensity distribution of the image . the performance of the classifications using one feature set only was 73% with the histogram features , 76% with the fast fourier transform coefficients , 80% with the gabor textures , and 83% with the pixel intensities . finally , in the problem of brain mri classification , the authors in used the wavelet coefficients as input to a support vector machine to classify normal and abnormal alzheimer 's disease images of size 256 256 pixels . more recently , the authors in used voxels to represent each brain mri of size 512 512 pixels . using cross - validated tests , the obtained correct classification rates of normal and alzheimer images were 90% , 92% , and 78% , respectively , when using classification by svm , nave bayes classifier , and voting feature intervals ( vfis ) . still more recently , the authors in employed the dwt to extract features from brain magnetic resonance images of size 256 256 pixels , and then principal component analysis was used to reduce the dimensions of the features space . the abnormal images included glioma , meningioma , alzheimer 's , alzheimer 's plus visual agnosia , pick 's disease , sarcoma , and huntington 's disease . the classification accuracies using backpropagation neural network ( bpnn ) were 100% using learning and testing sets of 33 images each . in this work , we are interested in how a dwt - gabor - based approach for feature extraction may provide better classification results than those reported in the previous works , particularly those based on the dwt alone . first , the dwt is applied to the biomedical image to obtain its high - frequency image component since it often contains most of the desired information about the biological tissue . indeed , sudden changes in the texture of the image are typical indicators of the presence of abnormal biological tissue . second , a bank of gabor filters with different scales and orientations is applied to the high - frequency image to obtain gabor - filtered images along different spatial orientations . finally , the svm is used to classify the resulting feature vector for final diagnosis . the block diagram of the dwt - gabor system is shown in figure 4 . the two - dimensional discrete wavelet transform ( 2d - dwt ) [ 1416 ] performs a subband coding of an image in terms of spectral spatial / frequency components , using an iterative and recursive process . figure 6 illustrates the case of two - level decomposition . the image is first represented by lh , hl , and hh subbands that encode the image details in three directions and an ll subband which provides an approximation of it . the obtained detail or approximation images can be decomposed again to obtain second - level detail and approximation images , and the process can be repeated for finer analysis as each iteration doubles the image scale . the computation of the 2d - dwt proceeds from that of the 1d - dwt , the discrete version of the one - dimensional continuous wavelet transform . the one - dimensional continuous wavelet transform of a signal x(t ) is defined by [ 7 , 8 ] ( 1)w(a , b)=+x(t)a , b(t)dt,(2)a , b(t)=1|a|(tba ) , where a , b( ) stands for a given wavelet function and a and b are the scale and translation parameters , respectively . the 1d - dwt is obtained by sampling a and b so that ( 1 ) becomes that of a sequence . in dyadic sampling , a and b are , respectively , a power of 2 and multiples thereof , and the sequence elements ( wavelet coefficients ) are given by(3a)cjk = w(2j,2jk),where j represents the discrete scale factor and k the discrete translation factor . in other words , a and b in ( 1 ) are replaced by 2 and 2k , respectively . the one - dimensional wavelet decomposition is extended to an image by applying it to the row variable first and then to the column variable of the obtained result . at each step , two subimages are created with half the number of pixels of the row or column that was processed . in the end , an m n image is decomposed into 4 subimages , each with m/2 n/2 resolution and preserved scale . however , ( 1 ) has only theoretical merit due to the infinite ranges of a and b. for a practical implementation , the fact that ( 1 ) is essentially a measure of correlation between a signal and various wavelets derived from a mother is exploited , and the dwt decomposition is turned into a filtering operation with a sequence of high - pass and low - pass filters . following the notation in [ 7 , 8 ] , the discrete form of ( 1 ) can then be written as ( 4)caj , k[x(t)]=ds[x(t)gj(t2jk)],cdj , k[x(t)]=ds[x(t)hj(t2jk ) ] , where coefficients caj , k and cdj , k specify approximation and details components provided by the g(n ) low - pass and h(n ) high - pass impulse responses , respectively , and the ds operator performs downsampling by a factor of 2 . the one - dimensional wavelet decomposition is extended to two - dimensional objects by using row and column decompositions as shown in figure 6 . in our work , the most frequently used wavelet ( daubechies-4 ) is considered to extract the hh image component . the two - dimensional ( 2d ) gabor filter decomposes an image into components corresponding to different scales and orientations , thus capturing visual properties such as spatial localization , orientation selectivity , and spatial frequency . the 2d gabor filter consists of a complex exponential centered at a given frequency and modulated by a gaussian envelope . because of the complex exponential , the filter has both real and imaginary parts . the general form of the real part is defined as follows : ( 5)g(x , y,x,y , f,)=exp[12((xx)2+(yy)2)]cos(2fx ) , where ( 6)x=xcos()+ysin(),y=ycos()xsin( ) and where x and y are the standard deviations of the gaussian envelope along the x and y axes . the parameters f and are , respectively , the central frequency and the rotation of the gabor filter . to obtain the gabor - filtered image f(x , y ) of a given image i(x , y ) the 2d convolution operation ( ) is performed : ( 7)f(x , y)=g(x , y,x,y , f,)i(x , y ) . the selection of parameters x , y , f , and plays an important role in the filter 's operation . however , no formal technique exists for choosing them and experience - guided intuition , trial and error , or heuristic search must be used . for retina digital images and brain mr images , x and y were arbitrarily set to unity . in the case of mammograms , x and y consequently , we used x = /2.35 , where is the full width at half - maximum of the gaussian and y = 8x . no values of x and y other than the previous ones were tried since optimality was not the primary concern of this work and we obtained satisfactory results with these values . four orientations , = 0 , /4 , /2 , and 3/4 , were used as in [ 22 , 33 ] . these values seemed reasonable as a first try since they covered both image axis directions and the forward and backward diagonals . finally , the central frequency f was set to 2 , 2.5 , and 3 . given that the gabor filter is modulated by the cosine of f , large values of f lead to a compressed cosine and , consequently , the filter output is more likely to show fast or frequent changes in biological tissue texture . this in turn would help verify our hypothesis that abnormal images are characterized by sudden and frequent variations in image texture . in the end , the application of the gabor filter bank to the hh image component obtained with the 2d - dwt leads to twelve gabor - filtered hh images components , for each choice of f and . statistical measures are employed to extract features from both the dwt hh subband image and the real gabor - filtered hh image components . more precisely , the entropy ( e ) and uniformity ( u ) of the coefficients of each one are computed . entropy and uniformity were selected as features because previous works on mammograms have shown that uniformity is correlated with suspicious malignancy and that entropy can successfully characterize breast biological tissue . in this study , the entropy and uniformity statistics are hypothesized to also characterize retina and brain mr images with similar contrast information ( i.e. , abrupt and/or frequent variations in texture ) . entropy ( e ) and uniformity ( u ) are defined by ( 8)e=p(z)log(p(z)),u=p2(z ) , where z is a random variable that represents a coefficient in the gabor filtered image and p(z ) is its probability of occurrence as estimated by its relative frequency . to investigate the performance of the previous approaches , the image features were extracted from hh at both level - one dwt decomposition ( hh1 ) and level - two dwt decomposition ( hh2 ) , with and without filtering by a gabor filter bank . we also applied the gabor filter directly to the original image without the dwt for comparison purpose . for each dwt hh subband image , the feature vector is given by ( 9)vdwt , a=[ea , ua ] , where a is the level of wavelet analysis ( decomposition ) . similarly , for each of the twelve outputs generated by the gabor filter bank ( 4 angles 3 central frequencies ) , the entropy and uniformity are computed and a twenty - four component feature vector is formed to represent the initial image . we thus have ( 10)vgabor , a=[e1,a , e2,a, ,e12,a , u1,a , u2,a, ,u12,a ] . . introduced by vapnik , the support vector machine ( svm ) classifier is based on statistical learning theory . it implements the principle of structural risk minimization and has excellent generalization ability as a result , even when the data sample is small . it has been used with great success in various applications , including speech emotion recognition , card - sharing traffic detection , fault diagnosis , cardiac decision making , parkinson 's disease diagnosis , and alzheimer 's disease detection . the support vector machine performs classification tasks by constructing an optimal separating hyperplane that maximizes the margin between the two nearest data points belonging to two separate classes . given a training set { ( xi , yi ) , i = 1,2 , , m } , where the input xi r and class labels yi { + 1 , 1 } , the separation hyperplane for a linearly separable binary classification problem is given by ( 11)f(x)=wx+b , where w is a weight vector and b is a bias . the optimal separation hyperplane is found by solving the following optimization problem : ( 12 ) minimizew , b, 12ww+ci=1mi subject to : yi(wxi+b)+i10 , i0 , where c is a penalty parameter that controls the tradeoff between the complexity of the decision function and the number of misclassified training examples and is a positive slack variable . the previous optimization model can be solved by introducing lagrange multipliers and using the karush - kuhn - tucker theorem of optimization to obtain the solution as ( 13)w=i=1miyixi . the xi values corresponding to positive lagrange multipliers i are called support vectors which define the decision boundary . once the optimal solution i is found , the optimal hyperplane parameters w and b are determined . then , the discriminant function of the svm for a linearly separable binary classification problem is ( 14)g(x)=sign(i=1myiixix+b ) . in the nonlinearly separable case , the svm classifier nonlinearly maps the training points to a high - dimensional feature space using a kernel function , where linear separation can be possible . the scalar product (xi ) (xj) is computed by mercer kernel function k as k(xi , xj ) = (xi ) (xj). then , the nonlinear svm classifier has the following form : ( 15)g(x)=sign(i=1myiikx , xi+b ) . in this study , a polynomial kernel of degree 2 was used for the svm . as a global kernel , it allows data points that are far away from each other to also have an influence on the kernel values . the general polynomial kernel is given by ( 16)k(x , xi)=((xix)+1)d , where d is the degree of the polynomial to be used . as mentioned previously , mammograms and retina and brain mr images corresponding to given pathologies are considered in this work , and the aim is to classify normal versus abnormal images for each image category . to do so , one hundred digital mammograms ( 171 364 pixels ) consisting of fifty normal images and fifty cancer images were taken from the digital database for screening mammography ( ddsm ) . for retina , a set of 69 color images ( 150 130 pixels ) from the stare database were employed including 23 normal images , 24 with microaneurysms , and 22 with circinate . finally , a collection of 56 axial , t2-weighted , and mr brain images ( 256 256 pixels ) were taken from the aanlib database of the harvard medical school . they consisted of 7 normal images , 9 with alzheimer 's disease , 13 with glioma , 8 with herpes encephalitis , 8 with metastatic bronchogenic carcinoma , and 14 with multiple sclerosis . it is unfortunate that the number of images was not constant across pathologies , but we had no control over this and used what was available , with tenfold cross - validation or leave - one - out cross - validation of the results depending on sample size . many kinds of biomedical images could be considered for our experiments ; we focused on mammograms , retina , and brain magnetic resonance images mainly because of public availability . an example of the processing of a normal retina and a retina with circinate is illustrated in figures 7 and 8 , respectively . for each image type , the average and standard deviation of the correct classification rate ( ccr ) , sensitivity , and specificity were computed to evaluate the performance feature extraction technique when used in conjunction with the svm classifier . the three performance measures are defined by ( 17)ccr = classified samplestotal number of samples , sensitivity = correctly classified positive samplestrue positive samples , specificity = correctly classified negative samplestrue negative samples , where positive samples and negative samples are , respectively , abnormal and normal images . finally , all experiments were performed with tenfold cross - validation , except those for mr images which used leave - one - out cross - validation due to the small sample size of each brain image category . table 1 shows the obtained average results for the three types of images that were investigated . the performance of the svm classifier improved for all types of images and all levels of hh decomposition by the dwt . at level one , the average correct classification rate increased by , respectively , 0.69 , 25.31 , and 9.56 percentage points for mammograms , retina , and brain magnetic resonance images when using the dwt - gabor approach . at level - two decomposition , tables 2 and 3 provide the average results for classifier sensitivity and specificity . at level - one dwt decomposition , the dwt - gabor approach improved classification specificity for mammograms and retina images to make it reach 100% , while it improved it by 78.03 percentage points for brain mr images . at level - two dwt decomposition , the improvement was 3.24 percentage points for mammograms , 97.68 percentage points for retina images ( 100% specificity ) , and 97.24 percentage points for mr images . regarding sensitivity , the results were mixed . at level - one dwt decomposition , the values were about the same for the dwt - gabor and dwt - only approaches for mammograms and retina images , with , respectively , 0.81 and 0 percentage points differences , but there was a degradation of 11.33 and 46.16 percentage points for brain mr images at level one and level two of decomposition , respectively . following the same cross - validation protocol , we also conducted classification experiments with features extracted from a gabor filtered image of the original biomedical image . the purpose was to check whether gabor - based features alone help characterize images better than dwt or dwt - gabor - based features . the obtained correct classification rate of mammograms , retina , and brain magnetic resonance images is , respectively , 68.03% ( 0.01 ) , 50.00% ( 0.00 ) , and 86.61% ( 0.03 ) . the average results for classifier specificity and sensitivity for all images are 100% and 0% . this finding indicates that gabor - based features are suitable to detect pathological images , but fails to detect normal images . in sum , the results show that gabor - based features do not perform better than dwt and dwt - gabor - based features . these findings confirm the superiority of combining the dwt and gabor filter banks for feature extraction . based on the previous results , it appears that the dwt - gabor approach for feature extraction is effective for detecting the abrupt changes in biological tissue that characterize the pathological patterns that were investigated and it yields better classification accuracy and specificity than the dwt - only approach . it also offers equal of better sensitivity , except for brain mris . for brain mris , the obtained specificity and sensitivity results with the dwt - gabor approach show improved true negative detection , but lower true positive performance . finally , the obtained results reveal also that level - one dwt decomposition is preferable to level - two decomposition . finally , table 5 compares the results obtained with the dwt - gabor approach to other work that we surveyed . in many cases , the dwt - gabor method yields higher classification rates , particularly for mammograms and retina . for the problem of brain mri classification , our obtained performance is better than the results of , but less than what is reported in [ 6 , 8 ] . however , these comparisons should be viewed with caution as not all the results stem from a common image database and the different authors use different sample and image sizes . moreover , many authors use no cross - validation and simply perform a single arbitrary split of their data into training and test sets to obtain their accuracy results . obviously , one can not generalize or draw definite conclusion from such efforts , and comparisons between works can not be made other than in general terms . in this respect , it can only be concluded from our results that the dwt - gabor for feature extraction is effective for obtaining high image classification accuracy by an svm and that it may outperform other feature extraction and classification techniques reported in the literature , at least those based on dwt - only image decomposition . unfortunately , a more definite conclusion is impossible without gaining access to the image databases used by the other authors . finally , the computational complexity of the dwt , gabor , contourlet , and curvelet for an n n image is , respectively , o(n ) , o(n m ) with m being the width of gabor ( gaussian ) mask filter , o(n ) , and o(nlog(n ) ) . as a result , the computational complexity of the combination of the dwt and gabor filter is o(n ) + o(n m ) . in terms of features extraction processing time , the average time required to process a brain , a mammogram , and a retina image with the dwt approach ( dwt - gabor ) was , respectively , 0.19 ( 0.31 ) , 0.17 ( 0.32 ) , and 0.15 seconds ( 0.35 ) using matlab r2009a on a 1.5 ghz core2 duo processor . we proposed a supervised system for biomedical images classification that uses statistical features obtained from the combination of the discrete wavelet transform and gabor filter to classify normal images versus cancer images , using support vector machines as classifiers . our experimental results show that such a hybrid processing model achieves higher accuracy in comparison to using dwt or gabor filter banks alone . therefore , the proposed image processing and features extraction approach seem to be very promising for the detection of certain pathologies in biomedical images . for future works , it is recommended to consider a larger set of features and a selection process to identify the most discriminant ones . in addition , the gabor parameters will be adjusted for each type of image separately to improve the accuracy . furthermore , the dwt - gabor will be directly compared to the dual - tree complex wavelet , curvelet , and contourlet using the same databases and images in order to draw general conclusions . also , multilabels classifications will be considered in future works to investigate the discriminative power of our approach for each type of pathology . finally , more experiments on the effect of kernel choice and its parameter on classification accuracy will be investigated .

a new methodology for automatic feature extraction from biomedical images and subsequent classification is presented . the approach exploits the spatial orientation of high - frequency textural features of the processed image as determined by a two - step process . first , the two - dimensional discrete wavelet transform ( dwt ) is applied to obtain the hh high - frequency subband image . then , a gabor filter bank is applied to the latter at different frequencies and spatial orientations to obtain new gabor - filtered image whose entropy and uniformity are computed . finally , the obtained statistics are fed to a support vector machine ( svm ) binary classifier . the approach was validated on mammograms , retina , and brain magnetic resonance ( mr ) images . the obtained classification accuracies show better performance in comparison to common approaches that use only the dwt or gabor filter banks for feature extraction .

1. Introduction 2. Related Works 3. Methodology 4. Experimental Results 5. Computational Complexity 6. Conclusion

although tumor immunosurveillance was first hypothesized more than four decades ago , the observed absence of spontaneous tumors in several immune - compromised mouse strains initially dampened enthusiasm for this idea . mabs specific for immunoregulators have sometimes proven useful in establishing the importance of such molecules in immunity against experimental tumors ( 13 ) . production of gene - targeted mice for specific effector molecules has greatly aided this endeavor ( 414 ) . nevertheless , experimental tumor models examined to date have provided limited information on tumor initiation and development . ifn- ( 15 ) and perforin ( pfp ; references 5 , 14 , and 16 ) , key molecules of both the innate and adaptive immune systems , contribute to tumor prevention in mice treated with the chemical carcinogen , methylcholanthrene ( mca ) , or in mice deficient for p53 expression . in mca- ( 10 ) and other carcinogen - induced cutaneous malignancy models ( 17 ) , the importance of antitumor immunity mediated by nk cells , nk t cells , and t cells has also been illustrated . however , in most of these models tumor development involves the transformation of many cells at one time and only a defined spectrum of tumors ( lymphomas and cutaneous malignancies ) is observed . an alternative model of tumor induction is that spontaneous tumors can arise from a single cell of any tissue that has acquired the appropriate genetic mutations with age in any given environment . herein , that environment has been controlled , and spontaneous tumor initiation compared in wt and gene - targeted mice of the same genetic background to illustrate the key role pfp and ifn- play in tumor immunosurveillance of lymphoma and lung adenocarcinoma . inbred c57bl/6j and balb / c wt mice were purchased from the walter and eliza hall institute of medical research , melbourne , australia . the following gene - targeted mice were bred and maintained at the austin research institute biological research laboratories , heidelberg , australia and the peter maccallum cancer institute , east melbourne , australia : c57bl/6j pfp - deficient ( b6 pfp ) ( targeted in c57bl/6j es cells and provided by d. kagi [ amgen institute , toronto , canada ] ; reference 18 ) ; c57bl/6j tnf - deficient ( b6 tnf ) ( targeted in c57bl/6j es cells [ reference 19 ] and provided by dr . sedgwick and originally from the centenary institute of cancer medicine and cell biology , sydney , australia ) ; c57bl/6 ifn--deficient ( b6 ifn-)(backcrossed to c57bl/6j for 10 generations from genentech , inc . ; reference 20 ) ; c57bl/6 il-12p40-deficient ( b6 il-12)(backcrossed to c57bl/6j for 10 generations from hoffmann - la roche ; reference 21 ) ; c57bl/6 il-18deficient ( b6 il-18 ) ( backcrossed to c57bl/6j for eight generations and provided by dr . s. akira , osaka university , japan ; reference 22 ) ; balb / c pfp ( backcrossed to balb / c for eight generations using microsatellite mapping ) , balb / c ifn- ( backcrossed to balb / c for 10 generations from genentech , inc . , reference 20 ) ; and balb / c pfp ifn-. f1 heterozygote progeny of some gene - targeted mice and their corresponding wt were bred as follows : ( b6 b6 ifn-)f1 ; ( balb / c balb / c ifn-)f1 ; and ( balb / c balb / c pfp)f1 . all aging mice were routinely screened for viruses , parasites , and other microbes and tested negative over the entire course of the experiment . any mouse with an abnormality ( palpable mass , abdominal distension , weight loss > 10% , ruffled fur ) was killed , its age recorded , and a postmortem performed . mean lifespan sem was calculated and probability of significance determined using a mann - whitney rank sum u - test . the significance of proportions of tumors and , in particular , disseminated lymphomas , was determined by a fisher 's exact test . a full autopsy was performed at sacrifice and tumor ( macroscopically detected ) , spleen , liver , thymus , and lymph nodes were routinely examined by histology after fixing these tissues in formalin and on occasions also fresh frozen . the preparation and staining of sections for histology were performed by the department of anatomical pathology , austin and repatriation medical centre , heidelberg , australia . the following reagents were used : anti tcr - apc ( h57597 ; bd pharmingen ) ; nk1.1-pe ( pk136 ; bd pharmingen ) ; cd4-fitc ( ct4 ; caltag laboratories ) ; cd8-apc ( 53.6.7 ; bd pharmingen ) ; cd8-biotin ( 53.5.8 ; bd pharmingen ) ; cd11c - fitc ( hl3 ; bd pharmingen ) ; b220-pe ( ra36b2 ; caltag laboratories ) ; thy-1-pe ( 30-h12 ; bd pharmingen ) ; cd45.2-fitc ( 104 ; bd pharmingen ) ; tcr - biotin ( clone gl3 ; bd pharmingen ) ; mac-1-biotin ( m170 ; caltag laboratories ) ; goat anti mouse ig - fitc ( silenus laboratories ) ; and streptavidin - percp ( bd pharmingen ) . anti - fc receptor ( 2.4g2 ) was used to prevent nonspecific binding by mab . fresh splenocytes from 6-wk - old b6 or balb / c mice were always used as labeling controls . a number of disseminated lymphomas were transplanted directly from b6.pfp , balb / c pfp , or b6 two representative experiments are shown using a b cell lymphoma from b6.pfp mice , pnk7 ( b220igtcr- ) , and a t cell lymphoma from b6 ifn- mice , bg18 ( cd8-cd4 tcr-ig ) . groups of five wt or gene - targeted mice were injected intraperitoneally with increasing numbers of lymphoma cells and observed daily for tumor growth for > 100 d. the supplemental figures are a ( fig . s2 ) flow cytometric analysis of lymphomas arising in balb pfp and b6 pfp mice . inbred c57bl/6j and balb / c wt mice were purchased from the walter and eliza hall institute of medical research , melbourne , australia . the following gene - targeted mice were bred and maintained at the austin research institute biological research laboratories , heidelberg , australia and the peter maccallum cancer institute , east melbourne , australia : c57bl/6j pfp - deficient ( b6 pfp ) ( targeted in c57bl/6j es cells and provided by d. kagi [ amgen institute , toronto , canada ] ; reference 18 ) ; c57bl/6j tnf - deficient ( b6 tnf ) ( targeted in c57bl/6j es cells [ reference 19 ] and provided by dr . sedgwick and originally from the centenary institute of cancer medicine and cell biology , sydney , australia ) ; c57bl/6 ifn--deficient ( b6 ifn-)(backcrossed to c57bl/6j for 10 generations from genentech , inc . ; reference 20 ) ; c57bl/6 il-12p40-deficient ( b6 il-12)(backcrossed to c57bl/6j for 10 generations from hoffmann - la roche ; reference 21 ) ; c57bl/6 il-18deficient ( b6 il-18 ) ( backcrossed to c57bl/6j for eight generations and provided by dr . s. akira , osaka university , japan ; reference 22 ) ; balb / c pfp ( backcrossed to balb / c for eight generations using microsatellite mapping ) , balb / c ifn- ( backcrossed to balb / c for 10 generations from genentech , inc . , reference 20 ) ; and balb / c pfp ifn-. f1 heterozygote progeny of some gene - targeted mice and their corresponding wt were bred as follows : ( b6 b6 ifn-)f1 ; ( balb / c balb / c ifn-)f1 ; and ( balb / c balb / c pfp)f1 . all aging mice were routinely screened for viruses , parasites , and other microbes and tested negative over the entire course of the experiment . any mouse with an abnormality ( palpable mass , abdominal distension , weight loss > 10% , ruffled fur ) was killed , its age recorded , and a postmortem performed . mean lifespan sem was calculated and probability of significance determined using a mann - whitney rank sum u - test . the significance of proportions of tumors and , in particular , disseminated lymphomas , was determined by a fisher 's exact test . a full autopsy was performed at sacrifice and tumor ( macroscopically detected ) , spleen , liver , thymus , and lymph nodes were routinely examined by histology after fixing these tissues in formalin and on occasions also fresh frozen . the preparation and staining of sections for histology were performed by the department of anatomical pathology , austin and repatriation medical centre , heidelberg , australia . the following reagents were used : anti tcr - apc ( h57597 ; bd pharmingen ) ; nk1.1-pe ( pk136 ; bd pharmingen ) ; cd4-fitc ( ct4 ; caltag laboratories ) ; cd8-apc ( 53.6.7 ; bd pharmingen ) ; cd8-biotin ( 53.5.8 ; bd pharmingen ) ; cd11c - fitc ( hl3 ; bd pharmingen ) ; b220-pe ( ra36b2 ; caltag laboratories ) ; thy-1-pe ( 30-h12 ; bd pharmingen ) ; cd45.2-fitc ( 104 ; bd pharmingen ) ; tcr - biotin ( clone gl3 ; bd pharmingen ) ; mac-1-biotin ( m170 ; caltag laboratories ) ; goat anti mouse ig - fitc ( silenus laboratories ) ; and streptavidin - percp ( bd pharmingen ) . anti - fc receptor ( 2.4g2 ) was used to prevent nonspecific binding by mab . fresh splenocytes from 6-wk - old b6 or balb / c mice were always used as labeling controls . a number of disseminated lymphomas were transplanted directly from b6.pfp , balb / c pfp , or b6 two representative experiments are shown using a b cell lymphoma from b6.pfp mice , pnk7 ( b220igtcr- ) , and a t cell lymphoma from b6 ifn- mice , bg18 ( cd8-cd4 tcr-ig ) . groups of five wt or gene - targeted mice were injected intraperitoneally with increasing numbers of lymphoma cells and observed daily for tumor growth for > 100 d. s2 ) flow cytometric analysis of lymphomas arising in balb pfp and b6 pfp mice . we undertook to monitor spontaneous tumor development in wt c57bl/6 ( b6 ) and balb / c mice or those that were deficient in ifn- and/or pfp . b6 mice deficient in il-12 , il-18 , or tnf were also examined , as all of these cytokines have been shown to regulate the expression of ifn- and other effector molecules in lymphocytes ( 2327 ) . b6 ifn- mice developed disseminated lymphomas ( 16/32 ( 50% ) and mean lifespan 491 104 d ) ( fig . 1 a ) . a few mice developed thymic lymphomas ( n = 2 ) or sarcoma ( n = 1 ) . like wt b6 mice ( 0/39 mice ) , b6 mice that were deficient in tnf ( 0/36 ) , il-12 ( 0/29 ) , or il-18 ( 0/27 ) did not develop any tumors over the same observation period ( fig . 1 a ) . these data illustrate spontaneous lymphoma formation in ifn-deficient mice and suggest that more than one of the upstream mediators of ifn- or an alternative cytokine must be required to regulate the protective effects of ifn-. consistent with our previous study ( 16 ) , b6 pfp mice died from aggressive disseminated lymphomas affecting the spleen , liver , and lymph nodes from 300 d onwards , with 57% ( 12/21 ) succumbing by the end of the experiment ( mean lifespan = 510 119 d ; fig . the appearance of tumors was recorded in mice of ( a ) c57bl/6 and ( b ) balb / c backgrounds as indicated . groups of mice ( number in parentheses ) were evaluated on a weekly basis and , when moribund , tumor type ( white squares , thymic lymphoma ; black squares , disseminated lymphoma ; black triangles , sarcoma ; white triangles , lung adenocarcinoma ; and white circles , other tumors ) recorded against the age at the time of death / autopsy ( in days ) . no tumors were observed in heterozygote control groups of ( b6 b6 ifn-)f1(n = 17 ) , ( balb / c balb / c ifn-)f1 ( n = 12 ) , and ( balb / c balb / c pfp)f1 ( n = 18 ) mice over a 750-d period . a small number of the oldest surviving balb / c ifn- mice ( 700750 d ) also developed nonmalignant mucosal hamartomas in the stomach . of great interest was a considerably distinct pattern of tumor development observed in similar gene - targeted mice on a balb / c background ( fig . c ifn- mice did not develop disseminated lymphoma ( 0/19 , p = 0.0001 ) . the finding that ifn- was not essential for host protection from this type of tumor in balb / c mice illustrated that strain - specific genetic factors can influence the nature of the immune response to these spontaneous lymphomas . by contrast , pfp was critical in host protection from disseminated lymphoma in both strains ( fig . 9 of 25 balb / c pfp mice developed disseminated lymphoma ( mean lifespan = 597 82 d ) , and a few mice developed thymic lymphomas ( n = 2 ) or sarcoma ( n = 1 ; fig . 1 b ) . the earlier onset ( mean lifespan = 368 120 d ) ( p < 0.0001 , compared with pfp mice ) and greater frequency ( 14/20 ) of disseminated lymphoma in balb / c mice deficient in both pfp and ifn- , revealed that in the absence of pfp , ifn- did play an important role in delaying the development of disseminated lymphoma ( fig . these data supported a general role for ifn- in protection from spontaneous lymphoma , but suggested that the relative role of ifn- was strain specific . lymphocyte - mediated immunosurveillance of epithelial malignancy has never been demonstrated , except where viral infection may contribute to oncogenesis ( e.g. , ebv , hpv transplant recipients ) or when carcinogens were also administered , and we therefore felt the development of spontaneous epithelial tumors in gene - targeted mice warranted particular attention . while ifn- and pfp were especially important for the control of disseminated lymphoma , a significant incidence of lung adenocarcinoma was detected in balb / c ifn- mice ( 3/19 , p = 0.0269 ) and balb / c pfp mice ( 3/25 , p = 0.048 ) ( fig . notably , it is relevant that the onset of lung adenocarcinomas was very late in balb / c ifn- ( 681 43 d ) and balb / c pfp ( 693 67 d ) mice . indeed , the development of all tumors , including disseminated lymphomas was significantly delayed in balb / c mice compared with b6 mice . far fewer balb / c pfp ifn- , b6 ifn- , and b6 pfp mice remained alive ( free of lymphoma ) over the first 600 d of life and therefore larger numbers of these will likely be required to observe adenocarcinomas . f1 progeny of gene - null mice and wt mice lacking either one allele of pfp or ifn- did not develop any spontaneous tumors ( fig . 1 ) . determining whether the immune system controls other epithelial malignancies is an important issue for future long - term studies and only a similar study of far larger groups of mice ( 100 ) will reveal whether significant numbers of other epithelial malignancies emerge in ifn- and pfp mice . 6 of the 16 lymphomas arising in b6 ifn- mice were analyzed for phenotype and while five were tcr- , no unique lymphoma phenotype was apparent ( unpublished data ) . several previous studies have indicated that t cell death after antigen - driven expansion may be regulated by ifn- ( 28 , 29 ) , and therefore possibly disrupted homeostasis and failed immunosurveillance by ifn- loss , may be the cause of t cell lymphoma in b6 ifn- mice . all of the disseminated lymphomas in b6 pfp mice were of b cell origin ( b220 sig cd4cd8 tcr ) , or plasmacytomas ( b220 cd4 cd8 tcr sig ; also defined histologically , unpublished data ) . our previous study of pfp mice with additional p53 loss indicated a majority of lymphomas of b cell origin , however some p53pfp mice also developed lymphomas of non - b cell origin ( 16 ) . one explanation for the development of b cell lymphomas in pfp mice is the recognized role for pfp in controlling the survival of some apcs , like b cells ( 30 , 31 ) . however , it remains unclear why only lymphomas of b cell origin developed in b6 pfp mice when enhanced expansions of cd8 t cells were observed in these mice , particularly after challenge with foreign antigens ( e.g. , viral or bacterial infection ; references 28 , and 3234 ) . most of the lymphomas in b6 pfp and b6 ifn- mice were diffuse large cell lymphomas ( see online supplemental materials ) . flow cytometric analysis of disseminated lymphomas in balb / c pfp and balb / c pfpifn- mice revealed that all were also b220 sig cd4cd8 tcr ( unpublished data ) . importantly , these data supported a role for ifn- in also suppressing the earlier onset of b cell lymphomas . interestingly , in the balb / c strain , a small number of the lymphomas arising in pfp and pfp ifn- mice showed an unusual histiocytic appearance ( fig . 1 , asterisk ) with a pale eosinophilic cytoplasm . these tumors additionally expressed cd11c and mac-1 antigens ( see online supplemental materials ) , but did not all express other markers of b1 cells ( e.g. , cd5 , unpublished data ) . of note , this type of histopathology occurs only rarely in humans , and interestingly the accumulations of activated histiocytes ( macrophages ) observed in pfp - deficient humans displaying familial hemophagocytic lymphohistiocytosis ( 35 ) are one example . all the lung adenocarcinomas in both balb / c pfp and balb / c ifn- mice were well - differentiated papillary adenocarcinomas ( see online supplemental materials ) . the malignancy of primary tumors arising was additionally confirmed by secondary transfer into mice of the same strain . two representative experiments of > 10 using different disseminated lymphomas transferred from either b6 pfp or b6 ifn- mice are depicted in fig . 2 . the transfer of three different t cell lymphomas from b6 ifn- mice ( one shown ) demonstrated that these tumors grew at a similar rate in b6 ifn- and b6 wt mice ( fig . 2 ) . by contrast , and in concert with our previous data ( 16 ) , all b cell lymphomas from b6 pfp mice grew at low cell numbers in b6 pfp mice , but were avidly rejected when transferred into b6 wt mice ( fig . thus far , all lymphomas of b cell origin arising in pfp mice , including five described herein , were rejected in wt mice by cd8 t cells ( reference 16 and unpublished data ) and in this study the donor tumor cells and recipient mice were genetically matched as closely as possible . similar experiments with three different b cell lymphomas from balb / c pfp mice have also demonstrated rejection in balb / c wt mice and ( balb / c balb / c . pfp)f1 heterozygous mice , but not balb / c pfp mice ( unpublished data ) . in concert with our previous lymphoma transfer studies ( 16 ) , it is likely that the lymphomas of b cell origin are generally being detected by cd8 t cells independently of any alloantigens potentially expressed by the tumor . it is probable that the lack of detectable immunogenicity of lymphomas from ifn- mice indicates an important functional distinction between the activities of ifn- and pfp in eliminating potentially transformed cells . consistent with the immune system functioning as a tumor - suppressor system , our results indicated that the immunoselection pressure of pfp on tumor cells was strong . lymphomas from pfp - deficient mice have emerged in the absence of this significant immunoselection pressure , and they are avidly rejected in wt mice upon transfer . immunoselection by ifn- appears far weaker from our spontaneous lymphoma transplant data , however it has been demonstrated in an experimental system using mca fibrosarcomas arising in ifn- receptor - deficient mice ( 36 ) . primary lymphomas arising in b6 pfp ( top ) and b6.ifn- ( bottom ) were secondarily transplanted intraperitoneally ( 1010 cells in 0.2 ml pbs , as indicated ) into groups of five untreated b6 wt ( black circles or squares ) , b6 pfp ( white circles ) or b6 ifn- ( white squares ) mice . the results are representative of seven primary lymphomas and three primary lymphomas transplanted from b6 pfp and b6 ifn- mice , respectively . top , pnk-7 ( b cell lymphoma from b6 pfp mouse ) ; bottom , bg18 ( t cell lymphoma from b6 ifn- mouse ) . previous studies have demonstrated that t cells ( 2 ) , nk t cells ( 37 ) , and nk cells ( 37 ) can all produce ifn- with antitumor activity . it remains unclear which type of lymphocytes use ifn- to control spontaneous lymphomas or lung adenocarcinomas , and this issue is very difficult to address without mice conditionally deficient ( e.g. , nk cell , nk t cell , or t cell ) in ifn-. defining exactly how a pleiotropic cytokine like ifn- mediates host protection from spontaneous tumors will also be a challenging goal for future investigation given that the lymphomas arising in b6 ifn- mice appear nonimmunogenic when transferred into wt mice . many potential direct and indirect activities of ifn- could suppress spontaneous tumor formation . although ifn- can suppress tumor angiogenesis , it is unlikely that this is the main mode of action in preventing the development of nonsolid tumors such as disseminated lymphomas . it also remains feasible that loss of ifn- may result in tumor promotion by another factor normally suppressed by ifn-. gene - targeted mice have been extremely valuable when used in models where tumors are induced by viruses or carcinogens or simply when the mice were aged for spontaneous tumor formation . the value of using several types of tumor induction models is no better illustrated by the lack of tumor development in tnf mice , but the reduced predisposition of these mice to carcinogen - induced skin tumors ( 38 ) . although some other recent studies in aging mice have suggested that tumor immunosurveillance may exist ( 16 , 36 ) , our study directly demonstrated the specific importance of ifn- in the development of lymphomas and adenocarcinomas . recently , a study of aging 129/sv mice doubly deficient for recombination activating gene ( rag)-2 and signal transducer and activator of transcription ( stat)-1 , the transcription factor mediating signaling by ifn- and ifn-/ receptors ( rag-2stat-1 ) , suggested that lymphocytes and ifn- may play an important role in host protection from breast and colon carcinomas ( 36 ) . mice deficient in stat-1 alone displayed a low incidence of mammary carcinoma , however stat-1 regulates far more genes than ifns alone , including the mammary tumor suppressor , brca-1 ( 39 ) . the lack of lymphoma reported in 129/sv stat-1 mice ( 32 ) may be because 129/sv behave like the balb / c strain . nevertheless , we did not observe colon or breast adenocarcinomas in either balb / c or c57bl/6 strains of ifn--deficient mice . the development of mammary and colon adenocarcinomas in 129/svev rag-2stat-1 mice may be a consequence of intestinal hyperplasia peculiar to 129 rag mice . by contrast , the lung adenocarcinomas observed in balb / c pfp and balb / c ifn- mice occurred in strains of mice with no evidence of predisposing epithelial hyperplasia . the concept of whether lymphocytes play a key role in immunosurveillance beyond their own regulation ( i.e. , of nonlymphoid tissues ) has been debated for years ( 40 ) . the adenocarcinomas observed in mice in our study suggested that future studies , monitoring spontaneous tumor development in gene - targeted mice alone and bred with other strains genetically prone to epithelial malignancies , will be most informative .

the immunosurveillance of transformed cells by the immune system remains one of the most controversial and poorly understood areas of immunity . gene - targeted mice have greatly aided our understanding of the key effector molecules in tumor immunity . herein , we describe spontaneous tumor development in gene - targeted mice lacking interferon ( ifn)- and/or perforin ( pfp ) , or the immunoregulatory cytokines , interleukin ( il)-12 , il-18 , and tumor necrosis factor ( tnf ) . both ifn- and pfp were critical for suppression of lymphomagenesis , however the level of protection afforded by ifn- was strain specific . lymphomas arising in ifn--deficient mice were very nonimmunogenic compared with those derived from pfp - deficient mice , suggesting a comparatively weaker immunoselection pressure by ifn-. single loss of il-12 , il-18 , or tnf was not sufficient for spontaneous tumor development . a significant incidence of late onset adenocarcinoma observed in both ifn- and pfp - deficient mice indicated that some epithelial tissues were also subject to immunosurveillance .

Introduction Materials and Methods Mice. Histopathology and Surface Phenotyping of Tumors. Tumor Transplantation Experiments. Online Supplemental Materials. Results and Discussion

primary immunodeficiency diseases ( pidd ) are a heterogeneous group of more than 150 disorders that result from many different congenital , or even acquired , genetic defects affecting the innate and adaptive immune systems [ 1 , 2 ] . despite major advances over the last 20 years in the clinical and molecular characterization of pidd , many patients remain undiagnosed or have a substantially delayed diagnosis with adverse results on morbidity and mortality . the diagnosis of pidd is most often suggested by recurrent or unusual infections and inflammatory or autoimmune conditions . several lists of warning signs for children or adults are based on these main clinical presentations and , in some cases , family history [ 36 ] . arkwright and gennery concluded that the 10 warning signs promoted by the jeffrey modell foundation have a sensitivity of 56 % and a specificity of 16 % . of secondary relevance were the use of intravenous antibiotics for sepsis , and failure to thrive in children with t lymphocyte primary immunodeficiency disease . observed that 20 % of hospitalized patients with suspected immune defects would not be included for immunologic investigation according to the 10 warning signs . all currently available lists of warning signs are based on family history and descriptions of infections suggestive of an immunodeficiency . however , there are many specific warning signs that certain specialists need to be aware of . because of the diversity of immune defects , their respective clinical presentations , and the difficulties in reaching many non - immunology subspecialists with these general warning signs , many signs that should raise suspicion of pidd are missed by physicians and a large proportion of patients are not properly diagnosed [ 1012 ] . here , we provide lists of specific warning signs in tables that can be used to familiarize subspecialists with signs and symptoms of pidd that they are likely to see in patients referred to their practices . the tables will provide quick summaries of the many warning signs and diagnoses encountered by different specialists , with suggestions for appropriate screenings , immunological evaluations , and proper referrals to clinical immunologists . the information offered in this article is based on a review of the pertinent literature and the clinical experience of all authors . tables i and ii list the tests that are recommended for screening of the main immunologic functions , along with histories and infections that all physicians and health care workers need to recognize as warning signs of a possible immunodeficiency . subsequent tables list special clinical presentations that should alert specific groups of specialists . when presenting information about pidd from this article , it is important to always include tables i and ii , and the pertinent tables prepared for various specialties.table iscreening laboratory tests for the non - immunology specialist in order to detect patients with possible piddpossible piddscreening testsantibody mediated immunity ( ami) cbc and differential serum igg , iga , igm antibody titers to protein and polysaccharide vaccinescellular mediated immunity ( cmi) cbc and differential lymphocytes : cd3 , cd4 , and cd8 ; cd19 ; cd16/56 chest x - raycomplement ( c) c4 ( if angioedema without urticaria) ch50phagocytosis ( p) neutrophil countsneutropenia oxidative burst by dhr testneutrophil functionautoimmunity ana , rcpinnate immunity ( ii) specialized tests ( consult an immunologist)the screening lab tests should be part of any initial immune evaluation . only the abbreviations in parentheses will be listed as suggested screening tests in all subsequent tables . hiv testing should be a routine test to exclude aids abbreviations : ana antinuclear antibodies , cbc complete blood count , dhr dihydrorhodamine , rcp reactive c proteintable iiinfections that are general warning signs of pidd for all clinicians otitis media* early onset < 34 months of age recurrence after antibiotic treatment complications : mastoiditis association with invasive infections recurrence after ear tubes change to sinusitis after ear tubes repeated ear tube placement * the number of otitis episodes that suggest pidd varies with age : 3 episodes / year under 5 years ; 2 episodes / year 5 years chronic recurrent rhinosinusitis association with persistent asthma requirement for sinus surgery due to fungal infections pneumonia evaluate after a single pneumonia if the patient has: a personal history of recurrent upper respiratory infections ( uri ) including recurrent otitis media a personal history of other immune problems ( autoimmunity , chronic diarrhea , periodic fevers , persistent skin rash , etc.) pneumonia that requires hospitalization ( any : icu or regular service) persistent pneumonia after adequate therapy with antibiotics pneumonia requiring iv antibiotics bilateral pneumonias necrotizing pneumonia interstitial pneumonitisevaluate patients with 2 or more pneumonias: all patients ( preferred option)or evaluate only if: x - ray proven pneumonias in different lung sites positive family history for early death or primary immunodeficiency ( pidd) pneumonia that is complicated by pneumatocele or bronchiectasis uncommon infections or uncommon presentations in hiv - negative patients*atypical mycobacteriosistuberculosis resistant infectionhistoplasmosisneurocryptococcosisaspergilosisleishmaniasisblastomycosis * more relevant signs in developed countries or non - endemic countries for these diseases chronic diarrhea or colitis evaluate if the patient has : rotavirusenteroviruses campylobacter cryptosporidium persistent salmonella clostridium difficile recurrent giardiasis chronic dermatitis recurrent staphylococcal infectionsrecurrent or persistent candidiasis or fungal infections abscesses ( liver , lungs , cutaneous ) staphylococcus aureus infections of central nervous system ( cns ) meningococcal meningitisherpes encephalitisfungal infections complications due to live attenuated vaccines disseminated bcg ( mycobacterium bovis bacillus calmette - gurin)poliomyelitis due to poliovirus vaccinediarrhea due to rotavirus vaccinesee text under recurrent , severe , or unusual infectionsfor all the common infections listed in this table , evaluation starts with a complete blood count to rule out neutropenia and assessment of antibody mediated immunity screening laboratory tests for the non - immunology specialist in order to detect patients with possible pidd the screening lab tests should be part of any initial immune evaluation . only the abbreviations in parentheses will be listed as suggested screening tests in all subsequent tables . hiv testing should be a routine test to exclude aids abbreviations : ana antinuclear antibodies , cbc complete blood count , dhr dihydrorhodamine , rcp reactive c protein infections that are general warning signs of pidd for all clinicians see text under recurrent , severe , or unusual infections for all the common infections listed in this table , evaluation starts with a complete blood count to rule out neutropenia and assessment of antibody mediated immunity table i lists the tests that should be performed according to the main clinical presentations specified in the subsequent tables . in some situations the relevant evaluation is not part of generally available screening tests , and it is preferable to refer the patient directly to an immunologist so that routine and specialized tests can be performed simultaneously . the tables for specialists will provide data for further laboratory investigation . our recommendations are similar to those in published lists of warning signs that consider the frequency of various types of infections . however , it is also important to consider the infection type , the circumstances under which infections occur , and which organs and tissues they affect . in our experience , this is a more effective way to identify pidd than emphasizing exact numbers of different infections or special definitions of severity . when severity is considered , it is important to consider the pathogenicity of an infectious agent ; infections with any pathogen of low pathogenicity , such as atypical mycobacteria or toxoplasma , are suggestive of an immune defect even if the infection is not severe . for example , paracoccidioidomycosis should be more commonly identified in tropical countries than in european or north american countries ( table ii ) . a general condition that should make any infection suspicious is the concomitant presence of noninfectious complications commonly associated with pidd , including severe allergy , autoimmunity , unregulated inflammation , or malignancies . however , it is also important to consider certain negative family histories for example , a family in which only one of 4 siblings has recurrent infections . the fact that the other 3 siblings and parents are healthy rules out environmental factors as the factors predisposing an individual to recurrent infections ; it also highlights the immunopathogenic effects of genetic alterations that may have occurred in a single family member . recurrent infections are often attributed to the presence of non - immune abnormalities , such as down syndrome , muscular dystrophy , or various congenital heart defects . such patients may also have an immunodeficiency , and appropriate recognition and treatment may significantly improve their quality of life . many pidds have unique susceptibilities to some pathogens and/or sites of infections that are listed in table iii . antibody - mediated immunity needs to be evaluated in all severe or unusual infections , in addition to other possible concurring immunological defects . a practical point is to screen for combined immunodeficiencies.table iiiwarning signs of pidd for infectious disease specialistsclinical occurrencespiddlab screening testsinfections from extracellular bacteriaantibody deficienciesamicomplement deficienciesc , ananeutropeniaspirak-4 , myd88ii , rcpinfections due to neisseria meningitidis complement deficiency of terminal components ( membrane attack complex)c + ap50infection from s aureus , and gram - negative bacterias : serratia marcescens , burkholderia cepacia and gladioli , nocardia spp , chromobacterium violaceum , granulobacter bethesdensis.chronic granulomatous disease ( cgd)phyper ige syndrome ( hies)features : pneumonia from s aureus , eczema , fungal infection , joint hypermobility , coarse facial featuresserum ige , eosinophiliaspecific score infection from fungi : pneumocystis jiroveci ; aspergillus and candida albicans.t cell defectscmicd40 ligand ( l ) deficiencyamihiesserum ige , eosinophiliaspecific score cgdpinfection from candida albicans chronic mucocutaneous candidiasiscmi + t lymphocyte proliferation induced by candida infection by atypical mycobacteria / salmonella and/or bacillus calmette - gurin side effects ; paracoccidioides sp , leishmania , cryptococcus t cell deficienciescmisevere combined immunodeficiency ( scid)ami + cmimendelian susceptibility to mycobacterial diseasesp and/or iiinfections from herpes t and nk cell deficienciescmifulminant or chronic infection by epstein - barr virusfamilial hemophagocytic lymphohistiocytosis ( fhl ) syndromex - linked lymphoproliferative ( xlp ) syndromes , types 1 or 2cbc , triglycerides , ferritin , serology ebnarecurrent or persistent cryptosporidium , isospora cd40l deficiencyamicommon variable immunodeficiency ( cvid)ami giardiasis antibody deficienciesamicomplications due to bcg , rotavirus or varicella vaccinesscid , cgdcmi and/or ii and/or pcomplications due to oral polio vaccineantibody deficienciesamipersistent fever of unknown originautoinflammatory diseasesana , rcp , blood smear abbreviations : ami antibody mediated immunity , ana antinuclear antibodies , cbc complete blood count , cmi cellular mediated immunity , ebna epstein - barr nuclear antigen , ii innate immunity , p phagocytosis , rcp reactive c protein score for classical hyper ige diagnosis warning signs of pidd for infectious disease specialists abbreviations : ami antibody mediated immunity , ana antinuclear antibodies , cbc complete blood count , cmi cellular mediated immunity , ebna epstein - barr nuclear antigen , ii innate immunity , p phagocytosis , rcp reactive c protein score for classical hyper ige diagnosis infectious disease is the hallmark of pidd , and infectious disease specialists are the physicians most likely to encounter patients with pidd in their daily practice . recurrent otitis media is considered to be more than 4 episodes of acute otitis media during the past 12 months with complete resolution of the disease between episodes [ 1315 ] . any patient with severe or recurrent infections or infection by opportunistic or uncommon microorganisms must be investigated for pidd after discarding the possibility of infection with human immunodeficiency virus ( hiv ) . pyogenic encapsulated bacteria ( streptococcus pneumoniae , haemophilus influenzae ) are frequently associated with infections in patients with antibody or complement deficiencies . patients with complement deficiencies may also present with meningitis and sepsis associated with pathogens such as neisseria . agammaglobulinemic patients and , to a lesser extent , patients with common variable immunodeficiency ( cvid ) have an increased risk of developing bloodstream bacterial infections . a study of 201 patients with x - linked agammaglobulinemia ( xla ) indicated that 11 % had at least one episode of meningitis or encephalitis , 10 % had at least one episode of sepsis , and 8 % had at least one episode of septic arthritis . patients with xla or cvid are at an increased risk for severe and often fatal infections with enteroviruses , including echoviruses , polioviruses , and coxsackie viruses . the central nervous system is nearly always involved in these infections , and evidence of systemic involvement of muscle , liver , and/or joints is present in about 40 % of patients . other organisms , including staphylococcus aureus , pseudomonas spp , mycoplasma spp , enterobacteriaceae , campylobacter spp , giardia , and enteroviruses , may be isolated from patients with specific types of pidd . infections caused by serratia marcessens , nocardia spp , chrombacterium violaceum , granulobacter bethesdensis , burholderia cepacia , and gladioli are strongly suggestive of chronic granulomatous disease ( cgd ) . evaluation of 259 patients with the x - linked recessive form of cgd indicated that 21 % had a history of either bacteremia or fungemia . infections caused by mycobacteria , salmonella , leishmania , and cryptococcus suggest defects in the interferon-/interleukin-12 axis included in innate immunity ( table iii ) . as previously mentioned , the incidence of certain infectious agents must be considered according to their endemicity in particular geographical regions . side effects of bacillus calmette - gurin ( bcg ) occur in more than 50 % of the severe combined immunodeficiency ( scid ) patients in brazil and more than 17 % of cgd patients in latin america ; this is a relevant warning sign for pidd . adverse reactions to other live attenuated viral vaccines such as poliovirus and rotavirus should be carefully observed as well . several developing countries maintain campaigns with sabin immunization with the possibility to cause vaccine - derived disease . although the respiratory and gastrointestinal tracts are the most common systems for presenting illness , the authors observed lymphadenopathy as the most common manifestation in the group of combined t and b cell immunodeficiencies , and superficial abscesses and lymphadenopathy in the group of congenital defects of phagocyte number and/or function [ 22 , 25 , 26 ] ( table iii ) . high percentages of pediatric and adult patients with pidd have upper and/or lower respiratory tract bacterial infections ; thus , they may be referred to otorhinolaryngologists or pulmonologists . rezaei reported on 930 patients with pidd , 353 cases of which were identified in the last 5 years . the most common presenting feature of pidd was pneumonia , which was seen in 20.1 % of patients , followed by diarrhea ( 13.7 % ) , sinusitis ( 10.3 % ) , and otitis media ( 9.6 % ) . it is important to rule out the existence of cofactors that might be associated with upper respiratory infections ( e.g. , smoking , day care attendance , gastroesophageal reflux , allergy ) before performing extensive immunological evaluation . the most common of these infections are recurrent otitis media , chronic sinusitis , and pneumonia . recurrent sinus infections have been reported as a presenting occurrence in 19 % to 98 % of patients with pidd ( wide range reflects the variety of pidd ) [ 28 , 29 ] . an assessment of 103 pediatric and adult patients with a history of recurrent or chronic ear , nose , or throat infections indicated that 16.5 % had defects in antibody - mediated immunity : cvid ( n = 2 ) , iga deficiency ( n = 4 ) , igg subclass deficiency ( n = 3 ) , and specific antibody deficiency against polysaccharide antigens ( n = 8) . warning signs for patients with recurrent otitis include progressive infections leading to mastoiditis ; associated abscesses or systemic infections ; lack of responsiveness to adequate antibiotic treatment ; or occurrence of unusual , severe , or frequently relapsing infections in other sites ( table ii ) . pneumonia is a common infectious manifestation of pidd ; one study of patients with cvid indicated that at least two - thirds had one or more pneumonia diagnoses prior to diagnosis of pidd . according to some registries [ 33 , 34 ] , approximately 50 % of patients with humoral immunodeficiency suffered from upper respiratory tract infections , and 40 % of xla and cvid patients suffered from recurring pneumonia [ 27 , 35 ] . pathogens frequently associated with pneumonia in patients with antibody deficiencies include s pneumoniae , h influenzae type b , haemophilus parainfluenzae , mycoplasma spp , pseudomonas spp , and staphylococcus spp . patients with cellular deficiencies such as cd40 ligand ( cd40l ) deficiency present with pneumonia that is a result of opportunistic pathogens such as pneumocystis , fungal , or viral infections ( table iv).table ivwarning signs of pidd for pulmonologistsclinical occurrencespiddlaboratory testspneumonias due to extracellular bacteria + otitis and sinusitisantibody deficienciesamicomplement deficienciesc , anapulmonary abscesspneumatocelehyper ige syndrome ( hies)features : pneumonia by s aureus , eczema , fungal infection , joint hypermobility , coarse facial featuresserum ige , eosinophiliaspecific score pneumonias due to staphylococcus or fungichronic granulomatous disease ( cgd ) : susceptibility to infections by catalase positive microorganisms . other infections : adenitis , liver abscess , osteomyelitispglucose-6-phosphate dehydrogenase ( g6pd ) deficiencymyeloperoxidase deficiency ( common in diabetes)g6pd activityperoxidase levelhiesserum ige , eosinophiliaspecific score pneumonia due to p jiroveci t cell deficiencies / cd4 lymphopeniacmi , amilymphoproliferation assaycd40 ligand ( l ) deficiencyami , cmiwiskott - aldrich syndrome ( was ) , eczema + thrombocytopeniacbc including platelet number and size ( small sized platelets ) ; cmi , amipneumonia due to mycobacteria tuberculosis or atypical mycobacteriat cell deficiencies / cd40l deficiencycmi , amimendelian susceptibility to mycobacterial diseasesii abbreviations : ami antibody mediated immunity , ana antinuclear antibodies , cbc complete blood count , cmi cellular mediated immunity , ii innate immunity , p phagocytosis score for classical hyper ige diagnosis warning signs of pidd for pulmonologists abbreviations : ami antibody mediated immunity , ana antinuclear antibodies , cbc complete blood count , cmi cellular mediated immunity , ii innate immunity , p phagocytosis score for classical hyper ige diagnosis several observational studies have indicated that complications and sequelae of infectious diseases such as bronchiectasis ( 76 % ) , recurrent chest infections ( 21 % ) , and granulomatous lung disease ( 5 % ) should also raise suspicion of pidd . a systematic review of the literature conducted by wood et al . included results from 7 surveys showing that respiratory / chest infections were presenting symptoms for 37 % to 90 % of patients with pidd . reviewed 26 studies that included 587 patients with cvid and found that up to 73 % developed chronic structural pulmonary complications pulmonary imaging by chest computed tomography in 30 patients with pidd revealed abnormalities in 53 % ; among these were bronchiectasis ( 75 % ) , peribronchial thickening ( 19 % ) , air trapping ( 31 % ) , lung volume reduction ( 25 % ) , atelectasis ( 12 % ) , follicular bronchiolitis ( 12 % ) , ground - glass abnormality ( 12 % ) , and parenchyma nodules ( 6 % ) . in this study , pulmonary function testing also indicated abnormalities in 18 patients ; these included an obstructive ( 38.8 % ) , restrictive ( 44.4 % ) , or mixed ( 16.7 % ) pattern of lung function abnormalities . results from other studies have indicated that staphylococcus - associated pneumonia and bronchial aspergillosis are seen in cgd ; lung abscesses represent usual characteristics in hyper ige syndrome ( hies ) ; and interstitial pneumonia may be observed in patients with scid ( table iv ) . frequent gastrointestinal manifestations are malabsorption , diarrhea , hepatomegaly , and inflammatory bowel disease ( ibd ) and all of these are commonly associated with increased morbidity [ 4244 ] . in a systematic review , indicated that gi infections were a presenting symptom in 6 % to 19 % of patients with pidd . severe diarrhea can start early in life in patients with scid or immunodysregulation , polyendocrinopathy , and enteropathy , x - linked ( ipex ) ; both of these pidd are considered pediatric emergencies [ 4547 ] . rezei et al . , in their study of 930 patients with pidd , indicated that infectious diarrhea occurred in 40.4 % of patients . other pathogens in patients with infectious colitis include rotavirus , campylobacter , enteroviruses , cryptosporidium parvum , salmonella spp , and clostridium difficile . liver abscesses caused mainly by s aureus may also be seen in patients with cgd and hies ( table v).table vwarning signs of pidd for gastroenterologistsclinical occurrencespiddlaboratory testschronic diarrheaantibody deficienciesamiinflammatory bowel diseasecombined immunodeficiencies ( infants)cmi , amichronic giardiasisautoimmune enteropathy + severe intractable diarrhea . other diagnoses associated : hypothyroidism , eczema , thrombocytopenia , autoimmune hemolytic anemia , neonatal diabetesimmunodysregulation , polyendocrinopathy and enteropathy , x - linked ( ipex)cmi , coombs , glycemia , and tshanapersistent candidiasiscombined immunodeficienciescmit cell lymphoproliferative assaychronic mucocutaneous candidiasislymphoproliferation to candida autoimmune polyendocrinopathy - candidiasis - ectodermal dystrophy ( apeced)candidin testana and endocrine evaluationsevere abdominal pain emulating an acute abdomenhereditary angioedemadosage and/or functional activity assay of c1inh , c4 , c1qliver abscess mainly due to s aureus chronic granulomatous disease ( cgd)phyper ige syndrome ( hies)serum ige , eosinophiliaspecific score hepatobiliary infection due to c parvum cd40 ligand ( l ) deficiencyamiinflammatory bowel disease in infantscgdpinterleukin-10 ( il-10 ) or interleukin 10 receptor ( il-10r ) deficienciesii abbreviations : ami antibody mediated immunity , ana antinuclear antibodies , c1inh c1 esterase inhibitor , cmi cellular mediated immunity , cbccomplete blood count , ii innate immunity , p phagocytosis , tsh thyroid stimulating hormone score for classical hyper ige diagnosis warning signs of pidd for gastroenterologists abbreviations : ami antibody mediated immunity , ana antinuclear antibodies , c1inh c1 esterase inhibitor , cmi cellular mediated immunity , cbccomplete blood count , ii innate immunity , p phagocytosis , tsh thyroid stimulating hormone score for classical hyper ige diagnosis results from a study focusing mainly on pediatric patients with pidd indicated clinical evidence of liver disease , including hepatomegaly , in 35.5 % ; 8 patients ( 13 % ) had clinical and/or laboratory evidence of chronic liver disease . hepatobiliary infection , sclerosing cholangitis , nodular regenerative hyperplasia , and portal hypertension are frequent conditions in cd40l deficiency [ 48 , 49 ] . the risk for autoimmune diseases is elevated in patients with pidd and is sometimes the only clinical manifestation of their causative condition . results from a larger study of 248 patients with cvid indicated that 69 ( 27.8 % ) had autoimmune disease . the most common conditions were thrombocytopenia in 18 patients , hemolytic anemia in 12 , rheumatoid arthritis in 5 , and juvenile rheumatoid arthritis in 4 [ 50 , 51 ] . results from another study of 189 patients with systemic lupus erythematosus ( sle ) indicated that 6 % had iga deficiency . other pidd are the result of genetic defects that lead to immune dysregulation associated with autoimmune occurrences . patients with autoimmune lymphoproliferative syndrome ( alps ) develop generalized lymphadenopathy , hepatosplenomegaly , hypergammaglobulinemia , b cell lymphocytosis , and autoimmune manifestations that commonly include hemolytic anemia , glomerulonephritis , and idiopathic thrombocytopenic purpura . autoimmune polyendocrinopathy , candidiasis , and ectodermal dystrophy ( apeced ) are also associated with symptoms of autoimmune disease . patients with ipex have mutations in the foxp3 gene in regulatory t cells , which leads to severe autoimmunity with a high mortality rate . first components of complement deficiency have also been shown to be associated with conditions such as sle and are likely to prompt referral to a rheumatologist . an important red flag for sle and complement defects is negative serology to double - stranded dna . the most common of these is arthritis , which may be associated with antibody deficiencies , and , less often , with cgd and wiskott - aldrich syndrome ( was ) [ 58 , 59 ] . importantly , infections with ureaplasma urealyticum , and mycoplasma ssp may lead to erosive arthritis in patients with severe antibody deficiencies . these infections most often result in large - joint monoarthritis involving the knee , shoulder , elbow , or hip joints , and less often in symmetrical polyarthritis . patients with certain pidd , such as autoinflammatory syndromes , will present with generalized serositis that might be confused with infectious arthritis . this is often accompanied by a myriad of symptoms , including elevation of acute phase reactants , rash , deafness , joint deformity , and multifocal osteomyelitis . patients with pidd may have skin conditions including fungal , bacterial , or viral infections ; eczematous dermatitis ; erythroderma ; skin symptoms of autoimmune diseases such as sle or scleroderma ; vasculitis ; granuloma formation ; and/or problems characteristic of specific immunodeficiency syndromes ( e.g. , telangiectasia , gray hair , and depigmentation ) . an immunological assessment of 382,383 pediatric patients admitted to an outpatient dermatology group indicated that 130 had pidd . of these , 69 % had skin infections , 29 % had eczema - dermatitis , and 44 % had other associated cutaneous conditions . in 79 % of this cohort , cutaneous abnormalities preceded and were the basis for the clinical immunologic diagnosis , demonstrating the relevance and high frequency of dermatologic manifestations in pidd patients . results from a cross - sectional study of 210 pediatric patients with pidd ( 68 with antibody deficiencies , 22 with t cell and combined deficiencies , 57 with phagocytic defects , and 63 with other pidd ) indicated that 67 of the patients ( 31.9 % ) had cutaneous abnormalities preceding the diagnosis of pidd . candida infections that may be among the first signs of pidd include mucocutaneous candidiasis , candida paronychia , granuloma formation , and erythroderma . patients with phagocytic , cellular , combined , and other pidd exhibit immune deficits that confer increased susceptibility to fungal infections . infections associated with candida , aspergillus , cryptococcus , histoplasma , paecilomyces , scedosporium , trichosporon , penicillium , and other fungal organisms have been observed in patients with cgd , scid , hies , defects in the interferon-/interleukin-12 axis , digeorge syndrome/22q deletion syndrome , cd40l deficiency , was , and cvid . patients with chediak - higashi or griscelli syndromes present partial albinism and gray hair ; dermatologists are frequently the first consulted specialists . prompt diagnosis is established by demonstration of giant intracellular granules in eosinophils , basophils , and monocytes , and are pathognomonic of this disease . eczema and high serum ige levels in the first months of life frequently lead to referral to allergists due to suspicion of cow milk protein allergy . however , these occurrences are also present in patients with hypomorphic scid , ipex , was , dock8 , and hies ; it has been shown that differential diagnosis is crucial for prognosis . zhang et al . described dock8 mutations in patients with a variant of combined immunodeficiency characterized by unusual susceptibility to cutaneous viral infections and cancers . in addition , dock8 deficiency was found in a subgroup of patients who were previously thought to have autosomal recessive hies with severe allergic manifestations . these syndromes exemplify the need for improved knowledge of pidd for dermatologists ( table vi ) . family histories are helpful for such cases , and c4 levels could be a useful screening test .table viwarning signs of pidd for dermatologistsclinical occurrencespiddlaboratory testeczemawiskott - aldrich syndrome ( was)cbc including platelet number and size ( small sized platelets ) ; cmi , amihyper ige syndrome ( hies)serum ige , eosinophiliaspecific score immunodysregulation , polyendocrinopathy and enteropathy , x - linked ( ipex)cmi , ana , rcpcoombs , glycemia , and tshsevere combined immunodeficiency ( scid ) , erythrodermacmicutaneous lesions by mycobacteria combined immunodeficienciescmihyper - igm syndromesamimendelian susceptibility to mycobacterial diseasesiichronic granulomatous diseases ( cgd)ppartial albinism , gray hairchediak - higashi syndromeenlarged cytoplasm granules in blood smeargriscelli syndrometelangiectasiasataxia - telangiectasiaami ; serum alfa - feto proteindisseminated warts and molluscumwarts , hypogammaglobulinemia , infections , and myelokathexis ( whim ) syndromeami , cmi lymphoproliferation assaycutaneous herpes infectionsdedicator of cytokinesis 8 ( dock8 ) deficiencyidiopathic cd4 lymphopeniafragile hair , conic teethectodermal dysplasiaii abbreviations : ami antibody mediated immunity , ana antinuclear antibodies , cbc complete blood count , cmi cellular mediated immunity , ii innate immunity , p phagocytosis , rcp reactive c protein , tsh thyroid stimulating hormone score for classical hyper ige diagnosis warning signs of pidd for dermatologists abbreviations : ami antibody mediated immunity , ana antinuclear antibodies , cbc complete blood count , cmi cellular mediated immunity , ii innate immunity , p phagocytosis , rcp reactive c protein , tsh thyroid stimulating hormone score for classical hyper ige diagnosis autoimmune hemolytic anemia , thrombocytopenia , and/or neutropenia are also seen in patients with selective iga deficiency , cvid , and class switch recombination ( csr ) defects [ 50 , 71 ] . in some patients , wiskott - aldrich syndrome is associated with defects in both b - lymphocyte and t - lymphocyte function , and these patients also have intrinsic platelet abnormalities and significant thrombocytopenia with small platelets . in addition to congenital neutropenia , neutropenia is also associated with xla , cd40l deficiency , cvid , scid , and shwachman - diamond syndrome [ 72 , 73 ] . hemophagocytic lymphohistiocytosis ( hlh ) is characterized by multisystem inflammation , a reactive process resulting from hyperactivation of macrophages , histiocytes and cd8 t cells , and abnormalities in the function of natural killer cells ( nk cells ) . the most frequent clinical and laboratorial findings include fever , splenomegaly , cytopenias , hypertriglyceridemia , and elevated ferritin . both chediak - higashi and griscelli syndromes present as hemophagocytic , with an accelerated phase affecting all patients by late childhood . this accelerated phase is characterized by the infiltration of nonmalignant lymphoid and histiocytoid cells into the viscera , usually induced by epstein - barr virus . clinical findings include hepatosplenomegaly , lymphadenopathy , and pancytopenia . impaired humoral and cellular immune responses , including disturbances in b , t , nk , and dendritic cells , along with chronic inflammatory autoimmune diseases , recurrent bacterial infections , and persistent antigenic stimulation , are speculated to favor carcinogenesis in cvid patients . lymphoma and gastric cancer are the most frequent neoplasms related to this immunologic defect [ 76 , 77 ] . the risk of cancer in pidd patients mainly takes the form of t cell malignancies ( 70-fold and 250-fold increased risks of leukemia and lymphoma , respectively ) , and b cell malignancies are high when compared with the general population . the spanish registry identified that 1.3 % of the 1,069 patients developed cancer ; however , the survey was developed within reference hospitals . non - hodgkin lymphoma and hodgkin lymphoma are 2 of the most common pidd - associated malignancies . a recent large - scale assessment of 1,132 patients in the pidd registry of the australasian society of clinical immunology and allergy indicated that this group had a 1.6-fold excess relative risk of cancer . the relative risk was significantly increased for non - hodgkin lymphoma , leukemia , and stomach cancer ( table vii).table viiwarning signs of pidd for hematologistsclinical occurrencespiddlaboratory teststhrombocytopenia with small - sized plateletswiskott - aldrich syndrome ( was)cbc including platelet number and size ( small sized platelets ) ; cmi , amiother symptoms : eczema and recurrent infectionsx - linked thrombocytopeniaautoimmune cytopenias ( autoimmune anemia , thrombocytopenia and neutropenia)common variable immunodeficiencyami , anaother features : recurrent infectionsfever , splenomegaly without evidence of malignancy , cytopeniashemophagocytic lymphohistiocytosis ( hlh)cbc , triglycerides , ferritin , ebnalymphadenopathy + splenomegalyautoimmune lymphoproliferative diseaseincreased number of alpha beta double - negative t cells ( cd3 + cd4-cd8- ) , ana , rcpexcluding neoplasias and infectionsapoptosis defectsquantitative and qualitative defects of neutrophils ( neutropenia and neutrophilia)neutropeniaspchronic granulomatous disease ( cgd)leukocyte adhesion deficiencyleukocytosis , cd18 cellspartial albinism , chediak - higashi or griscelli syndromeenlarged cytoplasm granules abbreviations : ami antibody mediated immunity , ana antinuclear antibodies , cbc complete blood count , cmi cellular mediated immunity , ebna epstein - barr nuclear antigen , p phagocytosis , rcp reactive c protein warning signs of pidd for hematologists abbreviations : ami antibody mediated immunity , ana antinuclear antibodies , cbc complete blood count , cmi cellular mediated immunity , ebna epstein - barr nuclear antigen , p phagocytosis , rcp reactive c protein table i lists the tests that should be performed according to the main clinical presentations specified in the subsequent tables . in some situations the relevant evaluation is not part of generally available screening tests , and it is preferable to refer the patient directly to an immunologist so that routine and specialized tests can be performed simultaneously . the tables for specialists will provide data for further laboratory investigation . our recommendations are similar to those in published lists of warning signs that consider the frequency of various types of infections . however , it is also important to consider the infection type , the circumstances under which infections occur , and which organs and tissues they affect . in our experience , this is a more effective way to identify pidd than emphasizing exact numbers of different infections or special definitions of severity . when severity is considered , it is important to consider the pathogenicity of an infectious agent ; infections with any pathogen of low pathogenicity , such as atypical mycobacteria or toxoplasma , are suggestive of an immune defect even if the infection is not severe for example , paracoccidioidomycosis should be more commonly identified in tropical countries than in european or north american countries ( table ii ) . a general condition that should make any infection suspicious is the concomitant presence of noninfectious complications commonly associated with pidd , including severe allergy , autoimmunity , unregulated inflammation , or malignancies . however , it is also important to consider certain negative family histories for example , a family in which only one of 4 siblings has recurrent infections . the fact that the other 3 siblings and parents are healthy rules out environmental factors as the factors predisposing an individual to recurrent infections ; it also highlights the immunopathogenic effects of genetic alterations that may have occurred in a single family member . recurrent infections are often attributed to the presence of non - immune abnormalities , such as down syndrome , muscular dystrophy , or various congenital heart defects . such patients may also have an immunodeficiency , and appropriate recognition and treatment may significantly improve their quality of life . many pidds have unique susceptibilities to some pathogens and/or sites of infections that are listed in table iii . antibody - mediated immunity needs to be evaluated in all severe or unusual infections , in addition to other possible concurring immunological defects . a practical point is to screen for combined immunodeficiencies.table iiiwarning signs of pidd for infectious disease specialistsclinical occurrencespiddlab screening testsinfections from extracellular bacteriaantibody deficienciesamicomplement deficienciesc , ananeutropeniaspirak-4 , myd88ii , rcpinfections due to neisseria meningitidis complement deficiency of terminal components ( membrane attack complex)c + ap50infection from s aureus , and gram - negative bacterias : serratia marcescens , burkholderia cepacia and gladioli , nocardia spp , chromobacterium violaceum , granulobacter bethesdensis.chronic granulomatous disease ( cgd)phyper ige syndrome ( hies)features : pneumonia from s aureus , eczema , fungal infection , joint hypermobility , coarse facial featuresserum ige , eosinophiliaspecific score infection from fungi : pneumocystis jiroveci ; aspergillus and candida albicans.t cell defectscmicd40 ligand ( l ) deficiencyamihiesserum ige , eosinophiliaspecific score cgdpinfection from candida albicans chronic mucocutaneous candidiasiscmi + t lymphocyte proliferation induced by candida infection by atypical mycobacteria / salmonella and/or bacillus calmette - gurin side effects ; paracoccidioides sp , leishmania , cryptococcus t cell deficienciescmisevere combined immunodeficiency ( scid)ami + cmimendelian susceptibility to mycobacterial diseasesp and/or iiinfections from herpes t and nk cell deficienciescmifulminant or chronic infection by epstein - barr virusfamilial hemophagocytic lymphohistiocytosis ( fhl ) syndromex - linked lymphoproliferative ( xlp ) syndromes , types 1 or 2cbc , triglycerides , ferritin , serology ebnarecurrent or persistent cryptosporidium , isospora cd40l deficiencyamicommon variable immunodeficiency ( cvid)ami giardiasis antibody deficienciesamicomplications due to bcg , rotavirus or varicella vaccinesscid , cgdcmi and/or ii and/or pcomplications due to oral polio vaccineantibody deficienciesamipersistent fever of unknown originautoinflammatory diseasesana , rcp , blood smear abbreviations : ami antibody mediated immunity , ana antinuclear antibodies , cbc complete blood count , cmi cellular mediated immunity , ebna epstein - barr nuclear antigen , ii innate immunity , p phagocytosis , rcp reactive c protein score for classical hyper ige diagnosis warning signs of pidd for infectious disease specialists abbreviations : ami antibody mediated immunity , ana antinuclear antibodies , cbc complete blood count , cmi cellular mediated immunity , ebna epstein - barr nuclear antigen , ii innate immunity , p phagocytosis , rcp reactive c protein score for classical hyper ige diagnosis infectious disease is the hallmark of pidd , and infectious disease specialists are the physicians most likely to encounter patients with pidd in their daily practice . the burden of infectious disease is immense even in children with healthy immune systems . recurrent otitis media is considered to be more than 4 episodes of acute otitis media during the past 12 months with complete resolution of the disease between episodes [ 1315 ] . any patient with severe or recurrent infections or infection by opportunistic or uncommon microorganisms must be investigated for pidd after discarding the possibility of infection with human immunodeficiency virus ( hiv ) . pyogenic encapsulated bacteria ( streptococcus pneumoniae , haemophilus influenzae ) are frequently associated with infections in patients with antibody or complement deficiencies . patients with complement deficiencies may also present with meningitis and sepsis associated with pathogens such as neisseria . agammaglobulinemic patients and , to a lesser extent , patients with common variable immunodeficiency ( cvid ) have an increased risk of developing bloodstream bacterial infections . a study of 201 patients with x - linked agammaglobulinemia ( xla ) indicated that 11 % had at least one episode of meningitis or encephalitis , 10 % had at least one episode of sepsis , and 8 % had at least one episode of septic arthritis . patients with xla or cvid are at an increased risk for severe and often fatal infections with enteroviruses , including echoviruses , polioviruses , and coxsackie viruses . the central nervous system is nearly always involved in these infections , and evidence of systemic involvement of muscle , liver , and/or joints is present in about 40 % of patients . other organisms , including staphylococcus aureus , pseudomonas spp , mycoplasma spp , enterobacteriaceae , campylobacter spp , giardia , and enteroviruses , may be isolated from patients with specific types of pidd . infections caused by serratia marcessens , nocardia spp , chrombacterium violaceum , granulobacter bethesdensis , burholderia cepacia , and gladioli are strongly suggestive of chronic granulomatous disease ( cgd ) . evaluation of 259 patients with the x - linked recessive form of cgd indicated that 21 % had a history of either bacteremia or fungemia . infections caused by mycobacteria , salmonella , leishmania , and cryptococcus suggest defects in the interferon-/interleukin-12 axis included in innate immunity ( table iii ) . as previously mentioned , the incidence of certain infectious agents must be considered according to their endemicity in particular geographical regions . side effects of bacillus calmette - gurin ( bcg ) occur in more than 50 % of the severe combined immunodeficiency ( scid ) patients in brazil and more than 17 % of cgd patients in latin america ; this is a relevant warning sign for pidd . adverse reactions to other live attenuated viral vaccines such as poliovirus and rotavirus should be carefully observed as well . several developing countries maintain campaigns with sabin immunization with the possibility to cause vaccine - derived disease . although the respiratory and gastrointestinal tracts are the most common systems for presenting illness , the authors observed lymphadenopathy as the most common manifestation in the group of combined t and b cell immunodeficiencies , and superficial abscesses and lymphadenopathy in the group of congenital defects of phagocyte number and/or function [ 22 , 25 , 26 ] ( table iii ) . high percentages of pediatric and adult patients with pidd have upper and/or lower respiratory tract bacterial infections ; thus , they may be referred to otorhinolaryngologists or pulmonologists . rezaei reported on 930 patients with pidd , 353 cases of which were identified in the last 5 years . the most common presenting feature of pidd was pneumonia , which was seen in 20.1 % of patients , followed by diarrhea ( 13.7 % ) , sinusitis ( 10.3 % ) , and otitis media ( 9.6 % ) . it is important to rule out the existence of cofactors that might be associated with upper respiratory infections ( e.g. , smoking , day care attendance , gastroesophageal reflux , allergy ) before performing extensive immunological evaluation . the most common of these infections are recurrent otitis media , chronic sinusitis , and pneumonia . recurrent sinus infections have been reported as a presenting occurrence in 19 % to 98 % of patients with pidd ( wide range reflects the variety of pidd ) [ 28 , 29 ] . an assessment of 103 pediatric and adult patients with a history of recurrent or chronic ear , nose , or throat infections indicated that 16.5 % had defects in antibody - mediated immunity : cvid ( n = 2 ) , iga deficiency ( n = 4 ) , igg subclass deficiency ( n = 3 ) , and specific antibody deficiency against polysaccharide antigens ( n = 8) . warning signs for patients with recurrent otitis include progressive infections leading to mastoiditis ; associated abscesses or systemic infections ; lack of responsiveness to adequate antibiotic treatment ; or occurrence of unusual , severe , or frequently relapsing infections in other sites ( table ii ) . pneumonia is a common infectious manifestation of pidd ; one study of patients with cvid indicated that at least two - thirds had one or more pneumonia diagnoses prior to diagnosis of pidd . according to some registries [ 33 , 34 ] , approximately 50 % of patients with humoral immunodeficiency suffered from upper respiratory tract infections , and 40 % of xla and cvid patients suffered from recurring pneumonia [ 27 , 35 ] . pathogens frequently associated with pneumonia in patients with antibody deficiencies include s pneumoniae , h influenzae type b , haemophilus parainfluenzae , mycoplasma spp , pseudomonas spp , and staphylococcus spp . patients with cellular deficiencies such as cd40 ligand ( cd40l ) deficiency present with pneumonia that is a result of opportunistic pathogens such as pneumocystis , fungal , or viral infections ( table iv).table ivwarning signs of pidd for pulmonologistsclinical occurrencespiddlaboratory testspneumonias due to extracellular bacteria + otitis and sinusitisantibody deficienciesamicomplement deficienciesc , anapulmonary abscesspneumatocelehyper ige syndrome ( hies)features : pneumonia by s aureus , eczema , fungal infection , joint hypermobility , coarse facial featuresserum ige , eosinophiliaspecific score pneumonias due to staphylococcus or fungichronic granulomatous disease ( cgd ) : susceptibility to infections by catalase positive microorganisms . other infections : adenitis , liver abscess , osteomyelitispglucose-6-phosphate dehydrogenase ( g6pd ) deficiencymyeloperoxidase deficiency ( common in diabetes)g6pd activityperoxidase levelhiesserum ige , eosinophiliaspecific score pneumonia due to p jiroveci t cell deficiencies / cd4 lymphopeniacmi , amilymphoproliferation assaycd40 ligand ( l ) deficiencyami , cmiwiskott - aldrich syndrome ( was ) , eczema + thrombocytopeniacbc including platelet number and size ( small sized platelets ) ; cmi , amipneumonia due to mycobacteria tuberculosis or atypical mycobacteriat cell deficiencies / cd40l deficiencycmi , amimendelian susceptibility to mycobacterial diseasesii abbreviations : ami antibody mediated immunity , ana antinuclear antibodies , cbc complete blood count , cmi cellular mediated immunity , ii innate immunity , p phagocytosis score for classical hyper ige diagnosis warning signs of pidd for pulmonologists abbreviations : ami antibody mediated immunity , ana antinuclear antibodies , cbc complete blood count , cmi cellular mediated immunity , ii innate immunity , p phagocytosis score for classical hyper ige diagnosis several observational studies have indicated that complications and sequelae of infectious diseases such as bronchiectasis ( 76 % ) , recurrent chest infections ( 21 % ) , and granulomatous lung disease ( 5 % ) should also raise suspicion of pidd . a systematic review of the literature conducted by wood et al . included results from 7 surveys showing that respiratory / chest infections were presenting symptoms for 37 % to 90 % of patients with pidd . reviewed 26 studies that included 587 patients with cvid and found that up to 73 % developed chronic structural pulmonary complications most often bronchiectasis and bronchial wall thickening . in another study , pulmonary imaging by chest computed tomography in 30 patients with pidd revealed abnormalities in 53 % ; among these were bronchiectasis ( 75 % ) , peribronchial thickening ( 19 % ) , air trapping ( 31 % ) , lung volume reduction ( 25 % ) , atelectasis ( 12 % ) , follicular bronchiolitis ( 12 % ) , ground - glass abnormality ( 12 % ) , and parenchyma nodules ( 6 % ) . in this study , pulmonary function testing also indicated abnormalities in 18 patients ; these included an obstructive ( 38.8 % ) , restrictive ( 44.4 % ) , or mixed ( 16.7 % ) pattern of lung function abnormalities . results from other studies have indicated that staphylococcus - associated pneumonia and bronchial aspergillosis are seen in cgd ; lung abscesses represent usual characteristics in hyper ige syndrome ( hies ) ; and interstitial pneumonia may be observed in patients with scid ( table iv ) . frequent gastrointestinal manifestations are malabsorption , diarrhea , hepatomegaly , and inflammatory bowel disease ( ibd ) and all of these are commonly associated with increased morbidity [ 4244 ] . in a systematic review , indicated that gi infections were a presenting symptom in 6 % to 19 % of patients with pidd . severe diarrhea can start early in life in patients with scid or immunodysregulation , polyendocrinopathy , and enteropathy , x - linked ( ipex ) ; both of these pidd are considered pediatric emergencies [ 4547 ] . rezei et al . , in their study of 930 patients with pidd , indicated that infectious diarrhea occurred in 40.4 % of patients . other pathogens in patients with infectious colitis include rotavirus , campylobacter , enteroviruses , cryptosporidium parvum , salmonella spp , and clostridium difficile . liver abscesses caused mainly by s aureus may also be seen in patients with cgd and hies ( table v).table vwarning signs of pidd for gastroenterologistsclinical occurrencespiddlaboratory testschronic diarrheaantibody deficienciesamiinflammatory bowel diseasecombined immunodeficiencies ( infants)cmi , amichronic giardiasisautoimmune enteropathy + severe intractable diarrhea . other diagnoses associated : hypothyroidism , eczema , thrombocytopenia , autoimmune hemolytic anemia , neonatal diabetesimmunodysregulation , polyendocrinopathy and enteropathy , x - linked ( ipex)cmi , coombs , glycemia , and tshanapersistent candidiasiscombined immunodeficienciescmit cell lymphoproliferative assaychronic mucocutaneous candidiasislymphoproliferation to candida autoimmune polyendocrinopathy - candidiasis - ectodermal dystrophy ( apeced)candidin testana and endocrine evaluationsevere abdominal pain emulating an acute abdomenhereditary angioedemadosage and/or functional activity assay of c1inh , c4 , c1qliver abscess mainly due to s aureus chronic granulomatous disease ( cgd)phyper ige syndrome ( hies)serum ige , eosinophiliaspecific score hepatobiliary infection due to c parvum cd40 ligand ( l ) deficiencyamiinflammatory bowel disease in infantscgdpinterleukin-10 ( il-10 ) or interleukin 10 receptor ( il-10r ) deficienciesii abbreviations : ami antibody mediated immunity , ana antinuclear antibodies , c1inh c1 esterase inhibitor , cmi cellular mediated immunity , cbccomplete blood count , ii innate immunity , p phagocytosis , tsh thyroid stimulating hormone score for classical hyper ige diagnosis warning signs of pidd for gastroenterologists abbreviations : ami antibody mediated immunity , ana antinuclear antibodies , c1inh c1 esterase inhibitor , cmi cellular mediated immunity , cbccomplete blood count , ii innate immunity , p phagocytosis , tsh thyroid stimulating hormone score for classical hyper ige diagnosis results from a study focusing mainly on pediatric patients with pidd indicated clinical evidence of liver disease , including hepatomegaly , in 35.5 % ; 8 patients ( 13 % ) had clinical and/or laboratory evidence of chronic liver disease . hepatobiliary infection , sclerosing cholangitis , nodular regenerative hyperplasia , and portal hypertension are frequent conditions in cd40l deficiency [ 48 , 49 ] . the risk for autoimmune diseases is elevated in patients with pidd and is sometimes the only clinical manifestation of their causative condition . results from a larger study of 248 patients with cvid indicated that 69 ( 27.8 % ) had autoimmune disease . the most common conditions were thrombocytopenia in 18 patients , hemolytic anemia in 12 , rheumatoid arthritis in 5 , and juvenile rheumatoid arthritis in 4 [ 50 , 51 ] . results from another study of 189 patients with systemic lupus erythematosus ( sle ) indicated that 6 % had iga deficiency . other pidd are the result of genetic defects that lead to immune dysregulation associated with autoimmune occurrences . patients with autoimmune lymphoproliferative syndrome ( alps ) develop generalized lymphadenopathy , hepatosplenomegaly , hypergammaglobulinemia , b cell lymphocytosis , and autoimmune manifestations that commonly include hemolytic anemia , glomerulonephritis , and idiopathic thrombocytopenic purpura . autoimmune polyendocrinopathy , candidiasis , and ectodermal dystrophy ( apeced ) are also associated with symptoms of autoimmune disease . patients with ipex have mutations in the foxp3 gene in regulatory t cells , which leads to severe autoimmunity with a high mortality rate . first components of complement deficiency have also been shown to be associated with conditions such as sle and are likely to prompt referral to a rheumatologist . an important red flag for sle and complement defects is negative serology to double - stranded dna . the most common of these is arthritis , which may be associated with antibody deficiencies , and , less often , with cgd and wiskott - aldrich syndrome ( was ) [ 58 , 59 ] . importantly , infections with ureaplasma urealyticum , and mycoplasma ssp may lead to erosive arthritis in patients with severe antibody deficiencies . these infections most often result in large - joint monoarthritis involving the knee , shoulder , elbow , or hip joints , and less often in symmetrical polyarthritis . patients with certain pidd , such as autoinflammatory syndromes , will present with generalized serositis that might be confused with infectious arthritis . this is often accompanied by a myriad of symptoms , including elevation of acute phase reactants , rash , deafness , joint deformity , and multifocal osteomyelitis . patients with pidd may have skin conditions including fungal , bacterial , or viral infections ; eczematous dermatitis ; erythroderma ; skin symptoms of autoimmune diseases such as sle or scleroderma ; vasculitis ; granuloma formation ; and/or problems characteristic of specific immunodeficiency syndromes ( e.g. , telangiectasia , gray hair , and depigmentation ) . an immunological assessment of 382,383 pediatric patients admitted to an outpatient dermatology group indicated that 130 had pidd . of these , 69 % had skin infections , 29 % had eczema - dermatitis , and 44 % had other associated cutaneous conditions . in 79 % of this cohort , cutaneous abnormalities preceded and were the basis for the clinical immunologic diagnosis , demonstrating the relevance and high frequency of dermatologic manifestations in pidd patients . results from a cross - sectional study of 210 pediatric patients with pidd ( 68 with antibody deficiencies , 22 with t cell and combined deficiencies , 57 with phagocytic defects , and 63 with other pidd ) indicated that 67 of the patients ( 31.9 % ) had cutaneous abnormalities preceding the diagnosis of pidd . candida infections that may be among the first signs of pidd include mucocutaneous candidiasis , candida paronychia , granuloma formation , and erythroderma . patients with phagocytic , cellular , combined , and other pidd exhibit immune deficits that confer increased susceptibility to fungal infections . infections associated with candida , aspergillus , cryptococcus , histoplasma , paecilomyces , scedosporium , trichosporon , penicillium , and other fungal organisms have been observed in patients with cgd , scid , hies , defects in the interferon-/interleukin-12 axis , digeorge syndrome/22q deletion syndrome , cd40l deficiency , was , and cvid . patients with chediak - higashi or griscelli syndromes present partial albinism and gray hair ; dermatologists are frequently the first consulted specialists . prompt diagnosis is established by demonstration of giant intracellular granules in eosinophils , basophils , and monocytes , and are pathognomonic of this disease . eczema and high serum ige levels in the first months of life frequently lead to referral to allergists due to suspicion of cow milk protein allergy . however , these occurrences are also present in patients with hypomorphic scid , ipex , was , dock8 , and hies ; it has been shown that differential diagnosis is crucial for prognosis . zhang et al . described dock8 mutations in patients with a variant of combined immunodeficiency characterized by unusual susceptibility to cutaneous viral infections and cancers . in addition , dock8 deficiency was found in a subgroup of patients who were previously thought to have autosomal recessive hies with severe allergic manifestations . these syndromes exemplify the need for improved knowledge of pidd for dermatologists ( table vi ) . in addition , angioedema without urticaria is often referred to both allergists and dermatologists . family histories are helpful for such cases , and c4 levels could be a useful screening test .table viwarning signs of pidd for dermatologistsclinical occurrencespiddlaboratory testeczemawiskott - aldrich syndrome ( was)cbc including platelet number and size ( small sized platelets ) ; cmi , amihyper ige syndrome ( hies)serum ige , eosinophiliaspecific score immunodysregulation , polyendocrinopathy and enteropathy , x - linked ( ipex)cmi , ana , rcpcoombs , glycemia , and tshsevere combined immunodeficiency ( scid ) , erythrodermacmicutaneous lesions by mycobacteria combined immunodeficienciescmihyper - igm syndromesamimendelian susceptibility to mycobacterial diseasesiichronic granulomatous diseases ( cgd)ppartial albinism , gray hairchediak - higashi syndromeenlarged cytoplasm granules in blood smeargriscelli syndrometelangiectasiasataxia - telangiectasiaami ; serum alfa - feto proteindisseminated warts and molluscumwarts , hypogammaglobulinemia , infections , and myelokathexis ( whim ) syndromeami , cmi lymphoproliferation assaycutaneous herpes infectionsdedicator of cytokinesis 8 ( dock8 ) deficiencyidiopathic cd4 lymphopeniafragile hair , conic teethectodermal dysplasiaii abbreviations : ami antibody mediated immunity , ana antinuclear antibodies , cbc complete blood count , cmi cellular mediated immunity , ii innate immunity , p phagocytosis , rcp reactive c protein , tsh thyroid stimulating hormone score for classical hyper ige diagnosis warning signs of pidd for dermatologists abbreviations : ami antibody mediated immunity , ana antinuclear antibodies , cbc complete blood count , cmi cellular mediated immunity , ii innate immunity , p phagocytosis , rcp reactive c protein , tsh thyroid stimulating hormone score for classical hyper ige diagnosis autoimmune hemolytic anemia , thrombocytopenia , and/or neutropenia are also seen in patients with selective iga deficiency , cvid , and class switch recombination ( csr ) defects [ 50 , 71 ] . in some patients , wiskott - aldrich syndrome is associated with defects in both b - lymphocyte and t - lymphocyte function , and these patients also have intrinsic platelet abnormalities and significant thrombocytopenia with small platelets . in addition to congenital neutropenia , neutropenia is also associated with xla , cd40l deficiency , cvid , scid , and shwachman - diamond syndrome [ 72 , 73 ] . hemophagocytic lymphohistiocytosis ( hlh ) is characterized by multisystem inflammation , a reactive process resulting from hyperactivation of macrophages , histiocytes and cd8 t cells , and abnormalities in the function of natural killer cells ( nk cells ) . the most frequent clinical and laboratorial findings include fever , splenomegaly , cytopenias , hypertriglyceridemia , and elevated ferritin . both chediak - higashi and griscelli syndromes present as hemophagocytic , with an accelerated phase affecting all patients by late childhood . this accelerated phase is characterized by the infiltration of nonmalignant lymphoid and histiocytoid cells into the viscera , usually induced by epstein - barr virus . clinical findings include hepatosplenomegaly , lymphadenopathy , and pancytopenia . impaired humoral and cellular immune responses , including disturbances in b , t , nk , and dendritic cells , along with chronic inflammatory autoimmune diseases , recurrent bacterial infections , and persistent antigenic stimulation , are speculated to favor carcinogenesis in cvid patients . lymphoma and gastric cancer are the most frequent neoplasms related to this immunologic defect [ 76 , 77 ] . the risk of cancer in pidd patients mainly takes the form of t cell malignancies ( 70-fold and 250-fold increased risks of leukemia and lymphoma , respectively ) , and b cell malignancies are high when compared with the general population . the spanish registry identified that 1.3 % of the 1,069 patients developed cancer ; however , the survey was developed within reference hospitals . non - hodgkin lymphoma and hodgkin lymphoma are 2 of the most common pidd - associated malignancies . a recent large - scale assessment of 1,132 patients in the pidd registry of the australasian society of clinical immunology and allergy indicated that this group had a 1.6-fold excess relative risk of cancer . the relative risk was significantly increased for non - hodgkin lymphoma , leukemia , and stomach cancer ( table vii).table viiwarning signs of pidd for hematologistsclinical occurrencespiddlaboratory teststhrombocytopenia with small - sized plateletswiskott - aldrich syndrome ( was)cbc including platelet number and size ( small sized platelets ) ; cmi , amiother symptoms : eczema and recurrent infectionsx - linked thrombocytopeniaautoimmune cytopenias ( autoimmune anemia , thrombocytopenia and neutropenia)common variable immunodeficiencyami , anaother features : recurrent infectionsfever , splenomegaly without evidence of malignancy , cytopeniashemophagocytic lymphohistiocytosis ( hlh)cbc , triglycerides , ferritin , ebnalymphadenopathy + splenomegalyautoimmune lymphoproliferative diseaseincreased number of alpha beta double - negative t cells ( cd3 + cd4-cd8- ) , ana , rcpexcluding neoplasias and infectionsapoptosis defectsquantitative and qualitative defects of neutrophils ( neutropenia and neutrophilia)neutropeniaspchronic granulomatous disease ( cgd)leukocyte adhesion deficiencyleukocytosis , cd18 cellspartial albinism , chediak - higashi or griscelli syndromeenlarged cytoplasm granules abbreviations : ami antibody mediated immunity , ana antinuclear antibodies , cbc complete blood count , cmi cellular mediated immunity , ebna epstein - barr nuclear antigen , p phagocytosis , rcp reactive c protein warning signs of pidd for hematologists abbreviations : ami antibody mediated immunity , ana antinuclear antibodies , cbc complete blood count , cmi cellular mediated immunity , ebna epstein - barr nuclear antigen , p phagocytosis , rcp reactive c protein during recent years , new pidd have been identified , including some with susceptibility to specific infectious agents and some diagnosed only in adulthood . training specialists to recognize pidd is important in order to identify the diverse pidd phenotypes . the majority of patients initially present to hospital doctors ; therefore , a focus on hospital specialists has been suggested . introducing other specialists and health care workers to the relevant warning signs will facilitate the early diagnosis of pidd in patients , leading to better treatments with less sequelae , or possible cures . in this review these warning signs are further influenced by the socioeconomic , climate - related , and living conditions found in different regions of the world . a review of warning signs that are specific to different areas of the world should eventually be undertaken by a worldwide pidd community of which lasid plays an important part . this work was supported by the latin american society of primary immunodeficiencies through an unrestricted educational grant from baxter bioscience . dr . grumach is on the advisory board of baxter , shire , and dyax pharmaceuticals , has consulted for and received grants , payments , and travel support from shire , and has received travel expenses from csl behring . franco is a board member and consultant for and has received payments and travel support from baxter . dr . condino - neto has received research grants from fundacao de amparo a pesquisa do estado de so paulo , conselho nacional de desenvolvimento cientfico e tecnolgico , brazil , and baxter bioscience research grants . the other authors had nothing to disclose .

purposepatients with primary immunodeficiency diseases ( pidd ) may present with recurrent infections affecting different organs , organ - specific inflammation / autoimmunity , and also increased cancer risk , particularly hematopoietic malignancies . the diversity of pidd and the wide age range over which these clinical occurrences become apparent often make the identification of patients difficult for physicians other than immunologists . the aim of this report is to develop a tool for educative programs targeted to specialists and applied by clinical immunologists.methodsconsidering the data from national surveys and clinical reports of experiences with specific pidd patients , an evidence - based list of symptoms , signs , and corresponding laboratory tests were elaborated to help physicians other than immunologists look for pidd.resultstables including main clinical manifestations , restricted immunological evaluation , and possible related diagnosis were organized for general practitioners and 5 specialties . tables include information on specific warning signs of pidd for pulmonologists , gastroenterologists , dermatologists , hematologists , and infectious disease specialists.conclusionsthis report provides clinical immunologists with an instrument they can use to introduce specialists in other areas of medicine to the warning signs of pidd and increase early diagnosis . educational programs should be developed attending the needs of each specialty .

Introduction Methods General Screening and Evaluation Recurrent, Severe, or Unusual Infections Infectious Diseases Otorhinolaryngology and Pulmonology Gastroenterology Rheumatology Dermatology Hematology/Oncology Conclusions Support Conflicts of interest and funding sources

the immune system consists of two closely related systems known as the innate and adaptive immune systems . the adaptive immune system responds to specific nonself antigens and generates immunological memory . in contrast , the innate immune system provides an immediate first line of defense against a diverse repertoire of invading microbial pathogens . the key components of innate immunity , cognate pattern recognition receptors ( prrs ) , are considered to act as sentinels against both invading organisms bearing pathogen - associated molecular patterns ( pamps ) and damage - associated molecular pattern molecules ( damps ) . toll - like receptors ( tlrs ) are good examples of these receptors . because of their wide - ranging impact upon both innate and adaptive immunity in several disease settings , tlrs and this review summarizes the main players in innate immune signaling and highlights possible drug targets in various disease settings . innate immune responses are triggered mainly by a spectrum of danger signals referred to as pamps and damps . pamps are exogenous molecules derived from both pathogenic and nonpathogenic microbes . in contrast , the vast majority of damps are endogenous molecules released from dying host cells molecules upon cellular stress or tissue damage [ 25 ] . the tlrs are a family of evolutionarily conserved prrs that play a key role in sensing the microbial world . different tlr members are reported to recognize and respond to different pamps and some endogenous damps , thus initiating innate immune responses and priming antigen - specific adaptive immunity , both in infectious and noninfectious disease scenarios . tlrs are type i transmembrane glycoproteins which are structurally characterized by extracellular leucine - rich repeats ( lprs ) and toll / il-1 receptor ( tir ) signaling domains . the first tlr to be characterized was tlr4 and the family has now been expanded to include 10 members in humans and 12 members in mice [ 2 , 3 ] . apart from the highly conserved pathogenic components of bacteria , viruses , fungi , and parasites , various tir domain - containing adaptors such as myd88 , tirap , trif , and tram become engaged or activated upon ligation of tlrs , as diagrammed in figure 1 . tlr1 , tlr2 , tlr4 , and tlr6 recruit tirap , which serves as an adaptor between the tir domain of tlrs and myd88 , while tlr5 , tlr7 , tlr9 , and tlr11 can recruit myd88 directly . however , the recruitment of trif by tlr4 needs the participation of tram ( figure 1 ) . the recruitment of these adaptors triggers a cascade of signaling molecules and ultimately activates the transcription factors , nf-b and irfs ( figure 1 ) . these transcription factors induce the expression of various inflammatory cytokines , type i interferons , and chemokines . nf-b is a central regulator of immune responses involved in cell proliferation and survival and induces the expression of many cytokine and chemokine genes including il-2 , il-6 , il-12 , mcp-1 , and tnf- . except for tlr3 , all tlr ligation events recruit the adaptor myd88 , followed by the irak family of protein kinases , leading to the activation of traf6 . nf-b or ap-1 is then activated by the ikk complex or map kinases , respectively ( figure 1 ) . on the other hand , tlr3 initiates a trif - dependent pathway that dictates the expression of inflammatory cytokines and type i ifns via two independent pathways . the n - terminal domain of trif interacts with traf6 while the c - terminal domain of trif interacts with rip1 and activates tak1 , both of which can activate nf-b , resulting in the expression of inflammatory cytokines . irf3 is phosphorylated and activated by the ikk - related kinase , tbk1 , facilitated by the recruitment of traf3 . tlr7 and tlr9 engagement induces the secretion of inflammatory cytokines through the activation of nf-b via myd88 . however , tlr7 and tlr9 can also induce the expression of type i ifns through the activation of irf7 , which is phosphorylated by irak1 , irak4 , or ikk , and then translocated to the nucleus where it induces the transcription of ifn- ( figure 1 ) . as is evident from figure 1 , the irak family of molecules plays a pivotal role in mediating almost all tlr - mediated functions . the irak family has four members : irak1 , irak2 , irakm , and irak4 . all irak family members contain an amino - terminal death domain and a serine - threonine kinase domain . the irak1 gene is composed of 14 exons and is located on the x chromosome . irak1 is the first member of this kinase family that was identified as a key component of the il-1r signaling pathway and plays a specific and essential role in ifn- induction downstream of tlr7 and tlr9 engagement . irak1 deficiency abrogates the production of ifn- , il-6 , il-12 , and tnf- when stimulated with tlr7 or tlr9 ligands [ 12 , 13 ] . tlrs constitute a primary defense mechanism in both infections and some noninfectious disease settings in mammals . activation of tlrs and the myd88 signaling pathway plays a protective role during infection with several pathogens , including protozoan parasites and pyogenic bacteria . patients with autosomal recessive myd88 deficiency have been reported to suffer from life - threatening , often recurrent pyogenic bacterial infections . interestingly , however , their clinical status improved in later life , alluding to the compensatory effect of adaptive immunity . consistent with their vital role in fighting infections , downregulation of tlr - related molecules or signaling has been associated with sepsis and autoimmune disease , as discussed below . on the other hand , upregulation of these molecules has been linked to cancer , allergy , other autoimmune diseases and immune abnormalities in hiv [ 16 , 17 ] , as detailed below . sepsis is a serious medical condition that is characterized by a systemic state and the presence of a known or suspected , which can lead to rapid tissue damage . the recognition of lps by tlr4 during an acute gram - negative bacterial infection plays a key role in the pathogenesis of sepsis . weighardt and colleagues found that myd88-deficient mice were protected from developing sepsis in a polymicrobial septic peritonitis disease model . in the irak4-deficient model , it was found that mice were severely impaired in their responses to viral and bacterial challenge . recently , ferwerda and coworkers reported that polymorphisms in human mal / tirap allele showed association to the pathogenesis of sepsis . on the other hand , others have provided proof that mkp-1 , a key negative regulator of tlr - induced inflammation , plays a critical role in the inhibition of innate immunity during gram - negative bacteria sepsis [ 21 , 22 ] . myd88 also plays a pivotal role in infectious diseases , as demonstrated by naiki et al . in another model of bacterial pneumonia . collectively , these studies revealed that the tlr pathway plays a pivotal role in the pathogenesis and control of sepsis . the importance of tlr expression and function in cancer cells and its association with tumorigenesis and tumor progression have recently been examined . tlr4 expression is linked to several cancers such as gastric cancer and human epithelial ovarian cancer . huang et al . reported that tlr4 was expressed in murine tumor cells and that the activation of tlr4 in these cells by lps induced the expression of various soluble factors including interleukin-6 , inducible nitric oxide synthase , interleukin-12 , b7-h1 , and b7-h2 and rendered tumor cells resistant to ctl attack . the factors in lps - stimulated tumor cell supernatants also had the capacity to inhibit t cell proliferation and natural killer cell activity . have reported that human epithelial ovarian cancer cells ( eoc ) ubiquitously express tlr4 and that the proliferation of eoc cells or enhanced cytokine / chemokine production by eoc cells was dependent upon expression of myd88 . myd88-dependent signaling controls the expression of several key modifier genes in tumorigenesis and has a critical role in both spontaneous and carcinogen - induced tumor development . others have reported that gene expression differences in the tlr gene cluster ( tlr10-tlr1-tlr6 ) were associated with a statistically significant reduced risk of prostate cancer . functionally active tlr9 is also known to be expressed on several human tumors , including lung cancer , burkitt lymphoma , cervical neoplasia , breast cancer , and prostate cancer [ 2832 ] . although the role of tlr9 in the development of these cancers is not fully understood , tlr9 appears to be playing an important modulatory role in several of these tumors [ 3235 ] . based on these findings , several tlr9 agonists , including cpg oligonucleotides , these agents have the capacity to upregulate the innate immune response , as well as a humoral immune response against the tumor , as reviewed elsewhere .thus , cpg - odn can activate plasmacytoid dcs ( pdcs ) to secrete type i interferon ( ifn ) and also promote the expression of costimulatory molecules such as cd80 and cd86 , on the dcs as well as on tlr9-expressing tumors . this can then lead to the secretion of various cytokines / chemokines , and also activate natural killer ( nk ) cells , th1 cells , and cytotoxic t lymphocytes ( ctls ) . engaging these diverse mechanisms , tlr9 agonists have been found to be therapeutically effective in various cancer trials [ 3640 ] . it was reported that inhibition of tlr4 suppressed the severity of experimental arthritis and resulted in lower il-1 expression in arthritic joints . roelofs found that a tlr4 variant ( asp299gly ) could reduce its potency to mediate signaling in rheumatoid arthritis patients . a tlr2 r753q polymorphism has been reported to be more prevalent among reactive arthritis patients . likewise , polymorphisms in tlr2 and tlr4 may also be important in the pathogenesis of sle in both patients and mouse models . moreover , tlr7 has been suggested to be functionally involved in autoantibody production , and hence closely linked to the pathogenesis of sle . indeed , the translocation of the x - linked tlr7 gene to the y - chromosome has been documented to facilitate the development of fatal lupus in mice with numerous immunological aberrations , hence constituting disease genes for murine lupus [ 47 , 48 ] . harada and coworkers reported that myd88-deficiency protected mrl / lpr mice from the development of autoimmune nephritis , marked by lower levels of serum anti - double - stranded dna ( anti - dsdna ) antibodies and reduced cytokines , including interferon- , interleukin-12 , il-6 , and ifn- . similarly it was reported that myd88-deficient mice were completely resistant to experimental autoimmune uveitis ( eau ) and had reduced th1 , but not th2 , responses upon immunization with retinal ag . in all of the above studies of sepsis , cancer , and autoimmunity , it has been documented that myd88-deficient mice show a loss of il-1- and il-18-mediated functions indicating that the phenotypes observed in myd88-deficient mice might arise in part because of defects in signaling downstream of the il-1 and il-18 receptors . autoimmunity also appears to be contingent upon the expression of another tlr signaling molecule , irak1 . it has been reported that irak1-deficient mice are resistant to experimental autoimmune encephalomyelitis ( eae ) , exhibiting little or no inflammation in the central nervous system . irak1-deficient mice exhibited impaired th1 cell development with minimal ifn- secretion during disease induction following immunization with myelin antigen . the authors suggested that the mechanism underlying this observation might relate to an impaired adjuvant effect on antigen presenting cells as a result of suboptimal tlr activation . our recent study has revealed that the irak1 gene was highly associated with both adult- and childhood - onset sle . using the irak1-deficient mice , we found that in mice bearing the lupus susceptibility loci , sle1 or sle3 , irak1 deficiency abrogated all lupus - associated phenotypes , including igm and igg autoantibodies , lymphocytic activation , and renal disease . in addition , the absence of irak1 reversed the dendritic cell hyperactivity associated with lupus . heme oxygenase-1 ( ho-1 ) , a key cytoprotective , antioxidant , and anti - inflammatory molecule , was reported to be involved in the activation of irf3 after tlr3 or tlr4 stimulation , or viral infection . ho-1 is necessary for the expression of primary irf3 target genes encoding rantes , ip-10 , and mcp-1 . in the experimental autoimmune encephalomyelitis model , mice with myeloid - specific ho-1 deficiency developed severe disease correlating with hyperactive antigen - presenting cells , enhanced infiltration of th17 cells , and nonregressing myelin - specific t cell reactivity . the overproduction of ifn- and ifn--dependent genes , such as irf5 , are additional factors that play a key role in the pathogenesis systemic lupus erythematosus . since the bulk of the data suggests that the tlr pathway plays a key role in multiple pathogenic processes , the targeting of either the tlrs themselves or the signals they generate is of great interest , as reviewed by o'neill . as myd88 is clearly involved in infectious disease , cancer , and autoimmune diseases , it is obviously an attractive target for intervention in these diseases . loiarro et al . have reported a myd88 inhibitor st2825 , a heptapeptide analog specifically designed to inhibit myd88 dimerization , which could control tlr mediated inflammatory responses . st2825 is reported to inhibit myd88 dimerization in co - immunoprecipitation experiments , and is specific for homodimerization of the tir domains but does not affect homodimerization of the death domains . they tested st2825 in experimental animal models of autoimmune , and inflammatory diseases , such as lupus , inflammatory bowel disease , and multiple sclerosis , and found that st2825 could interfere with the recruitment of irak1 and irak4 by myd88 , resulting in the inhibition of il-1-mediated activation of nf-b and il-6 . they also observed that st2825 suppressed b cell proliferation and differentiation into plasma cells in response to cpg - induced activation of tlr9 . compound 4a , which could interfere with the interaction between myd88 and il-1r at the tir domains . compound 4a inhibited il-1-induced phosphorylation of the mitogen - activated protein kinase p38 in el4 thymoma cells and in freshly isolated murine lymphocytes , and significantly attenuated il-1-induced fever response in vivo . these data suggest that myd88 might be a potential target for pharmaceutical usage in several disease settings where increased tlr signaling is driving disease pathogenesis . modulation of another signaling molecule in the tlr pathway , irak4 , has also been suggested to be an attractive therapeutic approach for the treatment of cancer , autoimmune and inflammatory diseases . a series of small - molecule compounds that inhibit irak4 has been developed by wang et al . . however , the effectiveness of these irak4 inhibitors in vitro and in vivo remains to be documented . another group has reported that a dual inhibitor of irak1 and irak4 ( ro0884 ) could efficiently inhibit both il-1 and tnf- induced p38 map kinase , c - jun n - terminal kinase activation , and il-6 production in human cells . through silencing irak1 or irak4 gene by sirna , they also found that irak4 was essential for response to il-1 but not to tnf- , whereas irak1 was essential for cellular responses to tnf- but not il-1. these data suggested that inhibition of both irak1 and irak4 kinases would be necessary to block proinflammatory cytokine production . collectively , these findings raise hope that irak1/irak4 inhibitors may be of therapeutic benefit in autoimmune and inflammatory diseases . administration of synthetic oligodeoxynucleotides with immunoregulatory sequences ( irs ) that specifically inhibit both tlr7 and tlr9 signaling ( irs 954 ) has been shown to decrease the production of ifn- by human pdcs after stimulation with dna or rna viruses . it has been reported that ( nzbnzw ) f1 mice showed decreased serum levels of antinuclear autoantibodies , proteinuria , and glomerulonephritis , with increased survival upon irs 954 treatment . another oligodeoxynucleotides inhibitor irs 661 , which is specific to tlr7 , had the capacity to significantly reduce disease in mrl / lpr mice , particularly the weights of spleen and lymph nodes and serum levels of tnf- as compared with saline - treated control . injection of irs 661 but not irs 954 can significantly decreased anti - dsdna and anti - sm antibodies in lupus - prone mice . most interestingly , both irs 661 and irs 954 could be detected in the kidneys and were found to be taken up by glomerular cells , tubular epithelial cells , and intrarenal macrophages . the mrna levels of inflammatory chemokines ccl2 and ccl5 and their respective chemokine receptors ccr2 and ccr5 were also decreased in the kidneys . consistent with these findings , the activity and chronicity scores for antibody - mediated nephritis in irs 661- or irs 954-treated mice were also reduced significantly . besides lupus , these inhibitors have also shown efficacy in other autoimmune diseases including arthritis and multiple sclerosis [ 6365 ] . based on a recent report suggesting that sle in patients is also contingent upon tlr signaling , antagonists of this pathway are expected to confer therapeutic benefit in patients as well . tlr molecules and their downstream signaling pathways play a critical role in activating innate immune cells and selected cell types in the adaptive arm of the immune system . given that this pathway is aberrantly expressed or activated in several diseases , this axis constitutes an attractive target for therapeutic intervention . there is mounting evidence documenting that the crippling of this pathway at the level of tlr , myd88 , or irak1/4 may confer therapeutic efficacy in autoimmunity and auto - inflammatory diseases . on the other hand , the total crippling of these pathways may compromise immune defense against invading infections and immune surveillance against cancers . in fact , agonists of these pathways appear to be useful in some of these disease settings . hence there is a need to carefully select the therapeutic target in the tlr signaling cascade , and closely regulate the degree of pathway activity so as to attain the desired therapeutic end - point . like many other molecules in biology , the tlr signaling axis emerges as yet another

toll - like receptors ( tlrs ) are transmembrane proteins acting mainly as sensors of microbial components . triggering tlrs results in increased expression of multiple inflammatory genes , which then play a protective role against infection . however , aberrant activation of tlr signaling has a significant impact on the onset of cancer , allergy , sepsis and autoimmunity . various adaptor proteins , including myd88 , iraks , tirap , trif , and tram , are involved in specific tlr signaling pathways . this article reviews the role of these molecules in tlr signaling , and discusses the impact of this pathway on various disease scenarios . given their important role in infectious and non - infectious disease settings , tlrs and their signaling pathways emerge as attractive targets for therapeutics .

1. IntroductionThe TLR Pathway 2. TLR Signaling and Disease 3. Potential Therapeutic Targets in the TLR Signaling Pathway 4. Conclusion

headache is a notable and common somatic complaint in paediatric age [ 1 , 2 ] . although migraine and tension - type headache show the highest incidence , other primary headaches are much rarer and poorly recognised , thus representing a clinical and therapeutic challenge . chronic paroxysmal hemicrania ( cph ) is a rare and well - characterised headache , classified amongst the trigeminal autonomic cephalgias ( tacs ) . cph is characterised by short - lasting ( 230 min ) and multiple pain attacks , with a typical attack frequency of more than 5 per day [ 36 ] . pain is unilateral , located on the fronto - orbital - temporal region , it has rapid onset and it is described as excruciating . attacks occur in combination with al least one ipsilateral autonomic symptom , such as conjunctival injection , lachrymation , miosis , ptosis , eyelid oedema , and nasal congestion or rhinorrhea [ 3 , 7 ] . the standard cph treatment is based on the use of indomethacin . the absolute pain responsiveness to indomethacin represents one of the diagnostic criteria for cph ( table 1).table 1diagnostic criteria for paroxysmal hemicrania ( ichd - ii)a . attacks completely prevented by therapeutic doses of indomethacin ( mg kg day)f . not attributed to another disorder diagnostic criteria for paroxysmal hemicrania ( ichd - ii ) because of its typical onset between 20 and 30 years of age [ 5 , 6 ] , cph has been extensively studied in adult population . the disease can occur , although more rarely , also in children [ 816 ] , as early as from the age of 3 [ 9 , 12 ] . however , few reports in paediatric age fulfilled the diagnostic criteria for cph [ 9 , 10 , 12 ] according to the international classification of headache disorders , 2nd edition ( ichd - ii ) ( table 2 ) . in particular , the presence of autonomic signs and response to indomethacin has not been discussed adequately [ 11 , 14 , 15 , 18 ] . some unilateral short - lasting paediatric headaches without autonomic signs have been diagnosed as cph [ 15 , 18 ] . moorjani described a case of unilateral , short - lasting and severe headache responsive to indomethacin , but the attack duration and the autonomic symptoms were not specified . despite the complete response to indomethacin is one of the diagnostic criteria , in some cph children , the response to indometachin was not tested [ 11 , 14 ] or this was done only in part . moreover , even one case of headache symptomatic of a cerebral infarction has been reported as cph .table 2characteristics of previously described children with paroxysmal hemicraniacase reportvieira talvik almeida gladstein klassen kudrow shabbir ( 2 cases)moorjani broeske seidel blankenbug ( 8 cases)age ( years)/gender9/m3/f10/f8/m6/m9/m13/f14/f4/f3/f17/m7 mean3f/5mattack duration ( min)53051515401530<101020 several min < 15not specified>4510151545attack frequency / daynot specified520not specified313 in a weekevery 1.5 h8922510520autonomic signs : absentyesyesyesyesyesnot specifiednot specifiedyesyesyes 1 miosis / ptosisabsentnononoyesyesnot specifiednot specifiedyesnoyestriggerabsentnot specifiednot specifiednot specifiedstressnot specifiednot specifiednot specifiednot specifiedquick head movementsstresssymptomatic phnonononononononocerebral infarctionnonoindomethacin responsenot administeredyesyesyesnot administerednot administered ( baby aspirin)incomplete ( + verapamil)yesyesyesyesf female , m malecph cases fulfilling the ichd - ii criteria characteristics of previously described children with paroxysmal hemicrania cph cases fulfilling the ichd - ii criteria here , we describe two children , referred to our headache centre , with typical cph features and a positive response to indomethacin therapy . l. , a 7-year - old boy , was referred to our headache center because of a headache that had started 1-year earlier . he suffered from unilateral pain , located in the right orbito - frontal region without side shift ; pain intensity was excruciating , and its quality was described as throbbing . headache occurred 13 times per day , during daytime and night - time , without circadian rhythm . pain was combined with ipsilateral autonomic symptoms , such as conjunctival injection , eyelid oedema and rhinorrhea . during the attacks , the patient cried and found difficult to lie still showing marked agitation and restlessness . moreover , skull ct scan , brain mri , also including the angiographic sequences , and eeg , were normal . . l. was submitted to psychological screening tests ( safa a , d , s scales ) which showed normal anxiety , depression and somatization indices . during the headache attack , pain intensity was not affected by most non - steroidal anti - inflammatory drugs ( nsaids ) , including acetaminophen , ketoprofen , and aspirin . a prophylactic treatment with amitriptyline 16 mg / day was attempted , with no improvement in attack frequency , nor in pain intensity . when considering the headache characteristics , the clinical history and the failure of most analgesic drugs , a prophylactic treatment with indomethacin was started , at the initial dose of 25 mg / day . the attack frequency immediately showed a strong reduction , decreasing from 1 to 3 attack / day to 23 attacks / month . moreover , the child s parents were invited to use indomethacin 25 mg for each single pain attack , and they reported a strong pain relief . after 6 months , the parents discontinued the treatment voluntarily , and the headache worsened considerably . over the following 9 months , flunarizine 5 mg / day was attempted , as suggested by another headache centre , without any amelioration . conversely , a satisfactory improvement was obtained after the resumption of indomethacin 25 mg / day ( attack frequency of 34 per months ) . a further reduction in headache attack frequency up to 12 months was obtained when topiramate was added at the dose of 45 mg / day ( corresponding to 1.5 mg / kg / day ) . however , due to an attentive performance reduction , probably caused by topiramate , this drug was discontinued and replaced by sodium valproate at the dose of 600 mg / day . when l. was 9 years , the dose of indomethacin was increased up to 50 mg / day , with a mean attack frequency of 1 per month and without any side effect , as also confirmed by periodic blood examinations . a. , a 11-year - old romanian boy , presented with a 3-year history of recurrent daily headache . he described episodes of severe throbbing pain attacks in the left fronto - orbital - temporal region . attacks were short - lasting ( ranging from 5 to 40 min ) , and they occurred 34 times per day . attacks occurred during daytime and night - time , without nocturnal predominance , nor circadian rhythm , and most of them were combined with ipsilateral lachrymation , conjunctival injection , ptosis and nasal congestion . pain was not accompanied by nausea , vomit nor other migraine - like features , but the child reported osmophobia during some headache attacks . owing to his headache , the child had stopped practicing sport . at the time of the first consultation in our centre , a. was assuming paracetamol 500 mg / day as a prophylactic treatment , without any positive effect . moreover , his parents referred that other nsaids were attempted , unsuccessfully . neurological examination was normal ; brain mri and blood tests did not show any abnormality . a. had a positive familiar history of headache , as his mother suffered from migraine attacks . paracetamol and ibuprophen treatment were attempted without any positive effect , because pain resolved in 3040 min also without medication . to reduce the frequency of pain attacks , a prophylactic treatment with topiramate ( 50 mg / day ) the clinical characteristics of our child s headache and the failure of the traditional symptomatic and prophylactic treatments led us to consider the diagnosis of cph . therefore , indomethacin 50 mg / day was started , soon affecting the frequency of the attacks , which were no longer present after the second administration . a. , who is continuing the indomethacin therapy at the same dose , has not suffered from any headache attack for the last 6 months . clinical symptoms and pain characteristics of our children are similar to those found in typical adult cph . l. and a. presented with a long history of severe and unilateral pain , which occurred in the fronto - orbital region without side shift . as required by the current cph definition , attacks were accompanied by at least one autonomic symptom , ipsilateral to pain . during the attacks , besides conjunctival injection , eyelid oedema and rhinorrhea , l. also presented vomiting and bilateral photophobia , which are typical migraine features , common in cph [ 5 , 7 , 10 ] . , there is a considerable overlapping between the diagnostic criteria for cph and ch , mostly concerning headache duration ( from 15 to 30 min ) and attack frequency ( from 5 to 8) , thus complicating the differential diagnosis . moreover , also the behavioural characteristics may not be useful in distinguishing cph from ch , since agitation , restlessness and aggressive behaviour during pain attacks , typical of ch , have been described also in some cph cases [ 7 , 12 ] . pain site and associated autonomic phenomena are similar in both headaches , but the higher frequency of the attacks , their shorter duration and the absolute response to indomethacin may help in distinguishing cph from ch . although cph is commonly considered to prevail amongst female patients , many cph cases in paediatric age are males [ 911 , 13 ] , as the children we described . in line with the previous cases of cph reported during developmental age , our patients showed some atypical features , not fully meeting the ichd - ii criteria . first , although the ichd - ii criteria require an attack frequency higher than 5 attacks per day , in our patients , the attack frequency was lower . this could be in favour of a diagnosis of ch , but an attack frequency lower than the one required by the ichd - ii criteria has been described in cph of both childhood [ 11 , 13 , 18 ] and adulthood [ 46 ] . moreover , according to icdh - ii criteria , the attack frequency needed for diagnosing cph should be higher than 5 per day , not constantly , but for at least half the time of observation . second , attack duration was variable in both our children , but in a. it was sometimes longer than 30 min , which represents the maximal duration for a cph attack , according to the ichd - ii criteria . however , several reports showed cph patients with attack duration longer than 30 min in both adults [ 5 , 7 ] and children [ 8 , 9 ] . frequency and duration of the attacks , nevertheless , are commonly different between paediatric and adult population also in more common primary headaches , such as migraine and tension - type headache . if attack duration and frequency can make the diagnosis more difficult , especially in paediatric age , the absolute response to indomethacin represents the diagnostic key for cph in both adults and children ( indotest ) [ 3 , 21 ] . indeed , it is noteworthy that our children showed a dramatic response to indomethacin . in particular , l. did not respond to amitriptyline , as a prophylactic drug , nor to common nsaids . moreover , after an initial response to indomethacin , the headache attacks increased their frequency after the voluntary discontinuation of the drug . they were markedly reduced when indomethacin was resumed . to avoid a prolonged treatment with a high indomethacin dose , topiramate was added , with a satisfactory clinical response , as suggested by previous studies on cph [ 9 , 22 , 23 ] . owing to cognitive adverse events , topiramate was replaced with valproate , which was effective , as well . to our knowledge , there are no studies showing the efficacy of valproate in cph . a. did not show any improvement when treated with paracetamol and topiramate , whereas headache attacks disappeared after indomethacin was started . in none of the patients , triptans were tested , since both of them are under 12 , and at their age the use of triptans is not allowed in italy . long - term therapy with indomethacin is generally well tolerated , but adverse side effects may occur both in adulthood and childhood [ 9 , 22 ] . the characteristics of our children s headache , particularly the positive response to indomethacin , led us to consider the diagnosis of cph . however , the frequency and duration of our patient attacks did not fulfil the ichd - ii criteria . cph diagnosis can be difficult in developmental age , due to the frequency and duration of attacks . although attacks should not be longer than 30 min , in children s cph , the headache duration can exceed 40 min [ 8 , 9 , 16 ] . moreover , although the ichd - ii criteria for cph require an attack frequency higher than 5 per day , many cph children show a lower attack frequency ( 23 per day or less ) [ 8 , 11 , 13 , 18 ] . these elements suggest that a revision of the current cph diagnostic criteria , possibly with the inclusion of special notes for developmental age , would be necessary .

chronic paroxysmal hemicrania ( cph ) is a rare primary headache syndrome , which is classified along with hemicrania continua and short - lasting unilateral neuralgiform headache attacks with conjunctival injection and tearing ( sunct ) as trigeminal autonomic cephalalgia ( tacs ) . cph is characterised by short - lasting ( 230 min ) , severe and multiple ( more than 5/day ) pain attacks . headache is unilateral , and fronto - orbital - temporal pain is combined with cranial autonomic symptoms . according to the international classification of headache disorders , 2nd edition , the attacks are absolutely responsive to indomethacin . cph has been only rarely and incompletely described in the developmental age . here , we describe two cases concerning a 7-year - old boy and a 11-year - old boy with short - lasting , recurrent headache combined with cranial autonomic features . pain was described as excruciating , and was non - responsive to most traditional analgesic drugs . the clinical features of our children s headache and the positive response to indomethacin led us to propose the diagnosis of cph . therefore , our children can be included amongst the very few cases of this trigeminal autonomic cephalgia described in the paediatric age .

Introduction Case 1 Case 2 Discussion

surfactant has become standard therapy in preterm infants with respiratory distress syndrome ( rds ) 1 . surfactant lowers the alveolar surface tension and prevents expiratory alveolar collapse increasing compliance , reducing pulmonary vascular resistance and improving ventilation - perfusion mismatching . surfactant can be inactivated in term infants with pneumonia , meconium aspiration syndrome , lung hypoplasia and pulmonary hemorrhage . this inactivation may be caused by various plasma proteins and local inflammatory cytokines that pass into the airways , damage type ii pneumocytes and alveolar cells rendered vulnerable by mechanical ventilation . case reports and studies from literature suggest that surfactant might be beneficial to infants with sepsis and pneumonia 2 . this study aimed to evaluate the effects of exogenous surfactant on ventilation parameters and oxygenation of term infants with respiratory failure . this is a case series study in which mechanically ventilated infants diagnosed with early neonatal sepsis and respiratory failure that had received a single dose of exogenous bovine surfactant ( survanta ) between january 2008 and december 2010 , with a gestational age above 37 weeks were retrospectively studied . diagnoses like meconium aspiration syndrome , persistent pulmonary hypertension of the newborn , asphyxia , and pulmonary hemorrhage were excluded . all infants suffered from severe respiratory failure according to definition of the european - american consensus conference with arterial oxygen partial pressure ( mmhg ) ( pao2)/ fraction of inspired oxygen ( fio2 ) below 200 mmhg 3 . diagnosis was confirmed with antenatal history , clinical , radiological and laboratory findings . because signs of sepsis are nonspecific , any term newborn infant with sudden onset of respiratory distress or other signs of illness was evaluated for sepsis including complete blood count , blood smear , c - reactive protein , blood cultures and tracheal aspirates . all infants were followed with synchronized intermittent mandatory ventilation ( simv ) mode of ventilator ( sle 2000 ) . early outcomes of surfactant treatment were evaluated via respiratory indices including oxygenation index ( oi ) , mean alveolar pressure ( map ) , fio2 , pao2 , arterial oxygen saturation ( sao2 ) and pao2/fio2 values . these values were carried out using arterial blood gas values and ventilator parameters of the infants before and six hours after surfactant administration . oi and map were calculated using the following equation : oi=(map fio2 100)/ pao2 map=[(pipxti)+(peepxte)]/ti + te pip : peak inspiratory pressure ( cmh20 ) ti : time of inspiration ( sec ) peep : positive end expiratory pressure ( cmh2o ) te : time of expiration ( sec ) comparisons between groups were done with wilcoxon signed ranks test and mann - whitney u test . mean gestational age of the infants was 38.31 weeks , mean birth weight was 3088 grams and median hospitalization day was 1 day ( table 1 ) . although gbs screening was negative in all mothers , eight mothers had clinical or histological chorioamnionitis , seven had prolonged ruptured membranes ( > 24 hours ) and three had fever ( > 38.3c ) before labor . all infants had leucocytosis ( 165026704/mm ) , elevated crp ( 33.03.4 mg / l ) , and bilateral alveolar densities with air bronchograms on chest x - rays ; however , tracheal and blood cultures were negative in all patients so that patients were diagnosed as suspected sepsis . demographic characteristics of infants meanstandard deviation ; median ( minimum , maximum ) in all of the 18 infants pulmonary gas exchange was critically impaired with median pao2/fio2 below 100 mmhg prior to surfactant administration . all patients received a single dose ( total phospholipid dose 100mg / kg ) surfactant when mechanical ventilation failed to improve oxygenation . of these infants , one suffered from pneumothorax within 24 hours after surfactant administration and two infants died due to multiorgan failure within the first week postpartum . before the surfactant treatment , median ( minimum and maximum ) oi , map , fio2 , pao2 , sao2 and pao2/fio2 values were as follows ; 15.3 ( 10.0 - 52.8 ) ; 8.5 ( 7.0 - 15.0 ) ; 80.0 ( 50.0 - 100.0 ) ; 45.4 ( 21.5 - 56.9 ) ; 83.5 ( 42.0 - 97.0 ) ; 60.3 ( 21.5 - 99.2 ) . following surfactant treatment these values were ; 3.4 ( 1.5 - 35.0 ) ; 8.0 ( 6.0 - 14.0 ) ; 40.0 ( 21.0 - 100.0 ) ; 83.5 ( 39.0 - 118.0 ) ; 97.0 ( 50.0 - 99.5 ) ; 234.0 ( 39.0 - 422.0 ) respectively . oi , map and fio2 values significantly decreased . whereas pao2 , sao2 and pao2/fio2 values significantly increased ( p<0,001 ) . respiratory indices of patients before and after surfactant treatment1 median ( minimum - maximum ) preterm and late preterm infants have increased risk of rds so that surfactant treatment guidelines mostly focuses on these group of patients 4 . term infants are also at risk of rds and any lung disease affecting these infants that involves surfactant dysfuncion or deficiency constitutes a potential target for replacement therapy . anadkat et al , have demonstrated that male sex and white race ethnicity increase the risk of rds in late preterm and term infants 5 . surfactant treatment response starts with improvement in po2 as also shown in our study , followed by improvement in compliance and oxygenation a more prolonged effect over hours that we evaluated by oi , then finally prolonged effect of the surfactant as substrate for surfactant metabolism persisting for weeks 6 . preterm infants diagnosed with rds that have not responded to surfactant treatment as expected have been found to suffer from a significantly higher rate of infection and birth asphyxia 7 . pneumonia in term infants might result in secondary surfactant deficiency or dysfunction as surfactant can be inactivated . secondary surfactant deficiency can result in respiratory deterioration and this may potentially affect the severity of illness in infants with pneumonia . these infants might also need ventilatory support or extracorporeal membrane oxygenation ( ecmo ) during their hospitalization . if an infant is intubated and exhibits a deteriorating condition during this ventilatory support , exogenous surfactant may improve infant 's respiratory status because surfactant can be easily inactivated by the inflammatory products of lung injury caused by mechanical ventilation , supplemental oxygen or infection 8 . in a multicenter trial it was shown that surfactant treatment of term infants with severe respiratory failure also can decrease the need for ecmo 9 . in this study infants were randomized to receive exogenous surfactant or placebo . the infants responded to surfactant better if the surfactant had been given at the early stages of respiratory failure evaluated by oi . in our study it is also shown that surfactant treatment rapidly improved gas exchange and oxygenation in infants with respiratory failure . surfactant has important antimicrobial properties and since endogenous surfactant can potentially be inactivated by inflammation and edema in pneumonia patients , exogenous surfactant may be useful in the treatment . herting et al , studied the effects of surfactant on 8 children diagnosed with acute respiratory distress triggered with pneumonia and found that pao2/fio2 was immediately increased after surfactant instillation 10 . it is also shown that surfactant therapy improves gas exchange in the majority of infants with streptococcal pneumonia but response was found to be slower compared with infants with rds 11 . referring to this study jobe concluded that there should be no concerns about treating infants with sepsis / pneumonia syndromes and respiratory failure with surfactant 12 . in our study we also demonstrated that infants presenting with respiratory failure and early neonatal sepsis benefit from surfactant administration in the early hours of treatment . first this was a retrospective study ; second , there was no control group ; thus there may have been confounding factors which contributed to the outcome . although , there is still no consensus on the efficacy of surfactant in infants with suspected or proven pneumonia 13 , this study shows that surfactant treatment can improve respiratory indices in infants diagnosed with early neonatal sepsis and respiratory failure . however larger randomized controlled trials are necessary to determine and conclude whether improved oxygenation is due to surfactant .

objectivethe objective of the study was to evaluate the effects of exogenous surfactant on respiratory indices in term infants with respiratory failure.methodsconsecutive 18 mechanically ventilated term infants , who received a single dose of exogenous surfactant were retrospectively included into the study . the respiratory outcome of surfactant rescue therapy was evaluated by comparing respiratory indices before and six hours after surfactant administration.findingsmedian oxygenation index ( oi ) , mean alveolar pressure ( map ) and fraction of inspired oxygen ( fio2 ) values were significantly decreased ( p<0.001 ) ; median arterial oxygen partial pressure ( pao2 ) , arterial oxygen saturation ( sao2 ) and pao2/fio2 values were significantly increased six hours after surfactant treatment ( p<0.001).conclusionrescue therapy with surfactant was found to be effective in the improvement of early respiratory indices in term infants with respiratory failure .

Introduction Subjects and Methods Findings Discussion Conclusion Conflict of Interest

although multiple factors have contributed to the decline of pacific salmon ( oncorhynchus spp . ) across portions of their natural range , dams have arguably played a major role in many locations ( nrc 1996 ; lichatowich 1999 ; ruckelshaus et al . large dams ( > 15 m tall)designed to generate hydropower , control floods , or facilitate navigation have had unintended side effects on the quantity and quality of habitat used by salmon . specifically , these dams have decreased the area for spawning and rearing , and have altered flows , sediments , and temperatures downstream ( pfmc 1979 ; ward and stanford 1979 ; ligon et al . 1995 ) . the effects of large dams on thermal conditions have been documented worldwide , and include changes in the mean and variance of temperature at several temporal scales ( petts 1984 ; crisp 1987 ; usace 2000a ; preece and jones 2002 ; steel and lange 2007 ) . these changes have significant biologic consequences because temperature influences the growth and reproduction of aquatic organisms ( ward and stanford 1979 ; schlosser et al . the spawning of adults , the emergence of fry , and the migration of smolts coincide with thermal cues in natal lakes and streams ( e.g. , brett 1971 ; brannon 1987 ; roper and scarnecchia 1999 ) . although changes in thermal regimes might have contributed to the declines of salmon ( nrc 1996 ) , we have much to learn about the underlying mechanisms ( see reviews in mccullough 1999 ; and richter and kolmes 2005 ) . 2006 ) , increase mortality ( smith et al . 2003 ; crozier and zabel 2006 ; gilhousen 1990 ) , perturb energetics ( brett 1995 ; rand and hinch 1998 ; macdonald et al . 2000 ) , slow growth ( bisson and davis 1976 ; pearcy 1992 ) , and create morbidity ( bartholomew 1998 ; materna 2001 ) . indirect effects can also result from shifts in the timing of or phenotypes at life - history transitions ( quinn and adams 1996 ) . for example , high temperatures during embryonic development can lead to premature emergence , which could expose fry to peak flows , scarce resources , or greater predation ( brannon 1987 ; murray and mcphail 1988 ; jensen and johnsen 1999 ; usace 2000a ) . since the first large dams in the pacific northwest were built in the early 1900s , many generations of salmon have spawned , developed , emigrated , and returned to spawn in the waters downstream . possibly , these fish have begun to adapt to the new thermal regimes caused by dams ( waples et al . although we can not infer adaptation from the available data , we can estimate the potential evolutionary impacts of dams and discuss the types of data that would be necessary to make such inferences . under this premise , we present an exploratory analysis of chinook salmon ( oncorhynchus tshawytscha ) in four parts . second , we review the effects of water temperature on physiological performance at specific life stages . third , we estimate the degree to which changes in physiological performance would influence the fitness of genotypes . finally , we assess the possibility of an evolutionary response to selective pressures imposed by dams . quantifying changes in water temperature downstream of a dam requires several types of data , including the location of the dam , the date of construction , and records of temperature . linking thermal changes to the performance of chinook salmon , in particular , requires additional knowledge of the distribution of populations . we compiled these data from a variety of sources ( willingham 1983 ; myers et al . 1998 ; usace 2000b ; streamnet 2005 ; city of tacoma 2005 ; kimbrough et al . 2006 ; usgs 2007 ) , and then imposed five criteria to identify several dams for our analysis . first , the dam had to reside upstream of a gauge that recorded water temperatures . second , the gauge s period of record had to span multiple years before and after the construction of the dam . third , the dam had to occur on a river or large creek that supported chinook salmon in washington , oregon , california or idaho ( usa ) . fourth , the distance between the dam and the gauge had to be < 30 km , to minimize the attenuation of thermal effects downstream ( usace 2000a ) . finally , no other large dam could exist upstream of the focal dam prior to its construction . using these criteria four dams selected for analysis based , in part , on the existence of water temperature gauges downstream . names , types and locations of these dams are provided , along with the approximate dates when the dams became operational ( i.e. , the reservoirs upstream were created ) . gauge identification numbers , distances between gauges and dams , and periods of temperature records are also provided . er , epilimnetic release ; mr , multiple depth release ; hr , hypolimnetic release . for details , see usgs ( 2007 ) . if only the year was known , the month was assumed to be january . in all cases , the first day of the month was assumed to be the starting point of the dam s operation . a map of washington and oregon , usa , depicting the locations of four dams selected for analysis : ( a ) mayfield , ( b ) fall creek , ( c ) hill creek , and ( d ) lost creek . the gauges downstream of these dams contained two types of gaps in their records of temperature . the first type comprised days when the minimal and maximal temperatures were recorded but the mean temperature was not recorded . for these days , we estimated the mean temperature by averaging the minimal and maximal temperatures . the second type comprised days when both the mean temperatures and extreme temperatures were missing . in these cases , we estimated the means by regression or interpolation ( see appendix a ) . we assessed the changes in temperature associated with each dam by comparing daily mean temperatures , averaged among the years before and after construction of the dam . based on our findings , the effect of a dam on water temperature depends on the time of year ( fig . conversely , during the fall and early winter , water was generally warmer in the presence of a dam . this reversal from summer cooling to fall warming was evident as early as august on the rogue river and as late as october on the cowlitz river ( fig . 2 ) . mean water temperatures before and after dam construction on the cowlitz river , fall creek , the middle fork of the willamette river , and the rogue river . bold lines depicting the mean water temperatures are surrounded by thinner lines depicting the upper and lower bounds of the 95% confidence intervals . in theory , the effect of a dam on water temperature should depend not only on the time of year , but also on thermal stratification upstream and the depth of water released at the dam ( ward 1982 ; crisp 1987 ; poff and hart 2002 ) . if the reservoir upstream is thermally stratified and the dam draws water from below the thermocline ( i.e. , the hypolimnetic layer ) , the water downstream of the dam should be unseasonably cold during summer and unseasonably warm during fall and winter . if the dam draws water from above the thermocline ( i.e. , the epilimnetic layer ) , the water downstream should be unusually warm during the summer . other dams draw water from multiple depths , enabling finer control over temperatures downstream . given that our sample contained three types of dams ( table 1 ) , we were surprised to discover these dams affected water temperatures similarly ( fig . a rich literature describes the influence of water temperature on the development , growth , and survival of salmon at various life stages ( reviewed by mccullough 1999 ) . generally , embryonic development proceeds more rapidly at higher temperatures . in chinook salmon , a doubling of water temperature within tolerable limits will halve the time between the fertilization of an egg and the emergence of a fry ( beacham and murray 1990 ) . embryonic survivorship remains high throughout a wide range of temperatures , but decreases precipitously outside that range . for example , the survivorship of chinook embryos exceeds 90% between 5 and 15c ; however , below 2c and above 18c the rate drops to < 5% ( fig . once salmon emerge from the gravel , their growth and survival continue to depend on temperature . for example , the growth rate of fry increases between 10 and 15c ( banks et al . 1971 ) , but survivorship decreases at higher temperatures ( fig . because these data pertain to salmon raised at constant temperatures , we must assume variation in temperature does not alter these conclusions a dubious assumption . some salmon tolerate acute exposure to temperatures that would prove lethal during prolonged exposure ( murray and beacham 1987 ; tang et al . 1998 ) , but we need more research to define the general limits of tolerance in variable environments . embryos and fry of chinook salmon survive well over a range of approximately 10c this conclusion was based on models that best fit published data for embryos ( velsen 1987 ; beacham and murray 1989 ) and fry ( olson et al . 1970 ) . model selection was performed according to angilletta ( 2006 ) ; details are provided in appendix b. the thermal sensitivities of physiological performances can mediate the direct and indirect effects of dams on the fitness of salmon . because temperatures in the fall can approach those that stress chinook salmon ( e.g. , see fall creek in fig . 2 ) , warming by a dam could directly increase the mortality of embryos . in the laboratory , brief exposure to 17c during embryonic development caused more than 98% mortality between the fertilization and emergence ( geist et al . more likely , however , dams indirectly influence fitness by advancing the timing of emergence . emergence describes a developmental transition in which fry leave the interstitial gravel and enter the water column , thereby initiating a sustained , free - swimming residence in a stream or river . at this point in development , the energy stored as yolk has been virtually exhausted and the juvenile must begin to feed exogenously . early or late emergence could lead to mortality from excessive flows , abundant predators , or insufficient resources ( jensen and johnsen 1999 ; einum and fleming 2000 ) . the warming caused by a dam in fall and winter would accelerate the development of embryos , leading to early emergence . thus , a dam could disrupt a match between the actual and the optimal dates of emergence . if salmon below the dam spawned later in the year , the temperatures experienced by their offspring would be lower because of seasonal cooling ( see fig . 2 ) . consequently , development would proceed more slowly and the timing of emergence would correspond to that before the dam s influence . alternatively , an evolutionary decrease in developmental rate would enable salmon to delay emergence despite the warm water below the dam . whether spawning behavior or developmental physiology would experience greater selective pressure depends on the cost of each strategy and the impact on other stages of the life cycle . the response to selection depends on the heritabilities of spawning behavior and embryonic physiology , and their genetic covariance . the relative timing of spawning seems highly heritable in salmon , with estimates of narrow - sense heritabilities ranging from 0.4 to 1.3 ( e.g. , see smoker et al . we know less about the heritability of thermal physiology in salmon , but additive genetic variance for developmental rate exists under some thermal conditions ( hebert et al . these data suggest behavior , physiology , or both could evolve in response to thermal change . the genetic covariance between spawning date and developmental rate could enhance or constrain adaptation , depending on its sign and magnitude ( lande and arnold 1983 ) . despite the ideas discussed above , current models of thermal adaptation can not generate deep insights about the responses of salmon to dams . first , these models focus on physiological responses to environmental temperature ( reviewed by angilletta et al . 2002 ; kingsolver and gomulkiewicz 2003 ) , ignoring the potential for behavioral responses that seem relevant to salmon ( e.g. , timing of spawning , placement of nests ) . second , these models assume that temperature influences either survivorship ( lynch and gabriel 1987 ) or fecundity ( gilchrist 1995 ) uniformly throughout the life of the organism . because both the thermal impacts of dams and the thermal tolerances of salmon vary throughout the life cycle , simple models will not accurately predict the strength of selection . in the next section , we use an age - structured model to explore the impact of physiological performance on the fitness of a genotype . matrix models of population growth provide a useful way to quantify the ecological and evolutionary consequences of environmental changes ( caswell 2001 ) , such as the thermal changes caused by dams . salmon and most other organisms have life cycles with distinct stages that occur in a characteristic seasonal cycle . matrix projections enable us to explore how a change in performance at a specific stage impacts the mean fitness of a population . matrix models have been applied to salmon by classifying the life cycle in terms of either stage or age ( e.g. , kareiva et al . 2000 ; greene and beechie 2004 ; scheuerell et al . 2006 ; zabel et al . ratner et al . ( 1997 ) developed an age - classified model for the spring run of chinook salmon from the south fork of the umpqua river ( oregon , usa ) . in this population , 90% of juveniles migrate to the ocean during their first spring ( ratner et al . therefore , the survivorship from oviposition to age 1 equals the product of survivorships for the embryo , fry , and smolt . thus , the matrix form of the model for a female is as follows : where sx equals the survivorship from age x-1 to age x , bx equals the propensity to breed at age x , mx equals the fecundity at age x , and equals the survivorship of adults during their migration to the spawning area . the leading eigenvalue of this matrix ( ) represents the long - term rate of population growth ( caswell 2001 ) . for the case represented by matrix a , we can not solve explicitly for , but we can calculate this rate numerically given a set of parameter values . using values for chinook salmon of the umpqua river ( see table 2 of ratner et al . 1997 ) , we computed a value of = 1.17 , which indicates a growing population ( > 1 ) . predicted dates of emergence for a range of spawning dates at each of four locations impacted by dams . emergence dates were predicted from water temperature recorded before and after the construction of dams . these dates are reported on a relative scale ( days after august 1 ) , along with the standard deviation of inter - annual variation in parentheses . analysis of variance was used to infer significant differences between predicted emergence dates before and after the construction of dams . assessing the effects of environmental change on population dynamics requires two steps . in the first step , we must relate changes in the environment to changes in life - history traits ( sx , mx , bx or ) . in the second step , we must relate changes in life - history traits to changes in mean fitness ( ) . the latter relationship is referred to as the demographic sensitivity ( sp ) : where p equals a life - history trait . for example , the demographic sensitivity for s1 , the survivorship from oviposition to age 1 , depends on da / ds1 : using estimates of these parameters for chinook salmon in the umpqua river , the demographic sensitivity of s1 ( 123.23 ) greatly exceeds the sensitivities of all other survivorships in the matrix ( 0.120.35 ) . in this framework , the impact of a change in water temperature on fitness equals the product of the demographic and thermal sensitivities : because survivorship changes nonlinearly with temperature , the effect on depends on the mean temperature , as well as the change in temperature . although dams have elevated the temperatures experienced by embryos and fry , temperatures during fall and winter usually lie within the tolerable limits ( compare figs 2 and 3 ) . therefore , the warming caused by dams likely imposes little direct thermal stress on embryos or fry . nevertheless , the winter warming caused by dams could have indirect effects on s1 , such as those believed to stem from early emergence ( see previous section ) . assume approximates the fitness of a genotype with the life cycle and vital rates described by the matrix a. then , we need only two pieces of data to connect early emergence to a decrement in fitness . first , we must know the degree to which a dam advances the date of emergence . as a first approximation , we can calculate this advance from records of water temperature and studies of developmental rate . second , we must know the relationship between the timing of emergence and the survivorship of juveniles ( s1 ) . unfortunately , this relationship has not been resolved for any population of chinook salmon , let alone the populations that spawn near our four dams . therefore , we conducted a sensitivity analysis by exploring a set of plausible relationships between the date of emergence and s1 . as a first step in our analysis , we calculated emergence dates in the rogue river for a set of potential spawning dates . we focused on the rogue river because it lies close to the umpqua river and the chinook salmon in these two rivers undergo similar life cycles ( myers et al . 1998 ; odfw 2003 ) ; thus , we felt comfortable using the parameter values reported by ratner et al . for each year for which we had thermal data , we used a degree - day model to estimate the timing of emergence associated with each spawning date ( ranging from august 1 to january 1 ) . we summed thermal units over successive days until we reached an empirically estimated threshold for emergence ( 889 units ; wedemeyer 2001 ) . using this procedure , we calculated emergence dates during the periods before and after the construction of a dam . although water temperatures recorded by the gauges ( fig . 2 ) probably differ from those experienced by embryos in the gravel , we assumed a similar relationship between recorded temperatures and actual temperatures existed before and after the construction of the dam . as a second step , we used predicted dates of emergence to estimate the potential impact on fitness . we used hypothetical functions describing the relationship between the date of emergence and s1 ; these functions differed in the optimal date of emergence and the strength of selection on the timing of emergence ( fig . optimal emergence dates were chosen by assuming development under predam conditions for three spawning dates : these hypothetical spawning dates span the temporal window of spawning within the rogue river ( myers et al . three strengths of selection were chosen based on a recent study of the empirical relationship between emergence date and juvenile survivorship in atlantic salmon ( einum and fleming 2000 ) . for all functions , the maximal value of s1 was set to 0.00267 ( ratner et al . , we computed for a genotype spawning before and after the construction of the dam by inserting s1 in the matrix model of ratner et al . nine hypothetical relationships between the timing of emergence and the survivorship of juveniles ( s1 ) used in our sensitivity analysis . these relationships vary in the optimal date of emergence ( early , intermediate , and late ) and the strength of selection for the optimal date ( strong , intermediate , and weak ) . first , the water temperatures imposed by dams appear to influence emergence dates in a complex manner ( table 2 ) . if salmon spawn in august , their fry would emerge at approximately the same time or slightly later than they would have emerged before the dam existed . however , if salmon spawn in september or later , their fry would emerge much earlier than they would have before the dam . because the thermal effects of dams change seasonally , the results of our sensitivity analysis depend strongly on the optimal date of emergence . in general , the presence of a dam shifts the optimal spawning date to a later time , such that offspring would emerge on the historical date despite their accelerated development . however , the optimal spawning date shifts more for salmon that historically spawned in october or november than it does for salmon that historically spawned in september ( fig . 5 ) . predicted fitnesses ( ) of chinook salmon before ( filled symbols ) and after ( open symbols ) the construction of the lost creek dam on the rogue river . each plot corresponds to one of the relationships between emergence date and juvenile survivorship depicted in fig . 4 . from left to right , plots show earlier to later optimal dates of emergence . from top to bottom , plots show decreasing strengths of selection for emergence on the optimal date . within the range of conditions we considered , the dam s influence on developmental rate should have caused the natural selection of spawning date . to illustrate this point , consider a hypothetical population in the rogue river whose optimal spawning date was september 1 before the construction of the lost creek dam . suppose that immediately after the dam began to influence water temperature , the mean spawning date was september 1 with a standard deviation of 12.4 days ( based on beechie et al . figure 6 illustrates the predicted relationships between the spawning date and the relative fitness for the conditions of our sensitivity analysis . the slope of each relationship represents a linear selection gradient ( ) , which quantifies the strength of directional selection ( range = 0.0240.036 day ) . predicted relationships between spawning date and relative fitness ( , standardized to a mean of 1 ) following the construction of the lost creek dam on the rogue river . each plot corresponds to one of the relationships between emergence date and juvenile survivorship depicted in fig . to right , plots show earlier to later optimal dates of emergence . from top to bottom , plots show decreasing strengths of selection for emergence on the optimal date . first , we can standardize the selection gradients by the standard deviation of spawning date ; a standardized selection gradient ( ) provides a useful metric to compare the strength of selection among traits and populations ( lande and arnold 1983 ; arnold and wade 1984 ) . in our analysis , standardized selection gradients ranged from 0.29 to 0.52 , indicating strong selection compared to values reported for other natural populations ( kingsolver et al . second , with knowledge of the phenotypic variance and the narrow - sense heritability of spawning time , we can predict the evolutionary response to selection ( i.e. , the change in the mean phenotypic value per generation ) . using a conservative estimate of the heritability of spawning date ( 0.2 ; hard 2004 ) , we predict an evolutionary change in the mean spawning time of 0.71.1 days per generation , depending on the relationship between emergence date and juvenile survivorship ( see fig . this calculation suggests the mean spawning date would approach the new optimum within 20 generations ( 85 years ) . 5 ) , evolution of the spawning date might have restored some of this loss during the last three decades and may continue to do so in the future . although dams have been altering the environments of salmon for decades , we still have much to learn about their ecological and evolutionary impacts . we explored some potential impacts by quantifying the thermal effects of dams and their consequences for the performance of chinook salmon . this exercise indicated that dams impose substantial loss of fitness and strong selection of phenotypes . 2005 ; neira et al . 2006 ) , the evolutionary responses to this selection should be rapid . furthermore , this framework for analyzing physiological , ecological , and evolutionary responses applies to other kinds of environmental change experienced by salmon . although our analysis offers insights on the potential for adaptation , we do not know whether chinook salmon have actually adapted to the thermal changes caused by dams . our calculations involved many assumptions to simplify the population dynamics of chinook salmon . for example , we assume juvenile survivorship depends only on emergence date ( i.e. , no density - dependence ) , spawning behavior does not influence other traits , and genetic variance remains constant during adaptation . for example , we might also consider processes that would ameliorate a negative effect of early emergence on juvenile survivorship . a phenological advance of the entire aquatic community could prevent a mismatch between emerging fry and their prey . furthermore , poor survivorship of embryos could lead to better survivorship of fry , through known mechanisms of density dependence ( einum et al . furthermore , we could consider the interaction of spawning date and developmental rate during adaptation . our focus on spawning behavior simply reflects the fact that heritabilities of phenological traits tend to exceed those of physiological traits ( reviewed by carlson and seamons 2008 ) . nonetheless , a change in the thermal sensitivity of developmental rate could also help salmon populations recover the loss of fitness caused by dams . indeed , physiological adaptation might proceed more rapidly than behavioral adaptation if genetic variance in spawning date erodes over time or if adults incur substantial costs of delayed spawning . for all of these reasons , one should interpret our analysis as a means of generating testable hypotheses about the impacts of dams and as a guide for identifying crucial data to confirm these impacts . based on our analysis , we should improve predictions about the impacts of dams by resolving certain empirical relationships . specifically , three factors contributed to the range of selective pressures reported in fig . 6 : ( i ) the thermal effects of dams ; ( ii ) the historical date of spawning ; and ( iii ) the relationship between emergence and survivorship . to precisely define selective pressures on salmon , all three factors must be quantified empirically for the population of interest . moreover , a general understanding of these selective pressures requires that these analyses be carried out for multiple dams . furthermore , additional data on the genetic variances of spawning date and developmental rate would provide better estimates of the response to selection . finally , we can use experimental approaches to establish whether populations have already responded to the selective pressure predicted by our analysis . common garden experiments , using eggs from populations affected by dams and populations unaffected by dams , could demonstrate whether developmental rate has adapted to the changes in thermal regimes . studies of spawning date in a common garden would also be informative yet more challenging , because of the need to rear large numbers of salmon to adulthood . still , only field studies can reveal how the thermal impacts of dams interact with predation risk , food availability , and hydrological conditions . reservoirs upstream of dams impact the timing of migration by juveniles , and reduced flows downstream of dams prolong the migration of both juveniles and adults ( waples et al . , dams prevent adults from reaching their historical spawning grounds , reducing the extent and variability of habitats that might favor one life - history type over another ( sheer and steel 2006 ) . changes in physical habitats both upstream and downstream of dams also create new opportunities for predators and competitors . dams are often mitigated by hatcheries , which present another source of competitors and an additional source of selection ( ford et al . construction of large dams has also facilitated massive withdrawals of water for agricultural and municipal purposes . runoff from these areas increases the concentrations of nutrients , contaminants , and salts in downstream reaches ( nrc 1996 ) . these changes in water quality adversely affect the development , growth , health , and survival of salmon ( regetz 2003 ; loge et al . given this combination of stressors , our analysis likely represents an overly optimistic outlook on the ability of salmon to adapt to the environmental changes imposed by dams .

dams designed for hydropower and other purposes alter the environments of many economically important fishes , including chinook salmon ( oncorhynchus tshawytscha ) . we estimated that dams on the rogue river , the willamette river , the cowlitz river , and fall creek decreased water temperatures during summer and increased water temperatures during fall and winter . these thermal changes undoubtedly impact the behavior , physiology , and life histories of chinook salmon . for example , relatively high temperatures during the fall and winter should speed growth and development , leading to early emergence of fry . evolutionary theory provides tools to predict selective pressures and genetic responses caused by this environmental warming . here , we illustrate this point by conducting a sensitivity analysis of the fitness consequences of thermal changes caused by dams , mediated by the thermal sensitivity of embryonic development . based on our model , we predict chinook salmon likely suffered a decrease in mean fitness after the construction of a dam in the rogue river . nevertheless , these demographic impacts might have resulted in strong selection for compensatory strategies , such as delayed spawning by adults or slowed development by embryos . because the thermal effects of dams vary throughout the year , we predict dams impacted late spawners more than early spawners . similar analyses could shed light on the evolutionary consequences of other environmental perturbations and their interactions .

Introduction Impacts of dams on water temperature Consequences of dams for the performance of salmon Modeling selective pressures imposed by dams Conclusions and future directions Supporting information

bladder exstrophy ( be ) is a rare condition with an incidence of 1 in 50.000 newborns [ 1 , 2 ] . it is a complex pathology with an unknown etiology and is characterized by a low - lying umbilicus , exposed bladder plate , pubic symphysis diastasis , divarication of the rectus abdominis and pelvic floor , anterior ectopia of anus and vagina and penile epispadias . bladder exstrophy ( be ) demands several surgical interventions to achieve satisfactory continence and genital appearance for a normal social , sexual and reproductive lifestyle . treatment of children born with classic be is one of the most technically challenging situations in pediatric urology . a better understanding of functional anatomy , together with improved surgical techniques , has reduced complications and the number of procedures required for satisfactory reconstruction in patients with be . the appearance and function of external genitalia are among the most important concerns during adolescence and adulthood [ 6 , 7 ] . quality of life ( qol ) is an elusive concept approachable at varying levels of generality , from the assessment of societal or community welfare to the specific evaluation of the situations of individuals or groups . qol integrates objective and subjective indicators , collectively reflecting a broad range of life domains by using an individual ranking of the relative importance of each domain . considering the possible repercussions of be , our aim was to study the long - term outcome of patients with be regarding quality of life comparing males and females . all patients treated for be in our institution since 1990 were recruited . in our study period , . only 51 were successfully contacted , and 43 agreed to participate in the study ( 29 males and 14 females ) , all of whom were over 15 years of age and were treated for be in our institution . those accepted to participate in the study were interviewed during an office visit using a validated questionnaire , short form 36 general health questionnaire ( sf-36 ) . the sf-36 measures a full range of health states and includes multi - item scales measuring each of eight health concepts : physical functioning , gender role limitations due to physical health , bodily pain , general health , vitality , social functioning , role limitations due to emotional problems , and mental health . patients were also asked about their social inclusion with general questions regarding education , work , income , siblings , family , continence , surgeries , sexual life and genital appearance , urinary diversion , and pregnancy . continence was defined as absence of urine loss or use of only 1 preventive pad daily . sexual life was assessed as the presence of at least one penile penetrative intercourse per month . genital appearance was assessed by asking the patients whether they would be satisfied if their genitalia looked like as it does in the moment of the survey for the rest of their life . questions addressing education , income , siblings and number of pregnancies had their answers grouped by the distribution in three scale groups . statistical analysis was performed using non - parametric tests with a 95% confidence interval for statistical significance ( p < 0.05 ) . the average age of men was 20.4 years ( 15 to 36 years ) , and the average age of women was 26.4 years ( 15 to 48 years ) . most patients were of a low - income class ( 30 out of 43 patients received less than 5 brazilian minimum wages ) . there was no statistically significant difference in the income of men and women ( p = 0.36 ) . both genders had similar number of surgical procedures ( p 0.27 ) ( table 1 ) . number of surgeries and bladder augment the report of incontinence was similar between groups ( p = 0.58 ) . satisfactory cosmesis was numerically better in the female group ( 71.4% ) than the male group ( 53.6% ) , but it was not significant ( p = 0.26 ) . the most common complaint after complete surgical repair was about penile length ( 26/29 patients , 89.6% ) . satisfactory cosmesis and incontinence in the patients treated nine out of fourteen ( 64.3% ) women and twenty out of twenty - nine ( 69.0% ) men related sexual activity . although most men reported normal sexual relations ( 70.0% ) , only three ( 10.3% ) were satisfied with their penile length . twenty - two ( 75.9% ) also reported ejaculation ( table 3 ) . in this study , six out of fourteen ( 42.85% ) women had a pregnancy , and the median number of pregnancies was 1 ; overall , one patient had 2 and two patients had 3 children . there was no difference in physical aspects , pain , functional capability , general health , vitality , social aspects , emotional aspects and mental health , thus not affecting the overall score ( p = 0.36 ) ( figure 1 ) . the qol was not age - related ( p = 0.63 ) . although no significant gender difference was found , clinical impairments such as urinary fistula , incontinence , penile length and infertility were associated with worse qol scores and were male - related ( p < 0.01 ) . there was no difference in physical aspects , pain , functional capability , general health , vitality , social aspects , emotional aspects and mental health , thus not affecting the overall score ( p = 0.36 ) ( figure 1 ) . the qol was not age - related ( p = 0.63 ) . although no significant gender difference was found , clinical impairments such as urinary fistula , incontinence , penile length and infertility were associated with worse qol scores and were male - related ( p < 0.01 ) . the perceptions of health status and quality of life , and its treatment , are being widely recognized as a topic of research in clinical and epidemiological studies . thus , the use of instruments to assess the qol becomes fundamental to understanding the difficulties faced by patients with be . patients with be have a wide range of qol scores , which was expected due to the disease having heterogeneous clinical manifestations and complications among patients . in most areas surveyed by the sf-36 , women reported worse qol scores than men . however , socio - economic differences between countries should be taken into account . in this study , majority of the patients were of a low - income class and were studying or working . previous studies suggested that female be patients require comparatively fewer surgeries to achieve a satisfactory cosmetic outcome and have fewer problems with sexuality and sexual intercourse . nevertheless , our findings did not show any difference between genders regarding the number of surgeries , physical aspects or sexual activity . the female mean age was about 6 years older than the male mean age . even though this difference was not significant , this could be a bias , since the self - perception of a person with 20 years of age is probably different than one with 26 years . male patients with be are severely bothered by their penile size , with anxiety and avoidance of sexual interaction . despite this , others studies report that only 40 - 50% of male patients are satisfied with their penile appearance . we found that 70.0% of men had regular sexual activity but that only 10.3% were satisfied with their penile length . regarding the points of strength of this study we should mention the sample size acquired for a rare condition such as be and the data obtained from a long follow - up period in a country without a national database system for diseases . we have presented positive results from a low - income population of a developing country , with very limited resources . among the limitations of this study we should mention it 's cross sectional design . a prospective and longitudinal study design would be appropriate to evaluate qol indices during the various stages of life and impact on the daily life of patients . moreover , there is no comparison between patients with be and regular population to define a control group . it should be noted that some issues covered in this study are not standardized in the sf-36 . the direct relationship between the sf-36 and variables such as number of surgeries , bladder augment and incontinence demands further studies to standardize the quality of life questionnaires for patients with be . although age and gender were not predictors for better qol score , any clinical impairment such as urinary leakage due to incontinence or fistula , penile length and infertility tended to significantly decrease the overall qol in male patients with be . incontinence had a widespread negative impact on almost all aspects of the quality of life questionnaire . the male genitalia seems to be a troublesome aspect post - adolescence in male patients treated for be , which has an important impact on the overall qol , mainly affecting these patients socially . physical , mental , and psychosocial problems have important clinical implications and underscore the need for further development of surgical and psychosocial interventions .

introductionbladder exstrophy ( be ) is a rare condition that requires complex surgical corrections to achieve the goals of bladder functionality , normal sexual function , continence , and finally cosmesis . the purpose of this study was to identify clinical parameters that predict better quality of life ( qol ) scores using a validated questionnaire ( sf-36 ) with young adults after completing surgical reconstruction.material and methodsforty - three young adults ( mean age 22.35 years , 29 men and 14 women ) treated for be were evaluated using the short form 36 general health questionnaire ( sf-36 ) . clinical assessment involved evaluation of the actual condition regarding continence , sexual function , genital satisfaction and overall cosmesis.resultsboth genders presented similar qol scores ( p = 0.36 ) . the qol was not age - related ( p = 0.63 ) . neither genders did not present any differences in the number of procedures ( p = 0.27 ) . although no significant gender difference was found , clinical impairments such as urinary fistula , incontinence , penile length and infertility were associated with worse qol scores and were male - related ( p < 0.01 ) . the most common complaint after complete surgical repair was about penile length ( 26/29 patients , 89.6%).conclusionsage and gender were not predictors of better qol scores . any clinical impairment , such as urinary leakage due to incontinence or fistula , penile length and infertility , tended to significantly decrease the overall qol in male patients with be . the male genitalia seems to be the most troublesome aspect post - adolescence in treated male patients with exstrophy - epispadias . it has an important impact on the overall qol , mainly having a social affect on those patients .

INTRODUCTION METHODS RESULTS Quality of Life (QOL) SF-36 assessment DISCUSSION CONCLUSIONS CONFLICTS OF INTEREST

supplementary material is available for this article at 10.1007/s13659 - 012 - 0040 - 1 and is accessible for authorized users .

five new mexicanolide - type limonoids , heytrijunolides a e ( 15 ) were isolated from the branches and leaves of heynea trijuga . the structures of these new compounds were elucidated on the basis of extensive spectroscopic analysis . compound 3 showed weak cytotoxicity against hl-60 , smmc-7721 and a-549 human tumor cell lines with the ic50 values of 21.88 , 20.66 and 12.70 m , respectively . electronic supplementary materialsupplementary material is available for this article at 10.1007/s13659 - 012 - 0040 - 1 and is accessible for authorized users .

Electronic Supplementary Material Electronic supplementary material

the influence of xylitol on the control of risk factors and prevention of dental caries has already been assessed in several studies . the outcomes of this strategy seem to depend on the detection of minimal salivary levels of that polyol along time . however , the rapid clearance of xylitol from the oral cavity can explain the short - term increase of salivary xylitol concentrations after the use of vehicles such as candies , chewing gums , dentifrices and solutions . high frequencies of intake of xylitol have been employed in most of the studies so that its anticariogenic effects could be observed in clinical trials . however , these protocols could cause discomfort to the patient and strongly rely on compliance , which turns difficult the use of xylitol in daily clinical practice . thus , the need of vehicles that could allow sustained xylitol release along time has been recognized . fluoride , chlorhexidine , copper and iron have been shown to be successfully delivered when incorporated into dental varnishes because their adherence to tooth surface leads to longer maintenance of these different agents in the oral cavity . however , to our knowledge , no attempt was made to include xylitol into a varnish formulation so far . hence , the present in vitro study aimed at analyzing the xylitol concentrations in artificial saliva along time after application of varnishes containing 10% and 20% xylitol . enamel specimens were obtained from lower bovine incisors . after visual inspection , stained and/or cracked teeth were excluded . besides , soft tissues were removed from the coronal and root surfaces with the aid of a periodontal curette ( duflex , sswhite , rio de janeiro , rj , brazil ) . fifteen enamel specimens ( 8x4 mm ) were obtained after two double sections of the widest portion of the dental crowns , as described by magalhes , et al.19 ( 2008 ) . subsequently , the specimens were numbered and randomly allocated to 3 different groups ( n=5/group ) , according to the type of varnish that would be applied : ( 1 ) 10% xylitol ( fgm / dentscare , joinville , sc , brazil ) ; ( 2 ) 20% xylitol ( fgm / dentscare ) and ( 3 ) no xylitol ( control ; fgm / dentscare ) . three different varnishes ( control , containing 10% and 20% xylitol ) were especially manufactured by fgm / dentscare for the present research . xylitol concentrations were determined by the maximum incorporation of that polyol into the varnish that would not lead to precipitation . varnishes used contain colophonium , synthetic resin , thickening polymer , essence and ethanol in their composition ( informed by manufacturer ) . xylitol was supplied by danisco ( xylitab 300 , danisco brasil ltda , cotia , sp , brazil ) . enamel specimens were weighed before and after varnish application to standardize the amount used . after a pilot study , 4 mg of the respective varnish were applied on a dry bovine enamel specimen with a microbrush . it was the maximum amount that could be applied considering the area of the specimens . after 10 min , each specimen was inserted into a microcentrifuge tube containing 500 l of artificial saliva ( 1.5 mmol / l ca(no3)2.h2o ; 0.9 mmol / l na2hpo4.2h2o ; 150 mmol / l kcl ; 0.1 mol / l h2nc(ch2oh)3 ( tris ) ; 0.05 g / ml naf , ph 7.0 ) . sample size and times of immersion of the specimens in artificial saliva were chosen based on results of a pilot study ( data not shown ) . after 1 , 8 , 12 , 16 , 24 , 48 and 72 h from the first immersion , each specimen was removed , washed with deionized water and placed into another microcentrifuge tube containing 500 l of fresh artificial saliva at room temperature . the analysis of xylitol concentrations in artificial saliva was performed by a colorimetric method , using a spectrophotometer ( ultrospec 2000 uv / visible spectrophotometer , pharmacia biotech , cambridge , usa ) and an enzymatic kit d - sorbitol / xylitol ( boehringer mannheim , r - biopharm , darmstadt , germany ) . a standard curve was obtained in triplicate using five different amounts of xylitol ( 0.5 , 2.0 , 5.0 , 8.0 and 10 g ) . a mathematical equation determined by the manufacturer of the enzymatic kit was used to convert the values of absorbance into xylitol concentration ( mg / l ) . all samples were analyzed in duplicate and the mean repeatability of the readings was 98.6% . graph pad instat version 3.0 for windows and graph pad prism version 4.0 for windows ( graph pad software inc . , san diego , usa ) were used . since data presented a normal distribution ( kolmogorov - smirnov test ) and homogeneity ( bartlett test ) , they were analyzed by two - way repeated - measures anova followed by bonferroni 's post hoc test for individual comparisons . enamel specimens were obtained from lower bovine incisors . after visual inspection , stained and/or cracked teeth were excluded . besides , soft tissues were removed from the coronal and root surfaces with the aid of a periodontal curette ( duflex , sswhite , rio de janeiro , rj , brazil ) . fifteen enamel specimens ( 8x4 mm ) were obtained after two double sections of the widest portion of the dental crowns , as described by magalhes , et al.19 ( 2008 ) . subsequently , the specimens were numbered and randomly allocated to 3 different groups ( n=5/group ) , according to the type of varnish that would be applied : ( 1 ) 10% xylitol ( fgm / dentscare , joinville , sc , brazil ) ; ( 2 ) 20% xylitol ( fgm / dentscare ) and ( 3 ) no xylitol ( control ; fgm / dentscare ) . three different varnishes ( control , containing 10% and 20% xylitol ) were especially manufactured by fgm / dentscare for the present research . xylitol concentrations were determined by the maximum incorporation of that polyol into the varnish that would not lead to precipitation . varnishes used contain colophonium , synthetic resin , thickening polymer , essence and ethanol in their composition ( informed by manufacturer ) . xylitol was supplied by danisco ( xylitab 300 , danisco brasil ltda , cotia , sp , brazil ) . enamel specimens were weighed before and after varnish application to standardize the amount used . after a pilot study , 4 mg of the respective varnish were applied on a dry bovine enamel specimen with a microbrush . it was the maximum amount that could be applied considering the area of the specimens . after 10 min , each specimen was inserted into a microcentrifuge tube containing 500 l of artificial saliva ( 1.5 mmol / l ca(no3)2.h2o ; 0.9 mmol / l na2hpo4.2h2o ; 150 mmol / l kcl ; 0.1 mol / l h2nc(ch2oh)3 ( tris ) ; 0.05 g / ml naf , ph 7.0 ) . sample size and times of immersion of the specimens in artificial saliva were chosen based on results of a pilot study ( data not shown ) . after 1 , 8 , 12 , 16 , 24 , 48 and 72 h from the first immersion , each specimen was removed , washed with deionized water and placed into another microcentrifuge tube containing 500 l of fresh artificial saliva at room temperature . the analysis of xylitol concentrations in artificial saliva was performed by a colorimetric method , using a spectrophotometer ( ultrospec 2000 uv / visible spectrophotometer , pharmacia biotech , cambridge , usa ) and an enzymatic kit d - sorbitol / xylitol ( boehringer mannheim , r - biopharm , darmstadt , germany ) . a standard curve was obtained in triplicate using five different amounts of xylitol ( 0.5 , 2.0 , 5.0 , 8.0 and 10 g ) . a mathematical equation determined by the manufacturer of the enzymatic kit was used to convert the values of absorbance into xylitol concentration ( mg / l ) . all samples were analyzed in duplicate and the mean repeatability of the readings was 98.6% . graph pad instat version 3.0 for windows and graph pad prism version 4.0 for windows ( graph pad software inc . , san diego , usa ) were used . since data presented a normal distribution ( kolmogorov - smirnov test ) and homogeneity ( bartlett test ) , they were analyzed by two - way repeated - measures anova followed by bonferroni 's post hoc test for individual comparisons . regardless of the time of immersion , xylitol could not be detected in artificial saliva samples of the control group . consequently , data obtained only after application of xylitol - containing varnishes are presented . mean xylitol concentrations in artificial saliva ranged between 63.42.7 mg / l ( 1 h ) and 32.70.9 mg / l ( 72 h ) after application of 10% xylitol varnish and between 169.01.5 mg / l ( 1 h ) and 12.70.4 mg / l ( 72 h ) after application of 20% xylitol varnish ( figure 1 ) . significant differences were detected for the variables varnishes ( f=33 , p=0.0004 ) and times of immersion in saliva ( f=2,466 , p<0.0001 ) , as well as a significant interaction between these criteria ( f=1,486 , p<0.0001 ) . mean accumulated xylitol concentrations ( mg / l ) in artificial saliva over time after application of 10% and 20% xylitol varnishes . distinct lowercase letters indicate significant differences among time points for each varnish ( two - way rm anova , p<0.05 ) in overview , a reduction of xylitol concentration was observed along time for both varnishes , except for 48 h after immersion in artificial saliva . nevertheless , a greater reduction could be noticed after application of 20% xylitol varnish than after application of 10% xylitol varnish ( figure 1 ) . in the short - term ( up to 8 h ) , xylitol concentrations in saliva were significantly higher after application of 20% xylitol varnish in comparison to its counterpart ( p<0.001 ) . this relationship was inverted after 12 h from application of the xylitol varnish during the remaining time ( p<0.001 ) . when the use of xylitol is aimed to prevent dental caries , important parameters such as frequency of administration , dose - response , and minimal salivary concentration must be regarded . however , such clinical conditions have not been established yet . the amount of 5 g of xylitol , divided in at least 3 times per day , is supported for a considerable number of studies as the minimum clinical effective dose to achieve prevention of dental caries . on the other hand , it has been recently reported that 0.01% xylitol was able to inhibit in vitro growth of mutans streptococci , but the extent to which this information can be transferred to the clinical situation demands further investigation . randomized clinical trials normally use daily xylitol amounts that vary between 2 and 15 g , divided in 3 to 5 times per day , delivered mainly by chewing gums it is desirable to develop new vehicles able to promote a sustained release of xylitol in saliva along time . hence , xylitol - containing varnishes were formulated and tested in the present study . dental varnishes can be used in lower frequencies and mastication is not required for their effectiveness which improves patient 's compliance . additionally , the varnishes were manufactured with a fruit flavor to make them more suitable for babies and young children , compared to chewing gums or solutions . it also has been recognized that the frequency of use of xylitol seems to be more important in the prevention of dental caries than the amount of that polyol . xylitol is rapidly removed from the oral cavity after application of the vehicles commonly available . ( 2006 ) observed that salivary xylitol levels can be significantly increased up to 16 min by mastication of chewing gums containing 1.32 g of xylitol or by mouth rinsing with 10% xylitol solution . tablets , candies and dentifrices were able to enhance salivary levels of that polyol up to 8 min after be used . this pioneer study evaluated the potential of a newly developed varnish to release xylitol in a medium that resembles the composition of natural saliva along time . therefore , a positive control group with xylitol delivered via another vehicle , such as chewing gum , was not included . bovine enamel specimens were immersed in artificial saliva and they were kept under gentle agitation to simulate the movements that occur in the mouth . conditions as salivary flow and temperature of the oral cavity were not simulated . in this study , the amount of varnish ( 4 mg ) applied on each specimen might contain 40 and 80 mg of xylitol in 10% and 20% varnishes , respectively , which is very low when compared to that is recommended for prevention of dental caries . however , the volume of varnish usually applied in the whole mouth is around 0.2 ml ( ~200 mg ) , nearly 50 times larger than that used in the present research . consequently , it could be clinically expected that around 2 and 4 g of xylitol would be found in the oral cavity after applying respectively 10% and 20% xylitol varnishes . higher concentrations of 20% xylitol could not be incorporated into the varnishes without precipitation . regarding safety considerations , high doses of xylitol ingested by adults and children can cause adverse gastrointestinal effects . adults can tolerate the intake of up to 200 g of xylitol per day , while children can ingest up to 45 g per day . then , even if the xylitol varnishes would be applied on all dental surfaces , the amount of xylitol that could be ingested is completely safe . we believe that more homogeneous varnishes might provide more sustained xylitol release along time and , perhaps , this hypothesis could explain the present results . significantly higher xylitol concentrations were released in saliva after application of the 20% xylitol varnish than of the 10% xylitol varnish on a short - term basis ( up to 8 h ) . thereafter , this trend was inverted from 12 h until the end of the experimental period ( 72 h ) . therefore , the 10% xylitol varnish was able to produce sustained degrees of xylitol release along time . however , both varnishes were not removed from the dental surfaces throughout the time . this situation is not observed under clinical conditions , when dental varnishes are usually removed from tooth surfaces after 6 to 12 h from application . taking this into account , a better clinical performance of the 20% xylitol varnish might be expected in relation to its 10% counterpart . before any speculative conclusion , the performance of these varnishes needs to be tested in vivo regarding xylitol release and their caries - preventive effect . considering the limitations of this pioneer in vitro study , it can be concluded that sustained xylitol releases can be obtained in artificial saliva after application of 10% xylitol varnish , although 20% xylitol varnish released larger amounts of that polyol in the short - term . these varnishes seem to be viable alternatives to increase salivary xylitol levels and , therefore , they should be clinically tested to confirm their effectiveness .

objective the present study analyzed xylitol concentrations in artificial saliva over time after application of varnishes containing 10% and 20% xylitol . material and methods fifteen bovine enamel specimens ( 8x4 mm ) were randomly allocated to 3 groups ( n=5/group ) , according to the type of varnish used : 10% xylitol , 20% xylitol and no xylitol ( control ) . after varnish application ( 4 mg ) , specimens were immersed in vials containing 500 l of artificial saliva . saliva samples were collected in different times ( 1 , 8 , 12 , 16 , 24 , 48 and 72 h ) and xylitol concentrations were analyzed . data were assessed by two - way repeated - measures anova ( p<0.05 ) . results colorimetric analysis was not able to detect xylitol in saliva samples of the control group . salivary xylitol concentrations were significantly higher up to 8 h after application of the 20% xylitol varnish . thereafter , the 10% xylitol varnish released larger amounts of that polyol in artificial saliva . conclusions despite the results in short - term , sustained xylitol releases could be obtained when the 10% xylitol varnish was used . these varnishes seem to be viable alternatives to increase salivary xylitol levels , and therefore , should be clinically tested to confirm their effectiveness .

INTRODUCTION MATERIAL AND METHODS Preparation of bovine enamel specimens Varnish application Times of immersion in artificial saliva Analysis of xylitol in artificial saliva Statistical analysis RESULTS DISCUSSION CONCLUSIONS

the anorectum plays an important role in regulation of defecation and in the maintenance of continence.1 the most widely used test for anorectal function is anorectal manometry . a comprehensive assessment of anorectal function consists of measuring at a minimum each of the following parameters : ( 1 ) anal sphincter function , ( 2 ) rectoanal reflex activity , ( 3 ) rectal sensation , ( 4 ) changes in anal and rectal pressures during attempted defecation , ( 5 ) rectal compliance and ( 6 ) performance of a balloon expulsion test.2 anal sphincter function is assessed by measurement of resting sphincter pressure , squeeze sphincter pressure , and the functional length of the anal canal . maximum resting anal canal tone predominantly reflects internal anal sphincter function , while voluntary anal squeeze pressure reflects external anal sphincter ( eas ) function . functional anal canal length is defined as the length of the anal canal over which resting pressure exceeds that of the rectum by greater than 5 mmhg or , alternatively , as the length of the anal canal over which pressures are greater than half of the maximal pressure at rest ( fig . maximal resting anal pressure is defined as the difference between intrarectal pressure and the highest recorded anal sphincter pressure at rest , and is generally recorded 1 - 2 cm from the anal verge . maximum squeeze pressure is defined as the difference between the intrarectal pressure and the highest pressure that is recorded at any level within the anal canal during the squeeze maneuver ( fig . rapid distention of the rectum induces a transient increase in rectal pressure , followed by a transient increase in anal pressure associated with eas contraction ( the rectoanal contractile reflex ) , and in turn , a more prolonged reduction in anal pressure due to relaxation of the internal anal sphincter ( the rectoanal inhibitory reflex , fig . the rectoanal contractile reflex is a compensatory guarding mechanism that allows a positive anorectal pressure gradient to be maintained during transient increases in intra - abdominal pressure ( such as coughing ) , which is essential for preserving continence . in fecal incontinence patients , anal sphincter pressure the lowest volume of air that evokes sensation and a desire to defecate , and the maximum tolerable volume are recorded.4 assessment of rectal sensation is useful in patients with fecal incontinence or rectal hyposensitivity . when an individual is requested to ' bear down , ' as if attempting to defecate , the normal response consists of an increase in rectal pressure that is coordinated with a relaxation of the eas ( fig . inability to perform this coordinated maneuver suggests a diagnosis of dyssynergic or obstructive defecation , a common cause of constipation.5 this response can be quantified using the defecation index = maximum rectal pressure during attempted defecation / minimum anal residual pressure during attempted defecation . a normal defecation index is > 1.5.5 three types of dyssynergic defecation are recognized.6 most patients show paradoxical increase in anal sphincter pressure during attempted defecation with normal adequate pushing force ( type 1 , fig . some patients are unable to generate an adequate pushing force , and exhibit a paradoxical anal contraction ( type 2 ) . in type 3 , the patient can generate an adequate pushing force , but has absent or incomplete ( < 20% ) sphincter relaxation . rectal compliance is calculated by plotting the relationship between balloon volume ( dv ) and steady state intrarectal pressure ( dp ) . the balloon expulsion test is used to assess rectoanal co - ordination during defecatory maneuvers.7 the test evaluates a patient 's ability to expel a filled balloon from the rectum , providing a simple and more physiologic assessment of defecation dynamics . most normal subjects can expel the balloon within 1 minute.5 if the patient is unable to expel the balloon within 3 minutes , dyssynergic defecation should be suspected .

anorectal manometry is the most well established and widely available tool for investigating anorectal function . anal sphincter tone can be quantified by anorectal manometry . the anorectal sensory response , anorectal reflexes , rectal compliance , and defecatory function are also assessed by anorectal manometry . this report will focus on defining parameters for measurement and interpretation of anorectal manometry tests .

Introduction Anal Sphincter Function Rectoanal Reflex Activity Rectal Sensation Changes in Anal and Rectal Pressures During Attempted Defecation Rectal Compliance Balloon Expulsion Test Conclusion

the microenvironment is a dynamic system that is largely composed of inflammatory cells and cytokines , including tumor cells and stroma ( the latter of which consists of immune cells and the surrounding extracellular matrix ) [ 1 , 2 ] . under specific stimulation conditions , inflammation can result in the production of antiangiogenic cytokines , which suppress tumor growth . despite being antagonistic to the action of tumor suppression , in most tumors , inflammatory cells can promote angiogenesis , growth , tumor cell migration , tissue degradation , and remodeling steps that are characteristic of the chronic inflammation associated with intense tissue disorders typical of acute inflammation [ 1 , 37 ] . the transcription factor nuclear kappa b ( nf-b ) plays a major role in innate immunity and inflammatory responses and it is considered the major link between inflammation and tumorigenesis . this factor activates antiapoptotic genes [ 911 ] and could promote the transcription of genes associated with growth , invasion , and metastasis , being the key factor that allows precancerous and malignant cells to escape apoptosis . interleukins ( ils ) could stimulate many signaling pathways and thus regulate the transcription of target genes involved in cellular proliferation and survival . besides that , they act as immune mediators and have been linked to cancer , playing a key role in the recruitment and positioning of leukocytes in carcinogenesis . the recruitment and activation of immune cells and the antitumor effects of il-4 , an anti - inflammatory cytokine , are involved in the inhibition of angiogenesis , inflammation , thrombosis , growth , and invasion in some cancers [ 16 , 17 ] . in addition to these effects on stromal cells , some studies have reported that il-4 has antitumor functions by inducing apoptosis in several types of tumor cells including breast cancer , renal cell carcinoma , and hepatocellular carcinoma . il-10 is a cytokine produced by th1 and th2 cells as well as by tumor cells and has potent immunosuppressive properties . originally known as an inhibitor factor of the synthesis of cytokines , il-10 suppresses the production of il-1 , il-1 , tumor necrosis factor- ( tnf- ) , il-6 , il-8 , il-12 , il-18 , granulocytes , inflammatory macrophages , and nf-b [ 7 , 21 , 22 ] . this cytokine acts concurrently with il-3 and il-4 to stimulate the proliferation of mast cells , peripheral blood lymphocytes , and macrophages , and their anti - inflammatory action has been suggested in tumor microenvironment . on the other hand , il-6 has strong procarcinogenic activity due to its role in tumor cell proliferation , survival , angiogenesis , inflammation , and metastasis [ 7 , 23 ] . in addition , il-6 is one of the activation signals for nf-b , promoting cell differentiation and subsequent metastasis , thus reinforcing the close link between inflammatory mediators and neoplastic progression . therefore , tnf- induces proinflammatory cytokines such as il-1 and il-6 [ 26 , 27 ] , during the inflammatory response [ 2628 ] . the interaction of this cytokine with its receptor ( tnfr ) induces apoptotic and antiapoptotic effects in different cell systems , and it can promote cell proliferation and inhibits apoptosis in the tumor microenvironment , at least indirectly , through the induction of nf-b . a better understanding of the complexity of these biological factors ' network , together with the characterization of new genes , aids in uncovering the molecular mechanisms of inflammatory diseases and cancer . in this way , the objective of this work was to ascertain the role of anti - inflammatory cytokines , il-4 and il-10 , and proinflammatory cytokines , il-6 and tnf- , in mammary tumors of female dogs and to correlate these results with the gene and protein expression of nf-b . in addition , we sought to verify the correlation of these cytokines with clinicopathological parameters , cancer progression , and overall survival , to determine their prognostic value in mammary tumors . peripheral blood and tumor samples were obtained from 30 female dogs with malignant mammary neoplasia ( test group ) attended to in so jos do rio preto , sp , brazil , veterinary clinics in the years 2011 and 2012 . animals with mammary tumors were included regardless of breed , weight , or age . these animals were followed up for 540 days after tumor excision and blood collection . for control group , peripheral blood samples from 30 female dogs and five samples of mammary tissue adjacent to mammary cancer were obtained from animals that underwent elective hysterectomy as a preventive measure , after completion of the consent form by their owners . the criteria for the control group were rigorously followed and included animals outside the estrus period , animals with no tumor history , and animals with no detectable disease / inflammation in the period or surgery in the next study period . in addition , animals that died of causes other than tumor recurrence or metastasis were excluded from the study . peripheral blood ( 3 ml ) was collected in a corvac serum separator tube ( labor import , so paulo , sp ) , stored at 4c , processed by centrifugation ( 1000 g , 25 min ) , and cryopreserved at 80c for posterior enzyme linked immunosorbent assay ( elisa ) and xmap protein analysis . the first one was fixed in 10% formaldehyde buffer for 24 hours and paraffin embedded for histopathology classification after hematoxylin - and - eosin ( he ) stain . tumor grading was ascertained according to misdorp et al . by the armed forces institute of pathology ( afip ) . the second piece was collected in a falcon tube containing the rna - stabilizing solution , rnalater ( life technologies ) , stored at room temperature for 24 hours , and posteriorly used for rna extraction and qpcr analysis . female dogs from the test group were evaluated with respect to physical ( age ) , pathological ( time course ( interval between tumor diagnosis and surgical removal ) , tumor nodules location , lymph node involvement , tumor mass size , clinical staging , ulceration , and vascularization ) , and clinical ( metastasis , local recurrence , and death ) characteristics ( table 1 ) . the parameters employed for the classification of clinical tumor grading were in accordance with the tnm ( size , lymph node involvement , and metastasis ) system established by the world health organization ( who ) for canine mammary gland tumors . the clinical grade was assigned as i , ii , iii , or iv according to the extent of the tumor and the prognosis . the age of the animals varied from 7 to 14 years ( mean = 10 years ) and there was predominance of poodle ( 19% ) and dachshund ( 24% ) races . in terms of the clinicopathological characteristics , most animals had a tumor for more than six months ( 54% ) and were of clinical stage iii or iv ( 54% ) . the proportions of animals with lymph node involvement , animals with metastasis / local recurrence , or animals that died during the study were 16% , 27% , and 27% , respectively ( table 1 ) . for total rna extraction , the tumor samples were manually processed into smaller pieces ( 100 mg / piece ) , and the pieces were then immersed in trizol reagent ( invitrogen life technologies ) and macerated with a politron ( dremel , mexico ) . previously , tumor samples were treated with dnase and 10x buffer ( 10 mm tris - hcl , ph 7.5 ; 50 mm cacl2 ; and 10 mm mgcl2 ) ( rneasy mini kit , qiagen ) followed by the addition of ethylenediaminetetraacetic acid ( edta ) to deactivate or eliminate proteins binding to template dna . the qpcr was performed in triplicate using the stepone plus real time pcr system ( applied biosystems ) and taqman universal master mix ( applied biosystems ) . the following specific primers were used : nf-b , cf02622551_m1 ( applied biosystems ) ; il-4 , cf02623112_m1 ( applied biosystems ) ; il-6 , cf02624153_m1 ( applied biosystems ) ; il-10 , cf02624265_m1 ( applied biosystems ) ; and tnf- , cf02628236_m1 ( applied biosystems ) . the following qpcr conditions were used : an initial step of 2 minutes at 50c ; denaturation for 10 minutes at 95c ; 40 cycles at 95c for 15 seconds and 60c for one minute to anneal primers and extend the chains , respectively ; and 35 seconds at 65c to collect the signal . the dissociation curve was generated after amplification and included a step of 15 seconds at 95c followed by 1 min at 60c . each transcript level was normalized to the expression of rps19 ( sense ( 5-gcc ttc ctc aaa aag tct ggg-3 ) , antisense ( 5-gct tgc tcc cta cga tga gaa c-3 ) , and probe ( 5-ccc tga atg ggt gga c-3 ) ( applied biosystems ) ) and rpl8 , cf02663820_m1 ( applied biosystems ) , which were used as endogenous control genes . the relative quantification ( rq ) value of the expression of interest genes was determined with dataassist v3.0 , by the quantification method related to the average of the normalizing genes used as endogenous controls ( ct ) and showed by log10 . the gene expression of each sample was separately analyzed and classified as underexpressed ( samples giving < 0 log10 measurement ) and overexpressed ( samples with quantification > 0 log10 ) . nf-b ( dog nuclear factor ( nf ) kappa b p105 subunit ( nf-b1 ) , cusabio biotech co. , hubei , china ) and il-4 ( uscn life science inc . , houston , tx , usa ) protein expression analysis was performed according to the manufacturers recommendations . first of all , the plate was precoated with a specific antibody for nf-b and il-4 . then , the standards and samples were added in each well and incubated for 2 hours at 37c . a biotin - conjugated antibody and a horseradish peroxidase- ( hrp- ) avidin conjugate were added to the plate to amplify the signal and to increase the detectability of the target molecule . a chromogenic substrate ( 3 - 3 - 5 - 5-tetramethylbenzidine , tmb ) finally , sulfuric acid ( stop solution ) was added to stabilize the color development to enable accurate measurement of the intensity at 450 nm . the analysis of il-6 , il-10 , and tnf- proteins was performed using the milliplex map kit ( ccytomag-90k ; millipore corporation , usa ) , and the protein levels were analyzed using the luminex xmap magpix ( millipore corporation , usa ) . the standard , control , and samples were added to the appropriate wells as were magnetic beads coated with specific antibodies for il-6 , il-10 , and tnf-. the plate was sealed and incubated overnight at 4c with agitation on a plate shaker . measurements were performed at 635 nm to excite the magnetic beads and 525 nm to detect phycoerythrin . the calculation of od was determined through the adjustment curve four - parameter logistic ( 4-pl ) using the skanit software for the multiskan fc 2.5.1 . clinicopathological characteristics , gene expression , and protein quantification were analyzed in the test and control groups , and the differences between these groups were determined by student 's t - test or anova followed by the bonferroni test . the cutoff points for the protein analyses were established by the receiver operating characteristic ( roc ) curve . for the roc curve , the protein expression levels from female dogs that died were compared with those that survived until the end of the followup period . survival curves were plotted by the kaplan - meier method and the differences between the curves were evaluated by a log - rank test and hazard function . spearman 's rank correlation analysis was performed to describe the correlation between proinflammatory and anti - inflammatory cytokines protein and gene expression with nf-b . in addition , the mutual relationship between proinflammatory and anti - inflammatory cytokines was analyzed . a multivariate logistic regression analysis was employed to evaluate the simultaneous influence of the prognostic factors on animal death . the nf-b gene was significantly overexpressed in animals with tumor time course greater than six months ( p < 0.0001 ) ; multiple tumor locations ( p = 0.005 ) ; abundant tumor vascularization ( p = 0.009 ) ; tumor mass size greater than three centimeters ( cm ) ( p = 0.005 ) ; lymph node involvement ( p = 0.008 ) ; metastasis ( p = 0.02 ) ; recurrence ( p = 0.01 ) ; and clinical stage iii or iv ( p < 0.0001 ) ( figure 1(a ) ) . in the same way , the nf-b protein expression was significantly higher in animals older than 10 years ( p = 0.0001 ) , as well as in animals with a tumor time course of less than six months ( p = 0.001 ) , multiple tumor locations ( p = 0.01 ) , abundant tumor vascularization ( p = 0.03 ) , and metastasis ( p = 0.0005 ) ( figure 1(b ) ) . the best cutoff point for nf-b established by the roc curve to discriminate the high risk of death was 219.5 pg / ml ( sensitivity ( 95% ci ) = 71% , specificity ( 95% ci ) = 41% ) . no correlation was observed between nf-b serum levels and survival time ( or 2.003 ; 95% ci = 0.5553 to 8.983 ; p = 0.29 ) ( data not shown ) . il-4 was significantly overexpressed in animals aged 10 years ( p = 0.02 ) and animals with tumor time course of less than six months ( p = 0.01 ) ; single tumor location ( p = 0.0001 ) ; moderate tumor vascularization ( p = 0.0002 ) ; tumor mass size less than three centimeters ( p = 0.0006 ) ; no lymph node involvement ( p = 0.001 ) ; metastasis ( p < 0.0001 ) ; recurrence ( p = 0.0003 ) ; and clinical stage i or ii ( p < 0.0001 ) and animals that were still alive at the end of the followup period ( p < 0.0001 ) ( figure 2(a ) ) . therefore , il-4 protein expression was significantly higher in animals aged 10 years ( p < 0.0001 ) and animals with tumor time course of less than six months ( p = 0.04 ) ; moderate tumor vascularization ( p = 0.03 ) ; tumor mass size of less than three centimeters ( p = 0.001 ) ; no metastasis ( p = 0.01 ) ; no recurrence ( p = 0.02 ) ; and clinical stage i or ii ( p = 0.008 ) and animals that were still alive at the end of the followup period ( p = 0.005 ) ( figure 2(b ) ) . using the roc curve , the best cutoff point for il-4 to discriminate the high risk of death was 368.4 pg / ml ( sensitivity ( 95% ci ) = 88% , specificity ( 95% ci ) = 95% ) . the kaplan - meier test demonstrated a positive correlation between il-4 level and survival time ( or 0.068184 ; 95% ci = 0.783029 to 1 ; p = 0.007 ) ( figure 3 ) . for il-10 , significant gene overexpression was also correlated with tumor time course of less than six months ( p = 0.003 ) ; moderate tumor vascularization ( p = 0.02 ) ; no lymph node involvement ( p = 0.0005 ) ; no metastasis ( p = 0.01 ) ; no recurrence ( p = 0.01 ) ; clinical stage i or ii ( p = 0.02 ) ; and animals that were still alive at the end of the followup period ( p = 0.009 ) ( figure 4(a ) ) . likewise , il-10 protein expression was significantly higher in animals with tumor time course of less than six months ( p = 0.003 ) ; single tumor location ( p = 0.0009 ) ; moderate vascularization ( p = 0.04 ) ; no recurrence ( p = 0.04 ) , and clinical stage i or ii ( p = 0.03 ) and animals that were still alive during the followup ( p = 0.04 ) ( figure 4(b ) ) . for the roc curve for il-10 , the best cutoff point to discriminate the high risk of death was 243 pg / ml ( sensitivity ( 95% ci ) = 100% , specificity ( 95% ci ) = 53% ) . no correlation was observed between il-10 protein expression and survival time ( or 0.3267 ; 95% ci = 0.06811 to 1.564 ; p = 0.16 ) ( data not shown ) . in contrast , il-6 was significantly overexpressed in animals aged > 10 years ( p = 0.03 ) and animals with multiple tumor locations ( p = 0.0005 ) ; abundant tumor vascularization ( p = 0.003 ) ; tumor mass size greater than three centimeters ( p = 0.01 ) ; lymph node involvement ( p = 0.04 ) ; metastasis ( p = 0.003 ) ; recurrence ( p = 0.03 ) ; and clinical stage iii or iv ( p = 0.02 ) and animals that died during followup ( p = 0.008 ) ( figure 5(a ) ) . similarly , il-6 protein expression was significantly higher in animals with tumor time course greater than six months ( p = 0.001 ) ; multiple tumor locations ( p = 0.01 ) ; abundant vascularization ( p = 0.02 ) ; tumor mass size greater than three centimeters ( p = 0.001 ) ; lymph node involvement ( p < 0.0001 ) ; metastasis ( p < 0.0001 ) ; recurrence ( p = 0.007 ) ; and clinical stage iii or iv ( p = 0.001 ) and animals that died during followup ( p = 0.04 ) ( figure 5(b ) ) . the best cutoff point for il-6 established by the roc curve to discriminate the high risk of death was 76.95 pg / ml ( sensitivity ( 95% ci ) = 75% , specificity ( 95% ci ) = 86% ) . no correlation was observed between il-6 protein expression and survival time ( or 1.719 ; 95% ci = 0.4045 to 9.580 ; p = 0.43 ) ( data not shown ) . tnf- was also overexpressed in animals with tumor time course greater than six months ( p = 0.01 ) ; multiple tumor locations ( p = 0.005 ) ; abundant tumor vascularization ( p = 0.007 ) ; tumor mass size greater than three centimeters ( p = 0.02 ) ; metastasis ( p = 0.001 ) ; recurrence ( p = 0.003 ) ; and clinical stage iii or iv ( p = 0.0002 ) and animals that died during followup ( p = 0.005 ) ( figure 6(a ) ) . the protein expression of tnf- was significantly higher in animals aged > 10 years ( p = 0.04 ) and animals with tumor time course greater than six months ( p = 0.004 ) ; abundant vascularization ( p = 0.01 ) ; lymph node involvement ( p = 0.0007 ) ; metastasis ( p = 0.02 ) ; and recurrence ( p = 0.01 ) and animals that died during followup ( p = 0.04 ) ( figure 6(b ) ) . the best cutoff point to discriminate the high risk of death established by the roc curve was 16.3 pg / ml ( sensitivity ( 95% ci ) = 87% , specificity ( 95% ci ) = 62% ) . this analysis demonstrated a negative correlation between tnf- protein expression and survival time ( or 5.906 ; 95% ci = 1.387 to 25.6 ; p = 0.01 ) ( figure 7 ) . spearman 's rank correlation demonstrated that proinflammatory cytokines il-6 and tnf- are mediated by nf-b . there was a positive correlation between the proinflammatory tnf- protein and gene expression when compared to nf-b protein and gene expression ( r = 0.5233 , p = 0.003 ; r = 0.6645 , p < 0.0001 ; figures 8(a ) and 8(b ) , resp . ) . in the same way , there was a positive correlation between il-6 and nf-b gene expression ( r = 0.4012 ; p = 0.02 ) ( figure 8(b ) ) . furthermore , the mutual balance between proinflammatory cytokines and anti - inflammatory ones was analyzed by spearman 's rank correlation and showed no correlation ( p > 0.05 ; data not shown ) . the multivariate analysis of high levels of the proinflammatory cytokines , low levels of the anti - inflammatory cytokines , and clinicopathological features of poor prognosis with regard to an increased risk of death showed no correlation ( p > 0.05 ) ( table 2 ) . nf-b is a crucial factor between chronic inflammation and cancer development and it regulates both anti- and proinflammatory cytokines at various stages of tumorigenesis and inflammation [ 35 , 36 ] . we observed high gene and protein expression levels of nf-b in animals with tumor progression for greater than six months , multiple tumor locations , abundant vascularization , and metastasis , indicating a role of this factor in angiogenesis support and inhibition of apoptosis in tumor cells . in accordance with our prediction , kiliccioglu et al . observed that nf-b inhibition , through proteasome inhibition , induces apoptosis in human prostate cancer cell line ( pc3 ) , resulting in significantly increased protein levels of caspase-3 , which plays a central role in the execution phase of cell apoptosis . similarly , chien et al . reported that pancreatic cancer cell growth is suppressed by inhibiting the nf-b pathway . in the same way , high protein levels of nf-b were observed in female dogs above 10 years old which could indicate the correlation between this nuclear factor and cell oxidative stress , leading to tissue damage and chronic inflammation . corroborating with this , a review of oxidative stress and breast cancer , recently published by nourazarian et al . , suggests that the increase of free radical levels in tumor cells occurs under the influence of the increased expression of cellular enzymes and enhanced activity of some dependent tumor cells , such as cancer - associated fibroblasts ( cafs ) and tumor - associated macrophages ( tams ) [ 39 , 40 ] . once nf-b has been activated through one of many pathways , crucial properties of the malignant phenotype are activated , promoting tumor cell adaptation to the tumor environment ; then , we also reported nf-b overexpressed in female dogs with tumor masses greater than three centimeters , lymph node involvement , recurrence , and clinical stage iii or iv , leading to a poor survival rate of the animals . it is known that nf-b is required for the expression of many cytokine genes that encode proteins associated with inflammatory mediators . then , through spearman 's rank correlation , we showed that high expression of nf-b modulates positively the il-6 and tnf- expression levels in mammary tumors . the proinflammatory cytokines il-1 , il-6 , and tnf- could be modulated by lipopolysaccharides ( lps ) administration as it occurs in inflammatory diseases . according to cheon et al . , some drugs , as saxifragin , inhibited the lps - induced nuclear translocation of p65 and the activation of caspase-1 in raw 264 cells , suggesting that the nf-b - regulated gene transcription is responsible for anti - inflammatory response of macrophages . in addition , osnes et al . affirmed that the inhibitory synthesis of tnf- by drugs as acetylsalicylic acid and sodium salicylate in human monocytes is due to interfering of nuclear translocation of nf-b / c - rel proteins . besides that , corroborating with our study , al - halabi et al . showed that the inhibition of nf-b significantly lowers microvessel density ( cd31 ) and mrna expression levels of il-6 , tnf- , and il-1 , as well as protein levels of proliferation marker ( ki-67 ) and vascular endothelial growth factor- ( vegf- ) a in human colon cancer , confirming the primordial role of nf-b in angiogenesis and , therefore , tumor progression . regarding anti - inflammatory interleukins , our study sustains a protector action of the anti - inflammatory cytokines il-4 and il-10 in female dogs ' mammary tumors . il-4 was highly expressed in gene and protein levels in female dogs with a better prognostic . this cytokine acts on endothelial cells , inhibiting tumor - induced vascularization and starving tumor cells , and also induces secretion of il-10 , the th2 cytokine with a strong suppressive effect on tumors [ 46 , 47 ] , which could explain high levels of this cytokine in animals that are still alive after the followup period and in overall survival . furthermore , according to okada and kuwashima , the sustained expression of il-4 may provide effective means for therapy of a variety of diseases , including cancer . in addition , high gene and protein expression of il-10 were correlated with characteristics of better prognosis . il-10 downregulates proinflammatory cytokine expression [ 48 , 49 ] and inhibits the expression of cd31 . lin and karin reported that il-10 could modulate apoptosis and suppress angiogenesis during tumor regression , downregulating vegf , tnf- , and il-6 production by tams . jindal and borges showed that il-10 inhibits tumor growth by preventing chemokine expression and angiogenesis when administered at higher doses in cancer animal models . correlated high il-10 expression in female dogs with no metastasis or recurrence and high overall survival . contrarily , gene overexpression of il-10 in animals that had metastasis could suggest posttranscriptional regulation of this cytokine , once the protein levels do not show this correlation . il-6 mediates a plethora of physiological functions , including the developmental differentiation of lymphocytes , cell proliferation , and cell survival during tumorigenesis [ 48 , 53 ] . this cytokine had high gene and protein expression correlated with poor prognosis and a negative correlation was found in animals that died during the followup . in the same way , a study by madeddu et al . demonstrated high expression of il-6 through activation of akt / pi3k / mtor cascade due to elevated oxidative stress , promoting oncogenesis and tumor progression . owing to its association with poor prognosis , researchers have proposed il-6 as a therapeutic target in cancer . several phase i / ii clinical trials are currently evaluating antibodies against il-6 or il-6r as therapeutic alternatives for prostate cancer [ 53 , 55 ] and renal cancer [ 56 , 57 ] . these results suggest that further studies are needed to determine the appropriate use of anti - interleukin monoclonal antibodies as a therapeutic treatment , and studies similar to ours , evaluating interleukin expression , may be useful as a foundation for further clinical trials of therapy using cytokines . increasing lines of evidence suggest that tnf- regulates many of the critical processes of tumor promotion and progression [ 48 , 58 ] . in our study , high protein levels of tnf- were found in animals that died during the followup and correlated with worse prognostic features . its cytokine has been implicated yet as a tumor promoter in different tumor types like ovarian cancer , gallbladder cancer , and oral squamous cell carcinoma . tnf- binds to two receptors , namely , the ubiquitously expressed tnf receptor 1 ( tnfr1 ) and hematopoietically restricted tnfr2 , and modulates a signaling cascade that induces mediators of transcriptional regulation that are key to cell survival , invasion , angiogenesis , and impairment of immune surveillance in tumor biology [ 49 , 58 , 59 , 62 ] . altogether , our data showed the effect of cytokines on tumor development and progression , as well as positive modulation of nf-b and proinflammatory interleukins in mammary tumors . analysis of the active genes and proteins in cancer can be used to target treatment more specifically , and our results suggest the use of these biological factors as prognostic biomarkers in cancer . our results are of utmost importance for a better understanding of the dynamic network of cytokines and the influence of nf-b on tumorigenesis . these cytokines could be employed as noninvasive prognostic markers in female dogs with mammary tumors and could be useful for predicting disease progression and tumor recurrence . there is lack of studies devoted to diagnostic , therapeutic , and prognostic markers in canine mammary tumors ; so , this study defines potential prognostic and predictive markers for routine use in the clinic , allowing the detection of recurrence and metastasis , which could allow the early adoption of more precise conduct to improve the overall survival of animals .

inflammation results in the production of cytokines , such as interleukin- ( il- ) 4 and il-10 with immunosuppressive properties or il-6 and tnf- with procarcinogenic activity . furthermore , nf-b is the major link between inflammation and tumorigenesis . this study verified the interaction between active inflammatory cytokines in the tumor microenvironment and serum of female dogs with mammary tumors and their correlation with the clinicopathological characteristics and overall survival . measurement of gene expression was performed by qpcr and protein levels by elisa / luminex . high gene and protein expression levels of nf-b , il-6 , and tnf- were found in association with characteristics that reflect worse prognosis and a negative correlation between tnf- protein expression and survival time was observed ( p < 0.05 ) . in contrast , high gene and protein expression levels of il-4 and il-10 were associated with characteristics of better prognosis and an increased level of il-4 and a longer survival time of animals were obtained ( p < 0.05 ) . in addition , there was a positive correlation between tnf- and il-6 expression in association with nf-b . the results show a significant correlation of these cytokines with tumor development , associated with nf-b expression and cytokines promodulation , showing that these biological factors could be used as predictive and prognostic markers in breast cancer .

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

breast - conserving therapy ( bct ) , including breast - conserving surgery ( bcs ) and postoperative radiotherapy ( rt ) , has become a standard treatment in early stages ( t1 , t2 ) breast cancer for the last 30 years [ 1 , 2 ] . breast - conserving therapy was determined as primary treatment at the national institutes for health consensus development conference in 1990 , for tumours up to 4 cm . many long - term studies confirm that breast - conserving surgery followed by radiotherapy results in outcomes comparable to mastectomy , allowing women to preserve their breast [ 1 , 48 ] . the standard postoperative rt after bcs consists of external beam radiotherapy of the whole breast ( whole breast irradiation wbi ) using two tangential fields up to a total dose of 45 - 50 gy . a significant role in reducing a risk of local recurrence rate is performed by an additional boost treatment to the tumour bed up to a dose of 10 - 25 gy [ 913 ] , although choice of an optimal technique of boost treatment between brachytherapy , photons beams or electrons remains controversial . this issue is a case of many studies , but none of them has definitely shown the superiority of any of these techniques [ 9 , 10 , 1419 ] . an advantage of brachytherapy is the delivery of a high dose in limited volume , due to a large dose gradient , minimizing the treated volume to the tumour bed and it s immediate surroundings , resulting in limitation of post radiation injuries . moreover , in case of brachytherapy , a dose intensity measured in minutes is higher than that of external radiotherapy measured in days , what increased a biological effectiveness against resistant tumour cells . a role of brachytherapy as a boost increases especially in case of lesions deeply seated in breast [ 2224 ] and modern , image - based brachytherapy planning systems allow for an assessment not only of a dose at a certain point , but doses received by respective volumes of structures . it is a huge advantage enabling to avoid a single point dose estimate uncertainty caused by a large dose gradient of brachytherapy sources . according to american brachytherapy society ( abs ) recommendations it is required to monitor maximum dose received by a skin , however , there is no unambiguous way of skin contouring provided [ 26 , 27 ] . there are two most often ways used to define maximum skin dose . the first way represents it as a dose at a point on the body surface ( determined by external patient contour , external ) [ 2835 ] . the second one defines maximum skin dose as the maximum dose to a relatively small volume ( 0.1 - 1 cc ) in the structure created by expanding an external patient contour by 5 - 10 mm [ 3638 ] . the structure defined this way is in fact placed outside the body and doses calculated should be treated as a dose at surface rather than a dose in volume . more corresponding to the anatomical model of skin is a structure made by reduction of external patient contour by some value representing breast skin thickness [ 39 , 40 ] . there are many studies focused on measuring breast skin thickness , both healthy and changed by disease or after radiotherapy [ 4147 ] . it has been shown that in case of tumour or when radiotherapy was applied , the skin is thickening [ 4143 , 46 , 47 ] . according to different studies , mean skin thickness in normal breast varies in range of 1.44 - 2.05 mm , while in breasts after radiotherapy 1.44 - 2.68 mm [ 4143 , 46 , 47 ] . this study compares doses received by different models of skin of 20 patients , who underwent interstitial brachytherapy as a boost in breast - conserving therapy . twenty patients with recognized breast cancer and treated with radiotherapy after breast - conserving surgery were examined . all patients underwent external beam radiotherapy ( wbi ) up to a dose of 45 - 54 gy and interstitial brachytherapy hdr as a boost . patients were treated with remote afterloading microselectron v2 hdr ( nucletron , an elekta company , elekta ab , stockholm , sweden ) using an ir stepping source with nominal activity of 10 ci . interstitial brachytherapy procedure were performed under local anaesthesia . from 4 to 9 metal needles in 1 or 2 planes ( 3 patients 1 plane , 17 patients 2 planes ) all patients with interstitial implant were scanned using ct scanner somatom sensation open ( siemens medical solutions inc . the ct images were sent to the brachytherapy planning system oncentra masterplan ( version 4.3 , nucletron , an elekta company , elekta ab , stockholm , sweden ) , where structures were outlined and treatment plan was made . planning target volume ( ptv ) and organ at risk : skin , most exposed rib , and ipsilateral lung were contoured . for this examination , ptv was defined by interstitial implant reaching 5 mm from needles in orthogonal plane and separated by 5 mm from the body surface , because the purpose of this study is not the evaluation of optimal treatment plan , but the examination of differences between doses received by skin based on the way of contouring of this structure . application of the standard 2d planning paris dosimetry system was perform to all patients , enabled invariable conditions for the entire analysed group . the skin was outlined in three different ways . the first model of skin , made by expansion of external patient contour by 4 mm , was named skin ext ( ext = external ) for the purpose of this study . it was created by an addition of a 4 mm bolus to the external contour . the second model , skin 2 mm , corresponds to the dermatological breast skin thickness . it was reaching 2 mm into an external patient contour and was created by contouring 2 mm inner margin of an external contour . the third model of skin , skin 4 mm , was made by inserting a 4 mm bolus onto the same contours as skin 2 mm ( external contour reduced by 2 mm margin ) . all models of skin were spatially limited to the treated breast in order to avoid a shift in dose - volume histograms ( dvh ) , caused by a large volume of this structure receiving only a insignificant dose . an evaluation of doses received by skin 2 mm was not performed in this study , because of an incorrect estimation of dose distribution for this structure , due to computing limitations of the planning system . during ct scanning , breasts of all patients were very tightly compressed by templates , causing a large contour gradient in proximity of templates . during the outlining of skin 2 mm , a certain discontinuity occurred in this structure ( despite using the biggest available accuracy structure set grid 1 mm ) in some particular configurations of templates relative to ct scans ( parallel or slightly sloped ) . the dose distribution was not calculated in these parts , so dose parameters for skin 2 mm did not correspond with the actual dose . in this connection , the analysis was done only for skin ext and skin 4 mm , both 4 mm wide . figure 2 shows the problem with skin 2 mm . a presentation of different skin models . skin ext ( orange ) = external + 4 mm , skin 2 mm ( yellow ) = external 2 mm , skin 4 mm ( green ) = external 2 mm a comparison of skin 2 mm ( yellow ) and skin 4 mm ( green ) in the same case . skin 2 mm with a certain discontinuity in places where template compressing the breast caused high contour gradient . in case of skin 4 mm , this problem does not occur all treatment plans were done according to paris dosimetry system . usage of a breast template enables an obtainment of a perfect implant , composed of parallel and equidistant needles ( distance between needles dose is specified at basal points , defined in the central plane of an implant , and placed between needles at points of lowest dose . in case of a single plane implant , basal points are placed midway between each pair of needles . in case of two or more planes , needles create set of equilateral triangles and basal points are in the center of gravity of each triangle . a reference dose is defined as 85% of basal dose in order to gain a reference isodose 5 mm from external needles . all treatment plans were optimized by geometrical distance or volume optimization , depending on type of interstitial implant ( one or two planes ) . a normality of the dose distribution was checked for all volumes of both skin models using the shapiro - wilk test . these variables were compared using the wilcoxon signed - rank test , while normally distributed variables ( skin d0.1cc , d1cc , d2cc ) were compared using the student s t - test for dependent samples . a difference between two variables was considered statistically significant when the p - value was less or equal than 0.05 . in all cases , minimal coverage of 90% of the prescription dose v90 exceeded 95.98% in all cases ( mean 97.94% ) . mean coverage of 100% of the prescription dose v100 was 91.83% ( 85.26 - 97.41% ) . mean volume of ptv encompassed by 150% and 200% ( v150 , v200 ) of the reference isodose was 30.98% ptv ( 8.10 cc ) and 15.47% ptv ( 3.99 cc ) , respectively . mean minimum dose at ptv ( d100 ) was 76.46% ( 62.83 - 87.65% ) , while mean value of a parameter more relevant for bt mean dose at 0.1 cc of the most exposed rib was 17.18% of the reference dose ( 9.86 - 33.10% ) , and mean dose at 2 cc of ipsilateral lung was 13.45% ( 9.28 - 22.88% ) . mean , median , minimum , maximum , and standard deviation of dose at 0.1 cc for skin ext and skin 4 mm were , respectively : 63.01% , 62.59% , 51.79% , 79.42% , 6.76% for skin ext , and 81.02% , 78.79% , 65.40% , 98.62% , 9.49% for skin 4 mm . parameters of target and oar dose distributions according to the abs recommendations , the values of ptv volumes encompassed by high doses v150 and v200 are given both as percentage and in absolute values . table 2 contains doses received by most exposed 0.1 cc , 1 cc , 2 cc , and the maximum dose for both models of skin . table 3 contains mean percentage differences between doses received by particular volumes of both models of skin , skin ext and skin 4 mm . a bold line on every figure points at the value of mean difference between doses received by both models of skin and equals , respectively : 50.35% ( 20.09 - 127.47% ) for maximum dose , 18.01% ( 13.27 - 27.84% ) for d0.1cc , 11.22% ( 6.69 - 14.35% ) for d1cc , and 9.78% ( 5.33 - 11.75% ) for d2cc . a bold , horizontal line at 18.01% indicates a mean percent difference percentage difference in d1cc of skin between skin ext and skin 4 mm . a bold , horizontal line at 11.22% indicates a mean percent difference percentage difference in d2cc of skin between skin ext and skin 4 mm . a bold , horizontal line at 9.78% indicates a mean percent difference percentage difference in dmax of skin between skin ext and skin 4 mm . a bold , horizontal line at 50.35% indicates a mean percent difference doses received by different skin volumes for two skin models percentage difference in doses received by skin ext and skin 4 mm for particular skin volumes the development of brachytherapy planning systems based on a three - dimensional imaging brings in many previously unavailable possibilities . image - based planning allows for an outlining of the planning target volume , and creation of a treatment plan based on the ptv instead of implementing standard dose distributions related to the geometry of applicators or interstitial implant . 3d - planning enables also a precise dose evaluation in defined volumes of organs at risk instead of an evaluation of a point dose . doses for the most important oars in the breast brachytherapy ( rib , lung , and skin ) have to be monitored according to abs recommendations . for a skin a few millimetre shifts may result in dose difference of a dozen up to several dozen percent , due to a large dose gradient in brachytherapy . huang et al . used breast ct acquisition techniques , combined with algorithms based on monte carlo method , designed for determining specific breast skin thickness metrics . he has examined 51 patients , determining mean breast skin thickness as 1.45 ( 1.0 - 2.2 mm ) among the cancer - free breasts , 1.53 mm ( 1.2 - 1.9 mm ) among those with benign findings , and 1.46 ( 0.9 - 2.3 mm ) among breasts with biopsy - confirmed breast cancer . , analysed 137 tomograms of breasts , both healthy and changed by a disease , determined breast skin thickness as 1.44 mm ( 0.87 - 2.34 mm ) and also no statistically significant difference between these groups . liu s ultrasound scanner research has shown a significant breast skin thickness difference between healthy and treated by radiotherapy breasts . he has noted mean skin thickness increased by 27.3% : from 2.05 mm ( 1.66 - 2.38 mm ) in untreated breasts to 2.61 mm ( 1.53 - 3.65 mm ) in treated breasts . a comparison of skin thickness in healthy breasts and those after radiotherapy was also made libshitz et al . according to his research , skin thickness in breasts after radiation treatment returned to normal ( defined as equal to the thickness of the healthy breast ) in 2 years for 60% of the patients , in 3 years for 80% of the patients , and in 4 years for all the patients . warszawski et al . also used ultrasonography for measurements comparing healthy breasts to breasts that undergone radiotherapy . their measurements of corium ( the deep vascular inner layer of the skin ) thickness for non - irradiated breast was on average 1.68 0.3 mm ) , increasing to the maximum value of 2.68 0.7 mm ) after 3 months ( early reactions ) , and then decreased to 2.31 mm ( 0.9 mm ) for late reactions ( 6 - 8 months later ) . basing on these studies , it seems reasonable to define mean breast skin thickness for irradiated patients as 2 mm . this thickness was assumed by gifford and his co - workers in their study comparing the doses received by skin in differently defined models in case of apbi brachytherapy provided by contura and savi balloons . gifford analysed a group of 70 patients and compared the doses received by the most exposed 0.1 cc ( d0.1cc ) of skin defined by two models . the first model , 2 mm thick , was created by 2 mm contraction of an external patient contour . the second one , named ext + 5 mm was also based on the external patient contour , but created by expanding this structure 5 mm outside . mean , median , maximum , and standard deviation of percentage difference between these two models for the whole examined group were 12.5% , 12.5% , 23.8% , and 3.6% , respectively . taking into consideration the treatment delivery method , smaller differences were recorded for contura balloon ( senorx , inc . , aliso viejo , ca , usa ) : 10.1% , 10.9% , 14.1% and 2.6% , respectively . the differences between doses for patients treated by savi applicator ( cianna medical , aliso viejo , ca ) were 14.4% , 14.4% , 23.8% , and 3.1% , respectively . our study compares the doses received by skin 4 mm , defined as 2 mm inside and 2 mm outside of the external contour , and skin ext , defined as a structure based on the external contour and expanding 4 mm outside . skin 4 mm replaced skin 2 mm , created earlier and representing actual breast skin thickness , which was not representative , because of discontinuities in its structure . however , skin 4 mm may successfully constitute an equivalent of two - millimetre skin used by gifford , because both of them were based on the same contour . d0.1cc , d1cc , d2cc , and dmax compares exactly the same volume inside the external patient contour both for our model and gifford s . moreover , skin ext can be an equivalent for ext + 5 mm from gifford s study , so the percentage difference between skin 4 mm and skin ext can be compared . mean , median , maximum , and standard deviation of percentage dose difference for the most exposed 0.1cc ( d0.1cc ) of skin in our study were 18.01% , 17.20% , 27.84% , and 4.01% , respectively . it must be remembered that the group examined by us underwent interstitial brachytherapy with needles stabilized by a template compressing the breast . the planning target volume was not limited to the tumour bed , but was defined by the needle implant on the full length of the needles preserving the 5 mm margin from external patient contour . probably extreme active source positions in needles were placed closer to patient s skin than in the case of balloons . it means that in our case both models of skin were in a region of higher dose gradient . this study compares also the maximum dose ( dmax ) and the doses for the most exposed 1 cc ( d1cc ) and 2 cc ( d2cc ) of skin . mean percentage difference between skin 4 mm and skin ext for dmax , d0.1cc , d1cc , and d2cc decreases with the increase of measured volume and was 50.35% , 18.01% , 11.22% , and 9.78% , respectively . calculations of dmax is based on single voxels , so this might not be authoritative . hence , the dose for the most exposed 0.1 cc ( d0.1cc ) is often treated as the maximum dose in brachytherapy . a comparison of different breast skin contouring methods was made by berger using a self - made phantom by inserting 7 needles in two planes . in his study he created three skin models contoured from the external contour inwards to the depth of 1 , 2 , and 3 mm , respectively , and three skin models based on the same contour , but extended 5 , 10 , and 15 mm outside of the phantom surface . all these structures were outlined in oncentra masterplan by means of an automatic contouring and transferred to the plato system . the values from both planning systems were compared to the real volumes of all six structures calculated , based on the dimensions of the phantom . in case of the inside structures , only the 5 mm one was within the tolerable 5% accuracy , whereas the models defined outside the phantom showed high accuracy of contouring . a similar tendency of discontinuities in structures of small thickness was found in our study ( skin 2 mm ) . in order to avoid unstable and uncertain parameters of histogram for structures inside the phantom , berger suggested to choose the structures outlined outside the phantom . moreover , berger considered the analysis of dose - surface histograms ( dsh ) , a better way to report skin dose than the dose - volume histogram ( dvh ) . only the position of the contour directed on the external patient contour is relevant in case of skin models outlined to outside , if an appropriate thickness is chosen and a histogram parameters for these skin models can be a good approximation of surface dose . berger analysed dmax , d0.1cc , d1cc , and d10cc for these skin models , but skipped the evaluation of doses received by the inside skin models . according to turesson and notter van limbergen noticed that 97% of the skin blood vessels , that are concerned by this complication , are located within the first 5 mm below the surface of the skin . these vessels receive about 40 - 80% dose from external beam radiotherapy ( 20 - 40 gy ) . the dose received by skin as the result of an additional brachytherapy , the results of this study suggest that it should be considered to introduce an additional skin model inside patient body and 5 mm thick . we have to remember that dose calculation algorithms in commercially available treatment planning software are still performing the calculations of the dose based on the tg-43 report , assuming that the radiation source is in the middle of a homogeneous phantom with a radius of 15 cm and full backscatter . in practice , the difference in density is especially observable near the body limits ( tissue - air ) . karaiskos et al . analyzed the difference in doses estimated theoretically and experimentally for points near the border of a water phantom containing a radiation source 192ir . compared breast doses computed by a treatment planning system with measurements based on the monte carlo method , and noticed that the doses for volumes encompassed by dose larger than 60% do not change , because of the proximity of a skin border or the presence of a lung . however , treatment planning systems overestimate the skin dose by 5 - 10% at points near body surface or lung and relatively far from implant compared to a monte carlo calculation . monitoring of doses received by this structure is necessary to avoid complications and to obtain a satisfactory cosmetic effect . it is difficult to assess the compatibility of skin doses of treatment plans with recommendations , while there is no unambiguous way of skin contouring provided . especially , if a mean difference of doses between two models of skin contouring is 18% ( 1.8 gy ) for the most exposed 0.1 cc and can reach almost 28% ( 2.8 gy ) in some cases . in order to evaluate the clinical usefulness of both skin models it would be necessary to analyze the actually used treatment plans for a larger group of patients and a much longer observation time is essential to assess the cosmetic effect after the radiotherapy .

purposeskin is a major organ at risk in breast - conserving therapy ( bct ) . the american brachytherapy society ( abs ) recommendations require monitoring of maximum dose received , however , there is no unambiguous way of skin contouring provided . the purpose of this study was to compare the doses received by the skin in different models.material and methodsstandard treatment plans of 20 patients who underwent interstitial breast brachytherapy were analyzed . every patient had a new treatment plan prepared according to paris system and had skin contoured in three different ways . the first model , skin 2 mm , corresponds to the dermatological breast skin thickness and is reaching 2 mm into an external patient contour . it was rejected in a further analysis , because of distinct discontinuities in contouring . the second model , skin 4 mm , replaced skin 2 mm , and is reaching 2 mm inside and 2 mm outside of the external contour . the third model , skin ext , is created on the external contour and it expands 4 mm outside . doses received by the most exposed 0.1 cc , 1 cc , 2 cc , and the maximum doses for skin 4 mm and skin ext were compared.resultsmean , median , maximum , and standard deviation of percentage dose difference between skin ext and skin 4 mm for the most exposed 0.1 cc ( d0.1cc ) of skin were 18.01% , 17.20% , 27.84% , and 4.01% , respectively . all differences were statistically significant ( p < 0.05).conclusionsmonitoring of doses received by skin is necessary to avoid complications and obtain a satisfactory cosmetic effect . it is difficult to assess the compatibility of treatment plans with recommendations , while there is no unambiguous way of skin contouring . especially , if a mean difference of doses between two models of skin contouring is 18% for the most exposed 0.1 cc and can reach almost 28% in some cases . differences of this magnitude can result in skin complications during bct .

Purpose Material and methods Results Discussion Conclusions Disclosure

proximal junctional kyphosis ( pjk ) is traditionally defined as a proximal junctional sagittal cobb angle change of 10 versus the preoperative value . although no consensus has been reached , the majority of authors have used kyphosis of 10 between the lower endplate of the uppermost instrumented vertebra ( uiv ) and the upper endplate of the two supra - adjacent vertebrae as a definition5 ) . pjk is a post - surgical radiographic status observed in the sagittal plane following multi - level fusion surgery for spinal deformity . in a broader sense , pjk is a type of adjacent segment disease associated with spinal fusion and often occurs after deformity correction . furthermore , pjk results in poor surgical results due to pain , deformity , instability , motion difficulties , and potential neurologic deficits . to minimize pjk , , several such factors have been described in the literature , namely , older age , inadequate restoration of global sagittal balance , combined anterior - posterior surgery , fusion to the sacrum , posterior only spinal fusion rather than anterior instrumentation , and low bone mineral density ( bmd)9,10,12,15,19 ) . skeletal muscles , including flexor and extensors , produce movements of the trunk and maintain body balance , and degeneration and atrophy of thoracolumbar extensor muscles lead to fatty replacement and back kyphosis as manifested by lumbar degenerative kyphosis or flat back syndrome . furthermore , sarcopenia may affect skeletal stability or surgical outcome , but the impact of thoracolumbar muscle volume on pjk has not been previously evaluated . the aim of this study was to identify the factors responsible for pjk after spinal instrumented fusion in adult spinal deformity with a minimum follow - up of 2 years . this study was conducted by retrospectively reviewing the medical charts and radiologic data of 49 patients with spinal deformity , including lumbar degenerative kyphosis , diffuse lumbar scoliosis , and post - operative flat - back syndrome , that underwent posterior fusion with instrumentation from january 2004 to december 2011 at two university hospitals . the minimum follow - up was 2 years and mean follow - up was 3.6 years . all 49 patients underwent multi - level fusion surgery at more than 4 levels from l5 or the sacral level . radiographic pjk was defined as angle change of > 10 on dynamic plain radiographs at the proximal end of a construct14 ) . preoperative evaluations included plain radiographic examinations and magnetic resonance imaging ( mri ) , a postoperative radiograph examination was conducted before discharge ( early postop , ep ) , and then annually . postoperative radiographs included standing antero - posterior , lateral whole spine , and dynamic flexion / extension views , and were used to determine the presence or not of pjk at proximal adjacent level from instrumentation . the 49 patients were divided in terms of two groups ( pjk and non - pjk groups ) based on the presence of pjk at final follow - up . these two groups were compared with respect to demographic factors , bmd , sagittal plane angle parameter , pelvic parameter , instrumentation factor , surgical approach , and the back muscle volume of thoracolumbar junction . group bmd values were compared using t scores determined by dual energy x - ray absorptiometry . c7pl was defined distance between c7 plumb line which is the vertical line originating the center of the c7 vertebral body and the posterior superior corner of s1 , and its length was measured preoperatively and at final follow - up . distance from the center of the upper instrumented vertebra ( uiv ) to c7pl was also measured at early postoperatively and final follow - up ( fig . pelvic incidence ( pi ) is defined as the angle between the perpendicular to the sacral plate at its midpoint and the line connecting this point to the middle axis of the femoral head . sacral slope ( ss ) is the angle between the superior endplate of s1 and the horizontal line . pelvic tilt ( pt ) is the angle between the line connecting the midpoint of the sacral plate to center of the axis of femoral heads , and the vertical line . data related to surgical procedures , including surgical approach ( concomitant anterior - posterior or not ) , cross - link usage , whether screws and rods were fractured or not , and uiv level , were also analyzed . contiguous t2-weighted mri scans were acquired in 5-mm slices using a 1.5-tesla ge ( general electric , milwaukee , wi , usa ) twin - speed scanner from t10 to l2 . patients were positioned supine on the imaging table and numbers of scan slices were adjusted to include the anatomic extent of muscles of interest . cross - section areas were calculated using software installed in the mri unit in the following way . after choosing a region of interest in the axial plane , the range of interest ( roi ) was enlarged on a monitor screen as much as necessary and subjected to planimetry . all measurements were obtained using a picture archiving and communication system and its software ( pacs , infinitt , seoul , korea ) ( fig . data were analyzed using spss for windows ( version 14.0 ; spss inc . , chicago , il , usa ) , and statistical significance was accepted for p values<0.05 . c7pl was defined distance between c7 plumb line which is the vertical line originating the center of the c7 vertebral body and the posterior superior corner of s1 , and its length was measured preoperatively and at final follow - up . distance from the center of the upper instrumented vertebra ( uiv ) to c7pl was also measured at early postoperatively and final follow - up ( fig . pelvic incidence ( pi ) is defined as the angle between the perpendicular to the sacral plate at its midpoint and the line connecting this point to the middle axis of the femoral head . sacral slope ( ss ) is the angle between the superior endplate of s1 and the horizontal line . pelvic tilt ( pt ) is the angle between the line connecting the midpoint of the sacral plate to center of the axis of femoral heads , and the vertical line . data related to surgical procedures , including surgical approach ( concomitant anterior - posterior or not ) , cross - link usage , whether screws and rods were fractured or not , and uiv level , were also analyzed . contiguous t2-weighted mri scans were acquired in 5-mm slices using a 1.5-tesla ge ( general electric , milwaukee , wi , usa ) twin - speed scanner from t10 to l2 . patients were positioned supine on the imaging table and numbers of scan slices were adjusted to include the anatomic extent of muscles of interest . cross - section areas were calculated using software installed in the mri unit in the following way . after choosing a region of interest in the axial plane , the range of interest ( roi ) was enlarged on a monitor screen as much as necessary and subjected to planimetry . all measurements were obtained using a picture archiving and communication system and its software ( pacs , infinitt , seoul , korea ) ( fig . data were analyzed using spss for windows ( version 14.0 ; spss inc . , chicago , il , usa ) , and statistical significance was accepted for p values<0.05 . the pjk group and non - pjk group contained 16 and 33 patients , respectively . women dominated in both groups 2 men and 14 women in pjk group and 4 men and 29 women in non - pjk group . mean overall age was 62.5 in pjk group ( range of 5669 ) and 61.9 in non - pjk group . sexual distributions and ages were non - significantly different ( table 1 ) . mean t score was 2.300.85 in pjk group and 1.010.67 in non - pjk group . bmd was significantly lower in the pjk group than in the non - pjk group ( p=0.027 ) ( table 1 ) . mean c7pl before surgery was 89.9551.60 mm in pjk group and 58.9766.98 mm in non - pjk group , and mean c7pl at final follow - up was 71.9658.04 mm in pjk group and 55.8337.00 mm in non - pjk group . mean distance from uiv to c7pl before surgery was 1.96.0 mm in pjk group and 0.53.0 mm in non - pjk group , and mean distance from uiv to c7pl at final follow - up was 5.55.4 mm in pjk group and 2.53.4 mm in non - pjk group . no intergroup difference was observed for mean c7pl at pre - op or at final follow - up and for mean uiv distance to c7pl distance at early post - op . however , mean uiv distance to c7pl distance at final follow - up was significantly greater in pjk group ( p=0.019 ) ( table 1 ) . mean preoperative pi was 57.9412.70 in pjk group and 58.458.37 in non - pjk group , and mean preoperative ss was 15.385.88 in pjk group and 16.916.59 in non - pjk group . mean preoperative pt was 42.0612.00 in pjk group and 41.219.16 in non - pjk group . pre - op mean pi , ss , and pt were not significantly different in the two groups ( table 1 ) . concomitant anterior - posterior surgery was performed in 6 of 16 ( 37.5% ) in pjk group and in 16 of 33 ( 48.4% ) in non - pjk group . cross - linkages were used in 13 of 16 ( 81.2% ) in pjk group and in 26 of 33 ( 78.8% ) in non - pjk group . instrument fractures occurred in 5 of 16 ( 31.2% ) in pjk group and in 10 of 33 ( 30.0% ) in non - pjk group . in pjk group , number of uiv levels were 2 of 16 ( t10 , 12.5% ) , 3 of 16 ( t11 , 18.8% ) , 5 of 16 ( t12 , 31.2% ) , 4 of 16 ( l1 , 25.0% ) , and 2 of 16 ( l2 , 12.5% ) , and in non - pjk group were 5 of 33 ( t10 , 15.1% ) , 4 of 33 ( t11 , 12.2% ) , 12 of 33 ( t12 , 36.3% ) , 6 of 33 ( l1 , 18.2% ) , and 6 of 33 ( l2 , 18.2% ) . surgical approaches , cross - link - usage , instrument fracture , and uiv level were non - significantly different in the two groups ( table 1 ) . mean thoracolumbar junction back muscle volumes in pjk and non - pjk group were 1042149 and 1315143 mm , respectively , at t10 , 1113101 and 1445176 mm at t11 , 1227111 and 1603230 mm at t12 , 133399 and 1783326 mm at l1 , and 1443175 and 1905303 mm , respectively , at l2 . thoracolumbar junction back muscle volume was significant lower in the pjk group than in the non - pjk group ( p<0.001 ) ( table 1 , fig . the correlation of thoracolumbar junction back muscle volume and preoperative sagittal imbalance in overall group was also evaluated . the mean muscle volume of t10 to l2 showed negative correlation with mean c7pl before surgery in the overall group . but the correlation was not strong with pearson correlation 0.313 ( p=0.029 ) ( fig . the pjk group and non - pjk group contained 16 and 33 patients , respectively . women dominated in both groups 2 men and 14 women in pjk group and 4 men and 29 women in non - pjk group . mean overall age was 62.5 in pjk group ( range of 5669 ) and 61.9 in non - pjk group . mean t score was 2.300.85 in pjk group and 1.010.67 in non - pjk group . bmd was significantly lower in the pjk group than in the non - pjk group ( p=0.027 ) ( table 1 ) . mean c7pl before surgery was 89.9551.60 mm in pjk group and 58.9766.98 mm in non - pjk group , and mean c7pl at final follow - up was 71.9658.04 mm in pjk group and 55.8337.00 mm in non - pjk group . mean distance from uiv to c7pl before surgery was 1.96.0 mm in pjk group and 0.53.0 mm in non - pjk group , and mean distance from uiv to c7pl at final follow - up was 5.55.4 mm in pjk group and 2.53.4 mm in non - pjk group . no intergroup difference was observed for mean c7pl at pre - op or at final follow - up and for mean uiv distance to c7pl distance at early post - op . however , mean uiv distance to c7pl distance at final follow - up was significantly greater in pjk group ( p=0.019 ) ( table 1 ) . mean preoperative pi was 57.9412.70 in pjk group and 58.458.37 in non - pjk group , and mean preoperative ss was 15.385.88 in pjk group and 16.916.59 in non - pjk group . mean preoperative pt was 42.0612.00 in pjk group and 41.219.16 in non - pjk group . op mean pi , ss , and pt were not significantly different in the two groups ( table 1 ) . concomitant anterior - posterior surgery was performed in 6 of 16 ( 37.5% ) in pjk group and in 16 of 33 ( 48.4% ) in non - pjk group . cross - linkages were used in 13 of 16 ( 81.2% ) in pjk group and in 26 of 33 ( 78.8% ) in non - pjk group . instrument fractures occurred in 5 of 16 ( 31.2% ) in pjk group and in 10 of 33 ( 30.0% ) in non - pjk group . in pjk group , number of uiv levels were 2 of 16 ( t10 , 12.5% ) , 3 of 16 ( t11 , 18.8% ) , 5 of 16 ( t12 , 31.2% ) , 4 of 16 ( l1 , 25.0% ) , and 2 of 16 ( l2 , 12.5% ) , and in non - pjk group were 5 of 33 ( t10 , 15.1% ) , 4 of 33 ( t11 , 12.2% ) , 12 of 33 ( t12 , 36.3% ) , 6 of 33 ( l1 , 18.2% ) , and 6 of 33 ( l2 , 18.2% ) . surgical approaches , cross - link - usage , instrument fracture , and uiv level were non - significantly different in the two groups ( table 1 ) . mean thoracolumbar junction back muscle volumes in pjk and non - pjk group were 1042149 and 1315143 mm , respectively , at t10 , 1113101 and 1445176 mm at t11 , 1227111 and 1603230 mm at t12 , 133399 and 1783326 mm at l1 , and 1443175 and 1905303 mm , respectively , at l2 . thoracolumbar junction back muscle volume was significant lower in the pjk group than in the non - pjk group ( p<0.001 ) ( table 1 , fig . the correlation of thoracolumbar junction back muscle volume and preoperative sagittal imbalance in overall group was also evaluated . the mean muscle volume of t10 to l2 showed negative correlation with mean c7pl before surgery in the overall group . but the correlation was not strong with pearson correlation 0.313 ( p=0.029 ) ( fig . the present study confirms preoperative bmd and thoracolumbar back muscle volume are related to the presence of pjk after segmental spinal instrumented fusion in adults with spinal deformity , and that although sagittal balance is achieved immediately after surgery , newly developed sagittal imbalance after deformity surgery is associated with increased pjk at final follow - up . many studies have identified risk factors of the postoperative development of pjk3,5,6,9,10,12,15,19 ) , such as , patient age , bone quality , and inadequate restoration of global sagittal balance requiring substantial correction to restore global alignment . furthermore , uiv level may play a role because the uiv in many deformity cases ends at the thoracolumbar junction , a transition zone between the mobile lumbar spine and the less mobile thoracic spine and rib cage . it is also possible that body mass index contributes to the development of pjk as the uiv may be biomechanically stressed in larger patients . takemitsu et al . measured the cross - sectional areas of paraspinal muscles in patients with lumbar degenerative kyphosis , which lumbar extensor muscles overwork to maintain secure balance by ct , and weakness of spinal extensors is caused by definite atrophy , which may produce round back kyphosis and more fatty replacement as compared with normal individuals8,18 ) . harrison et al . showed that anterior trunk translation in standing subjects increases extensor muscle activity and loads and stresses acting on intervertebral discs in the lower thoracic and lumbar regions7 ) . it has been established altered biomechanical stresses on an intervertebral disc may cause adjacent segment degeneration . in the present study , thoracolumbar back muscle volume was smaller in the pjk group than in the non - pjk group . we could know relation between preoperative thoracolumbar back muscle volume and pjk , even if this result could not answer what is the preceding between two factors . osteoporosis is a characterized by reduced bone strength , increased skeletal fragility , and fracture susceptibility . furthermore , a weak bone / screw interface is a known risk factor of screw pull - out after a spinal instrumentation procedure , and numerous studies have concluded low bone mineral density is a risk factor for adjacent segment disease after spinal fusion . terracciano et al.2 ) showed muscle atrophy is prominent in osteoporosis and preferentially affects type ii muscle fibers with little or no impact on type i fibers , and that this atrophy is associated with bmd , which suggests disease severity plays a central role in the pathogenesis of osteoporosis - related muscle atrophy . in the present study , patients allocated to two groups based on the presence of absence of pjk . osteoporosis and sarcopenia are aging related , but in the present study age was not found to be related with pjk . decreased skeletal muscle volume increases axial loading during the maintenance of skeletal stability , and osteoporotic bone can not sufficiently support skeletal loading , which means back muscles are exposed and a vicious cycle is initiated . furthermore , reduced screw pull - out strength imposes a heavy burden on back muscles and causes micro - motion of proximal instrumentation . resultantly , lower bmd in combination reduced thoracolumbar back muscle volume might in combination induce skeletal instability and facilitate proximal junctional kyphosis . line of gravity and center of gravity have been studied by several authors11,16 ) . in a stabilized standing position , the c7 plumb line is located behind the gravity line in the sagittal plane . however , in most pathological situations , the center of gravity is too far forward with a mechanical axis located in front of the femoral heads , and this imbalance will induce body reactions . the present study shows uiv distance from the c7 plumb line was greater in the pjk group . furthermore , center of gravity is located more frontally for greater uiv distances and stress loading on proximal instrumentation might be dependent on uiv distance , and thus , when compensatory mechanisms , such as , thoracic kyphosis , hip or knee bending can not tolerate a critical point , pjk might occur . second , fat volume could not be excluded from muscle mass measurements because of technical limitations . accordingly , we suggest a well designed prospective study be performed to identify more precisely factors that contribute to pjk , and that a biomechanical study be undertaken to confirm our results . nevertheless , the study provides surgeons with information useful for deciding on instrumentation level and surgical approach . the incidence of pjk following multi - level fusion surgery was higher related to preoperative lower bmd , thoracolumbar back muscle volume , and insufficient postoperative sagittal balance . surgeons should make treatment plan carefully to prevent pjk after multi - level fusion surgery , especially in cases of osteoporosis and sarcopenia . we believe the result of this study will be useful for preventing pjk after surgery , but more studies with long - term follow - up and more subjects are also needed in the future .

objectiveproximal junctional kyphosis ( pjk ) is radiologic finding , and is defined as kyphosis of > 10 at the proximal end of a construct . the aim of this study is to identify factors associated with pjk after segmental spinal instrumented fusion in adults with spinal deformity with a minimum follow - up of 2 years.methodsa total of 49 cases of adult spinal deformity treated by segmental spinal instrumented fusion at two university hospitals from 2004 to 2011 were enrolled in this study . all enrolled cases included at least 4 or more levels from l5 or the sacral level . the patients were divided into two groups based on the presence of pjk during follow - up , and these two groups were compared to identify factors related to pjk.resultspjk was observed in 16 of the 49 cases . age , sex and mean follow - up duration were not statistically different between two groups . however , mean bone marrow density ( bmd ) and mean back muscle volume at the t10 to l2 level was significantly lower in the pjk group . preoperatively , the distance between the c7 plumb line and uppermost instrumented vertebra ( uiv ) were no different in the two groups , but at final follow - up a significant intergroup difference was observed . interestingly , spinal instrumentation factors , such as , receipt of a revision operation , the use of a cross - link , and screw fracture were no different in the two groups at final follow-up.conclusionpreoperative bmd , sagittal imbalance at uiv , and thoracolumbar muscle volume were found to be strongly associated with the presence of pjk .

INTRODUCTION MATERIALS AND METHODS Sagittal plane angle parameters Pelvic parameters Surgical approach and instrumentation factors Thoracolumbar junction back muscle volume Statistical analysis RESULTS Demographic factors Bone mineral density Sagittal plane angle parameters Pelvic parameters Surgical approach and instrumentation factors Thoracolumbar junction back muscle volume DISCUSSION CONCLUSION

in studies spanning over three decades , antigen spread has been observed in the context of autoimmunity and infectious disease ( 2935 ) . cancer immunotherapy also appears to work , in part , through antigen spread , ie , beginning with an immune response to specific target antigens that broadens over time to additional antigens expressed within the tumor ( figure 1 ) ( 21 ) . mechanistically , this phenomenon likely occurs when an initial antitumor immune response ( eg , from a therapeutic anticancer vaccine or from t - cell reactivation by immune checkpoint blockade ) induces t - cell - mediated destruction of tumor cells . this destruction leads to the release of additional tumor - associated antigens ( taas ) that may be taken up locally by antigen - presenting cells ( apcs ) such as macrophages and dendritic cells . the taas are processed and presented by apcs to induce b - cell- and t - cell - mediated immune responses against the secondary antigens ( 21 ) . an additional / alternative hypothesis for the generation of secondary immune responses is that a successful immune response against the primary / targeted antigen of an immunotherapy may also overcome suppressive factors ( 17 ) . thus , immunotherapy may act to amplify preexisting antibody responses in addition to de novo generation of novel responses . in addition , radiation , chemotherapy , and other commonly used anticancer agents may kill tumor cells in an immunologically relevant manner . this treatment can lead to immunogenic cell death , which induces changes such as translocation of calreticulin to the surface of the dead tumor cells , which mediates the recognition and clearance of these cells by professional and nonprofessional phagocytes , allowing for tumor - antigen processing and presentation to the immune system ( 37 ) . however , this process may not be as efficient at generating an antitumor immune response as a therapeutic vaccine ( 38 ) . a ) an initial immune response is prompted following immunotherapy , releasing other antigens from dying tumor cells . b ) dendritic cells act as antigen - presenting cells ( apcs ) , processing the free antigens , including neoantigens , and presenting these to t - cells . c ) the apcs prime t - cells specific to antigens released from the tumor cells , increasing the breadth of the immune response . d ) the newly activated tumor - specific t - cells form in greater concentration and variation . thus , while the initial therapy may target one antigen , a broader adaptive antitumor immune response may ensue . muc-1 = mucin 1 ; pap = prostatic acid phosphatase ; psa = prostate - specific antigen ; psca = prostate stem cell antigen.figure 2.antigen spread following treatment with immunotherapy ( 36 ) . adapted ( with permission of springer ) from : jochems c , schlom j , gulley jl . tricom poxviral - based vaccines for the treatment of cancer . in : lukashevich is , shirwan h , eds . . 1 schematic overview of tricom vaccines showing the tumor antigen gene and the genes for the three costimulatory molecules lfa-3 , icam-1 , and b7 - 1 that are inserted within the virus . subcutaneous administration leads to antigen uptake by antigen - presenting cells ( apc ) in the skin . 2 : antigen presentation occurs in the draining lymph nodes , activating antigen - specific t cells . 3 : tumor sites are attacked by antigen - specific cytotoxic ( cd8 + ) t cells . 4 : tumor cell lysis leads to cross - presentation of multiple tumor antigens in the draining lymph nodes ( antigen spread / antigen cascade ) . 5 : antigen cascade leads to activation of additional antigen - specific t - cells , which increases the breadth and quite possibly the clinical activity of the antitumor response . a ) an initial immune response is prompted following immunotherapy , releasing other antigens from dying tumor cells . b ) dendritic cells act as antigen - presenting cells ( apcs ) , processing the free antigens , including neoantigens , and presenting these to t - cells . c ) the apcs prime t - cells specific to antigens released from the tumor cells , increasing the breadth of the immune response . d ) the newly activated tumor - specific t - cells form in greater concentration and variation . thus , while the initial therapy may target one antigen , a broader adaptive antitumor immune response may ensue . muc-1 = mucin 1 ; pap = prostatic acid phosphatase ; psa = prostate - specific antigen ; psca = prostate stem cell antigen . antigen spread following treatment with immunotherapy ( 36 ) . adapted ( with permission of springer ) from : jochems c , schlom j , gulley jl . . 1 schematic overview of tricom vaccines showing the tumor antigen gene and the genes for the three costimulatory molecules lfa-3 , icam-1 , and b7 - 1 that are inserted within the virus . subcutaneous administration leads to antigen uptake by antigen - presenting cells ( apc ) in the skin . 2 : antigen presentation occurs in the draining lymph nodes , activating antigen - specific t cells . 3 : tumor sites are attacked by antigen - specific cytotoxic ( cd8 + ) t cells . 4 : tumor cell lysis leads to cross - presentation of multiple tumor antigens in the draining lymph nodes ( antigen spread / antigen cascade ) . 5 : antigen cascade leads to activation of additional antigen - specific t - cells , which increases the breadth and quite possibly the clinical activity of the antitumor response . antigens released from the tumor may be more immunogenic than the antigens initially immunologically targeted ( eg , from a vaccine ) , and an ongoing , iterative process of antigen spread can initiate a broader and perhaps more clinically significant immune response ( 21 ) . furthermore , antigen spread may lead to an adaptive anticancer immune response that targets new mutations in tumor cell antigens as they occur ( 21 ) . as the immune response broadens over time , the immune system s durability and adaptability may mean that the kinetic profile of a clinical response following immunotherapy could differ from that following cytotoxic therapy . to overcome the diversity in both tumor - antigen expression and in immune repertoire , many cancer vaccines are designed to induce responses to multiple antigens , and there is evidence that breadth of response is an important determinant of vaccine efficacy . ima901 is a novel vaccine containing 10 tumor - associated peptides ( tumaps ) found in renal cell carcinoma ( rcc ) ( 39 ) . among rcc patients immunized with ima901 , those who responded to multiple tumaps had better disease control and improved os compared with patients who had no response or only responded to one tumap . while ima901 comprises multiple antigens , subsequent antigen spread could allow for broadening of the immune response regardless of the complexity of the agent used to generate the primary immune response . both preclinical and clinical data support the concept of antigen spread in cancer immunotherapy , and evidence continues to accumulate . one elegant preclinical study involved mice bearing tumors engineered to express carcinoembryonic antigen ( cea ) . here , cea - expressing ( cea+ ) tumors were implanted in one flank of experimental mice , while the opposite flank was implanted with parental ( cea ) tumors . mice were vaccinated with a cea - based vaccine , and substantial decreases in both the cea+ and cea tumors were observed . these responses were associated with induced cd8 + t - cell responses to both cea and other taas not included in the vaccine ( 40 ) . interestingly , immune responses to wild - type p53 and the endogenous retroviral epitope gp70 were detected , with the immune response to gp70 approximately 15 times greater than the response to cea , showing that responses to nontargeted antigens might be more important than those to the initial vaccine target ( 41 ) . several of these studies involve the agent sipuleucel - t . in the pivotal phase iii study , sipuleucel - t generated peripheral immune responses ( either t - cell or humoral ) to pa2024 ( the immunizing antigen ) and/or to pap ( the target antigen ) in 79% of patients treated with sipuleucel - t compared with 13% of patients receiving control ( 42 ) , demonstrating the ability of sipuleucel - t to induce immune responses to the original targeted antigen . antigen spread following treatment with sipuleucel - t has been most clearly demonstrated through studies of antibody responses in patients from the phase iii pivotal trial , as well as in a phase ii study ( 28 ) . these analyses involved two phases : an initial discovery phase followed by a confirmation phase . in the initial phase , protein microarrays were used to analyze patients sera for immunoglobulin g ( igg ) responses . following treatment with sipuleucel - t , most patients showed increased antibody responses , defined as twofold or greater elevation in antigen - specific igg after treatment , to a wide variety of nontarget proteins , whereas control patients showed no such responses . interestingly , the median number of nontarget antigens increased from 56 antigens at two weeks post - treatment to 162 at 10 weeks post - treatment , suggesting a cascading humoral response . in the confirmation phase of these analyses , an orthogonal assay ( luminex xmap , luminex corporation , austin , tx ) was used with a narrow pool of 10 antigens , five with the greatest fold increases in antibody levels after sipuleucel - t and five with known relevance to tumorigenesis or prostate cancer . the confirmation analyses showed that sipuleucel - t induced antibody increases to the secondary antigens psa , klk2 , lgals3 , and lgals8 , which have been shown to be expressed at elevated levels in prostate cancer and/or to play a role in prostate cancer development , as well as to k - ras and e - ras , which are known to have functional relevance in cancer ( 4349 ) . response rates to individual secondary antigens ranged from 28% to 44% in the phase iii study , with 25% of sipuleucel - t - treated patients responding to three or more nontarget antigens . antigen spread was observed two weeks after sipuleucel - t treatment and persisted for up to six months . clinically , antibody responses to psa and lgals3 were associated with improved os in sipuleucel - t - treated patients ( p .05 ) ( 28 ) . evidence of antigen spread in response to immunotherapy has also been reported in several other tumor types , including metastatic breast cancer ( 50 ) and melanoma ( 26,51,52 ) . women with metastatic breast cancer were treated with the monoclonal antibody trastuzumab in combination with a vaccine targeting human epidermal growth factor receptor 2 ( her2)/neu . as expected , most patients developed both helper and cytotoxic t - cell responses to the her2/neu peptides present in the vaccine . however , vaccinated patients also developed new or augmented immunity against two epitopes of her2/neu that were not included in the vaccine : p98.15 ( p = .0055 vs prevaccination ) and p776.15 ( p = .0006 ) . in a subset of patients , increased t - cell responses were also observed following vaccination against several nontarget antigens , including insulin - like growth factor - binding protein 2 ( p= .0973 ) , p53 ( p = .1282 ) , and topoisomerase ii- ( p = .0111 ) . these results are similar to the preclinical work described previously ; both intramolecular and intermolecular antigen spread is evident in treated patients . this study in patients with metastatic breast cancer also included a substantial follow - up phase . during a median follow - up of 36 months , most patients maintained the level of immunity reported at the end of the immunization period for both target and new antigens , and approximately 25% of patients developed further immune responses ( 50 ) . in a notable case study , a patient with metastatic melanoma had a durable remission after vaccination with melanoma - associated antigen ( mage ) , but had only a low - level anti - mage cytolytic t - cell ( ctl ) response ( 26,51 ) . using t - cell receptor ( tcr ) cdna libraries , very few vaccine - specific ctls were found in regressing metastases , but several tcr sequences were identified to correspond to nonvaccine tumor antigens . further tcr analyses of t - cells from tumor - infiltrating lymphocytes led to the identification of a cd8 clone that specifically lysed autologous melanoma cells . the target antigen was caseinolytic protein , a mitochondrial enzyme mutated in the tumor to produce a neoantigen . thus , in this patient , tumor rejection effectors were ctl responses to nonvaccine tumor - specific antigens , and this antigen spread was involved in tumor regression ( 26,51 ) . another recent study of adoptive cellular therapy combined with ctla-4 blockade demonstrated antigen spread in patients with a durable tumor response ( 52 ) . collectively , these data highlight both the persistence of an antitumor immune response and the concept of late , ongoing antigen spread . cancer immunotherapy also encompasses agents that function by blocking negative costimulatory molecules in the immunological synapse that inhibit an antitumor response . the immune response to these agents collectively referred to as immune checkpoint blockers , the monoclonal antibody ipilimumab was the first - in - class t - cell potentiator for metastatic melanoma ( 53 ) . ipilimumab ( anti - ctla-4 ) blocks the interaction between cytotoxic t - lymphocyte antigen 4 ( ctla-4 ) and b7 molecules , resulting in t - cell activation and subsequent objective antitumor responses in certain patients . to better understand the antigens targeted in patients treated with ipilimumab , potential cancer neoantigens were identified by dna sequencing of tissue samples from melanoma patients treated with ipilimumab ( 54 ) . a high mutational load was statistically significantly correlated with improved os , suggesting that the breadth of an antitumor immune response has clinical significance . to more closely quantify specific cd8 t - cell responses , two patient - specific neoantigens were assessed . for one of these neoantigens , there was no detectable t - cell response at seven weeks after initiation of ipilimumab ; at 24 weeks , however , a strong response was observed ( 54 ) . while it is unclear if this broader response is due to increased activation of immune cells ( direct broadening ) or more opportunities for immune cells to kill tumor cells and release antigens in an immunologically relevant manner ( tumor immunity cycle - mediated broadening ) , there are data suggesting that a broader response may be clinically relevant following immune checkpoint blockade as well as after vaccination ( 55,56 ) . a long - term class effect of immunotherapies is an improvement in os without short - term changes in conventional measures of disease progression ( 22 ) . this seemingly paradoxical dissociation between the two end points likely reflects the time it takes to mount an effective immune response and for that response to evolve into a more clinically relevant response , eg , the time it takes for antigen spread to occur ( figure 3 ) . schematic representing progression and broadening of response over time following treatment with cancer - targeting immunotherapy . antigen spread leads to more relevant targets ( eg , neoantigens ) for a given patient , and this highly individualized precision response could lead to improved clinical activity . furthermore , with subsequent treatment , the immune response may be further boosted as tumor cells are killed or modulated in an immunogenic manner , which translates into improving clinical activity over time . os = overall survival ; psa = prostate - specific antigen ; tdrp = time to disease - related pain ; tofa = time to first opioid analgesic . schematic representing progression and broadening of response over time following treatment with cancer - targeting immunotherapy . antigen spread leads to more relevant targets ( eg , neoantigens ) for a given patient , and this highly individualized precision response could lead to improved clinical activity . furthermore , with subsequent treatment , the immune response may be further boosted as tumor cells are killed or modulated in an immunogenic manner , which translates into improving clinical activity over time . os = overall survival ; psa = prostate - specific antigen ; tdrp = time to disease - related pain ; tofa = time to first opioid analgesic . although t- and b - cell responses to immunotherapy ( ie , immune responses ) can be detected in the blood relatively early in treatment , these initial responses may not be sufficient to delay radiographic progression and substantially increase pfs . the association of the kinetics of response to immunotherapy vs conventional therapy was examined using data from several clinical trials with either chemotherapy , androgen - deprivation therapy ( adt ) , or immunotherapy with a vaccine in mcrpc ( 5760 ) . these analyses showed that chemotherapy reduced tumor burden quickly after treatment initiation , but effects were frequently short - lived after treatment cessation ( figure 4a ) . psa kinetics during chemotherapy indicated tumor growth regression while on treatment ; however , upon treatment cessation , the regrowth trajectory reverted back to the pretreatment growth rate ( 57,58 ) . with immunotherapy , psa kinetics were not changed immediately by treatment ; however , there was an apparent slowing of disease progression as indicated by an os much greater than predicted by this model ( figure 4a ) . this modeling confirmed the clinical outcomes reported and suggested that , particularly if started earlier in the course of the disease , immunotherapy may lead to substantially longer os by slowing the disease trajectory ( figure 4b ) ( 60 ) . figure 4.tumor growth rates with immunotherapy ( vaccine ) and chemotherapy ( cytotoxic therapy ) . a ) tumor growth rate with no therapy ( dotted black line ) , with cytotoxic therapy ( blue line ) , and with vaccine ( red line ) , demonstrating the slow yet prolonged response with immunotherapy resulting from immune response activation ( red line ) and a short - term tumor reduction with chemotherapy ( blue line ) . b ) initiating immunotherapy in early - stage disease may enhance the effects of immunotherapy ( line b ) , whereas in later - stage disease the effects could be minimal ( line a ) . green arrows denote treatment initiation ; crosses denote death ( 60 ) . adapted ( with permission of oxford university press ) from : schlom j. therapeutic cancer vaccines : current status and moving forward . 2012;104(8):599613 . tumor growth rates with immunotherapy ( vaccine ) and chemotherapy ( cytotoxic therapy ) . a ) tumor growth rate with no therapy ( dotted black line ) , with cytotoxic therapy ( blue line ) , and with vaccine ( red line ) , demonstrating the slow yet prolonged response with immunotherapy resulting from immune response activation ( red line ) and a short - term tumor reduction with chemotherapy ( blue line ) . b ) initiating immunotherapy in early - stage disease may enhance the effects of immunotherapy ( line b ) , whereas in later - stage disease the effects could be minimal ( line a ) . green arrows denote treatment initiation ; crosses denote death ( 60 ) . adapted ( with permission of oxford university press ) from : schlom j. therapeutic cancer vaccines : current status and moving forward . j natl cancer inst the pivotal phase iii study of sipuleucel - t in mcrpc patients showed similar persistent treatment benefits . initial results showed a 4.1-month improvement in median survival ( 25.8 months in the sipuleucel - t group vs 21.7 months in the placebo group , hr = 0.78 , 95% ci = 0.61 to 0.98 , p = .03 ) ( 12 ) , and the 36-month survival probability was 31.7% in the sipuleucel - t group vs 23.0% in the placebo group , with differences in survival becoming apparent at six months following the start of treatment . as was the case for the studies above , no statistically significant improvement in time to objective disease progression was observed for sipuleucel - t . combined post hoc analyses of the three phase iii sipuleucel - t trials showed other measures consistent with a delayed treatment effect . while time to disease - related pain ( tdrp ) was not statistically significantly delayed between treated and control groups ( median tdrp = 5.6 months for sipuleucel - t vs 5.3 months for control , hr = 0.82 , 95% ci = 0.62 to 1.09 , p = .170 ) , the 12-month pain - free rates were 39.3% with sipuleucel - t vs 18.9% with control ( 61 ) . moreover , time to first use of opioid analgesics ( tfoa ) was statistically significantly delayed with sipuleucel - t ( median tfoa = 12.6 months for sipuleucel - t vs 9.7 months for control , hr = 0.76 , 95% ci = 0.58 to 0.99 , p = .038 ) . these data suggest an increasing impact of sipuleucel - t on end points that occur later in the disease course , as pain develops prior to the use of opioid analgesics ( figure 3 ) . studies with other immunotherapies have reported similar delayed treatment effects . data from 40 patients with mcrpc receiving psa - tricom ( a vector - based vaccine ) indicated that tumor growth rates , based on psa doubling time , were not reduced until day 80 relative to baseline ( median difference = 0.04 log psa / month , interquartile range = 0.08 to 0.01 , p = .02 ) ( 62 ) . a phase ii , randomized double - blind study in patients with mcrpc treated with psa - tricom failed to demonstrate an improvement in pfs compared with control ( 15 ) . however , a statistically significant difference favoring psa - tricom was reported for os ( 25.1 vs 16.6 months , respectively , estimated hr = 0.56 , 95% ci = 0.37 to 0.85 , p = .0061 ) . in patients with colorectal cancer , treatment with the panvac vaccine ( a poxviral - based vaccine targeting cea and muc-1 ) os improved with panvac vaccine after two years compared with controls ( median not reached vs 44.1 months , 95% ci = 36.2 to 63.4 , respectively , p < .0001 ) . however , the two - year recurrence - free survival rate was similar compared with controls ( 21.9 months , 95% ci = 16.9 to 38.8 , vs 25.7 months , 95% ci = 20.0 to 37.2 , respectively ) . similar delayed kinetics of benefit likely occur with immune checkpoint blocking agents . in a phase iii , placebo - controlled trial comparing ipilimumab plus dacarbazine compared with dacarbazine alone , median pfs was similar in the two groups based on the week 12 assessment . however , a statistically significant difference in os was observed ( 11.2 months , 95% ci = 9.4 to 13.6 , for ipilimumab + dacarbazine vs 9.1 months , 95% ci = 7.8 to 10.5 , for dacarbazine alone ; hr for death = 0.72 , 95% ci = 0.59 to 0.87 , p < .001 ) . the higher survival rates for the ipilimumab + dacarbazine group persisted at three years compared with dacarbazine alone ( 20.8% vs 12.2% ) ( 64 ) . consistent with this delayed and persistent benefit , a pooled analysis of 12 studies of ipilimumab for the treatment of melanoma reported a median os of 11.4 months ( 95% ci = 10.7 to 12.1 ) and noted that the os curve plateaus at around year 3 and extends to year 10 ( 65 ) . some patients initially categorized as partial responders ( n = 10 ) were later categorized as complete responders ( n = 5 ) , and the average time to a complete response was 30 months ( 66 ) . an improvement in os without improvement in median pfs has been seen with pd1 inhibition , also suggesting that this observation may be a class effect of immunotherapy ( 5 ) . these observations highlight the concept that traditional clinical trial end points ( tumor response by traditional response evaluation criteria in solid tumors , pfs ) may not adequately reflect the survival benefits gained from immunotherapy . consideration for extending the duration of clinical studies with immunotherapies may be necessary to adequately quantify their effects and/or adapt current response criteria , including immune - related response criteria ( 67 ) . because the clinical antitumor effects of immunotherapy may take time to evolve , it follows naturally that treatment with immunotherapy earlier in the disease course should yield greater clinical benefit . indeed , a subset analysis of the pivotal phase iii trial of sipuleucel - t demonstrated that with decreasing baseline psa ( used as a surrogate for disease severity ) , both os and os benefit relative to control improved ( 68 ) . the median os in patients in the lowest baseline psa quartile ( 22.1 ng / ml ) was 41.3 months ( sipuleucel - t ) vs 28.3 months ( placebo ; difference = 13.0 months , hr = 0.51 , 95% ci = 0.31 to 0.85 ) . for patients in the highest baseline psa quartile ( > 134 ng / ml ) , median os was 18.4 vs 15.6 months for sipuleucel - t and placebo , respectively ( difference = 2.8 months , hr = 0.84 , 95% ci = 0.55 to 1.29 ) ( 68 ) . therefore , patients in the lowest baseline psa quartile appeared to derive the greatest benefit from treatment with sipuleucel - t vs control . consistent with these observations , patients with lower lactate dehydrogenase values and better performance status also experienced greater benefit from sipuleucel - t compared with patients with more advanced disease . furthermore , patients with low tumor burden are generally less immunosuppressed both systemically and in the tumor microenvironment ( 69 ) . this concept is supported by a recent study of ipilimumab in prostate cancer ( 70 ) . in a post hoc analysis of this study , patients with favorable prognostic factors at baseline ( ie , alkaline phosphatase concentration < 1.5x upper limit of normal , hemoglobin levels 11.0 g / ml , no visceral metastases ) had statistically significantly prolonged os with ipilimumab compared with placebo , whereas in patients with at least one adverse prognostic factor , ipilimumab did not prolong os ( 70 ) . taken together , these data support the concept that patients with less advanced disease may have a more robust and effective immunologic response to therapy , allowing more time for antigen spread and associated survival benefits to be observed . further studies examining antigen spread following treatment with immunotherapy may be useful in not only understanding the full benefits to be gained from current treatments , but also in identifying other taas that may be appropriate targets for the development of immunotherapies . in addition , a detailed exploration of the clinical kinetics of immunotherapy should provide information of potential value in assisting clinical trial design and decision - making . if the hypothesis that immunotherapy has a delayed effect on the tumor growth curve is validated , treating earlier with immunotherapies may result in improved outcomes ( 60,71 ) . this observation is supported by analyses that indicate that patients with a lower tumor burden have a differentially better clinical response to immunotherapy than those with a high tumor burden ( 68,70,72,73 ) . as our understanding of the mechanisms associated with cancer immunotherapies expands , future research will likely focus on four key concepts : 1 ) combination regimens : the disparate properties of immunotherapy and conventional chemotherapy ( targeting the tumor vs the immune system ; timing of response and potential for resistance ) provide a strong rationale for combining immunotherapy with cytoreductive therapy . in addition , conventional therapy can lead to tumor cell death , which weakens the mechanisms of tumor immune evasion and can enhance the ability of an activated immune system to recognize and control subsequent tumor growth ( 37,74 ) . 2 ) sequence of agents : the concept that the relative sequence of therapy may have immunological implications was examined in a randomized phase ii study evaluating the optimal sequencing of sipuleucel - t and adt in men with biochemically recurrent prostate cancer at high risk of developing metastases ( sipuleucel - t combined with androgen deprivation therapy [ stand ] , nct01431391 ) ( 24 ) . data from stand indicate that cellular and humoral responses to pa2024 statistically significantly increased following sipuleucel - t treatment compared with baseline , and that these responses were sustained at all postdosing time points through 24 months ( p < .001 ) . sipuleucel - t given before , rather than after , adt initiation resulted in greater pa2024-specific t - cell proliferation . interestingly , sipuleucel - t - mediated antigen spread was observed with both treatments and was maintained through 12 months ( 24 ) . 3 ) combining vaccination with immune checkpoint blockade : while immune checkpoint blockers have shown promise , patients with no underlying immune response appear to be much less likely to benefit ( 75 ) . thus , vaccines may possibly convert a patient who is unlikely to respond into one more likely to respond to an immune checkpoint inhibitor ( 7678 ) . 4 ) biomarkers : identifying and validating biomarkers of response are important factors in early confirmation that patients are responding to treatment . such biomarkers may be useful both in guiding the timing and selection of subsequent therapy and in making more reliable determinations of the success or failure of novel immunotherapy agents in early - stage clinical trials . the trials with sipuleucel - t have identified antibody responses to the immunogen , and transient increases in serum eosinophil count and igg responses to psa and lgals3 have been linked to os ( 12,28,79 ) . in vitro measures of immune activation in the sipuleucel - t product ( cumulative apc activation , apc number , and total nucleated cell count ) and antigen - specific immune responses to sipuleucel - t have also been shown to correlate with os in patients with mcrpc ( 42 ) . similarly , an antibody response to a glycan is associated with improved os in patients treated with psa - tricom ( 80 ) . antigen spread following treatment with immunotherapy can evoke robust , durable , and adaptable immune responses against tumors over an extended time period ( 3,12 ) . understanding the process of antigen spread , and thus the mode of action of immunotherapies , may help explain the greater improvements in efficacy observed in longer - term clinical study outcomes compared with short - term studies ( eg , os vs pfs ) ( 3,12,42,70 ) . although immunotherapy has led to major clinical advances in the past several years , we continue to learn more about antigen spread and the mechanism of these treatments . in addition , we are exploring how to fully integrate and optimize their use with our current and emerging armory against cancer . thus , the coming years promise to accelerate our understanding of how to further optimize patient outcomes . dendreon personnel , who are not authors , also reviewed the paper and approved it for submission . medical writing and coordination support was provided by catherine lee and jackie phillipson of zoetic science , macclesfield , uk , an ashfield company .

immunotherapy is an important breakthrough in cancer . us food and drug administration - approved immunotherapies for cancer treatment ( including , but not limited to , sipuleucel - t , ipilimumab , nivolumab , pembrolizumab , and atezolizumab ) substantially improve overall survival across multiple malignancies . one mechanism of action of these treatments is to induce an immune response against antigen - bearing tumor cells ; the resultant cell death releases secondary ( nontargeted ) tumor antigens . secondary antigens prime subsequent immune responses ( antigen spread ) . immunotherapy - induced antigen spread has been shown in clinical studies . for example , in metastatic castration - resistant prostate cancer patients , sipuleucel - t induced early immune responses to the immunizing antigen ( pa2024 ) and/or the target antigen ( prostatic acid phosphatase ) . thereafter , most patients developed increased antibody responses to numerous secondary proteins , several of which are expressed in prostate cancer with functional relevance in cancer . the ipilimumab - induced antibody profile in melanoma patients shows that antigen spread also occurs with immune checkpoint blockade . in contrast to chemotherapy , immunotherapy often does not result in short - term changes in conventional disease progression end points ( eg , progression - free survival , tumor size ) , which may be explained , in part , by the time taken for antigen spread to occur . thus , immune - related response criteria need to be identified to better monitor the effectiveness of immunotherapy . as immunotherapy antitumor effects take time to evolve , immunotherapy in patients with less advanced cancer may have greater clinical benefit vs those with more advanced disease . this concept is supported by prostate cancer clinical studies with sipuleucel - t , psa - tricom , and ipilimumab . we discuss antigen spread with cancer immunotherapy and its implications for clinical outcomes .

Antigen Spread Evidence for Antigen Spread Mediated by Immunotherapy Delayed Clinical Response to Immunotherapy Immunotherapy in Early Disease States Future Considerations Conclusion Funding Notes

this study aimed to assess students perceptions of their educational environment in the faculty of medicine and pharmacy of rabat , morocco , using the dundee ready educational environment measure ( dreem ) . a cross - sectional survey was conducted in the faculty of medicine and pharmacy of rabat , morocco , in which medical students perceptions of their educational environment were assessed using the dreem criteria during the 2013 - 2014 academic years . the dreem inventory encompasses 50 items divided into five subdomains : perceptions of learning , perceptions of teaching , academic self - perceptions , perceptions of atmosphere , and social self - perceptions . the dreem has a maximum score of 200 , which would correspond to a perfect educational environment . the mean scores ( standard deviation ) of students responses were compared according to their year of study and gender . the responses of 189 postgraduate medical students were included . the mean total dreem score was 90.8 ( 45.4% ) . the mean total scores for five subdomains were 21.2/48 ( 44.2% ) , 21.8/44 ( 49.6% ) , 13.1/32 ( 40.9% ) , 19.0/48 ( 39.6% ) , and 15.6/28 ( 55.7% ) respectively . female students reported higher perceptions of teaching scores than males ( p=0.002 ) , and students in their fifth year of study reported significantly higher social self - perceptions scores than those in their fourth year ( p=0.03 ) . in this study of the oldest faculty of medicine in morocco , students perceived the educational environment as having many problems .

SUPPLEMENTARY MATERIAL

in healthy individuals , cell free dna ( cfdna ) is released into the circulation by cells undergoing apoptosis.13 , 14 apoptotic cells shed dna fragments approximately 185 to 200 bp in length.14 , 15 in contrast , cfdna of cancer patients is derived from both nonmalignant and malignant cells . interestingly , the percentage of circulating dna derived from cancer cells ranges from 3% to as much as 93%.16 since cancer cells die through multiple mechanisms that ultimately lead to dna cleavage such as apoptosis , necrosis and autophagy,17 ctdna displays less uniformity in size and integrity relative to cfdna in healthy individuals.14 , 15 , 17 some researcher groups report that ctdna has less integrity and is smaller relative to cfdna13 , 14 , 15 while others report the opposite.18 , 19 these conflicting results may be a result of the different dna isolation methods used and the sample sources tested . more recently , one study showed that decreased cfdna integrity and increased cfdna concentrations distinguish normal individuals from patients with primary and metastatic breast cancer.20 a second study used singlebase pair resolution sequencing to demonstrate that the shorter dna fragments in cancer patient plasma preferentially contained tumorassociated copy number alterations.21 collectively , these two reports suggest that the isolation and analysis of short dna fragments may improve the sensitivity of detection of ctdna . irrespective of dna fragment size , the ability to detect and quantify ctdna fragments carrying tumorspecific mutations has maintained interest in ctdna as a useful tool to monitor tumor growth . given that the frequency of mutant dna alleles in cfdna is as low as 0.01%,4 highly sensitive and specific detection methods are required for ctdna to serve as a clinically feasible approach . here we provide a brief description of the ctdna detection methods capable of such high specificity and sensitivity . initial studies on ctdna relied upon realtime allelespecific pcr to detect mutations of interest.22 however , primarily patients with high tumor burden , and thus higher ctdna levels in the plasma,23 were chosen for these studies as the sensitivity and specificity of realtime pcr approaches is limited.22 digital pcr ( dpcr ) has increased sensitivity and specificity relative to realtime pcr.22 with dpcr , a dna sample is segregated using limiting dilution such that individual dna molecules are captured within wateroil emulsion droplets or chambers.24 a portion of the droplets or chambers will contain no dna while others will contain individual dna molecules . the segregated individual dna molecules may be wild type or mutant in nucleotide sequence . using unique sets of primers and probes , mutant and normal dna sequences are amplified , quantified and the percent mutant allele frequency determined . for liquid biopsy samples , dpcr is capable of detecting mutant alleles with a fractional abundance of 0.005%25 to 0.04%.26 there are several advantageous features to dpcr including precision , sensitivity and increased resistance to pcr inhibitors.24 , 26 , 27 in addition , dpcr is capable of absolute measurements of rare dna alleles without the need for a standard curve.24 , 28 , 29 recently novel modifications to dpcr have been introduced with the intention of detecting highly infrequent mutant dna molecules in plasma.9 , 30 , 31 , 32 the ability to detect these extremely rare mutant dna molecules might allow for efficient screening and diagnosis of cancers at earlier stages with liquid biopsies . the analytical sensitivity of dpcr is one advantageous feature that is counterbalanced by the limited capacity of dpcr to interrogate multiple genomic alterations simultaneously.33 without prior and precise knowledge of the dna rearrangements , translocations and mutations of interest , dpcr is compromised in its utility to detect cancer and monitor tumor growth . massive parallel sequencing , also known as nextgeneration sequencing ( ngs ) , offers to circumvent this limitation as ngs has the ability of detect multiple somatic dna alterations simultaneously.33 , 34 , 35 early ctdna studies with ngs demonstrated the ability of ngs to detect chromosomal rearrangements and chromosomal copy number changes.35 , 36 in the setting of metastatic prostate cancer , heitzer and colleagues36 devised a genomewide ngs approach capable of identifying novel genomic alterations in ctdna associated with the emergence of metastatic tumor clones . impressively , this approach provided genomic cancer profiles in 2 days thus making it a more feasible test in a clinical setting.36 a shortcoming of most ngsbased approaches is the low sensitivity that renders them unfit to be reliable molecular diagnostic tests.34 , 35 , 37 more recently , newman and colleagues devised an ultrasensitive ngsbased approached termed cancer personalized profiling by deep sequencing ( cappseq ) with the ability to detect mutant allele fractions as low as 0.02% and with 96% specificity.38 importantly , cappseq reduces cost to approximately $ 200300 us dollars per test.33 further refinements to both dpcr and ngs may ultimately make ctdna cancer screening a reality . a small number of studies have examined the feasibility of ctdna in detecting early stage cancers and fewer still have considered ctdna for screening presymptomatic cancer patients.39 , 40 , 41 , 42 , 43 since effective screening of asymptomatic patients requires prior knowledge of mutations in genes commonly altered in low grade cancers , ctdna is most suited for the detection of advanced cancers with wellknown mutations . complicating matters further is the observation that healthy subjects have detectable mutations in their circulating dna . for instance , during minimalinvasive prenatal testing , amant and coworkers detected genomic alterations in 3 of 4,000 asymptomatic pregnant women that were later confirmed to have malignancies by magnetic resonance imaging and biopsy.40 in addition , the percentage of individuals with mutations in the blood increases with age44 , 45 at a rate that exceeds the clinical incidence of hematological malignancies in the general population.44 moreover , independent studies reported that mutations in kras and tp53 in the circulating dna of noncancer control subjects range from 1 to 3.6%.46 collectively these studies suggest that cfdna of healthy subjects contains mutations that might pose a significant challenge in accurately diagnosing and screening patients for cancer.47 on the other hand , presymptomatic testing for cancer might be more feasible with ctdna than originally thought . interestingly , sausen and coworkers demonstrated that pancreatic cancer recurrence was predicted 6.5 months earlier with ctdna detection relative to ct scanning48 ; thus supporting the notion that ctdna might be useful in screening and diagnosing asymptomatic patients . additional studies combining ctdna analysis with biomedical imaging will ultimately determine the benefits of ctdna in cancer surveillance as an adjunct diagnostic test . studies in multiple cancers have shown a pattern where ctdna levels buildup as tumors progress and then decline following surgery or drug treatment.23 , 32 , 37 , 49 , 50 thus it seems logical that ctdna levels might be used as a surrogate marker of tumor burden and therapeutic responses . conceptually , ctdna has clear advantages to monitor tumor burden and therapeutic response compared with protein cancer markers and biomedical imaging . for instance , ctdna has a halflive of approximately 2 h9 while proteins persist in the blood for weeks to months,51 , 52 thus analysis of ctdna should permit a more rapid assessment of tumor changes within hours rather than weeks to months.53 tumor specific protein markers have the limitation of being elevated in circumstances not related to tumor growth54 while elevated ctdna levels are associated with tumor changes . moreover , a direct comparison of ctdna , ctc and cancer antigen 153 levels in the same patients revealed that ctdna levels showed a greater correlation with tumor burden and the earliest measure of response to therapy,23 suggesting that ctdna levels more accurately predict tumor changes relative to protein markers55 ( fig . predicted tumor recurrence much earlier than conventional tumor imaging techniques.38 , 48 , 56 tumor burden and plasma ctdna levels show a direct correlation . as tumor burden increases , ctdna accumulates in the plasma . with therapeutic intervention , tumor burden and ctdna levels decrease . [ color figure can be viewed at wileyonlinelibrary.com ] the genetic basis of resistance by tumors to targeted molecular therapies has recently been determined.57 , 58 , 59 , 60 nonsmall cell lung cancers ( nsclc ) bearing activating mutations in the egfr gene are extremely sensitive to the small molecule inhibitors erlotinib and gefitinib.59 in approximately 50% of nsclc patients resistance to egfr inhibition is driven by a gatekeeper mutation in the egfr kinase domain that changes threonine 790 to a methionine ( t790 m ) . the t790 m mutation increases the affinity of the egfr kinase domain for atp while decreasing its affinity to erlotinib and gefitinib . in the case of colorectal cancers , mutations in kras ( g12r and g13d ) activate survival pathways that drive resistance to egfr monoclonal antibodies.56 with such precise knowledge of the molecular basis of response to targeted therapies , the possibility of detecting resistance in a minimalinvasive manner in the blood has been examined.61 , 62 as might be predicted , detection of the t790 m gatekeeper mutation was shown to correlate with therapy resistance and disease progression for nsclc patients treated with egfr inhibitors.11 , 63 impressively , in some cases , t790mdriven resistance was detected 16 weeks before tumor progression was detectable with radiographic imaging.63 in colon cancer , ctdna analysis revealed that the presence of mutant kras was associated either primary or acquired resistance to egfr inhibition.62 , 64 upon withdraw of antiegfr antibodies , mutant ras levels dropped and relatively novel mutations associated with either primary or acquired resistance were found in nras , met , erbb2 , flt3 , egfr and mapk21.62 thus far , several studies show that the analysis of ctdna levels is a powerful tool to monitor tumor dynamics and response to therapy . in analyzing ctdna , novel mutations associated with resistance to conventional and targeted therapy have been identified.62 , 65 these newly identified mutations might offer new avenues of treatment for cancer patients with advanced disease . studies in multiple cancers have shown a pattern where ctdna levels buildup as tumors progress and then decline following surgery or drug treatment.23 , 32 , 37 , 49 , 50 thus it seems logical that ctdna levels might be used as a surrogate marker of tumor burden and therapeutic responses . conceptually , ctdna has clear advantages to monitor tumor burden and therapeutic response compared with protein cancer markers and biomedical imaging . for instance , ctdna has a halflive of approximately 2 h9 while proteins persist in the blood for weeks to months,51 , 52 thus analysis of ctdna should permit a more rapid assessment of tumor changes within hours rather than weeks to months.53 tumor specific protein markers have the limitation of being elevated in circumstances not related to tumor growth54 while elevated ctdna levels are associated with tumor changes . moreover , a direct comparison of ctdna , ctc and cancer antigen 153 levels in the same patients revealed that ctdna levels showed a greater correlation with tumor burden and the earliest measure of response to therapy,23 suggesting that ctdna levels more accurately predict tumor changes relative to protein markers55 ( fig . 1 ) . with regard to imaging techniques , increases in ctdna levels predicted tumor recurrence much earlier than conventional tumor imaging techniques.38 , 48 , 56 tumor burden and plasma ctdna levels show a direct correlation . as tumor burden increases , ctdna accumulates in the plasma . with therapeutic intervention , tumor burden and ctdna levels decrease [ color figure can be viewed at wileyonlinelibrary.com ] the genetic basis of resistance by tumors to targeted molecular therapies has recently been determined.57 , 58 , 59 , 60 nonsmall cell lung cancers ( nsclc ) bearing activating mutations in the egfr gene are extremely sensitive to the small molecule inhibitors erlotinib and gefitinib.59 in approximately 50% of nsclc patients resistance to egfr inhibition is driven by a gatekeeper mutation in the egfr kinase domain that changes threonine 790 to a methionine ( t790 m ) . the t790 m mutation increases the affinity of the egfr kinase domain for atp while decreasing its affinity to erlotinib and gefitinib . in the case of colorectal cancers , mutations in kras ( g12r and g13d ) activate survival pathways that drive resistance to egfr monoclonal antibodies.56 with such precise knowledge of the molecular basis of response to targeted therapies , the possibility of detecting resistance in a minimalinvasive manner in the blood has been examined.61 , 62 as might be predicted , detection of the t790 m gatekeeper mutation was shown to correlate with therapy resistance and disease progression for nsclc patients treated with egfr inhibitors.11 , 63 impressively , in some cases , t790mdriven resistance was detected 16 weeks before tumor progression was detectable with radiographic imaging.63 in colon cancer , ctdna analysis revealed that the presence of mutant kras was associated either primary or acquired resistance to egfr inhibition.62 , 64 upon withdraw of antiegfr antibodies , mutant ras levels dropped and relatively novel mutations associated with either primary or acquired resistance were found in nras , met , erbb2 , flt3 , egfr and mapk21.62 thus far , several studies show that the analysis of ctdna levels is a powerful tool to monitor tumor dynamics and response to therapy . in analyzing ctdna , novel mutations associated with resistance to conventional and targeted therapy have been identified.62 , 65 these newly identified mutations might offer new avenues of treatment for cancer patients with advanced disease . although ctdna represents a promising real time tool for tumor characterization and is now being extensively studied , there are still many challenges that need to be overcome before it 's routinely used by clinicians . ctdna is not always detectable in peripheral blood due to the extremely low level presented in the blood . in addition , current digital pcr method preferably measures relatively longer fragments , i.e. , > 120 bp , while smaller fragments derived from tumors are not detected efficiently.66 therefore , the results from ctdna analysis might be false negative . many studies have shown an average of 7080% concordance between tumor somatic mutations and the presence of ctdna.67 , 68 this could be improved by the advances in genomic approaches that have higher sensitivity to identify all ctdna in the blood . additionally , we do not have a standard and validated procedure for samples handling and liquid biopsy analysis , both aspects would affect the final results . a standard operation procedure should be established and be strictly followed to ensure we get the right information from ctdna analysis . in addition to ctdna , ctcs , cell free rnas and exosomes also attract extensive attention for their potential clinical application . compared to these measurements , which can address tumor status at dna , rna and protein levels , ctdna results can only be reflected by mrna or protein expression analysis , such as arv7.69 in this situation , ctdna analysis may not be a good choice to monitor tumor development . in contrast to tumor tissues , ctdna represents mainly the genome of dying tumor cells , or the cells that respond to therapy , one can not ruled the exist of resistant cells or subpopulations that contain few or no dying cells based on ctdna analysis . selection of the appropriate time points for ctdna screening will be very important in some situation . also , results from ctdna analysis can not determine the exact origin of a mutation , i.e. , whether it is somatic or germline . the discovery of germline mutations might be an important indication for genetic screening for the patients ' family members as well . the shedding of ctdna into the circulation offers researchers and clinicians a novel target that carries the same dna mutations found in primary and metastatic cancer lesions . access to ctdna is minimalinvasive and simple as it involves a standard blood draw . with newly developed ultrasensitive and specific methods to detect and characterize genomic alteration present in ctdna have been shown to directly correlate to ctdna levels in patients and the frequency of ctdna molecules carrying mutations that confer resistance to cancer therapies . in some studies , detection of ctdna is superior in predicting tumor recurrence relative to conventional tumor imaging techniques . with further refinements to the methods to detect and quantify ctdna , it may be possible to screen asymptomatic patients with ctdna to detect cancers at their earliest stages ( fig . further studies should be launched to validate the potential of ctdna for routine clinical use . however , based on the large amounts of promising data and with the rapid advances and the decreased cost of nextgeneration sequencing , we anticipate there is much promise in influencing cancer patient treatment and survival with ctdna . in addition to ctdna , cfrna , ctcs , circulating exosomes or blood platelets are also promising as novel liquid biopsy candidates.70 , 71 , 72 the advantages and disadvantages of these technologies are worth to be further explored . ctdna can be obtained from plasma and in combination with digital pcr and nextgeneration sequencing ( nextgen seq ) might allow for detection of cancers at their earliest stages . in some reports , ctdna detection predicts cancer recurrence or progression months earlier than conventional cancer imaging methods . the shedding of ctdna into the circulation offers researchers and clinicians a novel target that carries the same dna mutations found in primary and metastatic cancer lesions . access to ctdna is minimalinvasive and simple as it involves a standard blood draw . with newly developed ultrasensitive and specific methods to detect and characterize genomic alteration present in ctdna the opportunity exists to monitor tumor progression and recurrence . tumor burden and recurrence have been shown to directly correlate to ctdna levels in patients and the frequency of ctdna molecules carrying mutations that confer resistance to cancer therapies . in some studies , detection of ctdna is superior in predicting tumor recurrence relative to conventional tumor imaging techniques . with further refinements to the methods to detect and quantify ctdna , it may be possible to screen asymptomatic patients with ctdna to detect cancers at their earliest stages ( fig . further studies should be launched to validate the potential of ctdna for routine clinical use . however , based on the large amounts of promising data and with the rapid advances and the decreased cost of nextgeneration sequencing , we anticipate there is much promise in influencing cancer patient treatment and survival with ctdna . in addition to ctdna , cfrna , ctcs , circulating exosomes or blood platelets are also promising as novel liquid biopsy candidates.70 , 71 , 72 the advantages and disadvantages of these technologies are worth to be further explored . clinical application of ctdna as a tool for cancer monitoring . ctdna can be obtained from plasma and in combination with digital pcr and nextgeneration sequencing ( nextgen seq ) might allow for detection of cancers at their earliest stages . in some reports , ctdna detection predicts cancer recurrence or progression months earlier than conventional cancer imaging methods .

for many decades it has been known that tumor dna is shed into the blood . as a consequence of technological limitations , researchers were unable to comprehensively characterize circulating dna . the advent of ultrasensitive and highly specific molecular assays has provided a comprehensive profile of the molecular characteristics and dynamics of circulating dna in healthy subjects and cancer patients . with these new tools in hand , significant interest has been provoked for an innovative type of tumor biopsy termed a liquid biopsy . liquid biopsies are obtained by minimal invasive blood draws from cancer patients . circulating cancer cells , exosomes and a variety of molecules contained within the liquid biopsy including cellfree circulating tumor dna ( ctdna ) can serve as promising tools to track cancer evolution . attractive features of ctdna are that ctdna isolation is straightforward , ctdna levels increase or decrease in response to the degree of tumor burden and ctdna contains dna mutations found in both primary and metastatic lesions . consequently , the analysis of circulating dna for cancerspecific mutations might prove to be a valuable tool for cancer detection . moreover , the capacity to screen for ctdna in serial liquid biopsies offers the possibility to monitor tumor progression and responses to therapy and to influence treatment decisions that ultimately may improve patient survival . here we focus on mutation detection in ctdna and provide an overview of the characteristics of ctdna , detection methods for ctdna and the feasibility of ctdna to monitor tumor dynamics . current challenges associate with ctdna will also be discussed .

Origins and Molecular Characteristics of ctDNA Detection of ctDNA Cancer Surveillance With ctDNA Monitoring tumor burden and responses to therapy using ctDNA Current Challenges and Limitations Conclusion and perspectives

it has been reported that over 50% of patients admitted for sah develop hyponatremia ( 1 - 3 ) . sah - associated hyponatremia can be caused by excessive intravenous fluid therapy , diuretic therapy , cerebral salt - wasting syndrome ( csws ) , and syndrome of inappropriate antidiuretic hormone ( siadh ) ( 4 ) . both csws and siadh cause hyponatremia by increasing renal excretion of sodium , and both are associated with serum hypo - osmolality and high urine osmolality ( 5 - 8 ) . ( 3 ) reported that siadh is more likely to occur than csws ( 35.4% vs. 22.9% , 71.4% vs. 0% ) in this patient population , yet csws has similar mortality rates and duration of hospital stay to siadh . the high urine sodium level observed in patients with csws is secondary to urine sodium wasting rather than volume expansion ( 11 ) . although the precise mechanism of salt wasting is poorly understood , several studies have begun to shed light on the pathophysiology of this disease . first , impaired sympathetic neural input to the juxtaglomerular apparatus may reduce proximal tubule sodium , urate , and water reabsorption and decrease renin and aldosterone release ( 12 ) . second , brain injury might increase the secretion of human atrial natriuretic peptide ( hanp ) and brain natriuretic peptide ( bnp ) , leading to an increase in sodium excretion and urine volume ( 1,10,13 ) . in contrast , hyponatremia secondary to siadh is a result of an excess of water rather than a deficiency of sodium . excessive secretion of antidiuretic hormone ( adh ) is caused by stimulation of the hypothalamus after trauma or ischemia . high levels of adh increase water reabsorption in the distal convoluted renal tubule , causing fluid retention and dilutional hyponatremia ( 14,15 ) . first , csws is characterized by volume depletion , while siadh is not ( 5,16 ) . however , the evaluation of extracellular fluid volume is notoriously difficult . second , changes in uric acid homeostasis may provide clues to the cause of hyponatremia . in siadh in contrast , in csws , hypouricemia and increased renal uric acid excretion persist after correction of serum sodium . furthermore , the fractional excretion of uric acid ( feua ) is greater than 10% after correction of hyponatremia in csws but is less than 10% after correction of hyponatremia in siadh ( 17 ) . it is important to distinguish between csws and siadh in patients with hyponatremia because treatment of these two syndromes is very different . he had been in good health until 4 days prior to presentation , at which point he fell and developed sah . two days following discharge , he developed a headache , nausea , thirst , and impaired consciousness , prompting readmission . on admission , his blood pressure was 127/72 mmhg , pulse rate was 61 beats per minute , and oxygen saturation was 98% while breathing ambient air . the laboratory findings included marked hyponatremia ( 106 mmol / l ) , plasma hypo - osmolality ( 220 mosm / l ) , urine hyper - osmolality ( 468 mosm / l ) , and elevated urine sodium ( 115 his serum arginine vasopressin peptide ( avp ) level was 2.2 pg / ml , and his serum creatinine , thyroid hormone , and adrenal cortical hormone levels were within normal limits . although his physical examination findings were normal , he reported increased urination and excessive thirst . furthermore , his inferior vena cava diameter was 8 mm on ultrasound , indicating volume depletion . together , the data suggested a diagnosis of csws , and he was treated with sodium repletion ( 300 to 600 meq daily ) . however , the serum sodium level did not improve ( 119 mmol / l ) , and he continued to excrete high levels of sodium . starting on hospital day 10 , he was treated with fludrocortisone ( 0.05 to 0.1 mg daily ) . shortly thereafter , the serum sodium level gradually improved ( 135 mmol / l ) , and the urine sodium level decreased ( figure ) . the fractional excretion of uric acid was 11.9% after correction of hyponatremia , which is consistent with a diagnosis of csws . his blood pressure was 148/78 mmhg , and his pulse rate was 64 beats per minute . he was again noted to be hyponatremic ( 118 mmol / l ) and was thus readmitted to our hospital . similar to his prior hospitalization , laboratory tests revealed hypo - osmolality ( 245 mosm / l ) , urine hyper osmolality ( 545 mosm / l ) , and increased renal excretion of sodium ( 82 mmol / l ) . his physical examination findings were unremarkable , but unlike at his previous hospitalization , the diameter of his inferior vena cava was normal ( 14 mm ) on ultrasound . he was initially treated with sodium repletion ( 300 to 400 meq daily ) and fludrocortisone ( 0.1 mg daily ) but his serum sodium level did not improve . he was subsequently treated with fluid restriction ( 800 ml / day ) , and his serum sodium level improved ( 140 mmol / l , figure ) . the fractional excretion of uric acid was 4.9% after correction of hyponatremia , which is consistent with a diagnosis of siadh . he continued to do well over the next four months , requiring no additional hospitalizations or medications . it has been reported that hyponatremia in patients with central nervous system ( cns ) diseases can be attributed to excessive fluid therapy , diuretic use , csws , or siadh ( 8) . although csws and siadh have similar laboratory findings , including hyponatremia , serum hypo - osmolality , and increased renal excretion of sodium , the pathophysiology and treatment of these syndromes are very different . hyponatremia due to csws improves with fluid repletion and fludrocortisone , while hyponatremia due to siadh improves with fluid restriction and vasopressin-2 receptor antagonists . thus , the correct etiology of hyponatremia must be identified before starting the appropriate therapy . csws is characterized by extracellular volume depletion , which stimulates adh release ( 1,10 ) . this is accompanied by increased production of hanp and bnp and impaired release of renin , despite fluid depletion . siadh , which is characterized by excessive extracellular volume , causes similar changes in renin , hanp and bnp . hence , changes in these serum markers can not be used to differentiate csws from siadh . other factors may be more useful in differentiating these two syndromes , including extracellular volume status , changes in uric acid homeostasis , and response to fluid restriction . table 2 shows the laboratory and physical findings during the patient 's first and second hospitalizations , leading to the diagnoses of csws and siadh . for example , the fractional excretion of uric acid was greater than 10% after the correction of hyponatremia during the first hospitalization , but it was less than 10% during the second hospitalization ( table 1 , 2 ) . furthermore , the response to fluid restriction differed between the first and second hospitalizations . the sensitivity and specificity of these findings for the diagnosis of csws and siadh are unknown . therefore , these differences may simply be diagnostic tools , rather than strict definitions of disease . nonetheless , the data suggest that his initial presentation was consistent with csws and his second presentation was consistent with siadh . there are no previous reports suggesting that csws may transform into siadh , and no reports of a temporal association between the two syndromes . siadh can occur hours to months after brain injury ; our patient may therefore have suffered from late - onset siadh , leading to the observed time difference between csws and siadh ( 18 ) . in our case , natriuretic factors such as anp and bnp were initially increased by direct damage to the brain . natriuretic factors would lead to a diminished activity of the renin - angiotensine - aldosterone system and increased natriuresis in the distal renal tubule ( 19 ) . inappropriate secretion of adh increases water reabsorption in the distal renal tubule , which is consistent with late - onset siadh , resulting in increased natriuresis . it is also possible that the patient developed both syndromes simultaneously but that the laboratory findings during his first admission were most consistent with csws . after fluid therapy and fludrocortisone treatment , the csws resolved , revealing the residual siadh . we might also need to consider how much the patient drank , particularly after the first hospital discharge , as excessive drinking can result in an excess of water , which would exacerbate siadh . however , the patient did not consume a large amount of water at his home . he reported that he consumed 1 to 1.5 liters of water per day , which would not cause an excess of water . without precise data on the amount he drank after the first hospital discharge , though , conclusions are difficult to make . we herein report a rare case of csws and siadh following sah . distinguishing between csws and siadh may help prevent complications resulting from inappropriate treatment .

hyponatremia is a common finding after subarachnoid hemorrhaging ( sah ) and can be caused by either cerebral salt - wasting syndrome ( csws ) or syndrome of inappropriate antidiuretic hormone ( siadh ) . distinguishing between these two entities can be difficult because they have similar manifestations , including hyponatremia , serum hypo - osmolality , and high urine osmolality . we herein report the case of a 60-year - old man who suffered from sah complicated by hyponatremia . during his initial hospitalization , he was diagnosed with csws . he was readmitted one week later with hyponatremia and was diagnosed with siadh . this is the first report of sah causing csws followed by siadh . these two different sources of hyponatremia require different treatments .

Introduction Case Report Discussion The authors state that they have no Conflict of Interest (COI).

although jean - martin charcot is credited with providing a comprehensive description of ms , reports of both ms and comorbid cognitive impairment precede charcot s 1868 lectures . friedrich von frerichs first cited ms - related cognitive impairment in 1849 , 25 years after the disease s initial clinical description . despite multiple early accounts of ms as a disease affecting cognition , reports on the incidence of cognitive impairment in patients with ms were mixed over the following century . while some late 19th and early 20th century physicians recognized deterioration of cognitive faculties in more than half of their ms patients , others reported that only two percent of their patients with ms experienced blunted intellectual function.1 discrepancies in these figures are probably due to the fact that the majority of neurologists did not ask patients with ms about their cognitive function , and those neurologists who did inquire had inconsistent means of measuring cognitive function . the minimal assessment of cognitive function in multiple sclerosis ( macfims ) battery a seven - test , 90-minute assessment of word fluency , visuospatial ability , learning , memory , processing , and executive function ( cognitive skills required to unite learning and memory with behavior)was not established until 2001 . the recent development of improved diagnostic tests for cognitive function has allowed researchers to reach a general consensus : cognitive impairment is a debilitating and widespread comorbidity of ms . today physicians recognize that ms affects more than 600,000 people in the united states and more than 2 million people worldwide , and 40 to 65 percent of these patients experience some degree of cognitive impairment.2 , 3 cognitive impairment substantially impacts the lives of patients with ms and their families . half to three - quarters of people with ms are unemployed within 10 years of diagnosis.4 cognitive impairment is the leading predictor of occupational disability , while physical disability , age , sex , and education contribute less than 15 percent to the likelihood of being employed.5 patients with impaired cognition participate in social activities less frequently . cognitive impairment due to ms may also place significant additional strain on the patient s caregiver , who must help the patient combat intellectual , social , and occupational disabilities . most cases of cognitive impairment in ms are relatively less severe than those observed in classically dementing neurological disorders , such as alzheimer s disease , in which the patient loses memory of previous experiences and is unable to respond properly to environmental stimuli . however , cognitive impairment in ms can be extremely debilitating , with substantial negative impacts on daily living . while some researchers conclude that patients with ms have trouble initially committing information to memory , the majority find that most patients have some difficulty remembering information learned in the past . in a study of 426 patients with ms , 66 percent of patients had deficits in at least one recall task , while only 14 percent had encoding impairments ( difficulties making new memories).6 the encoding difficulties could be due to decreased processing speed or the inability to make sense of incoming information , both of which are very difficult to measure without an extensive battery of neurocognitive tests . people with ms also frequently experience compromised attention , and performance on tasks requiring sustained attention can reveal deficits in patients with mild to moderate cognitive impairment . additionally , it might be difficult for a person with ms to remember information required to complete a task if other distractions are present a considerable impairment in our multitasking society . because the amount of cns damage and the locations of lesions in the brain vary among patients , cognitive impairment is a somewhat heterogeneous comorbidity of ms . however , studying the cognitive facilities most commonly affected in patients with ms can help us gain insight into effective coping strategies and reveal areas of the brain and signaling pathways that might be logical therapeutic targets . this has important implications for managing and compensating for the daily problems that cognitive impairment causes . although there are no predictors of which patients will suffer ms - related cognitive deficits , disease duration and subtype , race , sex , and cognitive reserve may all play a role . relapsing - remitting ms ( rr - ms ) is the most common ; this subtype is the initial diagnosis of approximately 85 percent of all people with ms . in rr - ms , patients experience flare - ups of disease symptoms for a period of time , followed by a complete recovery or remission . the majority of patients diagnosed with rr - ms develop secondary - progressive ms ( sp - ms ) within 10 to 20 years . in sp - ms , as in rr - ms , patients experience flare - ups or relapses of disease symptoms , but there is a steady increase in disease severity between the relapses . the second most common subtype diagnosed at initial presentation is primary - progressive ms ( pp - ms ) , in which a patient experiences a steady increase in symptom severity from the time of disease onset . the final and most rare subtype of ms , progressive - relapsing ms ( pr - ms ) , involves intermittent relapses punctuating a steady progression of the disease . while patients with progressive subtypes of ms are more likely to experience cognitive impairment in general , further studies of patients with ppms and pr - ms are needed . earlier onset of ms increases a patient s chance of developing ms - related cognitive decline . although ms disease incidence is highest in populations from the northern united states , northern europe , canada , new zealand , and southern australia,7 people from all countries and of all races have been diagnosed with the disease . for example , caucasians have delayed symptom onset compared to latin - american and african - american patients.8 it is possible that because clinical manifestations are more severe in african - american patients , the cognitive findings may be part of what is overall a more aggressive disease course . race also affects ms impact on cognition : adult african - american patients with ms develop cognitive deficits earlier in the disease course compared to adult caucasian patients.9 this difference is also observed in pediatric ms patients . a 2010 study from the university of alabama at birmingham reported that african - american children affected by pediatric - onset ms performed worse on tests of complex attention and language compared to caucasian children with ms matched by age , disease severity , gender , and socioeconomic status.10 a better understanding of the race - based differences in disease characteristics could help physicians tailor treatments to ensure optimal responses . ms occurs in women more frequently than it does in men ; ratios of incidence range from 2:1 to 3:1 , depending on the geographical region . despite the elevated frequency in women , studies have shown that disease severity is typically higher and progression more rapid in men compared to women . additionally , the incidence and severity of cognitive deficits are higher in men.11 intelligence and education history contribute to the formation of cognitive reserve , which affects the brain s resilience in the presence of injury . previous studies in alzheimer s disease ( ad ) have shown that individuals with higher cognitive reserve are less likely to develop dementia.12 as with ad , ms patients with high levels of cognitive reserve are less likely to experience cognitive impairment . a study following patients with ms over a five - year period showed that those with a high cognitive reserve at baseline experienced no loss of cognitive function , while those who started with a low cognitive reserve suffered a significant cognitive decline.13 inflammation , neuronal degeneration , and lesion formation are likely among the causes of cognitive impairment in people with ms . gray matter ( neuron ) loss in the brain , specifically in the cerebral cortex ( the thin layer of cells that makes up the outer layer of the brain ) and the thalamus ( the relay station between the brain and the spinal cord , through which nearly all motor and sensory information travels ) , correlates with cognitive impairment.14 however , some patients with extensive brain lesions remain cognitively intact , while others with a low lesion load experience cognitive impairment . for example , some patients experience relatively subtle cognitive problems , such as word - finding difficulty , while others are so debilitated that they can not navigate roads in their own neighborhood or remember important phone numbers that used to be familiar to them . while the exact causes of cognitive impairment in ms are unknown , two factors often further impair cognitive performance in patients with the disease : depression and physical disability . the lifetime prevalence of depression within the general population is approximately 20 percent , while the prevalence in patients with ms is around 50 percent . a host of studies have linked depression in ms to impairments in learning , memory , processing speed , and executive function . the lesion location in an ms patient can affect depressive symptoms , as patients with brain lesions are more likely to experience depression compared to patients with spinal cord lesions . furthermore , lesions in the temporal lobe elevate a patient s likelihood of experiencing depression compared to lesions in other areas of the brain . temporal lobe lesions could be the common thread linking depression and cognitive impairment , as brain structures involved in learning and memory function , such as the amygdala and the hippocampus , are located in the temporal lobes . depression is predominantly caused by inflammation in the brain , which is a hallmark of ms.15 although researchers do not fully understand the pathogenesis of ms , they think inflammation precedes neuron death and myelin loss . one might hypothesize that depression would arise due to early inflammation , to be followed by degeneration of neurons and lesion development , leading to cognitive impairment . physical and cognitive effects of ms can occur separately , but there are relationships between them . about 10 percent of patients suffer from benign ms ( that is , their score is two or below on the expanded disability status scale for at least 10 years of disease duration ) , in which physical disease symptoms are absent . approximately 20 percent of patients with clinically benign ms , with a relatively mild disease course and accumulation of little disability over time , have cognitive impairment , while more than half of all ms patients suffer from cognitive impairment . the relationships among psychological factors , fatigue , physical disability , and cognitive impairment raise some very important questions : which of these aspects of disease arise first , and how do they interact ? does depression lead to fatigue , lowered motivation , and decreased medication compliance , thus compromising physical ability ? does physical disability or cognitive impairment make a patient more likely to become depressed and fatigued ? a better understanding of disease pathogenesis and improved diagnostic tools will help researchers answer these important questions in the future . researchers recently evaluated four pharmacological interventions intended to reverse cognitive impairment in patients with ms in large - scale ( n > 40 ) , double - blind , placebo - controlled clinical studies . researchers likely chose the compounds ginkgo biloba , donepezil , rivastigmine , and memantine due to anecdotal evidence and clinical success in treating memory impairment in patients with alzheimer s disease ( ad).16 two of these drugs , donepezil and rivastigmine , are designed to increase brain levels of acetylcholine ( ach ) , a neurotransmitter ( or chemical messenger ) that facilitates learning and memory processes . the third , memantine , which prevents abnormal activation of signaling pathways between neurons in the brain , has demonstrated success in treating early ad . ad studies using ginkgo biloba , a plant often used in traditional chinese medicine and reported to affect neurotransmitter signaling and neuroprotection , have shown mixed results ; some demonstrate cognitive - enhancing effects , while others show no effect compared to placebo . unfortunately none of these compounds demonstrated beneficial , reproducible improvements in cognitive function in clinical trials with ms.16 cognitive rehabilitation therapy is a nonpharmacological method of improving a specific cognitive skill through practice and training . the brain is a dynamic organ , and practicing a specific cognitive task strengthens the communication between neurons required for that task . results from trials focusing on cognitive rehabilitation in ms are mixed.17 researchers did find , however , that neurocognitive rehabilitation alleviates fatigue in patients with ms , and this also might help restore cognitive facilities such as attention span and working ( short - term ) memory . if a patient has irreversible cognitive deficits , the focus shifts from restoration to compensation . emotion - focused strategies , which help a patient regulate the emotional consequences of cognitive deficits , include accepting the deficit and obtaining social support from peers or trained professionals . problem - focused strategies alleviate some of the stress that cognitive impairment places on the individual through solutions to specific problems , such as using a tape recorder in meetings or lectures to aid in recall . a 2010 study demonstrated that patients with ms are unlikely to use positive coping strategies . instead , many avoid situations in which their cognitive impairment might be evident or obvious to others.18 this is particularly true if the patient had deficits in attention and executive functioning , which indicates that educating patients with ms on the benefits of positive coping strategies is an important and unmet need . in addition , researchers found that physical activity affects cognition in some patients with ms . reported benefits of yoga in populations of patients with ms include reduced fatigue and improved attention.19 a 2011 study demonstrated a positive correlation between physical activity and cognitive processing speed in ambulatory patients with ms.20 while definite conclusions can not be drawn from these studies , the positive association between physical activity and cognitive function ( which also has been demonstrated in healthy and ad populations ) suggests that physical activity might be an efficacious nonpharmacological treatment for cognitive impairment in ms . the search for a marker or specific cause of cognitive impairment in patients with ms has proven unsuccessful , and not knowing the exact mechanism(s ) makes it extremely difficult to develop a treatment . the advancement of brain - imaging techniques and the development of more sophisticated experimental disease models have allowed for a more thorough understanding of pathogenesis in ms , but the exact cause or trigger is still unknown . less than five years ago , researchers identified a cell that significantly contributes to ms development and progression . these t helper 17 immune cells are thought to contribute to cns inflammation and are located within the brain lesions of people with ms . despite recent advances , much work is still required to understand the cause of ms , the triggers for disease pathogenesis , and the mechanisms behind loss of myelin and neuronal degeneration . before the advent of magnetic resonance imaging ( mri ) in the 1980s and computed tomography ( ct ) scans in the 1970s , only extremely crude brain - imaging techniques ( such as plain x - rays ) makeshift temperature tests were commonly used to assist in making an ms diagnosis , as uninsulated neurons conduct poorly at elevated temperatures . thus , in bygone eras , many patients who presented with symptoms suggestive of ms were told to go home and get into a hot bathtub , and if their condition worsened significantly , then the diagnosis was confirmed as well as possible . thankfully , diagnostic tools in neurology have improved , and techniques such as mri can safely and accurately aid in diagnosing ms . mri uses a powerful magnet without harmful radiation to view successive sections of the brain and spinal cord with remarkable detail in any desired plane , much as one would slice a loaf of bread or a vegetable . hyperintense on mri images , called t2 hyperintense areas or simply t2 lesions , are thought to correspond to regions of inflammation , swelling , or injury . dye is injected into the bloodstream of a patient , and leakage of dye into the brain indicates disruption of the protective barrier between the brain and the blood . this disruption occurs in patients with ms due to active inflammation , and immune cells rush into the brain to do battle with what is mistakenly perceived as an adversary . however , when it comes to the prediction of clinical status , course , or outcome , mri has proven to be a surprisingly poor indicator.21 perhaps the injury that results in clinical symptoms happens in a more general way throughout the brain , and the number of hyperintense lesions seen on mri is not directly related to the severity of a patient s deficits . alternatively , it is possible that the brain is particularly good at routing neural impulses around regions actively under attack by the immune system . although mri highlights sites of inflammation , it does not show the compensatory mechanisms mediated by brain changes in signal routing or electrochemical boosting . nowhere has the lack of a correlation between mri findings and disability been more pronounced than in the poor prediction of cognitive impairment . whatever the cause , the clinical - mri paradox ( the lack of correlation between findings on mri and the level of clinical disability ) has played a role in slowing the development of novel and potent therapies , especially those targeting cognitive preservation or improvement . researchers have investigated a number of related neuroimaging techniques in an effort to overcome the limitations of standard mri in predicting cognitive performance . general measurements of either whole - brain or regional atrophy ( brain shrinkage ) , the final outcome of demyelination and neuronal injury throughout the brain , correlate with cognitive impairment better than mri imaging does . two other techniques that indicate tissue damage have been used with some preliminary success in correlating with cognitive impairment in ms : magnetization transfer imaging , which measures how charged aspects of water interact with charges at the molecular level in the brain , and diffusion tensor imaging , which measures how water diffuses through the brain . we recently had preliminary success , which is not yet published , in correlating the cognitive function of human ms patients with magnetic resonance spectroscopy ( mrs ) . unlike mri , which determines the structural integrity of the brain based on the water distribution , mrs measures chemical compounds in specific areas of the brain . since the brain s hippocampus has a prominent role in learning and memory functions , we used mrs to investigate the chemistry of this brain region in people with ms . we found very strong positive correlations between cognitive function and levels of n - acetylaspartylglutamate ( naag ) , an abundant signaling molecule in the brain . although human studies of this chemical await the development of a drug that safely elevates naag levels in humans , we found that elevating the levels of naag in an animal model of ms resulted in a two - fold improvement in learning and memory functions compared to untreated animals . there may be hope on the horizon for the development of pharmacological interventions for ms cognitive impairment . today s method of drug development for cognitive impairment in patients with ms evaluating drugs that have improved cognition related to other neurodegenerative diseases does not work . while this approach was the obvious first step , other methodologies must be developed if effective treatments are to be found . a promising new avenue for cognition - enhancing drug development in ms involves the use of the animal model experimental autoimmune encephalomyelitis ( eae ) . eae is not a novel model of disease ; since 1933 , it has helped scientists to learn about the disease process and to test treatments to improve physical symptoms . in 2010 , researchers demonstrated that this model of ms , in addition to mimicking the disease with regard to lesion formation and induction of physical disability , also causes cognitive impairment.22 this was the first study that measured cognitive function in the eae model , and it provides a valuable new method for the evaluation of novel treatments for ms - related cognitive impairment . the awareness of cognitive impairment in ms is improving among physicians , researchers , and patients . although past efforts to develop treatments for cognitive impairment in ms have largely been minimal or ineffective , improved research tools and imaging modalities and the emergence of more studies focusing on this problem are causes for optimism .

editor s note : physicians first noted the presence of cognitive impairment in patients with multiple sclerosis ( ms ) more than 160 years ago , yet it took clinicians until 2001 to codify a standard test to measure cognitive function . we now know that cognitive impairment occurs in up to 65 percent of people with ms and usually lessens their ability to remember previously learned information . so far , trials of drugs formulated to treat cognitive impairment have failed , but the authors remain optimistic that new approaches to diagnosis and drug development could lead to effective therapies in the future .

Cognitive Impairment in MS: An Overview The Affected Cognitive Processes Risk Factors for Cognitive Impairment The Roles of Depression and Physical Disability Current Treatment Options The Role of Imaging Improving Treatment Development